EgSPE, a secreted effector from Epichloë gansuensis, modulates symbiotic establishment and host drought tolerance

EgSPE, a secreted effector from Epichloë gansuensis, modulates symbiotic establishment and host drought tolerance | 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 Research Article EgSPE, a secreted effector from Epichloë gansuensis, modulates symbiotic establishment and host drought tolerance Haijuan Zhang, Haotian Shi, Chunjie Li, Lei Lei This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9167048/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Background Epichloë endophytes form beneficial symbioses with cool-season grasses, enhancing host tolerance to abiotic stresses such as drought while maintaining normal plant growth. However, the molecular mechanisms underpinning this symbiosis, particularly the role of fungal-secreted effectors, remain largely unexplored. Results In this study, we identify EgSPE, a secreted effector from Epichloë gansuensis , as a key regulator of symbiotic establishment and host drought tolerance in drunken horse grass ( Achnatherum inebrians ). Transcriptome profiling during host colonization revealed EgSPE as a strongly induced gene encoding a secreted protein. Functional characterization facilitated by a substantially improved transformation system demonstrates that EgSPE is indispensable for fungal growth and efficient host colonization, as its deletion severely disrupted symbiotic establishment. Notably, EgSPE activates the host drought-responsive signaling by inducing the marker gene RD29A in a heterologous system ( Nicotiana benthamiana ) and upregulating stress-related genes ( AiRD22 , AiNAC5 , and AiABA1 ) in its native host ( A. inebrians ). Consistently, only the E. gansuensis wild-type and OE- EgSPE strains enhanced host drought tolerance, whereas the Δ egspe mutants failed to confer this benefit. Conclusions In summary, our research findings identify EgSPE as a fungal effector that plays an important role in the establishment of symbiosis and in the host's drought response, providing strong evidence for how E. gansuensis promotes abiotic stress tolerance in grasses. Epichloë endophyte transformation system secreted protein drought tolerance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 BACKGROUND Epichloë endophytes establish mutualistic symbioses with more than one hundred cool-season grass species, primarily within the subfamily Pooideae (e.g., Festuca , Lolium , and Poa spp.) [ 1 ]. These symbionts confer enhanced tolerance against a broad spectrum of biotic and abiotic stresses-including fungal pathogens [ 2 , 3 ], insect herbivory [ 4 ], drought [ 5 ], salinity [ 6 ], and heavy metal toxicity [ 7 ], without compromising host growth. Despite this agricultural potential, the association is typically host-specific [ 8 , 9 ], limiting its broader application in crop improvement. Expanding the utility of Epichloë symbionts will require a deeper mechanistic understanding of how these fungi establish symbiosis and confer stress tolerance-aspects that remain largely unresolved. Fungal colonization of plants, whether by pathogens or mutualists, is critically dependent on secreted proteins (SPs) that modulate host physiology to facilitate persistence within plant tissues [ 10 ]. In pathogenic interactions, SPs suppress host immunity and reprogram metabolism to promote infection. Similarly, mutualistic fungi must finely tune host defense responses to achieve stable colonization, a process in which secreted effectors play key roles. For instance, Efe-afpA , a secreted protein identified in the apoplast of E. festucae -colonized red fescue, exhibits antifungal activity against plant pathogens and may be required for symbiosis [ 2 , 11 ]. Although hundreds of putative SPs have been predicted in Epichloë genomes, functional characterization has been reported for only a single protein to date, representing a critical gap in our understanding of the molecular basis of Epichloë -grass symbioses [ 12 ]. Progress in functional genetic studies of Epichloë has been further constrained by the lack of efficient transformation systems. As slow-growing filamentous fungi, Epichloë species are recalcitrant to conventional protoplast-based methods developed for other fungi [ 13 , 14 ]. Establishing a high-efficiency genetic transformation system is therefore a prerequisite for accelerating functional genomics and dissecting the molecular mechanisms underlying Epichloë -grass interactions. In this study, we investigated the symbiosis between Epichloë gansuensis and drunken horse grass ( Achnatherum inebrians ), a well-characterized system in which endophyte colonization markedly enhances host drought tolerance [ 15 , 16 ]. Through transcriptomic profiling during host colonization, we identified EgSPE , a gene encoding a secreted protein that is strongly induced in planta, suggesting a potential role in symbiotic interaction. To enable functional analysis, we established a substantially improved genetic transformation system for Epichloë , incorporating optimized culture conditions and a split-marker deletion strategy. Using this platform, we demonstrate that EgSPE is indispensable for normal fungal growth and efficient host colonization, and that it contributes critically to endophyte-mediated drought tolerance. These findings provide new insight into the molecular mechanisms by which Epichloë endophytes promote abiotic stress tolerance in grasses. MATERIALS AND METHODS Plant material and growth conditions The seeds of the drunken horse grass ( Achnatherum inebrians ) were used as test material, which is collected from Qinghai-Tibet Plateau, China (97.27°E, 30.24°N). A sufficient amount of the grass seeds was disinfected with 75% ethanol for 1 min, then treated with 2% sodium hypochlorite for 30 min, and finally rinsed with sterile water 5 times. The seeds were cultured in 1/2MS medium and placed in a 23 ℃ climate chamber for growth, with a photoperiod of 16 h of light/8 h of darkness, and a relative humidity of 60%. The seeds were allowed to grow up to 15–20 d. Media and preparation of Epichloë gansuensis culture The fungal strain was isolated from A. inebrians and identified as E. gansuensis , exhibiting 99% sequence similarity to strain e7080 (NCBI Taxonomy ID: 447254) from the National Center for Biotechnology Information (NCBI). The endophyte was cultured on potato dextrose agar (PDA) medium in an artificial climate chamber (MGC-450HP-2, Bluepard) at 22°C under dark conditions. Before optimization, mycelia from the colony edge were transferred to 250 mL Erlenmeyer flasks, each containing 100 mL of yeast peptone dextrose medium (YPD), and incubated at 22°C with shaking at 200 rpm under dark conditions for 15 d. After