Molecular Espionage in Plant Immunity: A Pathogen Effector Eavesdrops on Salicylic Acid to Hijack the Host Transcriptional Mediator Hub

Molecular Espionage in Plant Immunity: A Pathogen Effector Eavesdrops on Salicylic Acid to Hijack the Host Transcriptional Mediator Hub | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Molecular Espionage in Plant Immunity: A Pathogen Effector Eavesdrops on Salicylic Acid to Hijack the Host Transcriptional Mediator Hub Qunqing Wang, Xinwei Tan, Yujing Sun, Zhaomei Qi, Yonghui Miao, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7305635/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Apr, 2026 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Pathogens disrupt transcriptional hubs to subvert host immunity, yet the spatiotemporal mechanisms remain enigmatic. Here, we report a pathogen effector hijacks the core eukaryotic transcriptional machinery by acting as a molecular mimic of host repressors, deploying this sabotage in synchrony with the plant immune rhythm. We discover the Phytophthora sojae nuclear effector directly targets the host Mediator complex. Crucially, PsAvh109 emulates the host repressor TOPLESS (TPL), competitively occupying the Mediator subunit MED21 and locks the MED21-MED6 interaction interface, repressing SA-responsive defense genes without triggering degradation. Strikingly, PsAvh109 expression is induced by host-derived SA, the very signal displaces TPL from MED21 to activate immunity. This enables the pathogen to deploy its molecular trap precisely when the host initiates defense, perpetuating transcriptional repression during a critical vulnerability window. This work establishes a paradigm-shifting 'sensing-response' model in plant-pathogen interactions, wherein Phytophthora pathogens deploy a dynamically synchronized virulence strategy. By eavesdropping on host-derived salicylic acid (SA), the pathogen induces nuclear effector PsAvh109 expression to precisely coincide with TPL dissociation from MED21. Crucially, PsAvh109 executes non-degradative steric blockade of the MED21-MED6 interface through high-fidelity molecular mimicry, perpetuating transcriptional repression without triggering host surveillance systems. This dual-layered strategy redefines effector function as a rhythm-matched molecular sabotage agent. Biological sciences/Microbiology/Pathogens Biological sciences/Plant sciences/Plant immunity/Effectors in plant pathology Salicylic acid Mediator PsAvh109 TOPLESS effector host immunity Phytophthora sojae Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION Plants deploy multi-layered innate immunity to thwart pathogen invasion, involving rapid recognition of microbial patterns and effectors, coupled with dynamic phytohormone signaling 1 , 2 . This defense hinges on transcriptional reprogramming that redirects resources from growth to immunity, involves the swift activation and deactivation of the transcription of numerous genes, which ultimately determines the fate of the plant 3 , 4 . Studies in plants have revealed extensive crosstalk between growth and defense signaling networks, coordinated largely by phytohormones such as salicylic acid (SA), jasmonic acid (JA), and auxin 5 . These hormones serve as key regulators that fine-tune transcriptional responses through a complex interplay of transcription factors (TFs) and repressors 6 . SA is particularly critical for resistance to biotrophic pathogens, acting through the activation of pathogenesis-related (PR) genes 7 , with several TF families, such as NPR1 (Nonexpressor of Pathogenesis-Related Genes 1) and TGA (TGACG-binding factors), identified as key regulators 8 . Among these, NPR1 functions as a central hub, integrating SA signals into downstream transcriptional networks. NPR1 forms a transcriptional activation complex with TGA factors, which bind to the TGACG motif (as-1 element), a conserved cis-element enriched in PR gene promoters 9 . This NPR1-TGA module bridges SA perception with transcriptional activation, and is essential for mounting effective immune responses against biotrophic pathogens 10 . Plants must balance growth and immunity to optimize fitness 5 . The defense response is tightly controlled under normal growth, while recognition of invading pathogens triggers a timely, accurate, and effective immune response, including transcriptional response to SA 11 . This shift is driven by transcriptional reprogramming, which governed by conserved regulators including the Mediator complex 12 . As an evolutionary bridge linking transcription factors (TFs) to RNA polymerase II in eukaryotes, Mediator orchestrates the assembly of pre-initiation complexes (PICs) to activate or repress defense genes 13 , 14 . Its core functional module, comprising head (MED6, MED8, MED11, MED17, MED18, MED19, MED20, MED22) and middle (MED1, MED4, MED7, MED9, MED10, MED21, MED31) subunits, facilitates signal integration to mediate immune transitions 15 – 19 . Central to this process is the physical interaction between MED6 and MED21, which facilitates PIC recruitment and is indispensable for transcriptional activation 20 – 23 . Studies in yeast have shown that the head module, particularly MED6, is essential for RNA Pol II activation by linking the head and middle modules to transmit regulatory signals 20 . MED6 adhering to the MED21 in the presence of RNA Pol II, and dissociating in its absence 21 – 23 . These interactions are indispensable for effective transcriptional regulation. Crucially, transcriptional repressors exploit this interface to enforce repression. In Arabidopsis , the corepressor TOPLESS (TPL) binds the N-terminal domain of MED21, which is the same region required for MED6 interaction, to obstruct Mediator assembly 24 , 25 . TPL integrates signals from multiple hormone pathways (e.g., JA via JAZ/NINJA 26,27 , auxin via AUX/IAA 28 , ethylene via ERF 29 , yet its role in SA signaling remains enigmatic. Although TPL interacts with SA-associated repressors (NPR3, NIMIN) 30 , 31 , direct evidence that TPL suppresses SA-responsive transcription is lacking, still is a critical knowledge gap in plant immunity. In the ongoing arms race between plants and pathogens, pathogens strategically target transcriptional hubs to subvert host defenses 32 . We unveil an evolutionary strategy where a pathogen effector mimics transcriptional corepressors to hijack the Mediator complex. Intriguingly, the nuclear-localized effector PsAvh109 from P. sojae , one of the most important pathogens of soybean ( Glycine max ), contributes to virulence by promoting biotrophic colonization and dampening host immunity. Avh109 has been shown to inhibit immune responses triggered by pathogen-associated molecular patterns (PAMPs) such as INF1 and XEG1 33,34 , but its virulence mechanism remains unknown. Here, we uncover a molecular mimicry strategy wherein PsAvh109 hijacks the TPL-MED21-MED6 regulatory node to lock defense transcription. The PsAvh109 functionally emulates the host transcriptional repressor TOPLESS (TPL), competitively displacing TPL from MED21 with > 3-fold higher affinity. This sabotage locks the MED21-MED6 interface to suppress defense transcription. Strikingly, this mechanism is conserved across Phytophthora species, revealing a paradigm for effector-mediated transcriptional sabotage. In the evolutionary arms race between plants and pathogens, effector-mediated disruption of transcriptional regulatory hubs represents a pivotal virulence strategy, but how pathogens achieve spatiotemporal precision in sabotaging these complexes remains enigmatic. Our p revious studies identified the transcriptional programming and functional synergy within the P. sojae RXLR effectors, an extraordinarily large effector superfamily with a conserved N-terminal RXLR (Arg-any amino acid-Arg-Leu) motif 33 . Avh109 and other representative “immediate-early” RXLR effectors was highly expressed even prior to infection and was further rapidly induced following infection, while some “early” RXLR effectors was weakly expressed prior to infection but induced 20- to 120-fold during the first 12 h of infection. Misexpression of key immediate-early or early effectors severely reduced the virulence of P. sojae transformants 33 . This also implies that such effectors are deliberately deployed in response to a certain infection stage. Here we report PsAvh109 is induced by host SA accumulation, uncovers a “sensing-response” paradigm wherein pathogens eavesdrop on host immune signals to synchronize effector deployment, redefining the co-evolutionary arms race. Thus, we uncover an unprecedented mechanism: the P. sojae nuclear effector PsAvh109 is induced by SA and hijacks the host Mediator subunit MED21 through molecular mimicry of the repressor TPL. This discovery establishes a SA-synchronized deployment of the effector to perpetuate immune suppression and a non-degradative steric blockade strategy that competitively locks the MED21-MED6 interaction interface. These insights redefine pathogen adaptation to host immunity and open avenues for engineering durable resistance in crops. Beyond canonical effector mechanisms involving enzymatic degradation or static interference, we unveil a sophisticated dynamically synchronized virulence strategy converging on two evolutionary innovations: First, the “sensing-response” synchronization, where pathogens exploit host immune signals as deployment cues, SA induction of PsAvh109 ensures effector injection coincides exactly with TPL displacement during defense activation. Second, a stealthy 'non-degradative interface lockdown' tactic, wherein PsAvh109 functionally mimics host repressors to sterically obstruct Mediator assembly without proteolytic turnover, evading immune detection while maximizing target conservation. This paradigm transforms our understanding of effector evolution from passive disruptors to active conductors orchestrating sabotage to the host’s defense melody. RESULTS SA induces PsAvh109 expression to synchronize effector deployment Numerous effector genes of P. sojae are expressed during the early stages of infection, coinciding with the critical window of salicylic acid (SA)-mediated resistance to biotrophic pathogens. To investigate whether SA functions as an inductive signal for RXLR effector gene expression, P. sojae germ tube was treated with 100 µM SA and followed RNA sequencing revealed that 2,608 genes were significantly upregulated and 2,993 downregulated following SA treatment compared to EtOH controls (Fig. S1a). Among previously annotated effectors, 64 were significantly upregulated, while only 6 were downregulated (Fig. S1b). PsAvh109 was among the most strongly induced effectors and was selected for further investigation due to its demonstrated ability to suppress immune responses triggered by pathogen-associated molecular patterns (PAMPs) such as INF1 and XEG1 33,34 . To monitor protein accumulation, we generated an in situ complementation strain (C-PsAvh109-6His-mCherry) by replacing the endogenous PsAvh109 gene. Exogenous SA treatment significantly increased PsAvh109-6His-mCherry protein levels relative to EtOH controls (Fig. 1 a, b). RT–qPCR analysis confirmed that SA induced PsAvh109 transcription in a concentration-dependent manner (Fig. 1 c). Furthermore, in soybean root hairs expressing GFP–NahG (which degrades endogenous SA 35 ), PsAvh109 transcript levels were markedly reduced (~ 43%) upon infection with strain P6497 (Fig. 1 d). Homologous genes in P. capsici (DVH05_001429) and P. parasitica (PPTG_19802) also exhibited SA-inducible expression (Fig. 1 e, f), indicating a conserved mechanism of SA responsiveness among oomycete pathogens. To investigate SA signal perception, we examined PsAvh109 expression in a PsGPA1 knockout mutant (KO-PsGPA1), which lacks the sole Gα subunit in P. sojae . SA treatment increased PsAvh109 expression by only 1.4-fold in the KO-PsGPA1 mutant, compared to 2.7-fold in the wild-type strain, implicating G-protein-coupled receptors (GPCRs) in SA-mediated effector activation (Fig. 1 g). Together, these findings demonstrate that P. sojae harbors an SA-sensing mechanism and that PsAvh109 expression is tightly regulated by host-derived SA. PsAvh109 requires nuclear localization to suppress immunity and promote the virulence of P. sojae PsAvh109 is conserved in Phytophthora species including P. infestans , P. parasitica , P. cactorum and P. capsici , in which all PsAvh109 homologs harbor a nuclear localization signal (NLS) (Fig. 2 a and S2 ). Mature PsAvh109 fused with green fluorescent protein (GFP) accumulated in the nucleus of Nicotiana benthamiana epidermal cells, co-localizing with nuclear marker histone 2B fused to mCherry protein (H2B-mCherry) (Fig. 2 b). NLS mutants, that mutated all conserved lysine (K) and arginine (R) residues to alanine (A) residues or replaced with a nuclear export signal (NES), named PsAvh109 AAA and PsAvh109 NES (Fig. 2 a), abolished nuclear targeting and seprated with H2B-mCherry (Fig. 2 b). In soybean hairy roots, PsAvh109—but not the NLS mutant PsAvh109 AAA —significantly increased P. sojae oospore production by 1.4-fold and biomass by 1.8-fold compared with the mCherry control (Fig. 2 c, d, e). Similarly, PsAvh109 enhanced P. capsici infection in N. benthamiana , resulting in a 1.3-fold increase in lesion diameter and a 2.3-fold increase in pathogen biomass, whereas PsAvh109 AAA showed no effect (Fig. S3a, b, c). PsAvh109 also suppressed INF1-triggered immune responses, a function lost in the NLS mutant (Fig. S3a). CRISPR/Cas9-mediated knockout of PsAvh109 (KO-PsAvh109) in P. sojae had no effect on mycelial growth or zoospore production (Fig. S3d, e, f), but markedly reduced virulence, as evidenced by smaller lesions on soybean hypocotyls and a 50% decrease in pathogen biomass compared with the wild-type strain. Pathogenicity was fully restored in the complementation strain (C-PsAvh109), but not in the nuclear localization-deficient mutant (C-PsAvh109 AAA ) (Fig. 2 f, g), indicating that PsAvh109 is essential for full virulence and that its function is dependent on nuclear localization. PsAvh109 targets the Mediator subunit MED21 to disrupt defense transcription Yeast two-hybrid screening identified soybean MED21 (GmMED21), a conserved subunit of the transcriptional mediator complex's middle module 36 , 37 , as a PsAvh109 interactor. Subsequent yeast two-hybrid assays confirmed that GmMED21 interacted with PsAvh109 but not with the PsAvh109 AAA mutant (Fig. 3 a). Notably, PsAvh109 selectively bound GmMED21, but not adjacent Mediator subunits (GmMED6, GmMED7, GmMED10, GmMED19, or GmMED25). Further analysis revealed that PsAvh109 also interacts with the Nicotiana benthamiana ortholog, NbMED21 (Fig. S4). In luciferase complementation imaging (LCI) assays, a strong luminescence signal was observed in N. benthamiana leaves co-expressing GmMED21-nLUC and cLUC-PsAvh109, but abolished in NLS mutants, confirming the interaction between GmMED21 and PsAvh109 (Fig. 3 b). Co-immunoprecipitation (Co-IP) experiments further corroborated the interaction, where GFP-PsAvh109 co-precipitated GmMED21-mCherry-3HA from N. benthamiana extracts. Neither GFP-PsAvh109 NES nor GFP-PsAvh109 AAA were detected in the immunoprecipitates, similar to the negative controls (Fig. 3 c). In contrast, in vitro pull-down assays showed that GmMED21-His interacted with GST-PsAvh109, as well as with the GST-PsAvh109 NES and GST-PsAvh109 AAA mutants (Fig. 3 d). These results indicate that PsAvh109 physically interacts with GmMED21 both in vivo and in vitro . However, the NLS mutations likely caused the protein translocation in vivo , rather than disrupting the physical interaction with GmMED21. To understand the roles of MED21 as the effector target, we inoculated the leaves with Agrobacterium tumefaciens strain carrying INF1 gene or P. capsici in RNAi- NbMED21 leaf tissues. The cell death triggered by INF1 was significantly attenuated in RNAi- NbMED21 (Fig. S5a). Compared with the wild-type control, silencing of NbMED21 resulted in a 1.2-fold increase in lesion diameter and a 2-fold increase in P. capsici biomass in infected leaves (Fig. S5a, b, c). Notably, RNAi-mediated silencing of NbMED21 abolished the virulence-promoting activity of PsAvh109, as PsAvh109 expression in RNAi- NbMED21 leaves failed to enhance P. capsici infection (Fig. S5d). Lesion diameter and pathogen biomass were comparable to those in GFP control tissues (Fig. S5e, f). These results suggest that PsAvh109 requires its interaction with MED21 to facilitate pathogen infection. Transcriptome analysis of PsAvh109-expressing soybean roots revealed significant suppression of SA-responsive genes. Compared with the GFP control group, a total of 841 differentially expressed genes (DEGs) were identified in the soybean overexpressing GFP-PsAvh109 (Fig. S6a). 157 downregulated genes enriched in defense response, response to salicylic acid, and the response to reactive oxygen species (ROS) (Fig. S6b). The up-regulated DEGs were significantly enriched in processes such as carbohydrate metabolic process and hydrogen peroxide catabolic process (Fig. S6b). Notably, PsAvh109 suppressed the expression of key SA-responsive immune genes in soybean, including members of the GmPR1 family and Salicylic acid (SA)-binding protein 2 ( GmSABP2 ), which are critical for SA-mediated defense (Fig. 3 e). RT–qPCR analysis confirmed that these genes were significantly upregulated in roots infected with the PsAvh109 knockout strain (KO-PsAvh109) compared to those infected with wild-type P. sojae (Fig. 3 f), while expression of the JA-responsive gene GmVSPa remained unchanged. Together, these results demonstrate that PsAvh109 hijacks MED21 to suppress SA-dependent transcriptional reprogramming. GmMED21 and GmMED6 act as the molecular switch of SA controlled gene transcription and blocked by PsAvh109 via competitive binding The head and middle modules constitute the functional core of the Mediator 38 , 39 . Functional validation showed that silencing GmMED21 or GmMED6 in soybean hairy roots reduced GmPR1 and GmSABP2 expression (Fig. S6c). The promoter regions (1 kb upstream of the start codon) of these genes all contained the as − 1 (TGACG) element, a conserved binding site for TGA transcription factors (Fig. S6d). Chromatin immunoprecipitation and qPCR (ChIP-qPCR) is employed to examine the MED21/MED6 recruitment to the GmPR1 promote in soybean transgenic hairy roots. The P1 (a sequence containing an as-1 element) region of the GmPR1 promoter showed strong binding affinity with both GmMED21 and GmMED6. No binding of GmMED21 or GmMED6 was detected with the promoter of control gene GmCYP2 (Fig. S6e). These results suggest that GmMED21 and GmMED6 are involved in the transcription of SA-responsive genes, positively regulating plant immunity. In addition, silencing of GmMED21 and GmMED6 led to a 1.7-fold increase in P. sojae oospore production and a 2-fold increase in pathogen biomass compared to the control (Fig. S6f, g, h). The interaction between MED21 and MED6 is crucial for the full activation of multiple activators 40 , 41 . In this study, we generated two GmMED21 truncation mutants: a N-terminal deletion mutant (GmMED21 16–139 ) and a short N-terminal fragment (GmMED21 1–15 ) (Fig. 4 a). Yeast two-hybrid and LCI assays mapped the MED21-MED6 interaction to the N-terminal 15-aa domain of MED21 (Fig. 4 b, c, d). Crucially, this same domain mediated binding to PsAvh109 (Fig. 4 b, d). The GmMED21 1–15 maintained interactions with PsAvh109 and MED6, whereas GmMED21 16–139 failed to interact with both (Fig. 4 b, c), indicating that the N-terminal 15 amino acids of GmMED21 might be the competitive binding of PsAvh109. In the LCI assays, a robust luminescence signal was observed in the leaves of N. benthamiana upon coinfiltration of MED21 and MED6. However, when additional GmMED21 1–15 -mCherry or full-length GmMED21-mCherry was expressed as a competitor, the luminescence intensity significantly decreased (Fig. S7a, b). Similarly, GST pull-down assays indicated that as the amount of GmMED21 1–15 -mCherry increased, the amount of GmMED21-His pulled down by GST-GmMED6 decreased (Fig. S7c). Functional assays showed that overexpression of GmMED21 1–15 in soybean root hairs significantly promoted P. sojae infection, as indicated by a 1.5-fold increase in oospore production and a 1.7-fold increase in pathogen biomass (Fig. S7d, e, f). Compared with EV-transformed roots, RT-qPCR showed that the expression levels of GmPR1-1, GmPR1-6, and GmSABP2 were lower in MED21 1–15 -overexpressing roots (Fig. S7g). Collectively, these results indicate that disrupting of MED21-MED6 interaction results in suppression of SA-responsive immunity. Given that both GmMED6 and PsAvh109 interact with the N-terminal domain of GmMED21, we hypothesized that PsAvh109 might compete with GmMED6 for binding to GmMED21, thereby disrupting the host resistance. Yeast three-hybrid (Y3H) assay indicates PsAvh109 (but not PsAvh109 AAA ) disrupted GmMED21-GmMED6 interaction (Fig. 4 e). Pull-down assays further validated that GST-PsAvh109 reduced MBP-GmMED6 binding to GmMED21-His in a dose-dependent manner (Fig. 4 f). Co-immunoprecipitation (Co-IP) assays revealed co-expression of PsAvh109-GFP decreased GmMED6-3Flag co-precipitation by GmMED21-HA (Fig. 4 g). In the LCI assay, co-expression of GFP–PsAvh109 with GmMED21–nLUC and cLUC–GmMED6 in N. benthamiana significantly reduced luminescence intensity compared to the GFP control, whereas GFP-PsAvh109 AAA had no effect (Fig. 4 h, i). ChIP-qPCR analysis revealed that PsAvh109 was significantly enriched at the P1 region of GmPR1 promoter (Fig. 4 j), consistent with that of GmMED21 (Fig. S6e). These findings suggest that PsAvh109 outcompetes MED6 for MED21 binding to perpetuate repression of SA-responsive genes. The N-terminal domain of MED21 is a hotspot for competitive binding by TPL and PsAvh109 Due to the growth defense trade-off in plants, the transcription of immunity-related genes is generally inhibited during the normal growth stage. In yeast, the general corepressor Tup1 hinders the recruitment of RNA polymerase II (Pol II) by competing with MED6 for binding to Srb7p, an analog of MED21, and that an intact N-terminus of Srb7p was needed for the interaction 24 . Notably, the residues of MED21 that interact with Tup1 are identical to those required for the transcriptional repression by the Arabidopsis corepressor TOPLESS (TPL) 24 , 25 . Previous reports have shown that the TPD domain of TPL (TPL N188 ) is the key domain responsible for the interaction with MED21, and repress the function of a transcriptional activator after auxin addition 22 . We hypothesized that repressors TPL might inhibit the interaction between MED21 and MED6, and the mechanism might be hijacked and exploited by Avh109. Y3H assays demonstrated that the interaction between GmMED21 and GmMED6 was evidently inhibited in the presence of GmTPL N188 (Fig. 5 a). Pull-down assays further confirmed that increasing