optimization, mycelia from the colony edge were transferred to 500 µL of YPD medium and homogenized using a ball mill at 30 m/s for 30 s until fully lysed. A 200 µL aliquot of the homogenate was spread onto PDA medium overlaid with cellophane membranes and incubated at 22°C in darkness for 4 d. Fresh mycelia were scraped from the cellophane membranes and transferred to 500 µL of YPD medium. The mixture was homogenized using a ball mill at 30 m/s for 30 s until fully lysed. The homogenate was then inoculated into four 250 mL Erlenmeyer flasks, each containing 100 mL of YPD medium, and incubated at 22°C with shaking at 200 rpm under dark conditions for 4 d. Protoplast isolation The fungal cultures were divided into batches and transferred to sterilized 50 mL centrifuge tubes. The tubes were then centrifuged at 4°C and 5,000 × g for 10 min. The pellet was washed three times with 0.7 mol/L NaCl, followed by final centrifugation at 4°C and 5,000 × g for 5 min, after which the supernatant was discarded. In case of enzymatic digestion, 0.2 g of fungal biomass was suspended in 1 mL of enzyme solution (prepared in 0.7 mol/L NaCl: 2.0% lysing enzymes, 1.5% Driselase basidiomycetes, 1.0% snailase, 1.0% cellulase, and 3 mg/ml Bovine serum albumin). The enzyme solution was filter-sterilized using a 0.45 µm filter column. The mixture was incubated in a water bath at 32°C for 4–5 h. The digested suspension was filtered through two layers of lens cleaning paper glass funnel (pre-wetted with 0.7 mol/L NaCl) into a 50 mL centrifuge tube. To the filtrate, 1 mL of STC buffer (1.2 M sorbitol, 10 mM Tris-HCl, 50 mM CaCl₂, pH 7.5) was added, and the mixture was centrifuged at 4°C and 3,000 × g for 20 min. The supernatant was discarded, and the pellet was resuspended in 1 mL of STC buffer, followed by centrifugation at 4°C and 2,000 ×g for 10 min. This washing step was repeated once. The final protoplasts pellet was gently resuspended in 500 µL of STC buffer. Protoplast concentration was quantified using a hemocytometer under a microscope, and the suspension was diluted with STC buffer to achieve a final density of ≥ 5 × 10⁶ protoplasts per mL. Knockout and overexpression vector construction The overexpression vector was constructed using the plasmid backbone pPCT74. To facilitate detection, an HA epitope tag was incorporated into the construct. The target gene was placed under the control of the ToxA promoter, a strong constitutive promoter in fungi, to ensure high expression levels. The native termination codon of the gene was removed, and an HA tag was fused to the C-terminal end of the protein, enabling efficient detection via PCR methods. The strategy based on the split-marker approach was used to obtain E. gansuensis secreted protein essential ( EgSPE ) gene knockout strains. In the obtained target gene sequence, an upstream primer was designed approximately 1,000 bp before the start codon (excluding the start codon), with the 5' end of the upstream primer incorporating a hygromycin linker homologous arm. A downstream primer was designed approximately 1,000 bp after the stop codon (excluding the stop codon), with the 3' end of the downstream primer incorporating a hygromycin linker homologous arm. The fused product was then purified and used as a template for amplification to obtain the knockout fragment. Additionally, we employed a strategy using hygromycin as the homologous arm, amplifying a fragment from approximately 1,000 bp before the start codon to the first two-thirds of the hygromycin sequence, and another fragment from the last two-thirds of the hygromycin sequence to approximately 1,000 bp after the stop codon. The overlap between the two truncated hph gene fragments was 500–700 bp. PEG-mediated protoplast transformation Transformation using 80 µL of protoplasts was mixed with 1 µg of linearized plasmid (overexpression and knockout fragments), then added 5 µL of 50 mM spermidine and stirred thoroughly. Subsequently, added 90 µL of 40% PEG 4,000 and incubated at room temperature for 10 min. Then added 2 volumes of STC (360 µL), centrifuged at 4°C and 2,000×g for 5 min, and repeated the centrifugation step three times. Finally, retained 100 µL of STC and gently resuspended the pellet by flicking. Protoplast regeneration and screening for stable transformants A 100 µL of the transformation product was spread onto regeneration medium (PDB, 0.6 M sucrose, and 1.0% agar). Hygromycin (100 µg/mL) (Solarbio: H8080) was used to screen the overexpression transformants and knockout transformants. Untransformed protoplasts were spread onto regeneration plates with and without antibiotics, followed by incubation at 22°C in the dark for 7–10 d. Then, the grown transformants were transferred to fresh antibiotic-resistant medium and DNA was extracted for identification (Fig. S2 A). The Hyg -F/R primer pairs were employed to detect the introduction of the hygromycin gene, EgSPE -F/R to verify gene knockout, and EgSPE -outF/R to confirm whether the target gene was replaced by hygromycin (Fig. S2 B). The PCR band sizes were used to determine the results (Fig. S2 C, D, E). Determination of fungal growth rate An inoculation pipette of 0.5 mm was used to inoculate the wild-type, overexpressed and knockout strains onto PDA medium. The wild-type E. gansuensis was inoculated onto an antibiotic-free medium, and the overexpressed and knockout strains were inoculated onto a medium containing hygromycin (100 µg/mL) (Solarbio: H8080). The colony diameter was recorded every three days using a vernier caliper, and measured continuously for 30 d. Each strain had three replicates, and the experiment was repeated three times. The growth rate was fastest on the 18th day. RT-qPCR analysis Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) was used to determine the expression level of the target gene of the overexpressed strain. RNA was extracted from a single (transformed) mycelium colony, reverse transcribed into cDNA, diluted to an appropriate concentration, and designed specific primers for the target gene with extension factor as the internal reference gene. ABclonal SYBR Green (RT-qPCR reagent No. RK21203) was used to perform RT-qPCR for the overexpressed transformed strain E. gansuensis , and the procedure was repeated three times. The RT-qPCR instrument automatically generated amplification and melting curve, through which the Ct value of target gene and internal reference gene in each sample was determined (Ct, cycle threshold, the number of cycles when the fluorescence signal reached the set threshold). Detection of endophytic fungal colonization rate Two months post-inoculation, the colonization rate of endophytic fungi in drunken horse grass was assessed for each transgenic strain treatment. Cut off a tiller and trim about 2 cm of the stem from the root upwards, and extract DNA for subsequent experiments. Fungal colonization was detected by PCR amplification of the indole alkaloid biosynthesis gene idtG . A total of 60 samples were analyzed per treatment. Transcriptome sequencing and data analysis The total RNA from inoculated stems with different time points inoculated and control was individually extracted to construct the RNA-seq library. Sequencing was performed using Illumina NovaSeq6,000 platform. DESeq2 (V1.22.2) /edgeR (V3.6.8) was used for differential expression significance analysis, and the screening threshold was FDR (false discovery rate) 1 or < -1. Analysis of drought resistance of transgenic strains in plants The genetic screen used the firefly luciferase reporter gene driven by the drought responsive RD29A promoter ( RD29A::LUC ). In N. benthamiana , each leaf was divided into two halves, with proRD29A::LUC co-expressed alongside EV on one side and EgSPE on the other. After 24 h of expression, the experimental group was treated with 20% PEG6,000 for 8 h, water treatment as a control. Leaves were cut and sprayed with D-Luciferin (GlpBio: GC11860) on the underside of the leaves, then observe and photographed using the Living Imaging system (PerkinElmer IVIS Lumina III). WGA-AF488 staining assay At 6 dpi, tissue segments (extending 1 cm above the inoculation site) were collected for assessment of fungal colonization. The samples were processed for fluorescence imaging according to the staining protocol described by [ 37 ], Wheat Germ Agglutinin-Alexa Fluor 488 (WGA-AF488) was used to stain fungal hyphae. Observation was carried out on a Leica SP8 laser scanning confocal microscope, with excitation/emission settings of 488/500–530 nm for eGFP (WGA-AF488). Validation of the secretion function of the predicted signal peptide of EgSPE The yeast signal sequence trap assay used the pSUC2T7M13Ori (pSUC2) vector, which carries a truncated SUC2 gene lacking the initiating methionine and the native signal peptide [ 38 ]. The secretion activity of the EgSPE signal peptide was verified. DNA fragments of the signal peptide were synthesized by SynbioB and inserted into pSUC2 through EcoRI and XhoI restriction sites, fused in-frame with the invertase gene [ 39 ]. Using the lithium acetate method, 20 ng of the plasmid was transformed into the enzyme-deficient yeast strain YTK12. After transformation, yeast cells were plated on CMD-W (minus-tryptophan) plates and transferred to fresh CMD-W plates, then stored at 30°C. PCR with vector-specific primers was used to confirm the transformation. To assess the secretion of the transformant enzyme, colonies were replicated onto YPRAA plates containing raffinose and lacking glucose (1% yeast extract, 2% peptone, 2% raffinose, and 2 µg/mL antibiotic A). The activity of the transformant enzyme was measured by TTC reduction to insoluble red triphenylformazan. Yeast cultures were inoculated into sucrose medium and incubated at 30°C for 24 h. The pellets were collected, washed, and resuspended in distilled water. The cultures were incubated with 0.1% TTC at 35°C for 35 min, followed by an additional 10 min at room temperature, and color change was monitored. Statistical analysis The Statistical Product and Service Solutions (SPSS) software was used to conduct the statistical analyzes. All experimental data were tested by Student’s t-test and Tukey post-test. Prism 10.1.2 software (GraphPad, San Diego, CA, USA) was used to generate figures. RESULTS Expression of the secreted protein EgSPE in E. gansuensis is highly induced during initial host colonization In order to explore the molecular mechanism of E. gansuensis colonization in A. inebrians , we performed transcriptomic analysis of the A. inebrians - E. gansuensis interaction at 0 and 96 hours post-inoculation (hpi). Differential expression analysis revealed 276 significantly upregulated and 1,468 downregulated fungal genes during host infection (Fig. 1 A). Functional enrichment analysis showed that upregulated genes were primarily associated with transmembrane export, immune regulation, DNA repair, and tubulin assembly (Fig. 1 B). Among these, a gene encoding a secreted protein, designated EgSPE ( E. gansuensis essential secreted protein), exhibited the most significant induction following host inoculation (Fig. 1 A). We further validated EgSPE expression under colonization. EgSPE transcript levels were markedly elevated during fungal inoculation (Fig. 1 C). Structural analysis predicted that EgSPE contains an N-terminal signal peptide and a carbohydrate-binding module (CBM) family 19 domain (Fig. 1 D). We then validated the functional secretion of the predicted signal peptide via a yeast signal sequence trap assay (Fig. 1 E). These results demonstrate that the N-terminal signal peptide effectively directs protein secretion in vivo , supporting the classification of EgSPE as a functional effector during the initial infection phase. Substantially improved transformation system enables functional analysis of EgSPE To functionally characterize EgSPE, efficient genetic manipulation of E. gansuensis was required. However, conventional transformation approaches for filamentous fungi are largely ineffective in Epichloë species, primarily due to their slow growth and limited protoplast yield. In particular, hyphal maturation leads to extensive chitin-glucan cross-linking in the cell wall, which markedly reduces enzymatic digestibility [ 17 – 19 ]. Consistent with this, 15-day-old mycelia grown on PDA-the standard condition for biomass accumulation-yielded only ~ 1.8 × 10⁶ protoplasts g⁻¹ after enzymatic treatment, with incomplete cell wall digestion (Fig. 2 A, Fig. S1 A, B). To overcome this bottleneck, we redesigned the hyphal culture strategy to obtain younger, more enzymatically susceptible mycelia. Briefly, colony fragments were homogenized and spread on cellophane-overlaid PDA to generate synchronized micro-colonies for 4 d, which were subsequently subcultured in YPD liquid medium for another 4 d (Fig. 2 B). This optimized approach dramatically improved cell wall digestibility, increasing protoplast yield to 6.9 × 10⁸ g⁻¹ with complete digestion (Fig. 2 C, D). Consequently, we observed a 9-fold increase in transformation efficiency, yielding over 175 transformants µg⁻¹ DNA (Fig. 2 E). Using this substantially improved system, we successfully generated both EgSPE overexpression and knockout mutants (Fig. S2 A-F). One overexpression line exhibiting ~ 70-fold elevated transcript levels was selected for further analyzes (Fig. 2 F). The application of a split-marker homologous recombination strategy further ensured precise gene targeting, achieving a knockout frequency of 12% for the Δ egspe mutants (Fig. 2 G, Fig. S2 C-F). Collectively, this optimized platform enables rapid and efficient genetic manipulation of E. gansuensis , thereby providing a robust foundation for dissecting the molecular mechanisms underlying Epichloë –grass symbiosis. EgSPE is required for mycelial growth of E. gansuensis . To determine whether EgSPE contributes to fungal development, we examined colony morphology and radial growth of overexpression and knockout strains in comparison with the wild type on PDA medium. While overexpression lines (OE- EgSPE ) displayed growth characteristics indistinguishable from the wild type, knockout mutants (Δ egspe ) exhibited reduced colony size, altered pigmentation and significantly slower radial expansion (Fig. 3 A, B). These results indicate that EgSPE is essential for maintaining normal vegetative growth and mycelial morphogenesis in E. gansuensis . EgSPE is essential for early fungal proliferation and systemic host colonization To determine the role of EgSPE in host-fungus interactions, we monitored the progression of fungal invasion in A. inebrians . At 6 days post-inoculation (dpi), the capacity for early hyphal expansion was assessed in tissues approximately 1 cm above the inoculation site (Fig. 4 A). Quantitative biomass analysis and WGA-AF488 fluorescence staining revealed that while OE- EgSPE strains exhibited proliferation levels comparable to the wild type, the Δ egspe mutants were significantly impaired in their ability to invade host tissues, showing a marked reduction in fungal biomass (Fig. 4 B, C). We further evaluated whether this early invasion defect impacts the long-term establishment of symbiosis. Analysis of two-month-old plants showed that OE- EgSPE did not significantly increase the colonization rate, whereas Δ egspe mutants resulted in a pronounced reduction compared with the wild type (Fig. 4 D). Collectively, these results demonstrate that EgSPE promotes both early fungal proliferation in host tissues and subsequent systemic colonization in the E. gansuensis - A. inebrians symbiosis. EgSPE activates host drought responses and enhances drought tolerance in A. inebrians The E. gansuensis-A. inebrians symbiosis is known to enhance host tolerance to multiple environmental stresses, particularly drought [ 16 ]. Consistent with this role, EgSPE expression was not only induced during endophyte-host interaction but further upregulated under drought stress (Fig. 5 A). Heterologous expression of EgSPE in N. benthamiana significantly enhanced the expression of the drought-responsive marker gene RD29A under drought treatment, suggesting a specialized role of EgSPE in endophyte-mediated drought tolerance (Fig. 5 B, C). To further confirm the functional role of EgSPE in the native host, we assessed drought responses of A. inebrians colonized by different E. gansuensis strains after verifying successful colonization via fungal marker detection (Fig. 5 D, S2 G). Under drought treatment, plants inoculated with the wild-type and OE- EgSPE strains exhibited significantly enhanced drought tolerance compared with endophyte-free plants (Eg-free). In contrast, plants colonized by Δ egspe mutants failed to confer any significant drought resistance (Fig. 5 E). We further analyzed the expression of downstream drought-responsive genes including AiRD22 , AiNAC5 , and AiABA1 . At 48 and 96 hpi, inoculation with Eg-wt, OE- EgSPE , and Δ egspe mutants all induced the expression of these downstream drought-responsive genes compared with Eg-free. Notably, the upregulation of these genes was more pronounced in plants colonized by the OE- EgSPE strains (Fig. 5 F). After 48 h of inoculation, treated with PEG6,000 for 48 h, OE- EgSPE colonization significantly enhanced the expression of AiRD22 , AiNAC5 , and AiABA1 relative to Eg-free. By contrast, only slight increases in the expression of these genes were observed in plants colonized by the Δ egspe mutants, and no significant induction was detected under PEG6,000 treatment (Fig. 5 F, G). Together, these results indicate that EgSPE plays a critical role in E. gansuensis -mediated enhancement of drought tolerance in A. inebrians , likely through activation of host drought-responsive pathways. DISCUSSION Epichloë endophytic fungal symbionts are widely recognized for conferring enhanced stress tolerance to their host plants [ 20 – 22 ]. Notably, these endophytes improve drought resistance in grasses without compromising host growth, a trait of considerable value for developing more resilient crops and forages under the pressures of climate change. Understanding the molecular mechanisms underlying Epichloë –grass interactions is therefore critical for the efficient and widespread application of this endophyte genus. In this study, we identified a protein effector, EgSPE, which plays multiple essential roles in E. gansuensis and its interaction with host grass, including the regulation of fungal morphology and growth rate, the establishment of systemic symbiosis, and the mediation of host drought tolerance. This effector may serve as a key entry point for elucidating the molecular crosstalk mechanisms in Epichloë –grass interactions. EgSPE encodes a small protein of 81 amino acids, contains a single carbohydrate-binding module (CBM) domain (Fig. 1 D). CBMs are non-catalytic domains that typically enhance polysaccharide-degrading efficiency by targeting enzymes to their substrates and disrupting substrate structure; they also participate in diverse biological processes-including pathogen defense and biosynthesis-through specific carbohydrate recognition [ 23 ]. For instance, LysM (CBM50) effectors such as Ecp6, Slp1, and RsLysM sequester chitin oligomers in the apoplast, competing with plant chitin receptors like CEBiP to prevent chitin perception and thereby dampening ROS bursts and defense-related gene expression [ 24 ]. Certain CBM containing proteins can enhance reactive oxygen species (ROS) bursts and contribute to drought-stress tolerance [ 25 ]. EgSPE contains a CBM 19 domain previously characterized as a chitin-binding module in fungal chitinases, suggesting a potential role in chitin-associated processes [ 26 ]. The phenotypic alterations observed in the Δ egspe mutants (Fig. 3 , 4 ) suggest that EgSPE contributes to fungal morphogenesis and symbiotic performance. One possibility is that EgSPE directly participates in cell wall organization through chitin binding, as proper chitin distribution is essential for hyphal growth and colony architecture [ 27 , 28 ]. Disruption of this process could alter cell wall structure and consequently affect colony morphology and pigment deposition, potentially explaining the white-to-pink transition observed in the mutants. Alternatively, EgSPE may function as a non-catalytic