amounts of GST-GmTPL N188 reduced the abundance of MBP-GmMED6 co-immunoprecipitated with GmMED21-His (Fig. 5 b). Co-IP assays showed that co-expressed GmTPL N188 -GFP significantly decreased GmMED6-3Flag co-precipitation by GmMED21-3HA (Fig. 5 c). In the LCI assays, the luminescence signal with GmMED21-nLUC and cLUC-GmMED6 was significantly attenuated when FLAG-GmTPL N188 was co - expressed (Fig. 5 d, e). This result suggest that GmTPL has a stronger interaction with GmMED21 compared to GmMED6 and thus suppresses the interaction between GmMED21 and GmMED6. ChIP–qPCR analysis revealed significant enrichment of GmTPL at the as-1 element (P1) of the GmPR1 promoter (Fig. 5 f), suggesting that GmTPL is recruited via its interaction with MED21. Given that both Avh109 and TPL N188 compete with MED6 for binding to MED21, we compared the interaction strengths between GmMED21 and its partners (GmMED6, GmTPL N188 , or PsAvh109). LCI assays revealed that co-expression of PsAvh109 with GmMED21 produced significantly stronger luminescence signals than co-expression of GmTPL N188 or GmMED6 with GmMED21 (Fig. 5 g, h). Furthermore, microscale thermophoresis (MST) measurements demonstrated that PsAvh109 binds GmMED21 with a dissociation constant (Kd) of 0.27 µM ± 0.04, whereas GmTPL N188 and GmMED6 exhibited higher Kd values of 0.76 µM ± 0.11 and 1.29 µM ± 0.38, respectively (Fig. 5 i). These data indicate that PsAvh109 exhibits higher binding affinity to GmMED21 than GmMED6 or GmTPL N188 . Hence, PsAvh109 might acts as a "molecular trap" locking MED21 by displacing TPL. Both N- and C-termini of PsAvh109 are required for its interaction with GmMED21 Phylogenetic analysis revealed the conservation of PsAvh109 across Phytophthora species, including P. parasitica, P. infestans , and P. capsici . All PsAvh109 homologs featured NLS, RxLR-dEER, and TP motifs. Moreover, the N-terminal 50 residues of PsAvh109 showed high conservation among the homologs, while the C-terminal 15 residues included three invariant residues and three additional highly conserved residues (Fig. S2b). To further delineate the domains of PsAvh109 required for interaction with GmMED21, we generated a series of PsAvh109 mutants by deleting specific motifs: the RxLR-dEER motif (designed as PsAvh109 − RxLR ), the TP motif (designed as PsAvh109 − TP ), the N-terminus of PsAvh109 (designed as PsAvh109 51–167 ), the C-terminus of PsAvh109 (designed as PsAvh109 21–152 ), or both termini (designed as PsAvh109 51–152 ) (Fig. S8a). We found that three mutants, PsAvh109 51–167 , PsAvh109 21–152 and PsAvh109 51–152 , lost their interactions with GmMED21 in yeast two-hybrid assays, but the other mutants did not (Fig. S8b), suggesting that the N-terminus and C-terminus of the mature effector protein were required for the interaction with MED21. Furthermore, LCI assays were conducted, revealing an undetectable luminescence signal in N. benthamiana leaves co-infiltrated with GmMED21-nLUC and cLUC-PsAvh109 51–152 , which lacks both termini. In contrast, a weak luminescence signal was observed from cLUC-PsAvh109 51–167 and cLUC-PsAvh109 21–152 (Fig. S8c). Additionally, all three mutants lost the ability to inhibit INF1-induced PCD, comparable to PsAvh109 AAA , while wild-type PsAvh109 strongly inhibited INF1-induced PCD (Fig. S8d). These results collectively indicate that both termini of PsAvh109 are necessary for full interactions with GmMED21. To identify the amino acid residues essential for the interaction between PsAvh109 and GmMED21, we conducted molecular modeling and docking analysis using AlphaFold3. The predicted 3D structure of GmMED21 exhibited high structural similarity to Arabidopsis MED21 (PDB ID: C0LU16). The analysis revealed that both the N-terminal (V23, R40, Y41, V54, N58, Q66, Y69, F71) and C-terminal (W154, F157, W160, Y161, D167) regions of PsAvh109 contribute to MED21 binding, forming a "headphone-like" structure that encircles the N-terminal region of MED21(Fig. S8e). This structural configuration likely accounts for the markedly higher binding affinity of PsAvh109 to MED21 compared to the native transcriptional repressor TPL. Notably, key interacting residues in the N-terminus (V23, R40) and C-terminus (F157, W160, D167) of PsAvh109 are highly conserved among its homologs, supporting the evolutionary conservation of its function in targeting MED21. Salicylic Acid releases the binding between TPL and MED21 without impacting Avh109 To explore whether SA alleviates TPL-mediated transcriptional repression of SA-responsive genes, we examined TPL protein stability in tobacco plants transiently expressing TPL following exogenous SA treatment. No significant difference in TPL accumulation was observed between SA-treated and EtOH control groups (Fig. 6 a), ruling out proteasomal degradation. Co-IP further demonstrated that exogenous SA significantly reduced the co-precipitation of GmMED21-3HA and GmTPL N188 -GFP compared to the EtOH control group (Fig. 6 b). In contrast, SA treatment had no effect on the co-precipitation of GmMED21-3HA and PsAvh109-GFP relative to the EtOH control group (Fig. 6 c). Additionally, LCI reporter assays revealed that SA treatment reduced LUC activity driven by the GmMED21-nLUC and cLUC-GmTPL N188 interaction to ~ 70% of the level observed in the EtOH control (Fig. 6 d). Conversely, SA treatment had no effect on LUC activity resulting from the interaction between GmMED21-nLUC and cLUC-PsAvh109 (Fig. 6 d). In ChIP-qPCR assays, SA treatment (100 µM) significantly reduced GmTPL enrichment at the as-1 sites (P1) of GmPR1 promoter compared to EtOH controls, PsAvh109 occupancy remained unchanged (Fig. 6 e). In summary, these findings demonstrate that SA mediates the disassembly of TPL-mediated blockade on MED21-MED6 interaction, whereas PsAvh109 competitively locks the MED21-MED6 interaction interface with higher affinity. Hence, we demonstrate this dynamic process (Fig. 7 ): TPL constitutively represses SA-responsive genes by occupying the MED21-MED6 interface; SA accumulation displaces TPL, enabling Mediator assembly and defense activation; PsAvh109 is induced by host SA and competitively binds MED21 with higher affinity than TPL or MED6, perpetuating transcriptional repression. DISCUSSION A paradigm for effector-mediated synchronized transcriptional sabotage Our study unveils a molecular strategy wherein Phytophthora effectors exploit the Mediator complex as an Achilles’ heel to suppress SA immunity. That is a three-tiered virulence strategy converging on the eukaryotic Mediator complex: (1) effector-as-repressor-mimic: PsAvh109 emulates TPL’s structure to occupy the MED21 N-terminus with higher affinity than its host counterpart; (2) non-degradative interface lockdown: steric blockade of MED21-MED6 assembly represses defense transcription without triggering proteolysis; (3) host-signal-synchronized deployment: SA induction of PsAvh109 ensures effector injection coincides precisely with TPL dissociation (Fig. 7 ). This “sensing-response” tactic redefines effector function: rather than static interference, PsAvh109 acts as a molecular spy that eavesdrops on host immunity to sabotage transcriptional activation at its most vulnerable moment. We demonstrate that PsAvh109 functions as a structural mimic of the host corepressor TPL, competitively occupying the N-terminal domain of MED21, a critical hub for Mediator assembly, with 3-fold higher affinity than TPL itself (Kd = 0.27 µM vs. 0.76 µM). This effectively "locks" the MED21-MED6 interface (Fig. 5 ), disrupting transcription of key defense genes (e.g., GmPR1 , GmSABP2 ). PsAvh109 operates through steric blockade without triggering degradation—a non-destructive tactic that may evade host surveillance. Conservation of this mechanism across P. capsici and P. parasitica (Fig. 1 e, f) suggests that targeting the Mediator hub represents a universal virulence strategy in oomycetes. Reconceptualizing pathogen adaptation to host immunity The induction of PsAvh109 by host-derived SA (Fig. 1 ) reveals a sophisticated "sensing-triggering" paradigm in pathogen evolution. Unlike traditional effectors constitutively expressed, PsAvh109 deployment is dynamically synchronized with host immune activation: as SA accumulates and displaces TPL from MED21 (Fig. 6 b, d), the pathogen exploits this temporal window to inject its molecular mimic, perpetuating transcriptional repression (Fig. 7 ). We propose that Phytophthora possesses SA receptors (possibly RLPs 42 or kinase cascades) that convert host signals into effector expression, which could be a frontier for future identification. This strategy is unprecedented in eukaryotes. The programmed deployment of Phytophthora effector repertoire is at least partly in response to the host's disease resistance rhythm, which enriches our model from over a decade ago 33 . Redefining pathogen tactics in the arms race Pathogens typically disrupt Mediator via degradative mechanisms (e.g., HaRxL44-mediated MED19a turnover). PsAvh109 exemplifies a distinct evolutionary solution: high-fidelity molecular mimicry enabling precision occupancy of a critical protein interface. This strategy minimizes host surveillance risks—unlike degradation, steric blockade leaves no proteolytic debris for immune detection—while maximizing target conservation across plant species. Crucially, synchronizing effector expression with host SA transforms virulence into a dynamic, rhythm-matched process, akin to pathogens “orchestrating sabotage to the host’s defense melody.” The structural emulation of host transcriptional repressors by PsAvh109 represents a highly evolved virulence strategy. PsAvh109 functions as a "pseudo-repressor" that physically occupies TPL’s binding site on MED21 (Fig. 4 ). Such strategy minimizes host surveillance risks while maximizing immune suppression efficiency. Crucially, both strategies exploit conserved eukaryotic regulatory nodes: Pre-initiation complex blockade in plants (steric inhibition of Mediator assembly) despite targeting distinct mediator subunitnes. Evolutionary convergence in transcriptional sabotage While little pathogen effectors are known to disrupt the plant Mediator complex, PsAvh109 employs a fundamentally distinct mechanism compared to the well-characterized downy mildew effector HaRxL44. HaRxL44 triggers proteasome-dependent degradation of MED19a, thereby shifting the balance of defence transcription from SA-responsive defence to JA/ET-signalling 32 . In contrast, the strategy employed by PsAvh109, via hijacking a transcriptional hub through molecular mimicry, unlike the enzymatic modifications. PsAvh109 operates through high-affinity competitive binding, a convergence toward similar functional outcomes (transcriptional repression) via divergent evolutionary paths. This comparison underscores a paradigm shift in effector evolution: whereas degradation-based effectors (e.g., HaRxL44) cause pleiotropic effects, PsAvh109 exemplifies "precision sabotage" through transient occupancy of a critical protein-protein interaction interface. The Mediator: A universal achilles’ heel in Eukaryotes The functional parallelism of PsAvh109 and HaRxL44 underscores transcription regulation as a universal Achilles’ heel across eukaryotes, repeatedly hijacked through pathogen-specific molecular mimicry. This highlights the Mediator complex as a universal vulnerability across kingdoms, targeted through pathogen-specific "molecular keys". The conservation of PsAvh109’s mechanism across Phytophthora species underscores MED21 targeting as a universal virulence strategy in oomycetes. Beyond plants, Mediator’s architectural conservation from yeast to humans suggests this hub could be similarly exploited by diverse eukaryotic pathogens. Fungal effectors often target transcriptional regulators, yet none demonstrate PsAvh109’s triad of mimicry, interface blockade, and signal synchronization. We propose that effector-mediated hijacking of Mediator’s core may extend to animal pathogens, positioning this complex as a universal vulnerability in eukaryotic infections. Decoding the SA-sensing paradigm: Future frontiers Our discovery that host-derived SA directly induces PsAvh109 expression (Fig. 2 a-f) unveils a sophisticated "danger sensing" capability in Phytophthora . How Phytophthora precisely senses SA remains enigmatic. While SA perception in plants is mediated by receptors like NPR1 9 , the SA sensor of pathogen remains enigmatic. We propose two mechanistic hypotheses for future investigation: Direct co-option of host receptors: Does Phytophthora exploit secreted ligands to manipulate NPR1-like pathways? Pathogen-encoded SA receptors: Are there oomycete homologs of host SA-binding proteins (e.g., SABP2) or novel receptors that transduce SA signals? Phytophthora likely possess SA receptors—potentially resembling P. sojae ’s isoflavone chemoreceptors 42 or or sterol-sensing receptor-like kinases 43 . Resolving these questions will illuminate how pathogens evolve to "eavesdrop" on host immunity-a critical step toward disrupting this communication channel. Identifying this sensor will unveil how pathogens “tap into” host signaling networks, which is a leap toward disrupting communication in the arms race. Potential receptors like G-protein coupled receptors 42 or receptor-like kinases 43 should be screened. Toward engineering broad-spectrum resistance: Engineering next-generation disease resistance by protecting the Mediator hub Convergent evolution of effector-mediated MED21 targeting underscores its role as a critical vulnerability in eukaryotic transcriptional regulation. Our mechanistic dissection of PsAvh109’s sabotage strategy exposes actionable targets for breeding Phytophthora -resistant crops. We propose two complementary approaches leveraging the MED21-MED6-TPL axis: 1. Competitive Decoys: Expression of engineered MED21 decoys to shield the Mediator hub could shield Mediator without pleiotropy. Notably, expression of the MED21 1–15 peptide competitively blocked PsAvh109 binding and also interfered with MED6 association (Fig. 4 ), prompting us to develop a refined peptide decoy that selectively binds PsAvh109 without perturbing MED6, aiming to mitigate Phytophthora pathogenicity. It should be noted that the virulence of the KO-Avh109 mutant of the P. sojae is significantly reduced (Fig. 2 f, g). This proof-of-concept suggests that engineered MED21 variants could generate durable resistance. Importantly, as Mediator architecture is conserved across crops 25 , this approach may extend beyond soybean. 2.Scalability across crops: Mediator architecture conservation: MED21 N-terminal domain shares 92–97% aa identity in maize/wheat. Search for effective variants of MED21 from natural genetic materials, maintaining the natural function while not binding to PsAvh109. This "molecular shield" strategy preserves native Mediator function while excluding effectors, overcoming pleiotropy risks of knocking down entire subunit Protecting the transcriptional core complex rewrites the rules of plant defense engineering—shifting from gene-for-gene resistance to fortress defense at the epicenter of immunity. Limitations of the study While we establish the role of PsAvh109 in suppressing SA responses, its impact on other hormone pathways (e.g., JA-ET crosstalk) warrants investigation. Does MED21 hijacking create an “immune polarization trap” favoring necrotrophy? The structural basis for PsAvh109’s superior affinity for MED21 also demands cryo-EM analysis. Resolving PsAvh109-MED21 and TPL-MED21 complexes via cryo-EM will reveal atomic determinants of mimicry and affinity divergence, and guiding decoy optimization. Materials and Methods Plant and microbe cultivation N. benthamiana plants were cultivated in a greenhouse at 25 °C with a cycle of 14-hours daylight and 10-hours darkness. Etiolated soybean seedlings were incubated at 25 °C for 5 days in darkness. P. sojae (P6497) and P. capsici (LT1534) strains were cultured in a 10% vegetable (V8) juice medium at 25 °C for 5-7 days. N. benthamiana transient expression and Virus-induced gene silencing The plasmid constructs were introduced into Agrobacterium tumefaciens GV3101 using chemical methods 34 . The A. tumefaciens transformants cell suspensions (OD 600 = 0.6) were infiltrated into the leaves of N. benthamiana . The Tobacco Rattle Virus (TRV) mediated VIGS system was employed to silence genes in N. benthamiana 44 . A gene-specific fragment of approximately 300 bp was cloned into the pTRV2 vector and then introduced into Agrobacterium strain GV3101. Subsequently, transformants were used to infiltrate four-leaved N. benthamiana plants with a 1:1 mixture of the GV3101 strain containing pTRV1. Fully expanded leaves of the silenced plants were then utilized for inoculation and subsequent quantitative PCR analysis. Induction of plant cell death and Phytophthora infection in N. benthamiana Target gene was cloned into pBIN or p1300 vectors and then introduced into Agrobacterium strain GV3101. The A. tumefaciens transformants cell suspensions (OD 600 = 0.6) were infiltrated into 6-week-old N. benthamiana leaves. Agrobacterium tumefaciens strain GV3101 carrying INF1 were co-infiltrated into the leaves of N. benthamiana . The ensuing cell death response of the leaves was then observed. Otherwise, the leaves were inoculated with mycelia of P. capsici at 12 hours after infiltration (hpi). P. capsici leaf lesions were photographed under UV light at 36 or 48 hpi, while leaf discs for RNA analysis following inoculation were collected at 6 hpi. The accumulation of the protein encoded by the infiltrated constructs was confirmed by immunoblotting, and plant transcript levels were assessed by RT-qPCR. P. sojae culture and transformation The wild type P. sojae strain P6497 was cultured in liquid 10% V8 medium for 3-5 days. The mycelium was then subjected to three washes with sterilized water to thoroughly remove any residual medium. Zoospores were generated from the mycelium by immersion in sterilized water at 25 °C for 9-12 hours. To produce infected tissue, seeds of the susceptible soybean variety Williams were germinated for 6 days at 25 °C in darkness indoors. The six-day-old seedlings were used for inoculation. One hundred zoospores of WT or the PsAvh109 mutants were inoculated onto soybean hypocotyls; the inoculated seedlings were then incubated at 25 °C in darkness for 2-3 days. More than 6 etiolated soybean hypocotyls were tested for each biological replicate. The infected seedlings were photographed at 2-3 days post-inoculation (dpi), and the samples were harvested and stored at -80 °C for P. sojae biomass detection. The CRISPR/Cas9 method was employed to generate mutants of PsAvh109 in P. sojae , along with PsAvh109-6His complementation strains, following established protocols 45 46 . RNA extraction and transcript level analysis To quantify gene transcript levels, total RNA from the P. sojae and plant samples was extracted following the protocol outlined in the Plant Total RNA Isolation Kit (Vazyme). First-strand cDNA was synthesized using HiScript III reverse transcriptase, following the manufacturer’s instructions provided with the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme). Real-time PCR was employed to measure gene transcript levels, following the manufacturer’s instructions for the 2×SYBR Premix URTAQ Kit (Vazyme). Specific primers, designed based on gene sequences, were utilized, and the actin gene served as an endogenous control. PsActin, NbEF1a , and GmCYP2 were employed as internal controls. Primer sequences are provided in Supplementary Table 1. RNA-seq analysis For RNA sequencing, total RNA was extracted from soybean hairy roots. The NEB Next Ultra II Directional RNA Library Prep Kit (NEB, E7760) was employed to generate cDNA libraries. Three biological replicates were conducted for both GFP and GFP-PsAvh109 overexpressing soybean hairy roots. Differentially expressed genes (DEGs) were identified using the criteria of a fold-change ≥ 2 or ≤ -2 across samples, together with a p-value < 0.05. To assess significant DEGs, soybean Gene Ontology (GO) term annotations were employed. The criteria for meaningful enrichment were set at q (false discovery rate) < 0.05 and p-value < 0.01. Fold enrichment was used to calculate the -Log 10 (p-value). Confocal Microscopy N. benthamiana leaf discs were placed in water and analyzed using an LSM 880 laser scanning microscope (Carl Zeiss, Germany). Excitation wavelengths of 488 nm and 580 nm were utilized to visualize GFP and mCherry fluorescence, respectively, with emission wavelengths ranging from 490 nm to 530 nm for GFP and from 590 nm to 620 nm for mCherry. Soybean Hairy Roots Transformation and Infection Assay Soybean (cv. Williams) seeds were grown under a 16-hour light and 8-hour dark photoperiod for 5-6 days. Explants for soybean hairy root transformation were selected from cotyledons that had not fully extended. Sterilized cotyledons were washed with autoclaved ddH 2 O five times. Under sterile conditions, a 3-5 mm small wound was created on the lower epidermis of the cotyledon near the hypocotyl with a sterile surgical knife. The wound was then inoculated with Agrobacterium rhizogenes strain K599 carrying the relevant overexpression plasmid. Subsequently, the cotyledons were cultured on MS medium at 25 °C for 3 to 4 weeks to induce the formation of healing tissue and the emergence of soybean hairy roots at the wound site. Production of stable transgenic soybean plants PsAvh109 was incorporated into a soybean overexpression vector (pDN018). The resulting construct was subsequently introduced into Agrobacterium tumefaciens strain EHA105 through heat shock. As reported previously, the soybean cultivar Williams served as the recipient for genetic transformation 47 . To confirm successful transformation, PCR was conducted using gene-specific primers, and RNA was extracted from leaf tissues to assess the expression of PsAvh109 in the transgenic plants. Protein Pull-Down and Co-Immunoprecipitation (Co-IP) Assays For in vitro pull-down assays, GmMED21 was cloned into pET28a with a C-terminal His-tag, while PsAvh109, PsAvh109 AAA , PsAvh109 NES , and GmMED6 without signal peptides were cloned into pGEX4T-1 fused with a N-terminal GST-tag. GmMED6 also was cloned into Pmal-c2X with an N-terminal MBP tag. GmMED21 1-15 was cloned into p1300 with a C-terminal mCherry-3xHA tag. The plasmids were transformed into E. coli BL21 and incubated at 25 °C. Each strain was induced to express the relevant protein using 0.5 mM IPTG for 10h at 25 ºC. For GST pull-downs, bacterial lysates containing GST-tagged proteins were purified with glutathione Sepharose 4B resin. Then the resin was incubated with bacterial lysates containing His-tagged proteins for 3 h at 4 °C, and then the resin was washed with PBS five times. Alternatively, for His pull-downs, bacterial lysates containing His-tagged proteins were purified with Ni-NTA resin, and then incubated with bacterial lysates containing GST-tagged proteins