accessory factor that facilitates the localization or activity of cell wall-modifying enzymes such as chitinases or glucanases. Consistent with this view, CBMs often enhance polysaccharide remodeling by coordinating enzyme–substrate interactions within insoluble cell wall matrices [ 29 , 30 ]. Together, these observations support a role for EgSPE in chitin-associated cell wall processes that influence fungal development and host interaction. In addition, EgSPE appears to exert a cross-kingdom function, as its secretion induces the expression of drought-responsive genes in plants. This observation suggests that EgSPE may act as a fungal-derived elicitor or microbe-associated molecular pattern (MAMP) that is perceived by plant pattern recognition receptors (PRRs), such as LysM receptor-like kinases involved in the recognition of fungal carbohydrate signals [ 31 , 32 ]. Activation of PRR-mediated signaling typically triggers early immune responses, including reactive oxygen species (ROS) accumulation, Ca²⁺ influx, and MAPK cascade activation, which subsequently regulate downstream transcription factors such as WRKY and DREB, leading to the induction of ABA-dependent and drought-responsive genes [ 33 – 35 ]. Consistent with this hypothesis, increasing evidence indicates that Epichloë endophytes enhance host tolerance to drought stress by modulating antioxidant systems and phytohormone signaling pathways. In particular, abscisic acid (ABA) plays a central role in coordinating stress-responsive gene expression and mitigating oxidative damage in endophyte-associated grasses [ 5 , 36 ]. The identification and functional characterization of EgSPE highlight the importance of secreted proteins in regulating the growth and development of Epichloë endophytes and their symbiotic interaction with host grasses. Our findings provide direct evidence that secreted proteins contribute to multiple aspects of the Epichloë –grass association, an area that has remained poorly understood. These results suggest that Epichloë -derived secreted proteins may represent valuable molecular tools for dissecting the complex molecular dialogue between endophytes and their host plants. Future studies should aim to identify additional effector or secreted proteins in Epichloë and to elucidate the molecular mechanisms underlying their secretion, perception and downstream signaling in host plants. The optimized fungal transformation system developed in this study will facilitate functional genetic analyzes and accelerate the discovery of symbiosis-related effectors, thereby advancing our understanding of the molecular basis of Epichloë -grass symbiosis. CONCLUSION In this study, we identify EgSPE as a secreted effector from E. gansuensis that contributes to symbiotic establishment and host drought tolerance in A. inebrians . Our results show that EgSPE is essential for normal fungal growth and efficient host colonization, and its absence abolishes the endophyte's ability to enhance drought tolerance in the host. Furthermore, EgSPE is associated with the activation of drought-responsive gene expression in both heterologous and native host systems. Together, these findings reveal a role for EgSPE in mediating mutualistic benefits in this grass–endophyte association and provide a mechanistic basis for further investigation of stress adaptation in symbiotic systems. Abbreviations EgSPE Epichloë gansuensis essential secreted protein OE- EgSPE EgSPE -overexpressed Epichloë gansuensis strain Δ egspe EgSPE knockout Epichloë gansuensis strain RD29A Responsive to Desiccation 29A SPs secreted proteins hpi hours post-inoculation dpi days post-inoculation CBM carbohydrate-binding module GO Gene Ontology ROS reactive oxygen species MAMP microbe-associated molecular pattern PRRs plant pattern recognition receptors ABA abscisic acid NCBI National Center for Biotechnology Information PDA potato dextrose agar YPD yeast peptone dextrose medium WGA-AF488 Wheat Germ Agglutinin-Alexa Fluor 488 Declarations Ethics approval and consent to participate The research was performed in accordance with Chinese law and international guidelines. The collection of the plant species was permitted by the University Ethical Committee. Lei Lei and Haijuan Zhang identified the studied plant using the WFO Plant List (https://www.wfoplantlist.org/) and deposited the voucher specimen in the Lanzhou University Herbarium (ID:00098860). Consent for publication Not applicable. Availability of data and materials The datasets analysed during the current study are available from the corresponding author ( [email protected] ) on reasonable request. Competing interests The authors declare no competing interests. Funding This research was supported by the National Natural Science Foundation of China (32300241, U21A20239); Gansu Province Intellectual Property Program (22ZSCQD01). Authors' contributions L.L. and C.J.L. conceived the study. L.L., H.J.Z. and H.T.S. designed the experiments and wrote the manuscript. L.L. and C.J.L. revised the manuscript to the present form. The final manuscript was confirmed by all authors and approved for submission. 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Yin WX, Wang YF, Hen T, Lin Y, Luo CX. Functional evaluation of the signal peptides of secreted proteins. Bio-Protocol. 2018;8(9):e2839. Additional Declarations No competing interests reported. Supplementary Files Supplementarydata.docx Supplementary data Table S1. Primer used in this study. Figure S1. Prolonged cultivation results in poor protoplast condition. (A) E. gansuensis was cultured in YPD liquid medium at 22°C with shaking at 200 rpm under dark conditions for 15 d. (B) E. gansuensis in S1A produces protoplasts with incompletely digested cell walls after 4-5 h of enzymatic hydrolysis. Figure S2. Identification of genetically modified strains. (A)The identification of overexpression strains, using wild-type E. gansuensis as a negative control, and positive clones were identified using primers ToxA- F /pCT74jianding-HA R. (B) Schematic diagram of knockout mutant construction and the primers involved. (C), (D), (E) PCR identification of the knockout mutant. Using wild-type E. gansuensis as a negative control, three pairs of primers- Hyg- F/R, EgSPEout- F/R, and EgSPE- F/R-were used to ensure the accuracy of the identification results. (F) RT-qPCR determined the expression level of knockout mutants. The elongation factor was used as the reference primer ( qFactor -F/R). (G) PCR identification of positive inoculated fungal. Using the alkaloid-producing gene unique to E. gansuensis as a primer ( idtG -F/R). Supplementarydata2.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 17 Apr, 2026 Reviews received at journal 13 Apr, 2026 Reviews received at journal 11 Apr, 2026 Reviewers agreed at journal 06 Apr, 2026 Reviewers agreed at journal 06 Apr, 2026 Reviewers invited by journal 01 Apr, 2026 Editor assigned by journal 01 Apr, 2026 Editor invited by journal 30 Mar, 2026 Submission checks completed at journal 28 Mar, 2026 First submitted to journal 28 Mar, 2026 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. 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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-9167048","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":617617086,"identity":"8384bf8c-0b3d-468d-935c-5913f7f01a0e","order_by":0,"name":"Haijuan Zhang","email":"","orcid":"","institution":"Lanzhou University","correspondingAuthor":false,"prefix":"","firstName":"Haijuan","middleName":"","lastName":"Zhang","suffix":""},{"id":617617087,"identity":"1344b0a6-c809-4f56-a6c2-fd77d0da2948","order_by":1,"name":"Haotian Shi","email":"","orcid":"","institution":"Lanzhou University","correspondingAuthor":false,"prefix":"","firstName":"Haotian","middleName":"","lastName":"Shi","suffix":""},{"id":617617088,"identity":"ebc04315-1780-42a0-8132-4fcc869961f4","order_by":2,"name":"Chunjie Li","email":"","orcid":"","institution":"Lanzhou University","correspondingAuthor":false,"prefix":"","firstName":"Chunjie","middleName":"","lastName":"Li","suffix":""},{"id":617617089,"identity":"6e04af60-8caf-48a9-b068-da83bedf2aa4","order_by":3,"name":"Lei Lei","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyUlEQVRIie3NsQrCMBCA4QuFdjl1jSjmFdLJpfgsKQVnJ+eTQl18gA76Dq5uCR3c6gO4tLtCR8EOFicXSd0c8g8HB/dxAC7XHzYEttEgo+l7wz7EB0YdWeIvBEADFL+QICXdrC4ocsWqewZibiVoyOTyiixXXnjIIDyRjfCYCuyIx5U/GWSgpLYRUVPRyhJ9roJnP8IZFSA1YvfF60cwJrOTCXKs0/G+5OHRRkbBua4e7WImtolpbutIWL98xKgbvP+9y+Vyub73ApB4OTmcGRjZAAAAAElFTkSuQmCC","orcid":"","institution":"Lanzhou University","correspondingAuthor":true,"prefix":"","firstName":"Lei","middleName":"","lastName":"Lei","suffix":""}],"badges":[],"createdAt":"2026-03-19 08:39:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9167048/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9167048/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106233413,"identity":"a3dd4f75-1ae4-4378-9101-3dcc803908c3","added_by":"auto","created_at":"2026-04-06 13:11:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":346469,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of a secreted protein EgSPE in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. gansuensis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e is highly induced during host colonization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eVolcano plot displaying differentially expressed genes between \u003cem\u003eE. gansuensis\u003c/em\u003e in culture versus inoculated into \u003cem\u003eA. inebrians\u003c/em\u003e at 96 hpi. Genes with a |log₂FC| \u0026gt; 1 are highlighted: up-regulated in orange, down-regulated in green. \u003cstrong\u003e(B)\u003c/strong\u003e Gene ontology (GO) analysis of differential proteins in \u003cem\u003eE. gansuensis\u003c/em\u003e biological process. Number of proteins in each GO terms indicated. \u003cstrong\u003e(C)\u003c/strong\u003e Expression levels of EgSPE were analyzed in inoculated with \u003cem\u003eE. gansuensis\u003c/em\u003e 0 and 96 hpi. The values represent the average of three biological replicates. Different letters indicate significant differences at \u003cem\u003eP\u003c/em\u003e≤0.05 (one-way ANOVA, Tukey post-test). \u003cstrong\u003e(D) \u003c/strong\u003eSchematic representation of EgSPE and the position of the signal peptide. \u003cstrong\u003e(E)\u003c/strong\u003e The secretion function of the predicted signal peptides was proved by employing a yeast secretion system with Saccharomyces cerevisiae strain YTK12. pSUC2 carrying signal peptides from EgSP1SPE and the positive control Avr1b could all make YTK12 survive on CMD-W and YPRAA medium, while YTK12 containing empty pSUC2 as a negative control failed to grow on YPRAA.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-9167048/v1/4060cbb354c85af152d94c6c.png"},{"id":106233436,"identity":"89ed63d2-5a20-42a6-a095-164ad4646064","added_by":"auto","created_at":"2026-04-06 13:11:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":466383,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOptimized transformation system enables functional analysis of EgSPE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003eGrow for18 d on PDA medium, 22°C, dark \u003cem\u003eE. gansuensis\u003c/em\u003e, Scale bars = 1 mm. \u003cstrong\u003e(B)\u003c/strong\u003e Grow for 4 d on PDA medium covered with cellophane, 22°C, dark \u003cem\u003eE. gansuensis\u003c/em\u003e, Scale bars = 10 mm. \u003cstrong\u003e(C)\u003c/strong\u003e \u003cem\u003eE. gansuensis\u003c/em\u003eprotoplasts, Scale bars = 10 μm. \u003cstrong\u003e(D)\u003c/strong\u003e Protoplasts yield under different treatment methods. YPD (15 d) refers to the cultivation of the YPD liquid medium for 15 d. PDA (4 d) + YPD (4 d) is culturing on a covered cellophane for 4 d, and then crushing the fungi and inoculating them onto YPD liquid medium for 4 d of cultivation.\u003cstrong\u003e (E)\u003c/strong\u003e Direct culture for 15 d and PDA (4 d) + YPD (4 d) transformation efficiency. \u003cstrong\u003e(F)\u003c/strong\u003e Expression level of different overexpressed strains. \u003cstrong\u003e(G)\u003c/strong\u003e Knockout efficiency of split-marker. Data in the figures are means ± SD (three replicates per treatment). **indicates significant differences at \u003cem\u003eP\u003c/em\u003e≤0.05 (one-way ANOVA, Tukey post-test).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9167048/v1/69c0d32484ef5706635ef6be.png"},{"id":106233415,"identity":"5b6b2171-20df-47a4-891b-44f4a569cb96","added_by":"auto","created_at":"2026-04-06 13:11:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":235738,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEgSPE is required for mycelial growth of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. gansuensis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003eColony morphology of wild-type, overexpressed and knockout strains on PDA medium, 22°C, dark culture for 18 d, Scale bars = 1 mm.\u003cstrong\u003e (B)\u003c/strong\u003e Colony growth rate on day-18 of growth on PDA. Data shown are means ± SD (three replicates per treatment), Different letters indicate significant differences at \u003cem\u003eP\u003c/em\u003e≤0.05 (one-way ANOVA, Tukey post-test).