for 3 h at 4 °C, and then the resin was washed with PBS five times. Outputs of both pull-down protocols were analyzed by Western blotting using anti-GST and anti-His antibodies, and in some experiments, anti-MBP and anti-HA antibodies. For in vivo Co-Immunoprecipitation (Co-IP) assays, mature PsAvh109, PsAvh109 AAA and PsAvh109 NES sequences were cloned into the pBIN vector fused with GFP at the N-terminus. GmMED21 was cloned into the p1300 plasmid fused with 3×HA-mCherry at the C-terminus. Transient co-expression of proteins in N. benthamiana leaves was achieved by Agrobacterium -mediated transformation. The total proteins of the N. benthamiana leaves were incubated with anti-HA magnetic beads at 4°C for 12 h. After washing five times with 1×PBS buffer, the magnetic beads with bound proteins were boiled with 1×loading buffer for 10 min. The co-precipitated proteins were detected using anti-GFP and anti-HA antibodies. Luciferase complementation imaging assays PsAvh109, PsAvh109 AAA and PsAvh109 NES without signal peptide were cloned into the p1300 plasmid fused with the C-terminus of luciferase. GmMED21 was cloned into the p1300 plasmid fused with the N-terminus of luciferase. The proteins were transiently co-expressed in N. benthamiana leaves by Agrobacterium -mediated transformation for 36 h, then the leaves were sprayed with 1 mM D-luciferin. Luminescence was observed using a plant live imaging system. To quantify luciferase activity, N. benthamiana leaf samples were collected at 36 hpi and incubated with 1 mM D-luciferin in 96-well plates. Luciferase activity was subsequently quantified using a microplate luminometer (Promega). Data were analyzed using GraphPad Prism 8.0 software, and statistical analysis was based on one-way ANOVA for multiple comparisons. Yeast two-hybrid (Y2H) screening and assay. The Y2H screen with pGBKT7-PsAvh109 was performed as follows. The PsAvh109 gene without the signal peptide was cloned into the yeast vector pGBKT7 (Clontech). The soybean cDNA library was constructed in pGADT7 using total RNA extracted from soybean hypocotyl tissues collected 12 and 24 h after inoculation with P. sojae zoospores (Clontech). More than 6 × 10 6 primary yeast clones (providing three-fold coverage) were screened using pGBKT7-PsAvh109 as the bait. Potential yeast transformants containing cDNA clones interacting with PsAvh109 were selected using the SD-TLHA selective medium (synthetic defined media (SD) without Trp, Leu, His, Ade). Yeast Three-hybrid (Y3H) assay The Y3H assay was conducted using the pBridge vector system following the instructions provided (Clontech) (Fig. S6). The pBridge and pGADT7 constructs were co-transfected into Y2H Gold cells and cultured on SD-TL plates at 28°C. After 3 days of incubation, positive colonies were screened and resuspended to OD 600 = 0.1, 0.01, and 0.001, respectively, in 0.9% NaCl. The diluted yeast strains were grown on SD/ -Trp -Leu, SD/ -Trp –Leu -His, and SD /-Trp –Leu –His -Met plates at 28°C for 5 days. Microscale thermophoresis (MST) Protein binding affinities between GmMED21 and PsAvh109, GmTPL, or GmMED6 were measured via MST using a Monolith NT.115 instrument (NanoTemper, Germany). Briefly, recombinant and purified His - GmMED21 was diluted in PBST buffer (10 mM Tris - HCl pH 7.5, 150 mM NaCl, 10 mM MgCl₂, and 0.05% [v/v] Tween 20). The buffer contained the Monolith His - tag labeling kit - RED - tris - NTA second - generation dye (NanoTemper, Cat. No.: MOL018). The mixture was incubated at room temperature for 30 min. RED - tris - NTA - labeled His - GmMED21 (concentration range from 50 to 0.00153 μM) was incubated with GST (concentration range from 150 to 0.00457 μM), GST - PsAvh109 (concentration range from 9.375 to 0.000286 μM), GST - GmTPL (concentration range from 37.5 to 0.00114 μM), or GST - GmMED6 (concentration range from 600 to 0.0183 μM) at room temperature for 5 min in equal volumes. Subsequently, the samples were loaded onto Monolith NT.115 capillaries and measured at 25 °C with 60% MST power using the Monolith NT.115 instrument (NanoTemper, Germany). The curves were fitted and the dissociation constant (Kd) values were calculated using Nano Temper analysis software (v.2.3). ChIP-qPCR assay Soybean hairy roots expressing 3Flag fused proteins were used for ChIP experiments 48 . ChIP - qPCR was performed on three biological replicates, and the results were normalized to the input DNA. Two independent ChIP - qPCR experiments were conducted, and the results were similar. The primers used for ChIP - qPCR are listed in Supplementary Table 1. Quantification and statistical analysis The quantification of protein abundance by Western blots was conducted using Image J software. Quantitative data from all assays were presented as mean ± standard deviation (SD). Statistical analyses were carried out using Student's t-test or one-way analysis of variance (ANOVA) test to determine the significance of observed differences. Alternatively, Tukey's test for multiple comparisons was performed, and significant differences (p < 0.05) were indicated by lower case letters. Declarations ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (32172387, 32172049, 31972249); Taishan Scholar Project (tsqn202211093); Natural Science Foundation of Shandong Province (ZR2021YQ20, SYS202206 and ZR2024MC195); Taishan Industrial Experts Program (No. tscy20221172), and Shandong Province First-class Discipline Construction "811" Project (SKL81121 and SKL81127). Shandong Guocangjian Biotechnology Co., Ltd., provide technical support for this research work. We are grateful to Professor Dajian Zhang for providing genetically modified soybeans. AUTHOR CONTRIBUTIONS Q.W and Q.X designed and supervised the study and wrote the manuscript. X.T performed the experiments, analyzed the data and wrote the manuscript. Y.M, X.H, Y.W, H.L and W.L performed the experiments and analyzed the data. Y.S, Z.Q and Y.C performed data analysis and participated in manuscript revision. Y.C constructed transgenic soybeans. Q.W, Y.C and Q.X revised the manuscript and provided the funding for this research. All authors contributed to the article and approved the submitted version. DECLARATION OF INTERESTS The authors declare no competing financial interest. References Wang, Y., Pruitt, R.N., Nürnberger, T., and Wang, Y. (2022). Evasion of plant immunity by microbial pathogens. Nat. Rev. Microbiol. 20 , 449-464. https://doi.org/10.1038/s41579-022-00710-3. Jones, J., and Dangl, J. (2006). The plant immune system. Nature 444 , 323-329. https://doi.org/10.1038/nature05286. Spoel, S.H., and Dong, X. (2012). How do plants achieve immunity? Defence without specialized immune cells. Nat. Rev. Immunol. 12 , 89-100. https://doi.org/10.1038/nri3141. Maleck, K., Levine, A., Eulgem, T., Morgan, A., Schmid, J., Lawton, K.A., Dangl, J.L., and Dietrich, R.A. (2000). The transcriptome of Arabidopsis thaliana during systemic acquired resistance. Nat. Genet. 26 , 403-410. https://doi.org/10.1038/82521. He, Z., Webster, S., and He, S.Y. (2022). Growth-defense trade-offs in plants. Curr. Biol. 32 , R634-R639. https://doi.org/10.1016/j.cub.2022.04.070. Couto, D., and Zipfel, C. (2016). Regulation of pattern recognition receptor signalling in plants. Nat. Rev. Immunol. 16 , 537-552. https://doi.org/10.1038/nri.2016.77. Durrant, W.E., and Dong, X. (2004). Systemic acquired resistance. Annu. Rev. Phytopathol. 42 , 185-209. https://doi.org/10.1146/annurev.phyto.42.040803.140421. Powers, J., Zhang, X., Reyes, A.V., Zavaliev, R., Ochakovski, R., Xu, S.L., and Dong, X. (2024). Next-generation mapping of the salicylic acid signaling hub and transcriptional cascade. Mol. Plant. 17 , 1558-1572. https://doi.org/10.1016/j.molp.2024.08.008. Mou, Z., Fan, W., and Dong, X. (2003). Inducers of plant systemic acquired resistance regulate NPR1 function through redox changes. Cell 113 , 935-944. https://doi.org/10.1016/s0092-8674(03)00429-x. Després, C., DeLong, C., Glaze, S., Liu, E., and Fobert, P.R. (2000). The Arabidopsis NPR1/NIM1 protein enhances the DNA binding activity of a subgroup of the TGA family of bZIP transcription factors. Plant Cell 12 , 279-290. https://doi.org/10.1104/pp.001057. Katagiri, F. (2004). A global view of defense gene expression regulation--a highly interconnected signaling network. Curr. Opin. Plant Biol. 7 , 506-511. https://doi.org/10.1016/j.pbi.2004.07.013. An, C., and Mou, Z. (2013). The function of the Mediator complex in plant immunity. Plant Signal. Behav. 8 , e23182. https://doi.org/10.4161/psb.23182. Kim, Y.J., Björklund, S., Li, Y., Sayre, M.H., and Kornberg, R.D. (1994). A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II. Cell 77 , 599-608. https://doi.org/10.1016/0092-8674(94)90221-6. Soutourina, J., Wydau, S., Ambroise, Y., Boschiero, C., and Werner, M. (2011). Direct interaction of RNA polymerase II and mediator required for transcription in vivo. Science 331 , 1451-1454. https://doi.org/10.1126/science.1200188. Kidd, B.N., Cahill, D.M., Manners, J.M., Schenk, P.M., and Kazan, K. (2011). Diverse roles of the Mediator complex in plants. Semin. Cell Dev. Biol. 22 , 741-748. https://doi.org/10.1016/j.semcdb.2011.07.012. Poss, Z.C., Ebmeier, C.C., and Taatjes, D.J. (2013). The Mediator complex and transcription regulation. Crit. Rev. Biochem. Mol. Biol. 48 , 575-608. https://doi.org/10.3109/10409238.2013.840259. Richter, W.F., Nayak, S., Iwasa, J., and Taatjes, D.J. (2022). The Mediator complex as a master regulator of transcription by RNA polymerase II. Nat. Rev. Mol. Cell Biol. 23 , 732-749. https://doi.org/10.1038/s41580-022-00498-3. Dotson, M.R., Yuan, C.X., Roeder, R.G., Myers, L.C., Gustafsson, C.M., Jiang, Y.W., Li, Y., Kornberg, R.D., and Asturias, F.J. (2000). Structural organization of yeast and mammalian mediator complexes. Proc. Natl Acad. Sci. USA. 97 , 14307-14310. https://doi.org/10.1073/pnas.260489497. Mathur, S., Vyas, S., Kapoor, S., and Tyagi, A.K. (2011). The Mediator complex in plants: structure, phylogeny, and expression profiling of representative genes in a dicot ( Arabidopsis ) and a monocot (rice) during reproduction and abiotic stress. Plant Physiol. 157 , 1609-1627. https://doi.org/10.1104/pp.111.188300. El Khattabi, L., Zhao, H., Kalchschmidt, J., Young, N., Jung, S., Van Blerkom, P., Kieffer-Kwon, P., Kieffer-Kwon, K.-R., Park, S., and Wang, X. (2019). A pliable mediator acts as a functional rather than an architectural bridge between promoters and enhancers. Cell 178 , 1145-1158. e1120. https://doi.org/10.1016/j.cell.2019.07.011. Kim, G.S., and Lee, Y.C. (2018). Postrecruitment Function of Yeast Med6 Protein during the Transcriptional Activation by Mediator Complex. Biochem. Res. Int. 2018 , 6406372. https://doi.org/10.1155/2018/6406372. Nayak, S., and Taatjes, D.J. (2022). SnapShot: Mediator complex structure. Cell 185 , 3458-3458.e3451. https://doi.org/10.1016/j.cell.2022.07.011. Zhang, H., Chen, D.H., Mattoo, R.U.H., Bushnell, D.A., Wang, Y., Yuan, C., Wang, L., Wang, C., Davis, R.E., Nie, Y., and Kornberg, R.D. (2021). Mediator structure and conformation change. Mol. Cell 81 , 1781-1788.e1784. https://doi.org/10.1016/j.molcel.2021.01.022. Gromöller, A., and Lehming, N. (2000). Srb7p is a physical and physiological target of Tup1p. Embo J. 19 , 6845-6852. https://doi.org/10.1093/emboj/19.24.6845. Leydon, A.R., Wang, W., Gala, H.P., Gilmour, S., Juarez-Solis, S., Zahler, M.L., Zemke, J.E., Zheng, N., and Nemhauser, J.L. (2021). Repression by the Arabidopsis TOPLESS corepressor requires association with the core mediator complex. eLife 10 . https://doi.org/10.7554/eLife.66739. An, C., Deng, L., Zhai, H., You, Y., Wu, F., Zhai, Q., Goossens, A., and Li, C. (2022). Regulation of jasmonate signaling by reversible acetylation of TOPLESS in Arabidopsis . Molecular Plant 15 , 1329-1346. https://doi.org/10.1016/j.molp.2022.06.014. Zhu, X., Zhao, Y., Shi, C.M., Xu, G., Wang, N., Zuo, S., Ning, Y., Kang, H., Liu, W., Wang, R., et al. (2024). Antagonistic control of rice immunity against distinct pathogens by the two transcription modules via salicylic acid and jasmonic acid pathways. Dev. Cell 59 , 1609-1622.e1604. https://doi.org/10.1016/j.devcel.2024.03.033. Szemenyei, H., Hannon, M., and Long, J.A. (2008). TOPLESS mediates auxin-dependent transcriptional repression during Arabidopsis embryogenesis. Science 319 , 1384-1386. https://doi.org/10.1126/science.1151461. Li, X., Chen, T., Li, Y., Wang, Z., Cao, H., Chen, F., Li, Y., Soppe, W.J.J., Li, W., and Liu, Y. (2019). ETR1/RDO3 Regulates Seed Dormancy by Relieving the Inhibitory Effect of the ERF12-TPL Complex on DELAY OF GERMINATION1 Expression. Plant Cell 31 , 832-847. https://doi.org/10.1105/tpc.18.00449. Consortium, A.I.M. (2011). Evidence for network evolution in an Arabidopsis interactome map. Science 333 , 601-607. https://doi.org/10.1126/science.1203877. Altmann, M., Altmann, S., Rodriguez, P.A., Weller, B., Elorduy Vergara, L., Palme, J., Marín-de la Rosa, N., Sauer, M., Wenig, M., Villaécija-Aguilar, J.A., et al. (2020). Extensive signal integration by the phytohormone protein network. Nature 583 , 271-276. https://doi.org/10.1038/s41586-020-2460-0. Caillaud, M.C., Asai, S., Rallapalli, G., Piquerez, S., Fabro, G., and Jones, J.D. (2013). A downy mildew effector attenuates salicylic acid-triggered immunity in Arabidopsis by interacting with the host mediator complex. PLoS. Biol. 11 , e1001732. https://doi.org/10.1371/journal.pbio.1001732. Wang, Q., Han, C., Ferreira, A.O., Yu, X., Ye, W., Tripathy, S., Kale, S.D., Gu, B., Sheng, Y., Sui, Y., et al. (2011). Transcriptional programming and functional interactions within the Phytophthora sojae RXLR effector repertoire. Plant Cell 23 , 2064-2086. https://doi.org/10.1105/tpc.111.086082. Ma, Z., Song, T., Zhu, L., Ye, W., Wang, Y., Shao, Y., Dong, S., Zhang, Z., Dou, D., Zheng, X., et al. (2015). A Phytophthora sojae Glycoside Hydrolase 12 Protein Is a Major Virulence Factor during Soybean Infection and Is Recognized as a PAMP. Plant Cell 27 , 2057-2072. https://doi.org/10.1105/tpc.15.00390. Liu, Y., Sun, T., Sun, Y., Zhang, Y., Radojičić, A., Ding, Y., Tian, H., Huang, X., Lan, J., Chen, S., et al. (2020). Diverse Roles of the Salicylic Acid Receptors NPR1 and NPR3/NPR4 in Plant Immunity. Plant Cell 32 , 4002-4016. https://doi.org/10.1105/tpc.20.00499. Baumli, S., Hoeppner, S., and Cramer, P. (2005). A conserved mediator hinge revealed in the structure of the MED7.MED21 (Med7.Srb7) heterodimer. J. Biol. Chem. 280 , 18171-18178. https://doi.org/10.1074/jbc.M413466200. Koschubs, T., Lorenzen, K., Baumli, S., Sandström, S., Heck, A.J., and Cramer, P. (2010). Preparation and topology of the Mediator middle module. Nucleic Acids Res. 38 , 3186-3195. https://doi.org/10.1093/nar/gkq029. Wang, X., Sun, Q., Ding, Z., Ji, J., Wang, J., Kong, X., Yang, J., and Cai, G. (2014). Redefining the modular organization of the core Mediator complex. Cell Res. 24 , 796-808. https://doi.org/10.1038/cr.2014.64. Nozawa, K., Schneider, T.R., and Cramer, P. (2017). Core Mediator structure at 3.4 Å extends model of transcription initiation complex. Nature 545 , 248-251. https://doi.org/10.1038/nature22328. Tsai, K.L., Yu, X., Gopalan, S., Chao, T.C., Zhang, Y., Florens, L., Washburn, M.P., Murakami, K., Conaway, R.C., Conaway, J.W., and Asturias, F.J. (2017). Mediator structure and rearrangements required for holoenzyme formation. Nature 544 , 196-201. https://doi.org/10.1038/nature21393. Larivière, L., Plaschka, C., Seizl, M., Wenzeck, L., Kurth, F., and Cramer, P. (2012). Structure of the Mediator head module. Nature 492 , 448-451. https://doi.org/10.1038/nature11670. Ji, P., Bao, Y., Zhou, H., Pei, Y., Song, W., Ou, K., Qiao, Z., Si, J., Zhong, Z., Xu, X., et al. (2025). Blocking the isoflavone chemoreceptor in Phytophthora sojae to prevent disease. Sci. Adv. 11 , eadt0925. https://doi.org/10.1126/sciadv.adt0925. Pei, Y., Ji, P., Miao, J., Gu, X., Wang, H., Zhao, Y., Song, W., Guo, Z., Zhou, H., Shen, D., et al. (2024). A receptor kinase senses sterol by coupling with elicitins in auxotrophic Phytophthora . Proc. Natl Acad. Sci. USA. 121 , e2408186121. https://doi.org/10.1073/pnas.2408186121. Liu, Y., Schiff, M., Marathe, R., and Dinesh-Kumar, S.P. (2002). Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required for N -mediated resistance to tobacco mosaic virus. Plant J. 30 , 415-429. https://doi.org/10.1046/j.1365-313x.2002.01297.x. Liu, C., Tan, X., Wang, J., Sun, Y., Xu, Q., Han, C., and Wang, Q. (2025). Upgrading of the genetic engineering toolkit accelerated the discovery process of the virulence effect of PsGH7d on Phytophthora sojae invasion. Physiol. Plant. 177 , e70083. https://doi.org/10.1111/ppl.70083. Fang, Y., and Tyler, B.M. (2016). Efficient disruption and replacement of an effector gene in the oomycete P hytophthora sojae using CRISPR/C as9. Molecular plant pathology 17 , 127-139. Jansing, J., Sack, M., Augustine, S.M., Fischer, R., and Bortesi, L. (2019). CRISPR/Cas9-mediated knockout of six glycosyltransferase genes in Nicotiana benthamiana for the production of recombinant proteins lacking β-1,2-xylose and core α-1,3-fucose. Plant Biotechnol. J. 17 , 350-361. https://doi.org/10.1111/pbi.12981. Kaufmann, K., Muiño, J.M., Østerås, M., Farinelli, L., Krajewski, P., and Angenent, G.C. (2010). Chromatin immunoprecipitation (ChIP) of plant transcription factors followed by sequencing (ChIP-SEQ) or hybridization to whole genome arrays (ChIP-CHIP). Nature ProtocolsMethods in Molecular Biology 5 , 457-472. https://doi.org/10.1038/nprot.2009.244. Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryFigures.docx Cite Share Download PDF Status: Published Journal Publication published 15 Apr, 2026 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7305635","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":528519311,"identity":"944a13f1-8275-4220-855d-034c2ec4cf3d","order_by":0,"name":"Qunqing Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYDADNgYGxgcJFTWkaWE2eHDmGIkWST5sYSaszOD42cMv3rbdke2Tbr9WkdjAxsDf3p2AX8uZvDTLuW3PjNtkzpTdSNwhwyBx5uwGvFrMDuSYGfO2HU5sk8hJu5F4ho3BQCKXgJbzbxBaChLbmInQciPH+DFES/oxBqK02N94Y8Y459xhY6AtzBIJZ47xEPSLZH+O8Yc3ZYdl589If/jxR0WNHH97L34tQMAmwQOM+QYGHgMQj4eQchBg/gDRwv6AGNWjYBSMglEwAgEAOXVOBw/SGLwAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-3424-8018","institution":"Shandong Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Qunqing","middleName":"","lastName":"Wang","suffix":""},{"id":528519312,"identity":"fe2cfbb7-11b0-47bb-953a-e2f3a44c7ab4","order_by":1,"name":"Xinwei Tan","email":"","orcid":"https://orcid.org/0009-0008-9921-1815","institution":"Shandong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xinwei","middleName":"","lastName":"Tan","suffix":""},{"id":528519313,"identity":"60ebf13b-4229-49ef-bca6-072b12a7a459","order_by":2,"name":"Yujing Sun","email":"","orcid":"","institution":"Shandong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yujing","middleName":"","lastName":"Sun","suffix":""},{"id":528519314,"identity":"6d2e8a30-c559-452f-ab10-162e45d58c70","order_by":3,"name":"Zhaomei Qi","email":"","orcid":"","institution":"Shandong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Zhaomei","middleName":"","lastName":"Qi","suffix":""},{"id":528519315,"identity":"4b787260-9f3e-46c9-a78f-754773fd6a0d","order_by":4,"name":"Yonghui Miao","email":"","orcid":"","institution":"Shandong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yonghui","middleName":"","lastName":"Miao","suffix":""},{"id":528519316,"identity":"96ef4438-6b40-45bf-a1f6-cfe6bb1ef02e","order_by":5,"name":"Yiman Wan","email":"","orcid":"","institution":"Shandong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yiman","middleName":"","lastName":"Wan","suffix":""},{"id":528519317,"identity":"a3241bc3-be7c-4dac-9840-be8e4d92cbff","order_by":6,"name":"Xiaoyuan Hou","email":"","orcid":"","institution":"Shandong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyuan","middleName":"","lastName":"Hou","suffix":""},{"id":528519318,"identity":"be43f1e6-145a-4e68-afb3-714c759b401b","order_by":7,"name":"Hengjing Li","email":"","orcid":"","institution":"Shandong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Hengjing","middleName":"","lastName":"Li","suffix":""},{"id":528519319,"identity":"8a636b4c-da1d-4af5-8e01-5878e978a966","order_by":8,"name":"Wenxiu Li","email":"","orcid":"","institution":"Shandong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Wenxiu","middleName":"","lastName":"Li","suffix":""},{"id":528519320,"identity":"bc849c28-5e87-4948-b1fe-54bac738e460","order_by":9,"name":"Yuan Chen","email":"","orcid":"","institution":"Peking University Institute of Advanced Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Chen","suffix":""},{"id":528519321,"identity":"0ab17dfd-3ef5-4705-b33b-caa869d675a5","order_by":10,"name":"Qian Xu","email":"","orcid":"https://orcid.org/0000-0002-7106-2714","institution":"Shandong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Qian","middleName":"","lastName":"Xu","suffix":""}],"badges":[],"createdAt":"2025-08-06 04:45:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7305635/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7305635/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-026-71909-8","type":"published","date":"2026-04-15T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":93623391,"identity":"a03d2f78-6ca3-41df-8d81-11d3be9b48d4","added_by":"auto","created_at":"2025-10-15 18:28:51","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6327743,"visible":true,"origin":"","legend":"","description":"","filename":"2025.8.6Avh109.docx","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/6940c9b53e80d8c5bd1bd762.docx"},{"id":93621502,"identity":"c3a2e6e2-6d78-47d4-be1f-54a76f929fa4","added_by":"auto","created_at":"2025-10-15 18:04:51","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":11311,"visible":true,"origin":"","legend":"","description":"","filename":"NCOMMS2566004T.json","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/a75dae1fdbda33ad28a34e63.json"},{"id":93621507,"identity":"b1835e55-583e-412c-b7ef-29147f6d7aae","added_by":"auto","created_at":"2025-10-15 18:04:51","extension":"xml","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":197036,"visible":true,"origin":"","legend":"","description":"","filename":"NCOMMS2566004T0enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/a7fcc6dd209312fc15512b07.xml"},{"id":93622529,"identity":"8faf4604-5906-46b2-a59c-7c03772a7aea","added_by":"auto","created_at":"2025-10-15 18:12:51","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":197904,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/bebf7e432285733a1a51e8c9.png"},{"id":93621505,"identity":"efebff65-40b3-4194-955c-3ff29027929a","added_by":"auto","created_at":"2025-10-15 18:04:51","extension":"jpeg","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":702452,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage10.