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9167048/v1/b6c76f5f3416d29682c9a4e8.png"},{"id":106233465,"identity":"c5fedd28-1665-49bd-acd3-ffcafd61032c","added_by":"auto","created_at":"2026-04-06 13:11:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":402923,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEarly fungal proliferation and systemic colonization require EgSPE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003eDiagram of endophytic fungal inoculation and sample collection. \u003cstrong\u003e(B)\u003c/strong\u003eMicrographs of \u003cem\u003eA. inebrians\u003c/em\u003e stems inoculated with wild-type, OE-\u003cem\u003eEgSPE\u003c/em\u003e, and Δ\u003cem\u003eegspe\u003c/em\u003e strains of \u003cem\u003eE. gansuensis\u003c/em\u003e to show the progression of tissue invasion. Samples were collected at 6 dpi and stained with WGA-AF488. Scale bars = 15 µm. \u003cstrong\u003e(C)\u003c/strong\u003e Fungal biomass was quantified in symbiotic samples collected 1 cm above the original inoculation site at 6 dpi. The elongation factor gene was used as the reference gene (\u003cem\u003eqFactor-\u003c/em\u003eF/R). The values represent the mean of three biological replicates. The \u003cem\u003eidtG\u003c/em\u003e gene was used as the target gene to quantify \u003cem\u003eE. gansuensis\u003c/em\u003e colonization. \u003cstrong\u003e(D)\u003c/strong\u003eEndophytic fungal hyphal colonization rate. Inoculation of Eg-wt, OE-\u003cem\u003eEgSPE\u003c/em\u003eand Δ\u003cem\u003eegspe\u003c/em\u003einto \u003cem\u003eA. inebrians \u003c/em\u003estems, and collect samples for PCR identification two months later. Data are means ± SD (three replicates per treatment). Different letters indicate significant differences at \u003cem\u003eP\u003c/em\u003e≤0.05 (one-way ANOVA, Tukey post-test).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9167048/v1/e18ec63ea75f9b107c2762eb.png"},{"id":106233453,"identity":"81234eb3-91d7-4b68-babf-587f8c45a89d","added_by":"auto","created_at":"2026-04-06 13:11:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":566663,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEgSPE activates host drought responses and enhances drought tolerance in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA. inebrians\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Expression levels of \u003cem\u003eEgSPE\u003c/em\u003e were analyzed in 20% PEG6,000 treatment for 48 h after 48 h of inoculation). \u003cstrong\u003e(B)\u003c/strong\u003e Luminescence after treatment with water (left) and 20% PEG6,000 (right) for 8 h. The color scale at right shows the luminescence intensity from dark blue (lowest) to red (highest). \u003cstrong\u003e(C)\u003c/strong\u003e Quantitation of the luminescence intensities shown in \u003cstrong\u003e(B)\u003c/strong\u003e was measured using the Image J software. \u003cstrong\u003e(D)\u003c/strong\u003e Positive seedling identification. \u003cstrong\u003e(E)\u003c/strong\u003e \u003cem\u003eA. inebrians \u003c/em\u003edrought phenotype. Eg-free\u003csub\u003e,\u003c/sub\u003e inoculated Eg wild type (Eg-wt), OE-\u003cem\u003eEgSPE\u003c/em\u003e and Δ\u003cem\u003eegspe\u003c/em\u003e after two months before treatment (left) and after natural drought stress (right). \u003cstrong\u003e(F)\u003c/strong\u003e RT-qPCR was used to verify the expression of downstream genes responsed to drought response. Eg-free\u003csub\u003e,\u003c/sub\u003e inoculated Eg-wt, OE-\u003cem\u003eEgSPE\u003c/em\u003e and Δ\u003cem\u003eegspe\u003c/em\u003e, collected separately at 48 h after inoculation, inoculated for 96 h (water treatment for 48 h after 48 h of inoculation), \u003cstrong\u003e(G)\u003c/strong\u003e 20% PEG6,000 treatment for 48 h after 48 h of inoculation. Data are means ± SD (three replicates per treatment), Different letters indicate significant differences at \u003cem\u003eP\u003c/em\u003e≤0.05 (one-way ANOVA, Tukey post-test).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-9167048/v1/91b586cb9fc37f97c18c80a4.png"},{"id":106959360,"identity":"934e6214-b934-45e4-9924-7eda8da2b20b","added_by":"auto","created_at":"2026-04-15 09:06:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3200723,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9167048/v1/ac7370eb-ec61-4f4b-97ba-0cb892a71c37.pdf"},{"id":106233435,"identity":"b58b2fcf-9524-4bf0-9c1e-bb31a39aae7b","added_by":"auto","created_at":"2026-04-06 13:11:40","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14409785,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S1\u003c/strong\u003e. Primer used in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S1. Prolonged cultivation results in poor protoplast condition.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e \u003cem\u003eE. gansuensis \u003c/em\u003ewas cultured in YPD liquid medium at 22°C with shaking at 200 rpm under dark conditions for 15 d. \u003cstrong\u003e(B)\u003c/strong\u003e \u003cem\u003eE. gansuensis\u003c/em\u003e in S1A produces protoplasts with incompletely digested cell walls after 4-5 h of enzymatic hydrolysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S2. Identification of genetically modified strains.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003eThe identification of overexpression strains, using wild-type \u003cem\u003eE. gansuensis\u003c/em\u003eas a negative control, and positive clones were identified using primers\u003cem\u003eToxA-\u003c/em\u003eF\u003cem\u003e/pCT74jianding-HA\u003c/em\u003eR. \u003cstrong\u003e(B)\u003c/strong\u003e Schematic diagram of knockout mutant construction and the primers involved. \u003cstrong\u003e(C), (D),\u003c/strong\u003e \u003cstrong\u003e(E)\u003c/strong\u003e PCR identification of the knockout mutant. Using wild-type \u003cem\u003eE. gansuensis\u003c/em\u003e as a negative control, three pairs of primers-\u003cem\u003eHyg-\u003c/em\u003eF/R, \u003cem\u003eEgSPEout-\u003c/em\u003eF/R, and \u003cem\u003eEgSPE-\u003c/em\u003eF/R-were used to ensure the accuracy of the identification results. \u003cstrong\u003e(F)\u003c/strong\u003e RT-qPCR determined the expression level of knockout mutants. The elongation factor was used as the reference primer (\u003cem\u003eqFactor\u003c/em\u003e-F/R). \u003cstrong\u003e(G)\u003c/strong\u003e PCR identification of positive inoculated fungal. Using the alkaloid-producing gene unique to \u003cem\u003eE. gansuensis\u003c/em\u003e as a primer (\u003cem\u003eidtG\u003c/em\u003e-F/R).\u003c/p\u003e","description":"","filename":"Supplementarydata.docx","url":"https://assets-eu.researchsquare.com/files/rs-9167048/v1/b462209114dd13a4bb65f32d.docx"},{"id":106233412,"identity":"a20a9327-b7dd-4b26-b601-e6912a8256ec","added_by":"auto","created_at":"2026-04-06 13:11:35","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":22299640,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata2.docx","url":"https://assets-eu.researchsquare.com/files/rs-9167048/v1/2e33dcbfdf195eb4a772f831.