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/5cac1b02d888ac11187d8c2d.jpeg"},{"id":93621511,"identity":"4f82ea34-3170-4a49-8b69-8a90949f699c","added_by":"auto","created_at":"2025-10-15 18:04:51","extension":"jpeg","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":477034,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage11.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/78810cc9521703971acfbf77.jpeg"},{"id":93622856,"identity":"759fb7ab-52b0-4968-a4f7-42409677bf59","added_by":"auto","created_at":"2025-10-15 18:20:51","extension":"jpeg","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":599218,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage12.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/85a7034e03da9a9b5b188828.jpeg"},{"id":93621527,"identity":"37d4e2b4-4ac0-475d-805b-60ed1d3d9418","added_by":"auto","created_at":"2025-10-15 18:04:51","extension":"jpeg","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":539731,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage13.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/771c77f6b81f971f40754b81.jpeg"},{"id":93623395,"identity":"f041cae0-d760-40a4-98f6-911feae4e758","added_by":"auto","created_at":"2025-10-15 18:28:51","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":383536,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage14.png","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/880b09ebde10b0ba036ac8a2.png"},{"id":93623568,"identity":"927cd3fd-8e7c-4917-b118-394e55b69c49","added_by":"auto","created_at":"2025-10-15 18:36:51","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":125825,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage15.png","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/c5cca49d48cdadcda8a46727.png"},{"id":93621515,"identity":"774ee0ce-c8ab-49a3-8674-418a55a2af71","added_by":"auto","created_at":"2025-10-15 18:04:51","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":213618,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/6f99bc45aedc57c2414d5cd5.png"},{"id":93623393,"identity":"10f4e705-96d0-4891-9be0-b8d542c7ee96","added_by":"auto","created_at":"2025-10-15 18:28:51","extension":"jpeg","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":774533,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/e7435c56e85cb2d24e491511.jpeg"},{"id":93621521,"identity":"ab7c9791-6c59-4db6-aba3-a594b583edcc","added_by":"auto","created_at":"2025-10-15 18:04:51","extension":"jpeg","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":529153,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/d524e0413af5e5372a168d49.jpeg"},{"id":93621517,"identity":"d531392b-4b9e-498e-ad6e-1cec4c21451b","added_by":"auto","created_at":"2025-10-15 18:04:51","extension":"jpeg","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":372418,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/7909d38659113058e4a11b5d.jpeg"},{"id":93622533,"identity":"e12389f1-81dc-4865-9490-09493b85bcc8","added_by":"auto","created_at":"2025-10-15 18:12:51","extension":"jpeg","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":498415,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/106480ea50a2d90681e5401c.jpeg"},{"id":93622543,"identity":"2ac5aae1-4580-4964-92ba-361682de52ee","added_by":"auto","created_at":"2025-10-15 18:12:51","extension":"jpeg","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":501658,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/c20486dde8cabde881e2fb5e.jpeg"},{"id":93622864,"identity":"6aad1f6f-eabf-4207-b358-c77025934474","added_by":"auto","created_at":"2025-10-15 18:20:51","extension":"jpeg","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":421512,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/eb556e615b7e40c7aeca43f3.jpeg"},{"id":93622862,"identity":"8b127499-f4b6-458f-a301-8e2748fdebac","added_by":"auto","created_at":"2025-10-15 18:20:51","extension":"jpeg","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":828916,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/d5b0200143f5608b89113bfb.jpeg"},{"id":93622859,"identity":"da4a1e37-69d9-4f22-b6f1-f12220221584","added_by":"auto","created_at":"2025-10-15 18:20:51","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":55635,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/f98bb93b4eced39f959bbb28.png"},{"id":93621520,"identity":"d7c299bf-3b87-46a5-8c56-d558f93b7bec","added_by":"auto","created_at":"2025-10-15 18:04:51","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":94595,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/dd05023216b4fcd9e48a55c1.png"},{"id":93622540,"identity":"2ae38dde-6e81-43ff-a774-5dfdea7709c5","added_by":"auto","created_at":"2025-10-15 18:12:51","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":66348,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/b4226fe8f208513b1b4e2d5a.png"},{"id":93621530,"identity":"cb07008c-ce31-49b5-ba57-6d771ed78bef","added_by":"auto","created_at":"2025-10-15 18:04:51","extension":"png","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":80761,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage12.png","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/2837de939c9903d76832e7df.png"},{"id":93622545,"identity":"b5fe61c6-7147-43e9-8d4f-5bba4c6715d3","added_by":"auto","created_at":"2025-10-15 18:12:51","extension":"png","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":132911,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage13.png","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/01dd4c2e76d5451c282e3b15.png"},{"id":93622865,"identity":"9ab45bee-c406-41b6-ad78-a87666766ee5","added_by":"auto","created_at":"2025-10-15 18:20:51","extension":"png","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":70505,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage14.png","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/dd3fcdf9d7cf0002d3bbc546.png"},{"id":93621534,"identity":"a2e9b385-c46b-42a7-a406-dfceef697b64","added_by":"auto","created_at":"2025-10-15 18:04:51","extension":"png","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":29646,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage15.png","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/eb0cc1435acf3fc657dea6b7.png"},{"id":93622547,"identity":"8271c947-4ac7-4b01-be71-c06ab786c441","added_by":"auto","created_at":"2025-10-15 18:12:51","extension":"png","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":38988,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/aee842e75be300ea9332614e.png"},{"id":93621539,"identity":"96aa1fa5-1a22-4c57-94ff-f72dbb14d79d","added_by":"auto","created_at":"2025-10-15 18:04:51","extension":"png","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":191049,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/54ec078dbcf50c2917010cd1.png"},{"id":93621535,"identity":"360e2759-363d-4750-9bca-f4ffd72ea6db","added_by":"auto","created_at":"2025-10-15 18:04:51","extension":"png","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":115315,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/7dcd6b014df917a631aeac0b.png"},{"id":93621537,"identity":"8c706ab1-4922-448c-8430-9cf71dc94e89","added_by":"auto","created_at":"2025-10-15 18:04:51","extension":"png","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":49487,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/128558e63bc44365de592486.png"},{"id":93621538,"identity":"9ee7031a-b8f5-4f81-9932-aefd887e6f25","added_by":"auto","created_at":"2025-10-15 18:04:51","extension":"png","order_by":29,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":109098,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/730a14dc3b65fcde8c89ba0f.png"},{"id":93621544,"identity":"f2941dd4-e24f-4f54-8c88-7b39de9d0219","added_by":"auto","created_at":"2025-10-15 18:04:52","extension":"png","order_by":30,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":73856,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/f8b8cf130796e51e1fecf8ba.png"},{"id":93621540,"identity":"a4e9a9d8-f378-48da-8f6d-8b3391dc9b09","added_by":"auto","created_at":"2025-10-15 18:04:51","extension":"png","order_by":31,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":51067,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/d4bef081cbe2c36edbf938ca.png"},{"id":93622549,"identity":"8e3939f8-2953-460d-9ff5-c30197c5678f","added_by":"auto","created_at":"2025-10-15 18:12:52","extension":"png","order_by":32,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":170340,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/93ed2fcbc675374be537bb10.png"},{"id":93622548,"identity":"cd56a33c-4688-451d-9112-f215cfe6bf3c","added_by":"auto","created_at":"2025-10-15 18:12:52","extension":"xml","order_by":33,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":192931,"visible":true,"origin":"","legend":"","description":"","filename":"NCOMMS2566004T0structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/69cd8041dc5d9ae0a8d41b14.xml"},{"id":93621541,"identity":"9b428342-20d2-4d3c-b5c2-c07942908475","added_by":"auto","created_at":"2025-10-15 18:04:52","extension":"html","order_by":34,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":211990,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/ce36fbe7b07fcfc011bf54c4.html"},{"id":93621503,"identity":"a1b3e40f-5df1-4efb-95c8-42d4a75668ba","added_by":"auto","created_at":"2025-10-15 18:04:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":448854,"visible":true,"origin":"","legend":"\u003cp\u003eSalicylic acid induces the production of effector Avh109 in \u003cem\u003ePhytophthora\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(a) Confocal microscopy analysis of Avh109 effector fluorescence in hyphae of \u003cem\u003eP. sojae\u003c/em\u003e wild-type strain P6497 and the C-PsAvh109-6His-mCherry gene-edited strain. Hyphae were treated with 100 μM salicylic acid (SA) for 30 minutes, with ethanol (EtOH) used as a solvent control. Scale bar: 20 μm.\u003c/p\u003e\n\u003cp\u003e(b) SA induces Avh109 protein accumulation in hyphae of \u003cem\u003eP. sojae\u003c/em\u003ewild-type strain P6497 and the C-PsAvh109-6His-mCherry gene-edited strain. Hyphae were treated with 100 μM SA for 30 minutes, with EtOH serving as a solvent control. PsAvh109 protein levels were assessed by Western blotting using an anti-His antibody. Ponceau S staining was used to verify equal protein loading.\u003c/p\u003e\n\u003cp\u003e(c) PsAvh109 transcript levels were quantified by RT-qPCR following treatment with SA (10μM, 50μM, 100μM, 1mM) for 30 min. EtOH served as a solvent control. Data were normalized to the reference gene PsACT and are expressed as means ± SD (n=3). Different letters indicate significant differences (P \u0026lt; 0.01; one-way ANOVA with Tukey's multiple comparisons test).\u003c/p\u003e\n\u003cp\u003e(d) \u003cem\u003ePsAvh109\u003c/em\u003e transcript levels in \u003cem\u003eP. sojae\u003c/em\u003e were quantified by RT-qPCR at 3 h post-infection of NahG transgenic soybean root hairs (SA-deficient) versus GFP-tagged wild-type root hairs. Data were normalized to the reference gene \u003cem\u003ePsACT\u003c/em\u003e and expressed as means ± SD (n = 3, Student's t - test).\u003c/p\u003e\n\u003cp\u003e(e) The transcription levels of the \u003cem\u003ePPTG_19802\u003c/em\u003e gene in \u003cem\u003eP. capsici\u003c/em\u003e were measured by RT-qPCR following induction with SA at the specified concentrations for 30 minutes. EtOH was used as a control. Relative transcription levels were normalized to the \u003cem\u003eP. capsici\u003c/em\u003e reference gene \u003cem\u003ePcUBC\u003c/em\u003e. Data are presented as the mean ± SD (n=3). Different letters indicate significant differences (P \u0026lt; 0.01; one-way ANOVA with Tukey's multiple comparisons test).\u003c/p\u003e\n\u003cp\u003e(f) The transcription levels of the \u003cem\u003eDVH05_001429\u003c/em\u003e gene in \u003cem\u003eP. parasitica\u003c/em\u003e were measured by RT-qPCR following induction with SA at the specified concentrations for 30 minutes. EtOH was used as a control. Relative transcription levels were normalized to the \u003cem\u003eP. parasitica\u003c/em\u003e reference gene \u003cem\u003ePpACT\u003c/em\u003e. Data are presented as the mean ± SD (n=3). Different letters indicate significant differences (P \u0026lt; 0.01; one-way ANOVA with Tukey's multiple comparisons test).\u003c/p\u003e\n\u003cp\u003e(g) The transcriptional level of \u003cem\u003ePsAvh109\u003c/em\u003ein wild-type and KO-PsGPA1 strains of \u003cem\u003eP. sojae\u003c/em\u003e was quantified by RT–qPCR 30 minutes following SA treatment, EtOH as the solvent control. Data were normalized to the reference gene \u003cem\u003ePsACT\u003c/em\u003e and expressed as means ± SD (n = 3, Student's t - test).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/a097f6b553e9afe488df82e3.png"},{"id":93622528,"identity":"7beabb5e-30fe-4589-99cd-5e6a992ea8ef","added_by":"auto","created_at":"2025-10-15 18:12:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":990761,"visible":true,"origin":"","legend":"\u003cp\u003ePsAvh109 is a nucleus-localized effector that is essential for \u003cem\u003eP. sojae\u003c/em\u003e virulence.\u003c/p\u003e\n\u003cp\u003e(a) Schematic representation of the PsAvh109 protein domain architecture, highlighting the nuclear localization signal (NLS) in green. The PsAvh109\u003csup\u003eAAA\u003c/sup\u003e mutant contains substitutions of lysine (K) and arginine (R) residues with alanine (A) within the NLS region (highlighted in blue), whereas the PsAvh109\u003csup\u003eNES\u003c/sup\u003e mutant features a replacement of the NLS with a nuclear export signal (NES), indicated in orange.\u003c/p\u003e\n\u003cp\u003e(b) Confocal microscopy analysis revealed the subcellular localization of PsAvh109 and its mutants. GFP-PsAvh109 colocalized with the nuclear marker H2B-mCherry after 36 hours of co-expression. Scale bar: 20 μm.\u003c/p\u003e\n\u003cp\u003e(c) Co-inoculation of soybean root hairs expressing mCherry, mCherry-PsAvh109, or mCherry-PsAvh109\u003csup\u003eAAA\u003c/sup\u003e with a GFP-expressing \u003cem\u003eP. sojae\u003c/em\u003e transformant (strain P6497). Fluorescence microscopy images were acquired at 48 hours post-inoculation (hpi). Scale bar: 200 μm.\u003c/p\u003e\n\u003cp\u003e(d) Quantification of \u003cem\u003eP. sojae\u003c/em\u003e oospore formation in soybean root hairs. Data are presented as mean ± standard deviation (SD) (n=10). The significance was calculated using one-way analysis of variance [ANOVA] with Dunnett's multiple comparisons test.\u003c/p\u003e\n\u003cp\u003e(e) Relative \u003cem\u003eP. sojae\u003c/em\u003e biomass in soybean root hairs, determined by quantitative PCR (qPCR) based on the ratio of \u003cem\u003eP. sojae\u003c/em\u003e to soybean genomic DNA. Data are presented as mean ± SD (n=3, one-way ANOVA with Dunnett's multiple comparisons test).\u003c/p\u003e\n\u003cp\u003e(f) Phenotypic analysis of \u003cem\u003eP. sojae\u003c/em\u003e virulence in soybean hypocotyls. Wild-type strain P6497, the PsAvh109 knockout mutant (KO-PsAvh109), and two complemented strains (C-PsAvh109 and C-PsAvh109\u003csup\u003eAAA\u003c/sup\u003e) were inoculated onto soybean hypocotyls. Representative images were taken at 48 hpi. The experiment was independently repeated three times with similar results.\u003c/p\u003e\n\u003cp\u003e(g) Quantification of \u003cem\u003eP. sojae\u003c/em\u003e biomass in infected soybean hypocotyls by qPCR, based on the ratio of \u003cem\u003eP. sojae\u003c/em\u003e to soybean genomic DNA. Data are shown as mean ±SD (n=3, one-way ANOVA with Dunnett's multiple comparisons test).\u0026nbsp;\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/c1b37c7eb80e4fc051b3b211.png"},{"id":93621510,"identity":"dac94722-edfe-4260-a8bd-2ecca35cc1c1","added_by":"auto","created_at":"2025-10-15 18:04:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":874583,"visible":true,"origin":"","legend":"\u003cp\u003eThe interaction between PsAvh109 and MED21 shapes plant transcriptional reprogramming\u003c/p\u003e\n\u003cp\u003e(a) Yeast two-hybrid analysis demonstrated that PsAvh109, but not the PsAvh109\u003csup\u003eAAA\u003c/sup\u003e mutant, interacts with GmMED21. Yeast transformants were serially diluted 10-fold and plated on synthetic dropout (SD) media lacking tryptophan and leucine (SD - TL) or lacking tryptophan, leucine, histidine, and adenine (SD - TLHA). Colonies were imaged after 4 days of incubation. AD and BD denote empty activation domain and binding domain vectors, respectively. The experiment was independently repeated at least three times with consistent results.\u003c/p\u003e\n\u003cp\u003e(b) Firefly luciferase complementation imaging assay confirming the interaction between PsAvh109 and GmMED21 in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. Agrobacterium strains expressing the target proteins were co-infiltrated into leaves, followed by D-luciferin application 2 days post-infiltration.\u003c/p\u003e\n\u003cp\u003e(c) Co-immunoprecipitation (Co-IP) assay validating the interaction between PsAvh109 and GmMED21 in \u003cem\u003eN. benthamiana\u003c/em\u003e. GFP, GFP-PsAvh109, GFP-PsAvh109\u003csup\u003eAAA\u003c/sup\u003e, and GFP-PsAvh109\u003csup\u003eNES\u003c/sup\u003e were co-expressed with GmMED21-3HA. Total protein extracts were incubated with anti-GFP-conjugated agarose beads. Western blotting was performed using anti-GFP and anti-HA antibodies. Ponceau S staining served as a loading control. Molecular weight markers (kDa) are indicated.\u003c/p\u003e\n\u003cp\u003e(d) In vitro pull-down assay of PsAvh109 and GmMED21 interaction. GST, GST-PsAvh109, GST-PsAvh109\u003csup\u003eAAA\u003c/sup\u003e, and GST-PsAvh109\u003csup\u003eNES\u003c/sup\u003e fusion proteins were incubated with His-tagged GmMED21 immobilized on glutathione Sepharose 4B resin. GST alone was used as a negative control. Input and eluted proteins were analyzed by Western blotting with anti-His and anti-GST antibodies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(e)\u003c/strong\u003e Heatmap showing transcriptomic profiles of soybean root hairs overexpressing GFP or GFP–PsAvh109 at 3 h post-inoculation with KO–\u003cem\u003ePsAvh109\u003c/em\u003e. Raw FPKM values were normalized to reference genes. Hierarchical clustering was performed using the average linkage method in TBtools-Ⅱ.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(f)\u003c/strong\u003e RT–qPCR analysis of \u003cem\u003eGmPR1-1\u003c/em\u003e, \u003cem\u003eGmPR1-6\u003c/em\u003e, \u003cem\u003eGmSABP2 \u003c/em\u003eand \u003cem\u003eGmVSPa\u003c/em\u003e transcript levels in soybean root hairs at 3 h post-inoculation with \u003cem\u003eP. sojae\u003c/em\u003e P6497 or KO–\u003cem\u003ePsAvh109\u003c/em\u003e. Expression was normalized to \u003cem\u003eGmCYP2\u003c/em\u003e. Data represent mean ± SD (n = 3; Student’s \u003cem\u003et\u003c/em\u003e-test).\u0026nbsp;\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/2c234e344a4484e92c1e086c.png"},{"id":93622531,"identity":"41324325-beb8-4b6b-9424-c8e85e34b5ab","added_by":"auto","created_at":"2025-10-15 18:12:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1223451,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe N-Terminus of MED21 Constitute the Crucial Domain for Interaction with MED6 and Avh109\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Schematic of structures of GmMED21 mutants.\u003c/p\u003e\n\u003cp\u003e(b) Interaction of GmMED21 or mutants GmMED21\u003csup\u003e16-139\u003c/sup\u003e, or GmMED21\u003csup\u003e1-15\u003c/sup\u003e with PsAvh109 or GmMED6 in yeast. Yeast transformants were serially diluted 10-fold and plated on synthetic dropout (SD) media lacking tryptophan and leucine (SD - TL) or lacking tryptophan, leucine, histidine, and adenine (SD - TLHA).\u003c/p\u003e\n\u003cp\u003e(c and d) Interactions of GmMED21 or mutants GmMED21\u003csup\u003e16-139\u003c/sup\u003e, or GmMED21\u003csup\u003e1-15\u003c/sup\u003e with PsAvh109, or GmMED6 in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves assayed by LCI. \u003cem\u003eAgrobacterium\u003c/em\u003e strains carrying constructs encoding the proteins were pairwise co-infiltrated into \u003cem\u003eN. benthamiana\u003c/em\u003e leaves, as indicated. Two days after infiltration, leaves were sprayed with D-luciferin, and luminescence of the leaves was observed using a plant live imaging system.\u003c/p\u003e\n\u003cp\u003e(e) The results of the Y3H assay demonstrate the impact of PsAvh109 on the interaction between GmMED21 and GmMED6. Yeast cells were co - transformed with three different combinations: pGAD - GmMED21 and pBridge - GmMED6 + EV, pGAD - GmMED21 and pBridge - GmMED6 + PsAvh109, or pGAD - GmMED21 and pBridge - GmMED6 + PsAvh109\u003csup\u003eAAA\u003c/sup\u003e. SD - TL represents synthetic defined medium lacking tryptophan and leucine; SD - TLH represents synthetic defined medium lacking tryptophan, leucine, and histidine; SD - TLHM represents synthetic defined medium lacking tryptophan, leucine, histidine, and methionine. The absence of methionine in the SD - TLHM medium induces the expression of Linker protein.\u003c/p\u003e\n\u003cp\u003e(f) In vitro pull - down experiments were carried out to verify that PsAvh109 competes with GmMED6 for binding to GmMED21. Fixed amounts of GmMED21 - His and MBP - GmMED6 were incubated with increasing amounts of purified GST - PsAvh109 or purified GST protein (used as a control). Subsequently, the proteins in the samples were pulled down by Ni - NTA beads, and then western blot assays were conducted using anti - GST, anti - MBP, or anti - His specific antibodies.\u003c/p\u003e\n\u003cp\u003e(g) PsAvh109 disrupts the interaction between GmMED21 and GmMED6 in co - IP assays. GmMED21 - 3HA and GmMED6 - 3Flag were co - transfected with either GFP - PsAvh109 or GFP in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. Total proteins were incubated with HA - Trap beads for co - IP detection, and immunoprecipitated proteins were analyzed using anti - GFP, anti - HA, or anti - Flag antibodies. Protein loading was indicated by Ponceau S staining of RBCL.\u003c/p\u003e\n\u003cp\u003e(h and i) Co-expression of PsAvh109 with cLUC-GmMED6 and GmMED21-nLUC reduced the expression of LUC, but co-expression with PsAvh109\u003csup\u003eAAA \u003c/sup\u003edid not. Luminescence was observed on the \u003cem\u003eN. benthamiana\u003c/em\u003e leaves using plant living imaging system. (i) Quantification of luciferase activity (integrated optical density, IOD) in treated leaves in (h). Data presented as mean ± SD (n=8). Different letters indicate significant differences (P \u0026lt; 0.01; one-way ANOVA with Tukey's multiple comparisons test).\u003c/p\u003e\n\u003cp\u003e(j) Occupancy of PsAvh109 in different promoter regions of selected immunity genes was evaluated by ChIP - qPCR in PsAvh109 - FLAG transgenic hairy root. IgG was used as a negative control. ChIP signals were normalized to the input. Data are presented as the mean ± SD (n=3, Student’s t-test). P1–P3 represent the promoter regions examined by ChIP - qPCR. The as - 1 elements in the promoters are shown as red triangles.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/417d5f2f1c0e1c31f888fcbe.png"},{"id":93622530,"identity":"4854442e-4ae8-4d67-9e98-7d2d64c92fcf","added_by":"auto","created_at":"2025-10-15 18:12:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1085826,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGmTPL competes with GmMED6 for binding sites on GmMED21\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) The results of the Y3H assay demonstrate the impact of GmTPL\u003csup\u003eN188\u003c/sup\u003e on the interaction between GmMED21 and GmMED6. Yeast cells were co - transformed with three different combinations: pGAD - EV and pBridge - GmMED6 + EV, pGAD - GmMED21 and pBridge - GmMED6 + EV, or pGAD - GmMED21 and pBridge - GmMED6 + GmTPL\u003csup\u003eN188\u003c/sup\u003e. SD - TL represents synthetic defined medium lacking tryptophan and leucine; SD - TLH represents synthetic defined medium lacking tryptophan, leucine, and histidine; SD - TLHM represents synthetic defined medium lacking tryptophan, leucine, histidine, and methionine. The absence of methionine in the SD - TLHM medium induces the expression of Linker protein.\u003c/p\u003e\n\u003cp\u003e(b) In vitro pull - down experiments were carried out to verify that GmTPL\u003csup\u003eN188\u003c/sup\u003e competes with GmMED6 for binding to GmMED21. Fixed amounts of GmMED21 - His and MBP - GmMED6 were incubated with increasing amounts of purified GST - GmTPL\u003csup\u003eN188\u003c/sup\u003e or purified GST protein (used as a control). Subsequently, the proteins in the samples were pulled down by Ni - NTA beads, and then western blot assays were conducted using anti - GST, anti - MBP, or anti - His specific antibodies.\u003c/p\u003e\n\u003cp\u003e(c) GmTPL\u003csup\u003eN188\u003c/sup\u003e disrupts the interaction between GmMED21 and GmMED6 in co - IP assays. GmMED21 - 3HA and GmMED6 - 3Flag were co - transfected with either GFP - GmTPL\u003csup\u003eN188\u003c/sup\u003e or GFP in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. Total proteins were incubated with HA - Trap beads for co - IP detection, and immunoprecipitated proteins were analyzed using anti - GFP, anti - HA, or anti - Flag antibodies. Protein loading was indicated by Ponceau S staining of RBCL.\u003c/p\u003e\n\u003cp\u003e(d and e) Co - expression of GmTPL\u003csup\u003eN188\u003c/sup\u003e with cLUC - GmMED6 and GmMED21 - nLUC led to a reduction in LUC expression, while co - expression with GFP did not have such an effect. Luminescence was detected on the\u003cem\u003e N. benthamiana\u003c/em\u003e leaves via a plant living imaging system. (e) Quantification of the luciferase activity (integrated optical density, IOD) in the treated leaves in (d). Data are presented as mean ± SD (n=8). Different letters indicate significant differences (P \u0026lt; 0.01; one-way ANOVA with Tukey's multiple comparisons test).\u003c/p\u003e\n\u003cp\u003e(f) Occupancy of GmTPL in different promoter regions of selected immunity genes was evaluated by ChIP - qPCR in GmTPL - FLAG transgenic hairy root. IgG was used as a negative control. ChIP signals were normalized to the input. Data are presented as the mean ± SD (n=3, Student’s t-test). P1–P3 represent the promoter regions examined by ChIP - qPCR. The as - 1 elements in the promoters are shown as red triangles.\u003c/p\u003e\n\u003cp\u003e(g and h) Detection of the interaction strengths between PsAvh109, GmMED6, GmTPL and GmMED21 respectively using the LUC bimolecular fluorescence complementation assay in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. (g) As shown below, the leaves of \u003cem\u003eN. benthamiana\u003c/em\u003e were infiltrated with Agrobacterium strains carrying the indicated constructs, and the LUC activity was quantified at 48 hpi. (h) Quantification of the luciferase activity (integrated optical density, IOD) in the treated leaves in (g). Data are presented as mean ± SD (n=8). Different letters indicate significant differences (P \u0026lt; 0.01; one-way ANOVA with Tukey's multiple comparisons test).\u003c/p\u003e\n\u003cp\u003e(i) Recombinant, purified GmMED21 protein was labeled with a fluorescent dye, while PsAvh109, GmTPL\u003csup\u003eN188\u003c/sup\u003e, GmMED6, and GST proteins were used as mobile analytes in a microscale thermophoresis (MST) assay. The dose-response curves for PsAvh109, GmTPL\u003csup\u003eN188\u003c/sup\u003e, GmMED6, and GST proteins were measured as they flowed over immobilized, labeled GmMED21 at varying concentrations. The dissociation constants (Kd) calculated were 0.27 μM ± 0.04 for PsAvh109, 0.76 μM ± 0.11 for GmTPL\u003csup\u003eN188\u003c/sup\u003e, and 1.29 μM ± 0.38 for GmMED6. No binding affinity was observed between GmMED21 and GST. These experiments were repeated three times, yielding consistent results.\u0026nbsp;\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/7d46a26075187737fa471360.png"},{"id":93623567,"identity":"877624d6-57e0-4707-b410-a50eea1f1a87","added_by":"auto","created_at":"2025-10-15 18:36:51","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":922186,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSalicylic Acid Disrupts the Interaction between TPL and MED21 without Impacting the Interaction between Avh109 and MED21\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Western blot analysis reveals that SA treatment has no impact on the accumulation of GmTPL - GFP in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves.\u003c/p\u003e\n\u003cp\u003e(b) The effect of SA on the interaction between GmTPL\u003csup\u003eN188\u003c/sup\u003e and GmMED21 was evaluated via in - vivo co - immunoprecipitation (Co - IP). Western blot (WB) analysis revealed that as the concentration of exogenously applied SA increased, the amount of GmMED21 protein pulled down by GmTPL\u003csup\u003eN188\u003c/sup\u003e gradually decreased. Ponceau S staining was used to indicate protein loading.\u003c/p\u003e\n\u003cp\u003e(c) The effect of salicylic acid (SA) on the interaction between PsAvh109 and GmMED21 was evaluated using co-immunoprecipitation (Co-IP). Western blot (WB) analysis revealed no significant change in the amount of GmMED21 protein co-precipitated with PsAvh109 as the concentration of exogenously added SA increased. Ponceau S staining was used as a loading control.\u003c/p\u003e\n\u003cp\u003e(d) LCI assays were employed to assess the impact of SA on the interaction strength between GmTPL and GmMED21, as well as between PsAvh109 and GmMED21, in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. \u003cem\u003eN. benthamiana\u003c/em\u003e leaves were infiltrated with Agrobacterium tumefaciens strains harboring the indicated constructs. Luciferase activity was quantified at 48 hours post-infiltration (hpi). Subsequently, EtOH or 100 μM SA was applied to both sides of the infiltrated leaves, followed by a second measurement of luciferase activity. Data are presented as mean ± SD (n=8, Student's t - test).\u003c/p\u003e\n\u003cp\u003e(e) The occupancy of GmTPL and PsAvh109 in the promoter regions of selected immunity genes was evaluated by ChIP - qPCR in GmTPL – Flag and PsAvh109-Flag transgenic hairy roots treated with 100 μM SA, EtOH served as a solvent control. GmCYP2 was used as a negative control, and the P1 region of the GmPR1-1 gene promoter was detected by ChIP-qPCR. ChIP signals were normalized to input signals. Data are presented as the mean ± SD (n=3, Student's t - test).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/4738b4464a7f353a6f7e0c07.png"},{"id":93622537,"identity":"f542b218-2ec3-47b2-a93c-aabc6c81b318","added_by":"auto","created_at":"2025-10-15 18:12:51","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":584689,"visible":true,"origin":"","legend":"\u003cp\u003eSalicylic acid induces the production of effector Avh109 in \u003cem\u003ePhytophthora\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eDuring infection, plants regulate transcriptional reprogramming by modulating Mediator complex assembly. The GmMED21–GmMED6 module functions as a key immune regulator. Under non-stress conditions, the transcriptional corepressor TOPLESS (TPL) binds the N-terminus of MED21, blocking GmMED6 recruitment and suppressing SA-responsive gene expression. Upon pathogen detection, elevated SA levels disrupt the TPL–MED21 interaction, enabling MED21–MED6 complex formation and activation of SA-responsive genes. Intriguingly, \u003cem\u003ePhytophthora\u003c/em\u003epathogens respond to SA by inducing expression of the RxLR effector PsAvh109, which suppresses host immunity by interfering with MED21–MED6-mediated transcriptional reprogramming of immune-related genes.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/810d16556bf27a466278ae39.png"},{"id":107044302,"identity":"1d51235d-5bd7-4fb4-9592-e627f7f896b8","added_by":"auto","created_at":"2026-04-16 07:07:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9086245,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/ca8ce9d7-2a65-42ae-9420-90d153ed1262.pdf"},{"id":93622535,"identity":"28f6bf5e-5ed0-40f4-bf57-89c2bc118ae4","added_by":"auto","created_at":"2025-10-15 18:12:51","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3504713,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-7305635/v1/4a1ee7169a1875937427bb3f.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Molecular Espionage in Plant Immunity: A Pathogen Effector Eavesdrops on Salicylic Acid to Hijack the Host Transcriptional Mediator Hub","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003ePlants deploy multi-layered innate immunity to thwart pathogen invasion, involving rapid recognition of microbial patterns and effectors, coupled with dynamic phytohormone signaling\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. This defense hinges on transcriptional reprogramming that redirects resources from growth to immunity, involves the swift activation and deactivation of the transcription of numerous genes, which ultimately determines the fate of the plant\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eStudies in plants have revealed extensive crosstalk between growth and defense signaling networks, coordinated largely by phytohormones such as salicylic acid (SA), jasmonic acid (JA), and auxin\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. These hormones serve as key regulators that fine-tune transcriptional responses through a complex interplay of transcription factors (TFs) and repressors\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. SA is particularly critical for resistance to biotrophic pathogens, acting through the activation of pathogenesis-related (PR) genes\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, with several TF families, such as NPR1 (Nonexpressor of Pathogenesis-Related Genes 1) and TGA (TGACG-binding factors), identified as key regulators\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Among these, NPR1 functions as a central hub, integrating SA signals into downstream transcriptional networks. NPR1 forms a transcriptional activation complex with TGA factors, which bind to the TGACG motif (as-1 element), a conserved cis-element enriched in \u003cem\u003ePR\u003c/em\u003e gene promoters\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. This NPR1-TGA module bridges SA perception with transcriptional activation, and is essential for mounting effective immune responses against biotrophic pathogens\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003ePlants must balance growth and immunity to optimize fitness\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The defense response is tightly controlled under normal growth, while recognition of invading pathogens triggers a timely, accurate, and effective immune response, including transcriptional response to SA\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. This shift is driven by transcriptional reprogramming, which governed by conserved regulators including the Mediator complex\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. As an evolutionary bridge linking transcription factors (TFs) to RNA polymerase II in eukaryotes, Mediator orchestrates the assembly of pre-initiation complexes (PICs) to activate or repress defense genes\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Its core functional module, comprising head (MED6, MED8, MED11, MED17, MED18, MED19, MED20, MED22) and middle (MED1, MED4, MED7, MED9, MED10, MED21, MED31) subunits, facilitates signal integration to mediate immune transitions\u003csup\u003e\u003cspan additionalcitationids=\"CR16 CR17 CR18\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Central to this process is the physical interaction between MED6 and MED21, which facilitates PIC recruitment and is indispensable for transcriptional activation\u003csup\u003e\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Studies in yeast have shown that the head module, particularly MED6, is essential for RNA Pol II activation by linking the head and middle modules to transmit regulatory signals\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. MED6 adhering to the MED21 in the presence of RNA Pol II, and dissociating in its absence\u003csup\u003e\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. These interactions are indispensable for effective transcriptional regulation.\u003c/p\u003e\u003cp\u003eCrucially, transcriptional repressors exploit this interface to enforce repression. In \u003cem\u003eArabidopsis\u003c/em\u003e, the corepressor TOPLESS (TPL) binds the N-terminal domain of MED21, which is the same region required for MED6 interaction, to obstruct Mediator assembly\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. TPL integrates signals from multiple hormone pathways (e.g., JA via JAZ/NINJA\u003csup\u003e26,27\u003c/sup\u003e, auxin via AUX/IAA\u003csup\u003e28\u003c/sup\u003e, ethylene via ERF\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, yet its role in SA signaling remains enigmatic. Although TPL interacts with SA-associated repressors (NPR3, NIMIN)\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, direct evidence that TPL suppresses SA-responsive transcription is lacking, still is a critical knowledge gap in plant immunity.\u003c/p\u003e\u003cp\u003eIn the ongoing arms race between plants and pathogens, pathogens strategically target transcriptional hubs to subvert host defenses\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. We unveil an evolutionary strategy where a pathogen effector mimics transcriptional corepressors to hijack the Mediator complex. Intriguingly, the nuclear-localized effector PsAvh109 from \u003cem\u003eP. sojae\u003c/em\u003e, one of the most important pathogens of soybean (\u003cem\u003eGlycine max\u003c/em\u003e), contributes to virulence by promoting biotrophic colonization and dampening host immunity. Avh109 has been shown to inhibit immune responses triggered by pathogen-associated molecular patterns (PAMPs) such as INF1 and XEG1\u003csup\u003e33,34\u003c/sup\u003e, but its virulence mechanism remains unknown. Here, we uncover a molecular mimicry strategy wherein PsAvh109 hijacks the TPL-MED21-MED6 regulatory node to lock defense transcription. The PsAvh109 functionally emulates the host transcriptional repressor TOPLESS (TPL), competitively displacing TPL from MED21 with \u0026gt;\u0026thinsp;3-fold higher affinity. This sabotage locks the MED21-MED6 interface to suppress defense transcription.\u003c/p\u003e\u003cp\u003eStrikingly, this mechanism is conserved across \u003cem\u003ePhytophthora\u003c/em\u003e species, revealing a paradigm for effector-mediated transcriptional sabotage. In the evolutionary arms race between plants and pathogens, effector-mediated disruption of transcriptional regulatory hubs represents a pivotal virulence strategy, but how pathogens achieve spatiotemporal precision in sabotaging these complexes remains enigmatic.\u003c/p\u003e\u003cp\u003eOur \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ep\u003c/span\u003erevious studies identified the transcriptional programming and functional synergy within the \u003cem\u003eP. sojae\u003c/em\u003e RXLR effectors, an extraordinarily large effector superfamily with a conserved N-terminal RXLR (Arg-any amino acid-Arg-Leu) motif\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Avh109 and other representative \u0026ldquo;immediate-early\u0026rdquo; RXLR effectors was highly expressed even prior to infection and was further rapidly induced following infection, while some \u0026ldquo;early\u0026rdquo; RXLR effectors was weakly expressed prior to infection but induced 20- to 120-fold during the first 12 h of infection. Misexpression of key immediate-early or early effectors severely reduced the virulence of \u003cem\u003eP. sojae\u003c/em\u003e transformants\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. This also implies that such effectors are deliberately deployed in response to a certain infection stage. Here we report PsAvh109 is induced by host SA accumulation, uncovers a \u0026ldquo;sensing-response\u0026rdquo; paradigm wherein pathogens eavesdrop on host immune signals to synchronize effector deployment, redefining the co-evolutionary arms race.\u003c/p\u003e\u003cp\u003eThus, we uncover an unprecedented mechanism: the \u003cem\u003eP. sojae\u003c/em\u003e nuclear effector PsAvh109 is induced by SA and hijacks the host Mediator subunit MED21 through molecular mimicry of the repressor TPL. This discovery establishes a SA-synchronized deployment of the effector to perpetuate immune suppression and a non-degradative steric blockade strategy that competitively locks the MED21-MED6 interaction interface. These insights redefine pathogen adaptation to host immunity and open avenues for engineering durable resistance in crops.\u003c/p\u003e\u003cp\u003eBeyond canonical effector mechanisms involving enzymatic degradation or static interference, we unveil a sophisticated dynamically synchronized virulence strategy converging on two evolutionary innovations: First, the \u0026ldquo;sensing-response\u0026rdquo; synchronization, where pathogens exploit host immune signals as deployment cues, SA induction of PsAvh109 ensures effector injection coincides exactly with TPL displacement during defense activation. Second, a stealthy 'non-degradative interface lockdown' tactic, wherein PsAvh109 functionally mimics host repressors to sterically obstruct Mediator assembly without proteolytic turnover, evading immune detection while maximizing target conservation. This paradigm transforms our understanding of effector evolution from passive disruptors to active conductors orchestrating sabotage to the host\u0026rsquo;s defense melody.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eSA induces PsAvh109 expression to synchronize effector deployment\u003c/h2\u003e\u003cp\u003eNumerous effector genes of \u003cem\u003eP. sojae\u003c/em\u003e are expressed during the early stages of infection, coinciding with the critical window of salicylic acid (SA)-mediated resistance to biotrophic pathogens. To investigate whether SA functions as an inductive signal for RXLR effector gene expression, \u003cem\u003eP. sojae\u003c/em\u003e germ tube was treated with 100 \u0026micro;M SA and followed RNA sequencing revealed that 2,608 genes were significantly upregulated and 2,993 downregulated following SA treatment compared to EtOH controls (Fig. S1a). Among previously annotated effectors, 64 were significantly upregulated, while only 6 were downregulated (Fig. S1b). PsAvh109 was among the most strongly induced effectors and was selected for further investigation due to its demonstrated ability to suppress immune responses triggered by pathogen-associated molecular patterns (PAMPs) such as INF1 and XEG1\u003csup\u003e33,34\u003c/sup\u003e. To monitor protein accumulation, we generated an in situ complementation strain (C-PsAvh109-6His-mCherry) by replacing the endogenous PsAvh109 gene. Exogenous SA treatment significantly increased PsAvh109-6His-mCherry protein levels relative to EtOH controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b). RT\u0026ndash;qPCR analysis confirmed that SA induced PsAvh109 transcription in a concentration-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Furthermore, in soybean root hairs expressing GFP\u0026ndash;NahG (which degrades endogenous SA\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e), PsAvh109 transcript levels were markedly reduced (~\u0026thinsp;43%) upon infection with strain P6497 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Homologous genes in \u003cem\u003eP. capsici (DVH05_001429)\u003c/em\u003e and \u003cem\u003eP. parasitica (PPTG_19802)\u003c/em\u003e also exhibited SA-inducible expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, f), indicating a conserved mechanism of SA responsiveness among oomycete pathogens.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo investigate SA signal perception, we examined PsAvh109 expression in a \u003cem\u003ePsGPA1\u003c/em\u003e knockout mutant (KO-PsGPA1), which lacks the sole Gα subunit in \u003cem\u003eP. sojae\u003c/em\u003e. SA treatment increased \u003cem\u003ePsAvh109\u003c/em\u003e expression by only 1.4-fold in the KO-PsGPA1 mutant, compared to 2.7-fold in the wild-type strain, implicating G-protein-coupled receptors (GPCRs) in SA-mediated effector activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eg). Together, these findings demonstrate that \u003cem\u003eP. sojae\u003c/em\u003e harbors an SA-sensing mechanism and that PsAvh109 expression is tightly regulated by host-derived SA.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePsAvh109 requires nuclear localization to suppress immunity and promote the virulence of\u003c/b\u003e \u003cb\u003eP. sojae\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePsAvh109 is conserved in \u003cem\u003ePhytophthora\u003c/em\u003e species including \u003cem\u003eP. infestans\u003c/em\u003e, \u003cem\u003eP. parasitica\u003c/em\u003e, \u003cem\u003eP. cactorum\u003c/em\u003e and \u003cem\u003eP. capsici\u003c/em\u003e, in which all PsAvh109 homologs harbor a nuclear localization signal (NLS) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Mature PsAvh109 fused with green fluorescent protein (GFP) accumulated in the nucleus of \u003cem\u003eNicotiana benthamiana\u003c/em\u003e epidermal cells, co-localizing with nuclear marker histone 2B fused to mCherry protein (H2B-mCherry) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). NLS mutants, that mutated all conserved lysine (K) and arginine (R) residues to alanine (A) residues or replaced with a nuclear export signal (NES), named PsAvh109\u003csup\u003eAAA\u003c/sup\u003e and PsAvh109\u003csup\u003eNES\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), abolished nuclear targeting and seprated with H2B-mCherry (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). In soybean hairy roots, PsAvh109\u0026mdash;but not the NLS mutant PsAvh109\u003csup\u003eAAA\u003c/sup\u003e\u0026mdash;significantly increased \u003cem\u003eP. sojae\u003c/em\u003e oospore production by 1.4-fold and biomass by 1.8-fold compared with the mCherry control (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, d, e). Similarly, PsAvh109 enhanced \u003cem\u003eP. capsici\u003c/em\u003e infection in \u003cem\u003eN. benthamiana\u003c/em\u003e, resulting in a 1.3-fold increase in lesion diameter and a 2.3-fold increase in pathogen biomass, whereas PsAvh109\u003csup\u003eAAA\u003c/sup\u003e showed no effect (Fig. S3a, b, c). PsAvh109 also suppressed INF1-triggered immune responses, a function lost in the NLS mutant (Fig. S3a).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eCRISPR/Cas9-mediated knockout of \u003cem\u003ePsAvh109\u003c/em\u003e (KO-PsAvh109) in \u003cem\u003eP. sojae\u003c/em\u003e had no effect on mycelial growth or zoospore production (Fig. S3d, e, f), but markedly reduced virulence, as evidenced by smaller lesions on soybean hypocotyls and a 50% decrease in pathogen biomass compared with the wild-type strain. Pathogenicity was fully restored in the complementation strain (C-PsAvh109), but not in the nuclear localization-deficient mutant (C-PsAvh109\u003csup\u003eAAA\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003ef, g), indicating that PsAvh109 is essential for full virulence and that its function is dependent on nuclear localization.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePsAvh109 targets the Mediator subunit MED21 to disrupt defense transcription\u003c/h3\u003e\n\u003cp\u003eYeast two-hybrid screening identified soybean MED21 (GmMED21), a conserved subunit of the transcriptional mediator complex's middle module\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, as a PsAvh109 interactor. Subsequent yeast two-hybrid assays confirmed that GmMED21 interacted with PsAvh109 but not with the PsAvh109\u003csup\u003eAAA\u003c/sup\u003e mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Notably, PsAvh109 selectively bound GmMED21, but not adjacent Mediator subunits (GmMED6, GmMED7, GmMED10, GmMED19, or GmMED25). Further analysis revealed that PsAvh109 also interacts with the \u003cem\u003eNicotiana benthamiana\u003c/em\u003e ortholog, NbMED21 (Fig. S4).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn luciferase complementation imaging (LCI) assays, a strong luminescence signal was observed in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves co-expressing GmMED21-nLUC and cLUC-PsAvh109, but abolished in NLS mutants, confirming the interaction between GmMED21 and PsAvh109 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Co-immunoprecipitation (Co-IP) experiments further corroborated the interaction, where GFP-PsAvh109 co-precipitated GmMED21-mCherry-3HA from \u003cem\u003eN. benthamiana\u003c/em\u003e extracts. Neither GFP-PsAvh109\u003csup\u003eNES\u003c/sup\u003e nor GFP-PsAvh109\u003csup\u003eAAA\u003c/sup\u003e were detected in the immunoprecipitates, similar to the negative controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). In contrast, in vitro pull-down assays showed that GmMED21-His interacted with GST-PsAvh109, as well as with the GST-PsAvh109\u003csup\u003eNES\u003c/sup\u003e and GST-PsAvh109\u003csup\u003eAAA\u003c/sup\u003e mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). These results indicate that PsAvh109 physically interacts with GmMED21 both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e. However, the NLS mutations likely caused the protein translocation \u003cem\u003ein vivo\u003c/em\u003e, rather than disrupting the physical interaction with GmMED21.\u003c/p\u003e\u003cp\u003eTo understand the roles of MED21 as the effector target, we inoculated the leaves with \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain carrying \u003cem\u003eINF1\u003c/em\u003e gene or \u003cem\u003eP. capsici\u003c/em\u003e in RNAi-\u003cem\u003eNbMED21\u003c/em\u003e leaf tissues. The cell death triggered by INF1 was significantly attenuated in RNAi-\u003cem\u003eNbMED21\u003c/em\u003e(Fig. S5a). Compared with the wild-type control, silencing of NbMED21 resulted in a 1.2-fold increase in lesion diameter and a 2-fold increase in \u003cem\u003eP. capsici\u003c/em\u003e biomass in infected leaves (Fig. S5a, b, c). Notably, RNAi-mediated silencing of \u003cem\u003eNbMED21\u003c/em\u003e abolished the virulence-promoting activity of PsAvh109, as PsAvh109 expression in RNAi-\u003cem\u003eNbMED21\u003c/em\u003e leaves failed to enhance \u003cem\u003eP. capsici\u003c/em\u003e infection (Fig. S5d). Lesion diameter and pathogen biomass were comparable to those in GFP control tissues (Fig. S5e, f). These results suggest that PsAvh109 requires its interaction with MED21 to facilitate pathogen infection.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTranscriptome analysis of PsAvh109-expressing soybean roots revealed significant suppression of SA-responsive genes. Compared with the GFP control group, a total of 841 differentially expressed genes (DEGs) were identified in the soybean overexpressing GFP-PsAvh109 (Fig. S6a). 157 downregulated genes enriched in defense response, response to salicylic acid, and the response to reactive oxygen species (ROS) (Fig. S6b). The up-regulated DEGs were significantly enriched in processes such as carbohydrate metabolic process and hydrogen peroxide catabolic process (Fig. S6b).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNotably, PsAvh109 suppressed the expression of key SA-responsive immune genes in soybean, including members of the \u003cem\u003eGmPR1\u003c/em\u003e family and Salicylic acid (SA)-binding protein 2 (\u003cem\u003eGmSABP2\u003c/em\u003e), which are critical for SA-mediated defense (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). RT\u0026ndash;qPCR analysis confirmed that these genes were significantly upregulated in roots infected with the PsAvh109 knockout strain (KO-PsAvh109) compared to those infected with wild-type \u003cem\u003eP. sojae\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003ef), while expression of the JA-responsive gene \u003cem\u003eGmVSPa\u003c/em\u003e remained unchanged. Together, these results demonstrate that PsAvh109 hijacks MED21 to suppress SA-dependent transcriptional reprogramming.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGmMED21 and GmMED6 act as the molecular switch of SA controlled gene transcription and blocked by PsAvh109 via competitive binding\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe head and middle modules constitute the functional core of the Mediator\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Functional validation showed that silencing \u003cem\u003eGmMED21\u003c/em\u003e or \u003cem\u003eGmMED6\u003c/em\u003e in soybean hairy roots reduced \u003cem\u003eGmPR1\u003c/em\u003e and \u003cem\u003eGmSABP2\u003c/em\u003e expression (Fig. S6c). The promoter regions (1 kb upstream of the start codon) of these genes all contained the as \u0026minus;\u0026thinsp;1 (TGACG) element, a conserved binding site for TGA transcription factors (Fig. S6d). Chromatin immunoprecipitation and qPCR (ChIP-qPCR) is employed to examine the MED21/MED6 recruitment to the \u003cem\u003eGmPR1\u003c/em\u003e promote in soybean transgenic hairy roots. The P1 (a sequence containing an as-1 element) region of the \u003cem\u003eGmPR1\u003c/em\u003e promoter showed strong binding affinity with both GmMED21 and GmMED6. No binding of GmMED21 or GmMED6 was detected with the promoter of control gene \u003cem\u003eGmCYP2\u003c/em\u003e (Fig. S6e). These results suggest that GmMED21 and GmMED6 are involved in the transcription of SA-responsive genes, positively regulating plant immunity. In addition, silencing of GmMED21 and GmMED6 led to a 1.7-fold increase in \u003cem\u003eP. sojae\u003c/em\u003e oospore production and a 2-fold increase in pathogen biomass compared to the control (Fig. S6f, g, h).\u003c/p\u003e\u003cp\u003eThe interaction between MED21 and MED6 is crucial for the full activation of multiple activators\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. In this study, we generated two GmMED21 truncation mutants: a N-terminal deletion mutant (GmMED21\u003csup\u003e16\u0026ndash;139\u003c/sup\u003e) and a short N-terminal fragment (GmMED21\u003csup\u003e1\u0026ndash;15\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Yeast two-hybrid and LCI assays mapped the MED21-MED6 interaction to the N-terminal 15-aa domain of MED21 (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, c, d). Crucially, this same domain mediated binding to PsAvh109 (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, d). The GmMED21\u003csup\u003e1\u0026ndash;15\u003c/sup\u003e maintained interactions with PsAvh109 and MED6, whereas GmMED21\u003csup\u003e16\u0026ndash;139\u003c/sup\u003e failed to interact with both (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, c), indicating that the N-terminal 15 amino acids of GmMED21 might be the competitive binding of PsAvh109.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn the LCI assays, a robust luminescence signal was observed in the leaves of \u003cem\u003eN. benthamiana\u003c/em\u003e upon coinfiltration of MED21 and MED6. However, when additional GmMED21\u003csup\u003e1\u0026ndash;15\u003c/sup\u003e-mCherry or full-length GmMED21-mCherry was expressed as a competitor, the luminescence intensity significantly decreased (Fig. S7a, b). Similarly, GST pull-down assays indicated that as the amount of GmMED21\u003csup\u003e1\u0026ndash;15\u003c/sup\u003e-mCherry increased, the amount of GmMED21-His pulled down by GST-GmMED6 decreased (Fig. S7c). Functional assays showed that overexpression of GmMED21\u003csup\u003e1\u0026ndash;15\u003c/sup\u003e in soybean root hairs significantly promoted \u003cem\u003eP. sojae\u003c/em\u003e infection, as indicated by a 1.5-fold increase in oospore production and a 1.7-fold increase in pathogen biomass (Fig. S7d, e, f). Compared with EV-transformed roots, RT-qPCR showed that the expression levels of GmPR1-1, GmPR1-6, and GmSABP2 were lower in MED21\u003csup\u003e1\u0026ndash;15\u003c/sup\u003e-overexpressing roots (Fig. S7g). Collectively, these results indicate that disrupting of MED21-MED6 interaction results in suppression of SA-responsive immunity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eGiven that both GmMED6 and PsAvh109 interact with the N-terminal domain of GmMED21, we hypothesized that PsAvh109 might compete with GmMED6 for binding to GmMED21, thereby disrupting the host resistance. Yeast three-hybrid (Y3H) assay indicates PsAvh109 (but not PsAvh109\u003csup\u003eAAA\u003c/sup\u003e) disrupted GmMED21-GmMED6 interaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). Pull-down assays further validated that GST-PsAvh109 reduced MBP-GmMED6 binding to GmMED21-His in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). Co-immunoprecipitation (Co-IP) assays revealed co-expression of PsAvh109-GFP decreased GmMED6-3Flag co-precipitation by GmMED21-HA (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e4\u003c/span\u003eg). In the LCI assay, co-expression of GFP\u0026ndash;PsAvh109 with GmMED21\u0026ndash;nLUC and cLUC\u0026ndash;GmMED6 in \u003cem\u003eN. benthamiana\u003c/em\u003e significantly reduced luminescence intensity compared to the GFP control, whereas GFP-PsAvh109\u003csup\u003eAAA\u003c/sup\u003e had no effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e4\u003c/span\u003eh, i). ChIP-qPCR analysis revealed that PsAvh109 was significantly enriched at the P1 region of \u003cem\u003eGmPR1\u003c/em\u003e promoter (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e4\u003c/span\u003ej), consistent with that of GmMED21 (Fig. S6e). These findings suggest that PsAvh109 outcompetes MED6 for MED21 binding to perpetuate repression of SA-responsive genes.\u003c/p\u003e\n\u003ch3\u003eThe N-terminal domain of MED21 is a hotspot for competitive binding by TPL and PsAvh109\u003c/h3\u003e\n\u003cp\u003eDue to the growth defense trade-off in plants, the transcription of immunity-related genes is generally inhibited during the normal growth stage. In yeast, the general corepressor Tup1 hinders the recruitment of RNA polymerase II (Pol II) by competing with MED6 for binding to Srb7p, an analog of MED21, and that an intact N-terminus of Srb7p was needed for the interaction\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Notably, the residues of MED21 that interact with Tup1 are identical to those required for the transcriptional repression by the \u003cem\u003eArabidopsis\u003c/em\u003e corepressor TOPLESS (TPL)\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Previous reports have shown that the TPD domain of TPL (TPL\u003csup\u003eN188\u003c/sup\u003e) is the key domain responsible for the interaction with MED21, and repress the function of a transcriptional activator after auxin addition\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. We hypothesized that repressors TPL might inhibit the interaction between MED21 and MED6, and the mechanism might be hijacked and exploited by Avh109.\u003c/p\u003e\u003cp\u003eY3H assays demonstrated that the interaction between GmMED21 and GmMED6 was evidently inhibited in the presence of GmTPL\u003csup\u003eN188\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Pull-down assays further confirmed that increasing amounts of GST-GmTPL\u003csup\u003eN188\u003c/sup\u003e reduced the abundance of MBP-GmMED6 co-immunoprecipitated with GmMED21-His (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Co-IP assays showed that co-expressed GmTPL\u003csup\u003eN188\u003c/sup\u003e-GFP significantly decreased GmMED6-3Flag co-precipitation by GmMED21-3HA (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). In the LCI assays, the luminescence signal with GmMED21-nLUC and cLUC-GmMED6 was significantly attenuated when FLAG-GmTPL\u003csup\u003eN188\u003c/sup\u003e was co - expressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003ed, e). This result suggest that GmTPL has a stronger interaction with GmMED21 compared to GmMED6 and thus suppresses the interaction between GmMED21 and GmMED6.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eChIP\u0026ndash;qPCR analysis revealed significant enrichment of GmTPL at the as-1 element (P1) of the \u003cem\u003eGmPR1\u003c/em\u003e promoter (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003ef), suggesting that GmTPL is recruited via its interaction with MED21.\u003c/p\u003e\u003cp\u003eGiven that both Avh109 and TPL\u003csup\u003eN188\u003c/sup\u003e compete with MED6 for binding to MED21, we compared the interaction strengths between GmMED21 and its partners (GmMED6, GmTPL\u003csup\u003eN188\u003c/sup\u003e, or PsAvh109). LCI assays revealed that co-expression of PsAvh109 with GmMED21 produced significantly stronger luminescence signals than co-expression of GmTPL\u003csup\u003eN188\u003c/sup\u003e or GmMED6 with GmMED21 (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003eg, h). Furthermore, microscale thermophoresis (MST) measurements demonstrated that PsAvh109 binds GmMED21 with a dissociation constant (Kd) of 0.27 \u0026micro;M\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04, whereas GmTPL\u003csup\u003eN188\u003c/sup\u003e and GmMED6 exhibited higher Kd values of 0.76 \u0026micro;M\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 and 1.29 \u0026micro;M\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003ei). These data indicate that PsAvh109 exhibits higher binding affinity to GmMED21 than GmMED6 or GmTPL\u003csup\u003eN188\u003c/sup\u003e. Hence, PsAvh109 might acts as a \"molecular trap\" locking MED21 by displacing TPL.\u003c/p\u003e\n\u003ch3\u003eBoth N- and C-termini of PsAvh109 are required for its interaction with GmMED21\u003c/h3\u003e\n\u003cp\u003ePhylogenetic analysis revealed the conservation of PsAvh109 across \u003cem\u003ePhytophthora\u003c/em\u003e species, including \u003cem\u003eP. parasitica, P. infestans\u003c/em\u003e, and \u003cem\u003eP. capsici\u003c/em\u003e. All PsAvh109 homologs featured NLS, RxLR-dEER, and TP motifs. Moreover, the N-terminal 50 residues of PsAvh109 showed high conservation among the homologs, while the C-terminal 15 residues included three invariant residues and three additional highly conserved residues (Fig. S2b).\u003c/p\u003e\u003cp\u003eTo further delineate the domains of PsAvh109 required for interaction with GmMED21, we generated a series of PsAvh109 mutants by deleting specific motifs: the RxLR-dEER motif (designed as PsAvh109\u003csup\u003e\u0026minus;\u0026thinsp;RxLR\u003c/sup\u003e), the TP motif (designed as PsAvh109\u003csup\u003e\u0026minus;\u0026thinsp;TP\u003c/sup\u003e), the N-terminus of PsAvh109 (designed as PsAvh109\u003csup\u003e51\u0026ndash;167\u003c/sup\u003e), the C-terminus of PsAvh109 (designed as PsAvh109\u003csup\u003e21\u0026ndash;152\u003c/sup\u003e), or both termini (designed as PsAvh109\u003csup\u003e51\u0026ndash;152\u003c/sup\u003e) (Fig. S8a). We found that three mutants, PsAvh109\u003csup\u003e51\u0026ndash;167\u003c/sup\u003e, PsAvh109\u003csup\u003e21\u0026ndash;152\u003c/sup\u003e and PsAvh109\u003csup\u003e51\u0026ndash;152\u003c/sup\u003e, lost their interactions with GmMED21 in yeast two-hybrid assays, but the other mutants did not (Fig. S8b), suggesting that the N-terminus and C-terminus of the mature effector protein were required for the interaction with MED21. Furthermore, LCI assays were conducted, revealing an undetectable luminescence signal in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves co-infiltrated with GmMED21-nLUC and cLUC-PsAvh109\u003csup\u003e51\u0026ndash;152\u003c/sup\u003e, which lacks both termini. In contrast, a weak luminescence signal was observed from cLUC-PsAvh109\u003csup\u003e51\u0026ndash;167\u003c/sup\u003e and cLUC-PsAvh109\u003csup\u003e21\u0026ndash;152\u003c/sup\u003e (Fig. S8c). Additionally, all three mutants lost the ability to inhibit INF1-induced PCD, comparable to PsAvh109\u003csup\u003eAAA\u003c/sup\u003e, while wild-type PsAvh109 strongly inhibited INF1-induced PCD (Fig. S8d). These results collectively indicate that both termini of PsAvh109 are necessary for full interactions with GmMED21.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo identify the amino acid residues essential for the interaction between PsAvh109 and GmMED21, we conducted molecular modeling and docking analysis using AlphaFold3. The predicted 3D structure of GmMED21 exhibited high structural similarity to Arabidopsis MED21 (PDB ID: C0LU16). The analysis revealed that both the N-terminal (V23, R40, Y41, V54, N58, Q66, Y69, F71) and C-terminal (W154, F157, W160, Y161, D167) regions of PsAvh109 contribute to MED21 binding, forming a \"headphone-like\" structure that encircles the N-terminal region of MED21(Fig. S8e). This structural configuration likely accounts for the markedly higher binding affinity of PsAvh109 to MED21 compared to the native transcriptional repressor TPL. Notably, key interacting residues in the N-terminus (V23, R40) and C-terminus (F157, W160, D167) of PsAvh109 are highly conserved among its homologs, supporting the evolutionary conservation of its function in targeting MED21.\u003c/p\u003e\n\u003ch3\u003eSalicylic Acid releases the binding between TPL and MED21 without impacting Avh109\u003c/h3\u003e\n\u003cp\u003eTo explore whether SA alleviates TPL-mediated transcriptional repression of SA-responsive genes, we examined TPL protein stability in tobacco plants transiently expressing TPL following exogenous SA treatment. No significant difference in TPL accumulation was observed between SA-treated and EtOH control groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e6\u003c/span\u003ea), ruling out proteasomal degradation. Co-IP further demonstrated that exogenous SA significantly reduced the co-precipitation of GmMED21-3HA and GmTPL\u003csup\u003eN188\u003c/sup\u003e-GFP compared to the EtOH control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). In contrast, SA treatment had no effect on the co-precipitation of GmMED21-3HA and PsAvh109-GFP relative to the EtOH control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). Additionally, LCI reporter assays revealed that SA treatment reduced LUC activity driven by the GmMED21-nLUC and cLUC-GmTPL\u003csup\u003eN188\u003c/sup\u003e interaction to ~\u0026thinsp;70% of the level observed in the EtOH control (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). Conversely, SA treatment had no effect on LUC activity resulting from the interaction between GmMED21-nLUC and cLUC-PsAvh109 (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). In ChIP-qPCR assays, SA treatment (100 \u0026micro;M) significantly reduced GmTPL enrichment at the as-1 sites (P1) of \u003cem\u003eGmPR1\u003c/em\u003e promoter compared to EtOH controls, PsAvh109 occupancy remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). In summary, these findings demonstrate that SA mediates the disassembly of TPL-mediated blockade on MED21-MED6 interaction, whereas PsAvh109 competitively locks the MED21-MED6 interaction interface with higher affinity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eHence, we demonstrate this dynamic process (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e7\u003c/span\u003e): TPL constitutively represses SA-responsive genes by occupying the MED21-MED6 interface; SA accumulation displaces TPL, enabling Mediator assembly and defense activation; PsAvh109 is induced by host SA and competitively binds MED21 with higher affinity than TPL or MED6, perpetuating transcriptional repression.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eA paradigm for effector-mediated synchronized transcriptional sabotage\u003c/h2\u003e\u003cp\u003eOur study unveils a molecular strategy wherein \u003cem\u003ePhytophthora\u003c/em\u003e effectors exploit the Mediator complex as an Achilles\u0026rsquo; heel to suppress SA immunity. That is a three-tiered virulence strategy converging on the eukaryotic Mediator complex: (1) effector-as-repressor-mimic: PsAvh109 emulates TPL\u0026rsquo;s structure to occupy the MED21 N-terminus with higher affinity than its host counterpart; (2) non-degradative interface lockdown: steric blockade of MED21-MED6 assembly represses defense transcription without triggering proteolysis; (3) host-signal-synchronized deployment: SA induction of PsAvh109 ensures effector injection coincides precisely with TPL dissociation (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e7\u003c/span\u003e). This \u0026ldquo;sensing-response\u0026rdquo; tactic redefines effector function: rather than static interference, PsAvh109 acts as a molecular spy that eavesdrops on host immunity to sabotage transcriptional activation at its most vulnerable moment.\u003c/p\u003e\u003cp\u003eWe demonstrate that PsAvh109 functions as a structural mimic of the host corepressor TPL, competitively occupying the N-terminal domain of MED21, a critical hub for Mediator assembly, with 3-fold higher affinity than TPL itself (Kd\u0026thinsp;=\u0026thinsp;0.27 \u0026micro;M vs. 0.76 \u0026micro;M). This effectively \"locks\" the MED21-MED6 interface (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003e), disrupting transcription of key defense genes (e.g., \u003cem\u003eGmPR1\u003c/em\u003e, \u003cem\u003eGmSABP2\u003c/em\u003e). PsAvh109 operates through steric blockade without triggering degradation\u0026mdash;a non-destructive tactic that may evade host surveillance. Conservation of this mechanism across \u003cem\u003eP. capsici\u003c/em\u003e and \u003cem\u003eP. parasitica\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, f) suggests that targeting the Mediator hub represents a universal virulence strategy in oomycetes.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eReconceptualizing pathogen adaptation to host immunity\u003c/h3\u003e\n\u003cp\u003eThe induction of PsAvh109 by host-derived SA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e) reveals a sophisticated \"sensing-triggering\" paradigm in pathogen evolution. Unlike traditional effectors constitutively expressed, PsAvh109 deployment is dynamically synchronized with host immune activation: as SA accumulates and displaces TPL from MED21 (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, d), the pathogen exploits this temporal window to inject its molecular mimic, perpetuating transcriptional repression (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e7\u003c/span\u003e). We propose that \u003cem\u003ePhytophthora\u003c/em\u003e possesses SA receptors (possibly RLPs\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e or kinase cascades) that convert host signals into effector expression, which could be a frontier for future identification. This strategy is unprecedented in eukaryotes. The programmed deployment of \u003cem\u003ePhytophthora\u003c/em\u003e effector repertoire is at least partly in response to the host's disease resistance rhythm, which enriches our model from over a decade ago\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eRedefining pathogen tactics in the arms race\u003c/h2\u003e\u003cp\u003ePathogens typically disrupt Mediator via degradative mechanisms (e.g., HaRxL44-mediated MED19a turnover). PsAvh109 exemplifies a distinct evolutionary solution: high-fidelity molecular mimicry enabling precision occupancy of a critical protein interface. This strategy minimizes host surveillance risks\u0026mdash;unlike degradation, steric blockade leaves no proteolytic debris for immune detection\u0026mdash;while maximizing target conservation across plant species. Crucially, synchronizing effector expression with host SA transforms virulence into a dynamic, rhythm-matched process, akin to pathogens \u0026ldquo;orchestrating sabotage to the host\u0026rsquo;s defense melody.\u0026rdquo; The structural emulation of host transcriptional repressors by PsAvh109 represents a highly evolved virulence strategy. PsAvh109 functions as a \"pseudo-repressor\" that physically occupies TPL\u0026rsquo;s binding site on MED21 (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Such strategy minimizes host surveillance risks while maximizing immune suppression efficiency. Crucially, both strategies exploit conserved eukaryotic regulatory nodes: Pre-initiation complex blockade in plants (steric inhibition of Mediator assembly) despite targeting distinct mediator subunitnes.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eEvolutionary convergence in transcriptional sabotage\u003c/h2\u003e\u003cp\u003eWhile little pathogen effectors are known to disrupt the plant Mediator complex, PsAvh109 employs a fundamentally distinct mechanism compared to the well-characterized downy mildew effector HaRxL44. HaRxL44 triggers proteasome-dependent degradation of MED19a, thereby shifting the balance of defence transcription from SA-responsive defence to JA/ET-signalling\u003csup\u003e32\u003c/sup\u003e. In contrast, the strategy employed by PsAvh109, via hijacking a transcriptional hub through molecular mimicry, unlike the enzymatic modifications. PsAvh109 operates through high-affinity competitive binding, a convergence toward similar functional outcomes (transcriptional repression) via divergent evolutionary paths. This comparison underscores a paradigm shift in effector evolution: whereas degradation-based effectors (e.g., HaRxL44) cause pleiotropic effects, PsAvh109 exemplifies \"precision sabotage\" through transient occupancy of a critical protein-protein interaction interface.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eThe Mediator: A universal achilles\u0026rsquo; heel in Eukaryotes\u003c/h2\u003e\u003cp\u003eThe functional parallelism of PsAvh109 and HaRxL44 underscores transcription regulation as a universal Achilles\u0026rsquo; heel across eukaryotes, repeatedly hijacked through pathogen-specific molecular mimicry. This highlights the Mediator complex as a universal vulnerability across kingdoms, targeted through pathogen-specific \"molecular keys\". The conservation of PsAvh109\u0026rsquo;s mechanism across \u003cem\u003ePhytophthora\u003c/em\u003e species underscores MED21 targeting as a universal virulence strategy in oomycetes. Beyond plants, Mediator\u0026rsquo;s architectural conservation from yeast to humans suggests this hub could be similarly exploited by diverse eukaryotic pathogens. Fungal effectors often target transcriptional regulators, yet none demonstrate PsAvh109\u0026rsquo;s triad of mimicry, interface blockade, and signal synchronization. We propose that effector-mediated hijacking of Mediator\u0026rsquo;s core may extend to animal pathogens, positioning this complex as a universal vulnerability in eukaryotic infections.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eDecoding the SA-sensing paradigm: Future frontiers\u003c/h2\u003e\u003cp\u003eOur discovery that host-derived SA directly induces PsAvh109 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-f) unveils a sophisticated \"danger sensing\" capability in \u003cem\u003ePhytophthora\u003c/em\u003e. How \u003cem\u003ePhytophthora\u003c/em\u003e precisely senses SA remains enigmatic. While SA perception in plants is mediated by receptors like NPR1\u003csup\u003e9\u003c/sup\u003e, the SA sensor of pathogen remains enigmatic. We propose two mechanistic hypotheses for future investigation:\u003c/p\u003e\u003cp\u003eDirect co-option of host receptors: Does \u003cem\u003ePhytophthora\u003c/em\u003e exploit secreted ligands to manipulate NPR1-like pathways?\u003c/p\u003e\u003cp\u003ePathogen-encoded SA receptors: Are there oomycete homologs of host SA-binding proteins (e.g., SABP2) or novel receptors that transduce SA signals? \u003cem\u003ePhytophthora\u003c/em\u003e likely possess SA receptors\u0026mdash;potentially resembling \u003cem\u003eP. sojae\u003c/em\u003e\u0026rsquo;s isoflavone chemoreceptors\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e or or sterol-sensing receptor-like kinases\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eResolving these questions will illuminate how pathogens evolve to \"eavesdrop\" on host immunity-a critical step toward disrupting this communication channel. Identifying this sensor will unveil how pathogens \u0026ldquo;tap into\u0026rdquo; host signaling networks, which is a leap toward disrupting communication in the arms race. Potential receptors like G-protein coupled receptors\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e or receptor-like kinases\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e should be screened.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eToward engineering broad-spectrum resistance: Engineering next-generation disease resistance by protecting the Mediator hub\u003c/h2\u003e\u003cp\u003eConvergent evolution of effector-mediated MED21 targeting underscores its role as a critical vulnerability in eukaryotic transcriptional regulation. Our mechanistic dissection of PsAvh109\u0026rsquo;s sabotage strategy exposes actionable targets for breeding \u003cem\u003ePhytophthora\u003c/em\u003e-resistant crops. We propose two complementary approaches leveraging the MED21-MED6-TPL axis:\u003c/p\u003e\u003cp\u003e1. Competitive Decoys: Expression of engineered MED21 decoys to shield the Mediator hub could shield Mediator without pleiotropy.\u003c/p\u003e\u003cp\u003eNotably, expression of the MED21\u003csup\u003e1\u0026ndash;15\u003c/sup\u003e peptide competitively blocked PsAvh109 binding and also interfered with MED6 association (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e4\u003c/span\u003e), prompting us to develop a refined peptide decoy that selectively binds PsAvh109 without perturbing MED6, aiming to mitigate \u003cem\u003ePhytophthora\u003c/em\u003e pathogenicity. It should be noted that the virulence of the KO-Avh109 mutant of the \u003cem\u003eP. sojae\u003c/em\u003e is significantly reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003ef, g). This proof-of-concept suggests that engineered MED21 variants could generate durable resistance. Importantly, as Mediator architecture is conserved across crops\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, this approach may extend beyond soybean.\u003c/p\u003e\u003cp\u003e2.Scalability across crops:\u003c/p\u003e\u003cp\u003eMediator architecture conservation: MED21 N-terminal domain shares 92\u0026ndash;97% aa identity in maize/wheat. Search for effective variants of MED21 from natural genetic materials, maintaining the natural function while not binding to PsAvh109. This \"molecular shield\" strategy preserves native Mediator function while excluding effectors, overcoming pleiotropy risks of knocking down entire subunit\u003c/p\u003e\u003cp\u003eProtecting the transcriptional core complex rewrites the rules of plant defense engineering\u0026mdash;shifting from gene-for-gene resistance to fortress defense at the epicenter of immunity.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eLimitations of the study\u003c/h2\u003e\u003cp\u003eWhile we establish the role of PsAvh109 in suppressing SA responses, its impact on other hormone pathways (e.g., JA-ET crosstalk) warrants investigation. Does MED21 hijacking create an \u0026ldquo;immune polarization trap\u0026rdquo; favoring necrotrophy?\u003c/p\u003e\u003cp\u003eThe structural basis for PsAvh109\u0026rsquo;s superior affinity for MED21 also demands cryo-EM analysis. Resolving PsAvh109-MED21 and TPL-MED21 complexes via cryo-EM will reveal atomic determinants of mimicry and affinity divergence, and guiding decoy optimization.\u003c/p\u003e\u003c/div\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003ePlant and microbe cultivation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eN. benthamiana\u003c/em\u003e plants were cultivated in a greenhouse at 25 °C with a cycle of 14-hours daylight and 10-hours darkness. Etiolated soybean seedlings were incubated at 25 °C for 5 days in darkness. \u003cem\u003eP. sojae\u003c/em\u003e (P6497) and \u003cem\u003eP. capsici\u003c/em\u003e (LT1534) strains were cultured in a 10% vegetable (V8) juice medium at 25 °C for 5-7 days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eN. benthamiana\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e transient expression and Virus-induced gene silencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe plasmid constructs were introduced into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e GV3101 using chemical methods\u003csup\u003e34\u003c/sup\u003e. The \u003cem\u003eA. tumefaciens\u003c/em\u003e transformants cell suspensions (OD\u003csub\u003e600\u003c/sub\u003e = 0.6) were infiltrated into the leaves of \u003cem\u003eN. benthamiana\u003c/em\u003e. \u003c/p\u003e\n\u003cp\u003eThe Tobacco Rattle Virus (TRV) mediated VIGS system was employed to silence genes in \u003cem\u003eN. benthamiana\u003c/em\u003e \u003csup\u003e44\u003c/sup\u003e. A gene-specific fragment of approximately 300 bp was cloned into the pTRV2 vector and then introduced into \u003cem\u003eAgrobacterium\u003c/em\u003e strain GV3101. Subsequently, transformants were used to infiltrate four-leaved \u003cem\u003eN. benthamiana\u003c/em\u003e plants with a 1:1 mixture of the GV3101 strain containing pTRV1. Fully expanded leaves of the silenced plants were then utilized for inoculation and subsequent quantitative PCR analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInduction of plant cell death and \u003cem\u003ePhytophthora\u003c/em\u003e infection in \u003cem\u003eN. benthamiana\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTarget gene was cloned into pBIN or p1300 vectors and then introduced into \u003cem\u003eAgrobacterium\u003c/em\u003e strain GV3101. The \u003cem\u003eA. tumefaciens\u003c/em\u003e transformants cell suspensions (OD\u003csub\u003e600\u003c/sub\u003e = 0.6) were infiltrated into 6-week-old \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain GV3101 carrying INF1 were co-infiltrated into the leaves of \u003cem\u003eN. benthamiana\u003c/em\u003e. The ensuing cell death response of the leaves was then observed. \u003c/p\u003e\n\u003cp\u003eOtherwise, the leaves were inoculated with mycelia of \u003cem\u003eP. capsici\u003c/em\u003e at 12 hours after infiltration (hpi). \u003cem\u003eP. capsici\u003c/em\u003e leaf lesions were photographed under UV light at 36 or 48 hpi, while leaf discs for RNA analysis following inoculation were collected at 6 hpi. The accumulation of the protein encoded by the infiltrated constructs was confirmed by immunoblotting, and plant transcript levels were assessed by RT-qPCR.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eP. sojae\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e culture and transformation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe wild type \u003cem\u003eP. sojae\u003c/em\u003e strain P6497 was cultured in liquid 10% V8 medium for 3-5 days. The mycelium was then subjected to three washes with sterilized water to thoroughly remove any residual medium. Zoospores were generated from the mycelium by immersion in sterilized water at 25 °C for 9-12 hours. To produce infected tissue, seeds of the susceptible soybean variety Williams were germinated for 6 days at 25 °C in darkness indoors. The six-day-old seedlings were used for inoculation. One hundred zoospores of WT or the PsAvh109 mutants were inoculated onto soybean hypocotyls; the inoculated seedlings were then incubated at 25 °C in darkness for 2-3 days. More than 6 etiolated soybean hypocotyls were tested for each biological replicate. The infected seedlings were photographed at 2-3 days post-inoculation (dpi), and the samples were harvested and stored at -80 °C for \u003cem\u003eP. sojae\u003c/em\u003e biomass detection.\u003c/p\u003e\n\u003cp\u003eThe CRISPR/Cas9 method was employed to generate mutants of PsAvh109 in \u003cem\u003eP. sojae\u003c/em\u003e, along with PsAvh109-6His complementation strains, following established protocols\u003csup\u003e45\u003c/sup\u003e \u003csup\u003e46\u003c/sup\u003e. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA extraction and transcript level analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo quantify gene transcript levels, total RNA from the \u003cem\u003eP. sojae\u003c/em\u003e and plant samples was extracted following the protocol outlined in the Plant Total RNA Isolation Kit (Vazyme). First-strand cDNA was synthesized using HiScript III reverse transcriptase, following the manufacturer’s instructions provided with the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme).\u003c/p\u003e\n\u003cp\u003eReal-time PCR was employed to measure gene transcript levels, following the manufacturer’s instructions for the 2×SYBR Premix URTAQ Kit (Vazyme). Specific primers, designed based on gene sequences, were utilized, and the actin gene served as an endogenous control. \u003cem\u003ePsActin, NbEF1a\u003c/em\u003e, and \u003cem\u003eGmCYP2\u003c/em\u003e were employed as internal controls. Primer sequences are provided in Supplementary Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA-seq analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor RNA sequencing, total RNA was extracted from soybean hairy roots. The NEB Next Ultra II Directional RNA Library Prep Kit (NEB, E7760) was employed to generate cDNA libraries. Three biological replicates were conducted for both GFP and GFP-PsAvh109 overexpressing soybean hairy roots. Differentially expressed genes (DEGs) were identified using the criteria of a fold-change ≥ 2 or ≤ -2 across samples, together with a p-value \u0026lt; 0.05. To assess significant DEGs, soybean Gene Ontology (GO) term annotations were employed. The criteria for meaningful enrichment were set at q (false discovery rate) \u0026lt; 0.05 and p-value \u0026lt; 0.01. Fold enrichment was used to calculate the -Log\u003csub\u003e10\u003c/sub\u003e(p-value).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConfocal Microscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eN. benthamiana\u003c/em\u003e leaf discs were placed in water and analyzed using an LSM 880 laser scanning microscope (Carl Zeiss, Germany). Excitation wavelengths of 488 nm and 580 nm were utilized to visualize GFP and mCherry fluorescence, respectively, with emission wavelengths ranging from 490 nm to 530 nm for GFP and from 590 nm to 620 nm for mCherry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSoybean Hairy Roots Transformation and Infection Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSoybean (cv. Williams) seeds were grown under a 16-hour light and 8-hour dark photoperiod for 5-6 days. Explants for soybean hairy root transformation were selected from cotyledons that had not fully extended. Sterilized cotyledons were washed with autoclaved ddH\u003csub\u003e2\u003c/sub\u003eO five times. Under sterile conditions, a 3-5 mm small wound was created on the lower epidermis of the cotyledon near the hypocotyl with a sterile surgical knife. The wound was then inoculated with \u003cem\u003eAgrobacterium rhizogenes\u003c/em\u003e strain K599 carrying the relevant overexpression plasmid. Subsequently, the cotyledons were cultured on MS medium at 25 °C for 3 to 4 weeks to induce the formation of healing tissue and the emergence of soybean hairy roots at the wound site.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProduction of stable transgenic soybean plants \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePsAvh109 was incorporated into a soybean overexpression vector (pDN018). The resulting construct was subsequently introduced into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain EHA105 through heat shock. As reported previously, the soybean cultivar Williams served as the recipient for genetic transformation\u003csup\u003e47\u003c/sup\u003e. To confirm successful transformation, PCR was conducted using gene-specific primers, and RNA was extracted from leaf tissues to assess the expression of \u003cem\u003ePsAvh109\u003c/em\u003e in the transgenic plants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein Pull-Down and Co-Immunoprecipitation (Co-IP) Assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor \u003cem\u003ein vitro\u003c/em\u003e pull-down assays, \u003cem\u003eGmMED21\u003c/em\u003e was cloned into pET28a with a C-terminal His-tag, while \u003cem\u003ePsAvh109, PsAvh109\u003csup\u003eAAA\u003c/sup\u003e\u003c/em\u003e, \u003cem\u003ePsAvh109\u003csup\u003eNES\u003c/sup\u003e,\u003c/em\u003e and\u003cem\u003e GmMED6 \u003c/em\u003ewithout signal peptides were cloned into pGEX4T-1 fused with a N-terminal GST-tag. \u003cem\u003eGmMED6\u003c/em\u003e also was cloned into Pmal-c2X with an N-terminal MBP tag. \u003cem\u003eGmMED21\u003csup\u003e1-15\u003c/sup\u003e\u003c/em\u003e was cloned into p1300 with a C-terminal mCherry-3xHA tag. The plasmids were transformed into \u003cem\u003eE. coli\u003c/em\u003e BL21 and incubated at 25 °C. Each strain was induced to express the relevant protein using 0.5 mM IPTG for 10h at 25 ºC. For GST pull-downs, bacterial lysates containing GST-tagged proteins were purified with glutathione Sepharose 4B resin. Then the resin was incubated with bacterial lysates containing His-tagged proteins for 3 h at 4 °C, and then the resin was washed with PBS five times. Alternatively, for His pull-downs, bacterial lysates containing His-tagged proteins were purified with Ni-NTA resin, and then incubated with bacterial lysates containing GST-tagged proteins for 3 h at 4 °C, and then the resin was washed with PBS five times. Outputs of both pull-down protocols were analyzed by Western blotting using anti-GST and anti-His antibodies, and in some experiments, anti-MBP and anti-HA antibodies.\u003c/p\u003e\n\u003cp\u003eFor \u003cem\u003ein vivo\u003c/em\u003e Co-Immunoprecipitation (Co-IP) assays, mature \u003cem\u003ePsAvh109, PsAvh109\u003csup\u003eAAA\u003c/sup\u003e\u003c/em\u003e and \u003cem\u003ePsAvh109\u003csup\u003eNES\u003c/sup\u003e\u003c/em\u003e sequences were cloned into the pBIN vector fused with \u003cem\u003eGFP\u003c/em\u003e at the N-terminus. \u003cem\u003eGmMED21\u003c/em\u003e was cloned into the p1300 plasmid fused with \u003cem\u003e3×HA-mCherry\u003c/em\u003e at the C-terminus. Transient co-expression of proteins in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves was achieved by \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation. The total proteins of the \u003cem\u003eN. benthamiana\u003c/em\u003e leaves were incubated with anti-HA magnetic beads at 4°C for 12 h. After washing five times with 1×PBS buffer, the magnetic beads with bound proteins were boiled with 1×loading buffer for 10 min. The co-precipitated proteins were detected using anti-GFP and anti-HA antibodies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLuciferase complementation imaging assays \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePsAvh109, PsAvh109\u003csup\u003eAAA\u003c/sup\u003e\u003c/em\u003e and \u003cem\u003ePsAvh109\u003csup\u003eNES\u003c/sup\u003e\u003c/em\u003e without signal peptide were cloned into the p1300 plasmid fused with the C-terminus of luciferase. \u003cem\u003eGmMED21\u003c/em\u003e was cloned into the p1300 plasmid fused with the N-terminus of luciferase. The proteins were transiently co-expressed in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves by \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation for 36 h, then the leaves were sprayed with 1 mM D-luciferin. Luminescence was observed using a plant live imaging system.\u003c/p\u003e\n\u003cp\u003eTo quantify luciferase activity, \u003cem\u003eN. benthamiana\u003c/em\u003e leaf samples were collected at 36 hpi and incubated with 1 mM D-luciferin in 96-well plates. Luciferase activity was subsequently quantified using a microplate luminometer (Promega). Data were analyzed using GraphPad Prism 8.0 software, and statistical analysis was based on one-way ANOVA for multiple comparisons.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYeast two-hybrid (Y2H) screening and assay. \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Y2H screen with pGBKT7-PsAvh109 was performed as follows. The \u003cem\u003ePsAvh109\u003c/em\u003e gene without the signal peptide was cloned into the yeast vector pGBKT7 (Clontech). The soybean cDNA library was constructed in pGADT7 using total RNA extracted from soybean hypocotyl tissues collected 12 and 24 h after inoculation with \u003cem\u003eP. sojae\u003c/em\u003e zoospores (Clontech). More than 6 × 10\u003csup\u003e6\u003c/sup\u003e primary yeast clones (providing three-fold coverage) were screened using pGBKT7-PsAvh109 as the bait. Potential yeast transformants containing cDNA clones interacting with PsAvh109 were selected using the SD-TLHA selective medium (synthetic defined media (SD) without Trp, Leu, His, Ade).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYeast Three-hybrid (Y3H) assay \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Y3H assay was conducted using the pBridge vector system following the instructions provided (Clontech) (Fig. S6). The pBridge and pGADT7 constructs were co-transfected into Y2H Gold cells and cultured on SD-TL plates at 28°C. After 3 days of incubation, positive colonies were screened and resuspended to OD\u003csub\u003e600\u003c/sub\u003e = 0.1, 0.01, and 0.001, respectively, in 0.9% NaCl. The diluted yeast strains were grown on SD/ -Trp -Leu, SD/ -Trp –Leu -His, and SD /-Trp –Leu –His -Met plates at 28°C for 5 days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicroscale thermophoresis (MST) \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtein binding affinities between GmMED21 and PsAvh109, GmTPL, or GmMED6 were measured via MST using a Monolith NT.115 instrument (NanoTemper, Germany). Briefly, recombinant and purified His - GmMED21 was diluted in PBST buffer (10 mM Tris - HCl pH 7.5, 150 mM NaCl, 10 mM MgCl₂, and 0.05% [v/v] Tween 20). The buffer contained the Monolith His - tag labeling kit - RED - tris - NTA second - generation dye (NanoTemper, Cat. No.: MOL018). The mixture was incubated at room temperature for 30 min. RED - tris - NTA - labeled His - GmMED21 (concentration range from 50 to 0.00153 μM) was incubated with GST (concentration range from 150 to 0.00457 μM), GST - PsAvh109 (concentration range from 9.375 to 0.000286 μM), GST - GmTPL (concentration range from 37.5 to 0.00114 μM), or GST - GmMED6 (concentration range from 600 to 0.0183 μM) at room temperature for 5 min in equal volumes. Subsequently, the samples were loaded onto Monolith NT.115 capillaries and measured at 25 °C with 60% MST power using the Monolith NT.115 instrument (NanoTemper, Germany). The curves were fitted and the dissociation constant (Kd) values were calculated using Nano Temper analysis software (v.2.3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChIP-qPCR assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSoybean hairy roots expressing 3Flag fused proteins were used for ChIP experiments\u003csup\u003e48\u003c/sup\u003e. ChIP - qPCR was performed on three biological replicates, and the results were normalized to the input DNA. Two independent ChIP - qPCR experiments were conducted, and the results were similar. The primers used for ChIP - qPCR are listed in Supplementary Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantification and statistical analysis \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe quantification of protein abundance by Western blots was conducted using Image J software. Quantitative data from all assays were presented as mean ± standard deviation (SD). Statistical analyses were carried out using Student's t-test or one-way analysis of variance (ANOVA) test to determine the significance of observed differences. Alternatively, Tukey's test for multiple comparisons was performed, and significant differences (p \u0026lt; 0.05) were indicated by lower case letters.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eACKNOWLEDGMENTS\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (32172387, 32172049, 31972249); Taishan Scholar Project (tsqn202211093); Natural Science Foundation of Shandong Province (ZR2021YQ20, SYS202206 and ZR2024MC195); Taishan Industrial Experts Program (No. tscy20221172), and Shandong Province First-class Discipline Construction \u0026quot;811\u0026quot; Project (SKL81121 and SKL81127). Shandong Guocangjian Biotechnology Co., Ltd., provide technical support for this research work. We are grateful to Professor Dajian Zhang for providing genetically modified soybeans.\u003c/p\u003e\n\u003cp\u003eAUTHOR CONTRIBUTIONS\u003c/p\u003e\n\u003cp\u003eQ.W and Q.X designed and supervised the study and wrote the manuscript. X.T performed the experiments, analyzed the data and wrote the manuscript. Y.M, X.H, Y.W, H.L and W.L performed the experiments and analyzed the data. Y.S, Z.Q and Y.C performed data analysis and participated in manuscript revision. Y.C constructed transgenic soybeans. Q.W, Y.C and Q.X revised the manuscript and provided the funding for this research. All authors contributed to the article and approved the submitted version.\u003c/p\u003e\n\u003cp\u003eDECLARATION OF INTERESTS\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interest.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWang, Y., Pruitt, R.N., N\u0026uuml;rnberger, T., and Wang, Y. (2022). Evasion of plant immunity by microbial pathogens. Nat. Rev. Microbiol. \u003cem\u003e20\u003c/em\u003e, 449-464. https://doi.org/10.1038/s41579-022-00710-3.\u003c/li\u003e\n\u003cli\u003eJones, J., and Dangl, J. (2006). The plant immune system. Nature \u003cem\u003e444\u003c/em\u003e, 323-329. https://doi.org/10.1038/nature05286.\u003c/li\u003e\n\u003cli\u003eSpoel, S.H., and Dong, X. (2012). How do plants achieve immunity? Defence without specialized immune cells. Nat. Rev. Immunol. \u003cem\u003e12\u003c/em\u003e, 89-100. https://doi.org/10.1038/nri3141.\u003c/li\u003e\n\u003cli\u003eMaleck, K., Levine, A., Eulgem, T., Morgan, A., Schmid, J., Lawton, K.A., Dangl, J.L., and Dietrich, R.A. (2000). The transcriptome of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e during systemic acquired resistance. Nat. Genet. \u003cem\u003e26\u003c/em\u003e, 403-410. https://doi.org/10.1038/82521.\u003c/li\u003e\n\u003cli\u003eHe, Z., Webster, S., and He, S.Y. (2022). Growth-defense trade-offs in plants. Curr. Biol. \u003cem\u003e32\u003c/em\u003e, R634-R639. https://doi.org/10.1016/j.cub.2022.04.070.\u003c/li\u003e\n\u003cli\u003eCouto, D., and Zipfel, C. (2016). Regulation of pattern recognition receptor signalling in plants. Nat. Rev. Immunol. \u003cem\u003e16\u003c/em\u003e, 537-552. https://doi.org/10.1038/nri.2016.77.\u003c/li\u003e\n\u003cli\u003eDurrant, W.E., and Dong, X. (2004). Systemic acquired resistance. Annu. Rev. Phytopathol. \u003cem\u003e42\u003c/em\u003e, 185-209. https://doi.org/10.1146/annurev.phyto.42.040803.140421.\u003c/li\u003e\n\u003cli\u003ePowers, J., Zhang, X., Reyes, A.V., Zavaliev, R., Ochakovski, R., Xu, S.L., and Dong, X. (2024). Next-generation mapping of the salicylic acid signaling hub and transcriptional cascade. Mol. Plant. \u003cem\u003e17\u003c/em\u003e, 1558-1572. https://doi.org/10.1016/j.molp.2024.08.008.\u003c/li\u003e\n\u003cli\u003eMou, Z., Fan, W., and Dong, X. (2003). Inducers of plant systemic acquired resistance regulate NPR1 function through redox changes. Cell \u003cem\u003e113\u003c/em\u003e, 935-944. https://doi.org/10.1016/s0092-8674(03)00429-x.\u003c/li\u003e\n\u003cli\u003eDespr\u0026eacute;s, C., DeLong, C., Glaze, S., Liu, E., and Fobert, P.R. (2000). The Arabidopsis NPR1/NIM1 protein enhances the DNA binding activity of a subgroup of the TGA family of bZIP transcription factors. Plant Cell \u003cem\u003e12\u003c/em\u003e, 279-290. https://doi.org/10.1104/pp.001057.\u003c/li\u003e\n\u003cli\u003eKatagiri, F. (2004). A global view of defense gene expression regulation--a highly interconnected signaling network. Curr. Opin. Plant Biol. \u003cem\u003e7\u003c/em\u003e, 506-511. https://doi.org/10.1016/j.pbi.2004.07.013.\u003c/li\u003e\n\u003cli\u003eAn, C., and Mou, Z. (2013). The function of the Mediator complex in plant immunity. Plant Signal. Behav. \u003cem\u003e8\u003c/em\u003e, e23182. https://doi.org/10.4161/psb.23182.\u003c/li\u003e\n\u003cli\u003eKim, Y.J., Bj\u0026ouml;rklund, S., Li, Y., Sayre, M.H., and Kornberg, R.D. (1994). A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II. Cell \u003cem\u003e77\u003c/em\u003e, 599-608. https://doi.org/10.1016/0092-8674(94)90221-6.\u003c/li\u003e\n\u003cli\u003eSoutourina, J., Wydau, S., Ambroise, Y., Boschiero, C., and Werner, M. (2011). Direct interaction of RNA polymerase II and mediator required for transcription in vivo. Science \u003cem\u003e331\u003c/em\u003e, 1451-1454. https://doi.org/10.1126/science.1200188.\u003c/li\u003e\n\u003cli\u003eKidd, B.N., Cahill, D.M., Manners, J.M., Schenk, P.M., and Kazan, K. (2011). Diverse roles of the Mediator complex in plants. Semin. Cell Dev. Biol. \u003cem\u003e22\u003c/em\u003e, 741-748. https://doi.org/10.1016/j.semcdb.2011.07.012.\u003c/li\u003e\n\u003cli\u003ePoss, Z.C., Ebmeier, C.C., and Taatjes, D.J. (2013). The Mediator complex and transcription regulation. Crit. Rev. Biochem. Mol. Biol. \u003cem\u003e48\u003c/em\u003e, 575-608. https://doi.org/10.3109/10409238.2013.840259.\u003c/li\u003e\n\u003cli\u003eRichter, W.F., Nayak, S., Iwasa, J., and Taatjes, D.J. (2022). The Mediator complex as a master regulator of transcription by RNA polymerase II. Nat. Rev. Mol. Cell Biol. \u003cem\u003e23\u003c/em\u003e, 732-749. https://doi.org/10.1038/s41580-022-00498-3.\u003c/li\u003e\n\u003cli\u003eDotson, M.R., Yuan, C.X., Roeder, R.G., Myers, L.C., Gustafsson, C.M., Jiang, Y.W., Li, Y., Kornberg, R.D., and Asturias, F.J. (2000). Structural organization of yeast and mammalian mediator complexes. Proc. Natl Acad. Sci. USA. \u003cem\u003e97\u003c/em\u003e, 14307-14310. https://doi.org/10.1073/pnas.260489497.\u003c/li\u003e\n\u003cli\u003eMathur, S., Vyas, S., Kapoor, S., and Tyagi, A.K. (2011). The Mediator complex in plants: structure, phylogeny, and expression profiling of representative genes in a dicot (\u003cem\u003eArabidopsis\u003c/em\u003e) and a monocot (rice) during reproduction and abiotic stress. Plant Physiol. \u003cem\u003e157\u003c/em\u003e, 1609-1627. https://doi.org/10.1104/pp.111.188300.\u003c/li\u003e\n\u003cli\u003eEl Khattabi, L., Zhao, H., Kalchschmidt, J., Young, N., Jung, S., Van Blerkom, P., Kieffer-Kwon, P., Kieffer-Kwon, K.-R., Park, S., and Wang, X. (2019). A pliable mediator acts as a functional rather than an architectural bridge between promoters and enhancers. Cell \u003cem\u003e178\u003c/em\u003e, 1145-1158. e1120. https://doi.org/10.1016/j.cell.2019.07.011.\u003c/li\u003e\n\u003cli\u003eKim, G.S., and Lee, Y.C. (2018). Postrecruitment Function of Yeast Med6 Protein during the Transcriptional Activation by Mediator Complex. Biochem. Res. Int. \u003cem\u003e2018\u003c/em\u003e, 6406372. https://doi.org/10.1155/2018/6406372.\u003c/li\u003e\n\u003cli\u003eNayak, S., and Taatjes, D.J. (2022). SnapShot: Mediator complex structure. Cell \u003cem\u003e185\u003c/em\u003e, 3458-3458.e3451. https://doi.org/10.1016/j.cell.2022.07.011.\u003c/li\u003e\n\u003cli\u003eZhang, H., Chen, D.H., Mattoo, R.U.H., Bushnell, D.A., Wang, Y., Yuan, C., Wang, L., Wang, C., Davis, R.E., Nie, Y., and Kornberg, R.D. (2021). Mediator structure and conformation change. Mol. Cell \u003cem\u003e81\u003c/em\u003e, 1781-1788.e1784. https://doi.org/10.1016/j.molcel.2021.01.022.\u003c/li\u003e\n\u003cli\u003eGrom\u0026ouml;ller, A., and Lehming, N. (2000). Srb7p is a physical and physiological target of Tup1p. Embo J. \u003cem\u003e19\u003c/em\u003e, 6845-6852. https://doi.org/10.1093/emboj/19.24.6845.\u003c/li\u003e\n\u003cli\u003eLeydon, A.R., Wang, W., Gala, H.P., Gilmour, S., Juarez-Solis, S., Zahler, M.L., Zemke, J.E., Zheng, N., and Nemhauser, J.L. (2021). Repression by the \u003cem\u003eArabidopsis\u003c/em\u003e TOPLESS corepressor requires association with the core mediator complex. eLife \u003cem\u003e10\u003c/em\u003e. https://doi.org/10.7554/eLife.66739.\u003c/li\u003e\n\u003cli\u003eAn, C., Deng, L., Zhai, H., You, Y., Wu, F., Zhai, Q., Goossens, A., and Li, C. (2022). Regulation of jasmonate signaling by reversible acetylation of TOPLESS in \u003cem\u003eArabidopsis\u003c/em\u003e. Molecular Plant \u003cem\u003e15\u003c/em\u003e, 1329-1346. https://doi.org/10.1016/j.molp.2022.06.014.\u003c/li\u003e\n\u003cli\u003eZhu, X., Zhao, Y., Shi, C.M., Xu, G., Wang, N., Zuo, S., Ning, Y., Kang, H., Liu, W., Wang, R., et al. (2024). Antagonistic control of rice immunity against distinct pathogens by the two transcription modules via salicylic acid and jasmonic acid pathways. Dev. Cell \u003cem\u003e59\u003c/em\u003e, 1609-1622.e1604. https://doi.org/10.1016/j.devcel.2024.03.033.\u003c/li\u003e\n\u003cli\u003eSzemenyei, H., Hannon, M., and Long, J.A. (2008). TOPLESS mediates auxin-dependent transcriptional repression during \u003cem\u003eArabidopsis\u003c/em\u003e embryogenesis. Science \u003cem\u003e319\u003c/em\u003e, 1384-1386. https://doi.org/10.1126/science.1151461.\u003c/li\u003e\n\u003cli\u003eLi, X., Chen, T., Li, Y., Wang, Z., Cao, H., Chen, F., Li, Y., Soppe, W.J.J., Li, W., and Liu, Y. (2019). ETR1/RDO3 Regulates Seed Dormancy by Relieving the Inhibitory Effect of the ERF12-TPL Complex on DELAY OF GERMINATION1 Expression. Plant Cell \u003cem\u003e31\u003c/em\u003e, 832-847. https://doi.org/10.1105/tpc.18.00449.\u003c/li\u003e\n\u003cli\u003eConsortium, A.I.M. (2011). Evidence for network evolution in an \u003cem\u003eArabidopsis\u003c/em\u003e interactome map. Science \u003cem\u003e333\u003c/em\u003e, 601-607. https://doi.org/10.1126/science.1203877.\u003c/li\u003e\n\u003cli\u003eAltmann, M., Altmann, S., Rodriguez, P.A., Weller, B., Elorduy Vergara, L., Palme, J., Mar\u0026iacute;n-de la Rosa, N., Sauer, M., Wenig, M., Villa\u0026eacute;cija-Aguilar, J.A., et al. (2020). Extensive signal integration by the phytohormone protein network. Nature \u003cem\u003e583\u003c/em\u003e, 271-276. https://doi.org/10.1038/s41586-020-2460-0.\u003c/li\u003e\n\u003cli\u003eCaillaud, M.C., Asai, S., Rallapalli, G., Piquerez, S., Fabro, G., and Jones, J.D. (2013). A downy mildew effector attenuates salicylic acid-triggered immunity in \u003cem\u003eArabidopsis\u003c/em\u003e by interacting with the host mediator complex. PLoS. Biol. \u003cem\u003e11\u003c/em\u003e, e1001732. https://doi.org/10.1371/journal.pbio.1001732.\u003c/li\u003e\n\u003cli\u003eWang, Q., Han, C., Ferreira, A.O., Yu, X., Ye, W., Tripathy, S., Kale, S.D., Gu, B., Sheng, Y., Sui, Y., et al. (2011). Transcriptional programming and functional interactions within the \u003cem\u003ePhytophthora sojae\u003c/em\u003e RXLR effector repertoire. Plant Cell \u003cem\u003e23\u003c/em\u003e, 2064-2086. https://doi.org/10.1105/tpc.111.086082.\u003c/li\u003e\n\u003cli\u003eMa, Z., Song, T., Zhu, L., Ye, W., Wang, Y., Shao, Y., Dong, S., Zhang, Z., Dou, D., Zheng, X., et al. (2015). A \u003cem\u003ePhytophthora sojae\u003c/em\u003e Glycoside Hydrolase 12 Protein Is a Major Virulence Factor during Soybean Infection and Is Recognized as a PAMP. Plant Cell \u003cem\u003e27\u003c/em\u003e, 2057-2072. https://doi.org/10.1105/tpc.15.00390.\u003c/li\u003e\n\u003cli\u003eLiu, Y., Sun, T., Sun, Y., Zhang, Y., Radojičić, A., Ding, Y., Tian, H., Huang, X., Lan, J., Chen, S., et al. (2020). Diverse Roles of the Salicylic Acid Receptors NPR1 and NPR3/NPR4 in Plant Immunity. Plant Cell \u003cem\u003e32\u003c/em\u003e, 4002-4016. https://doi.org/10.1105/tpc.20.00499.\u003c/li\u003e\n\u003cli\u003eBaumli, S., Hoeppner, S., and Cramer, P. (2005). A conserved mediator hinge revealed in the structure of the MED7.MED21 (Med7.Srb7) heterodimer. J. Biol. Chem. \u003cem\u003e280\u003c/em\u003e, 18171-18178. https://doi.org/10.1074/jbc.M413466200.\u003c/li\u003e\n\u003cli\u003eKoschubs, T., Lorenzen, K., Baumli, S., Sandstr\u0026ouml;m, S., Heck, A.J., and Cramer, P. (2010). Preparation and topology of the Mediator middle module. Nucleic Acids Res. \u003cem\u003e38\u003c/em\u003e, 3186-3195. https://doi.org/10.1093/nar/gkq029.\u003c/li\u003e\n\u003cli\u003eWang, X., Sun, Q., Ding, Z., Ji, J., Wang, J., Kong, X., Yang, J., and Cai, G. (2014). Redefining the modular organization of the core Mediator complex. Cell Res. \u003cem\u003e24\u003c/em\u003e, 796-808. https://doi.org/10.1038/cr.2014.64.\u003c/li\u003e\n\u003cli\u003eNozawa, K., Schneider, T.R., and Cramer, P. (2017). Core Mediator structure at 3.4 \u0026Aring; extends model of transcription initiation complex. Nature \u003cem\u003e545\u003c/em\u003e, 248-251. https://doi.org/10.1038/nature22328.\u003c/li\u003e\n\u003cli\u003eTsai, K.L., Yu, X., Gopalan, S., Chao, T.C., Zhang, Y., Florens, L., Washburn, M.P., Murakami, K., Conaway, R.C., Conaway, J.W., and Asturias, F.J. (2017). Mediator structure and rearrangements required for holoenzyme formation. Nature \u003cem\u003e544\u003c/em\u003e, 196-201. https://doi.org/10.1038/nature21393.\u003c/li\u003e\n\u003cli\u003eLarivi\u0026egrave;re, L., Plaschka, C., Seizl, M., Wenzeck, L., Kurth, F., and Cramer, P. (2012). Structure of the Mediator head module. Nature \u003cem\u003e492\u003c/em\u003e, 448-451. https://doi.org/10.1038/nature11670.\u003c/li\u003e\n\u003cli\u003eJi, P., Bao, Y., Zhou, H., Pei, Y., Song, W., Ou, K., Qiao, Z., Si, J., Zhong, Z., Xu, X., et al. (2025). Blocking the isoflavone chemoreceptor in \u003cem\u003ePhytophthora sojae\u003c/em\u003e to prevent disease. Sci. Adv. \u003cem\u003e11\u003c/em\u003e, eadt0925. https://doi.org/10.1126/sciadv.adt0925.\u003c/li\u003e\n\u003cli\u003ePei, Y., Ji, P., Miao, J., Gu, X., Wang, H., Zhao, Y., Song, W., Guo, Z., Zhou, H., Shen, D., et al. (2024). A receptor kinase senses sterol by coupling with elicitins in auxotrophic \u003cem\u003ePhytophthora\u003c/em\u003e. Proc. Natl Acad. Sci. USA. \u003cem\u003e121\u003c/em\u003e, e2408186121. https://doi.org/10.1073/pnas.2408186121.\u003c/li\u003e\n\u003cli\u003eLiu, Y., Schiff, M., Marathe, R., and Dinesh-Kumar, S.P. (2002). \u003cem\u003eTobacco Rar1, EDS1\u003c/em\u003e and \u003cem\u003eNPR1/NIM1\u003c/em\u003e like genes are required for \u003cem\u003eN\u003c/em\u003e-mediated resistance to tobacco mosaic virus. Plant J. \u003cem\u003e30\u003c/em\u003e, 415-429. https://doi.org/10.1046/j.1365-313x.2002.01297.x.\u003c/li\u003e\n\u003cli\u003eLiu, C., Tan, X., Wang, J., Sun, Y., Xu, Q., Han, C., and Wang, Q. (2025). Upgrading of the genetic engineering toolkit accelerated the discovery process of the virulence effect of PsGH7d on \u003cem\u003ePhytophthora sojae\u003c/em\u003e invasion. Physiol. Plant. \u003cem\u003e177\u003c/em\u003e, e70083. https://doi.org/10.1111/ppl.70083.\u003c/li\u003e\n\u003cli\u003eFang, Y., and Tyler, B.M. (2016). Efficient disruption and replacement of an effector gene in the oomycete P hytophthora sojae using CRISPR/C as9. Molecular plant pathology \u003cem\u003e17\u003c/em\u003e, 127-139.\u003c/li\u003e\n\u003cli\u003eJansing, J., Sack, M., Augustine, S.M., Fischer, R., and Bortesi, L. (2019). CRISPR/Cas9-mediated knockout of six glycosyltransferase genes in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e for the production of recombinant proteins lacking \u0026beta;-1,2-xylose and core \u0026alpha;-1,3-fucose. Plant Biotechnol. J. \u003cem\u003e17\u003c/em\u003e, 350-361. https://doi.org/10.1111/pbi.12981.\u003c/li\u003e\n\u003cli\u003eKaufmann, K., Mui\u0026ntilde;o, J.M., \u0026Oslash;ster\u0026aring;s, M., Farinelli, L., Krajewski, P., and Angenent, G.C. (2010). Chromatin immunoprecipitation (ChIP) of plant transcription factors followed by sequencing (ChIP-SEQ) or hybridization to whole genome arrays (ChIP-CHIP). Nature ProtocolsMethods in Molecular Biology \u003cem\u003e5\u003c/em\u003e, 457-472. https://doi.org/10.1038/nprot.2009.244.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Salicylic acid, Mediator, PsAvh109, TOPLESS, effector, host immunity, Phytophthora sojae","lastPublishedDoi":"10.21203/rs.3.rs-7305635/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7305635/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePathogens disrupt transcriptional hubs to subvert host immunity, yet the spatiotemporal mechanisms remain enigmatic. Here, we report a pathogen effector hijacks the core eukaryotic transcriptional machinery by acting as a molecular mimic of host repressors, deploying this sabotage in synchrony with the plant immune rhythm. We discover the \u003cem\u003ePhytophthora sojae\u003c/em\u003e nuclear effector directly targets the host Mediator complex. Crucially, PsAvh109 emulates the host repressor TOPLESS (TPL), competitively occupying the Mediator subunit MED21 and locks the MED21-MED6 interaction interface, repressing SA-responsive defense genes without triggering degradation. Strikingly, PsAvh109 expression is induced by host-derived SA, the very signal displaces TPL from MED21 to activate immunity. This enables the pathogen to deploy its molecular trap precisely when the host initiates defense, perpetuating transcriptional repression during a critical vulnerability window. This work establishes a paradigm-shifting 'sensing-response' model in plant-pathogen interactions, wherein Phytophthora pathogens deploy a dynamically synchronized virulence strategy. By eavesdropping on host-derived salicylic acid (SA), the pathogen induces nuclear effector PsAvh109 expression to precisely coincide with TPL dissociation from MED21. Crucially, PsAvh109 executes non-degradative steric blockade of the MED21-MED6 interface through high-fidelity molecular mimicry, perpetuating transcriptional repression without triggering host surveillance systems. This dual-layered strategy redefines effector function as a rhythm-matched molecular sabotage agent.\u003c/p\u003e","manuscriptTitle":"Molecular Espionage in Plant Immunity: A Pathogen Effector Eavesdrops on Salicylic Acid to Hijack the Host Transcriptional Mediator Hub","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-15 18:04:46","doi":"10.21203/rs.3.rs-7305635/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b2f6711b-a6dc-4fe7-a1b8-609783361a87","owner":[],"postedDate":"October 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":56168883,"name":"Biological sciences/Microbiology/Pathogens"},{"id":56168884,"name":"Biological sciences/Plant sciences/Plant immunity/Effectors in plant pathology"}],"tags":[],"updatedAt":"2026-04-16T07:07:00+00:00","versionOfRecord":{"articleIdentity":"rs-7305635","link":"https://doi.org/10.1038/s41467-026-71909-8","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2026-04-15 04:00:00","publishedOnDateReadable":"April 15th, 2026"},"versionCreatedAt":"2025-10-15 18:04:46","video":"","vorDoi":"10.1038/s41467-026-71909-8","vorDoiUrl":"https://doi.org/10.1038/s41467-026-71909-8","workflowStages":[]},"version":"v1","identity":"rs-7305635","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7305635","identity":"rs-7305635","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}