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"EgSPE, a secreted effector from Epichloë gansuensis, modulates symbiotic establishment and host drought tolerance","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003e \u003cem\u003eEpichlo\u0026euml;\u003c/em\u003e endophytes establish mutualistic symbioses with more than one hundred cool-season grass species, primarily within the subfamily Pooideae (e.g., \u003cem\u003eFestuca\u003c/em\u003e, \u003cem\u003eLolium\u003c/em\u003e, and \u003cem\u003ePoa\u003c/em\u003e spp.) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. These symbionts confer enhanced tolerance against a broad spectrum of biotic and abiotic stresses-including fungal pathogens [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], insect herbivory [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], drought [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], salinity [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], and heavy metal toxicity [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], without compromising host growth. Despite this agricultural potential, the association is typically host-specific [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], limiting its broader application in crop improvement. Expanding the utility of \u003cem\u003eEpichlo\u0026euml;\u003c/em\u003e symbionts will require a deeper mechanistic understanding of how these fungi establish symbiosis and confer stress tolerance-aspects that remain largely unresolved.\u003c/p\u003e \u003cp\u003eFungal colonization of plants, whether by pathogens or mutualists, is critically dependent on secreted proteins (SPs) that modulate host physiology to facilitate persistence within plant tissues [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In pathogenic interactions, SPs suppress host immunity and reprogram metabolism to promote infection. Similarly, mutualistic fungi must finely tune host defense responses to achieve stable colonization, a process in which secreted effectors play key roles. For instance, \u003cem\u003eEfe-afpA\u003c/em\u003e, a secreted protein identified in the apoplast of \u003cem\u003eE. festucae\u003c/em\u003e-colonized red fescue, exhibits antifungal activity against plant pathogens and may be required for symbiosis [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Although hundreds of putative SPs have been predicted in \u003cem\u003eEpichlo\u0026euml;\u003c/em\u003e genomes, functional characterization has been reported for only a single protein to date, representing a critical gap in our understanding of the molecular basis of \u003cem\u003eEpichlo\u0026euml;\u003c/em\u003e-grass symbioses [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eProgress in functional genetic studies of \u003cem\u003eEpichlo\u0026euml;\u003c/em\u003e has been further constrained by the lack of efficient transformation systems. As slow-growing filamentous fungi, \u003cem\u003eEpichlo\u0026euml;\u003c/em\u003e species are recalcitrant to conventional protoplast-based methods developed for other fungi [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Establishing a high-efficiency genetic transformation system is therefore a prerequisite for accelerating functional genomics and dissecting the molecular mechanisms underlying \u003cem\u003eEpichlo\u0026euml;\u003c/em\u003e-grass interactions.\u003c/p\u003e \u003cp\u003eIn this study, we investigated the symbiosis between \u003cem\u003eEpichlo\u0026euml; gansuensis\u003c/em\u003e and drunken horse grass (\u003cem\u003eAchnatherum inebrians\u003c/em\u003e), a well-characterized system in which endophyte colonization markedly enhances host drought tolerance [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Through transcriptomic profiling during host colonization, we identified \u003cem\u003eEgSPE\u003c/em\u003e, a gene encoding a secreted protein that is strongly induced in planta, suggesting a potential role in symbiotic interaction. To enable functional analysis, we established a substantially improved genetic transformation system for \u003cem\u003eEpichlo\u0026euml;\u003c/em\u003e, incorporating optimized culture conditions and a split-marker deletion strategy. Using this platform, we demonstrate that EgSPE is indispensable for normal fungal growth and efficient host colonization, and that it contributes critically to endophyte-mediated drought tolerance. These findings provide new insight into the molecular mechanisms by which \u003cem\u003eEpichlo\u0026euml;\u003c/em\u003e endophytes promote abiotic stress tolerance in grasses.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant material and growth conditions\u003c/h2\u003e \u003cp\u003eThe seeds of the drunken horse grass (\u003cem\u003eAchnatherum inebrians\u003c/em\u003e) were used as test material, which is collected from Qinghai-Tibet Plateau, China (97.27\u0026deg;E, 30.24\u0026deg;N). A sufficient amount of the grass seeds was disinfected with 75% ethanol for 1 min, then treated with 2% sodium hypochlorite for 30 min, and finally rinsed with sterile water 5 times. The seeds were cultured in 1/2MS medium and placed in a 23 ℃ climate chamber for growth, with a photoperiod of 16 h of light/8 h of darkness, and a relative humidity of 60%. The seeds were allowed to grow up to 15\u0026ndash;20 d.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMedia and preparation of\u003c/b\u003e \u003cb\u003eEpichlo\u0026euml; gansuensis\u003c/b\u003e \u003cb\u003eculture\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe fungal strain was isolated from \u003cem\u003eA. inebrians\u003c/em\u003e and identified as \u003cem\u003eE. gansuensis\u003c/em\u003e, exhibiting 99% sequence similarity to strain e7080 (NCBI Taxonomy ID: 447254) from the National Center for Biotechnology Information (NCBI). The endophyte was cultured on potato dextrose agar (PDA) medium in an artificial climate chamber (MGC-450HP-2, Bluepard) at 22\u0026deg;C under dark conditions. Before optimization, mycelia from the colony edge were transferred to 250 mL Erlenmeyer flasks, each containing 100 mL of yeast peptone dextrose medium (YPD), and incubated at 22\u0026deg;C with shaking at 200 rpm under dark conditions for 15 d. After optimization, mycelia from the colony edge were transferred to 500 \u0026micro;L of YPD medium and homogenized using a ball mill at 30 m/s for 30 s until fully lysed. A 200 \u0026micro;L aliquot of the homogenate was spread onto PDA medium overlaid with cellophane membranes and incubated at 22\u0026deg;C in darkness for 4 d. Fresh mycelia were scraped from the cellophane membranes and transferred to 500 \u0026micro;L of YPD medium. The mixture was homogenized using a ball mill at 30 m/s for 30 s until fully lysed. The homogenate was then inoculated into four 250 mL Erlenmeyer flasks, each containing 100 mL of YPD medium, and incubated at 22\u0026deg;C with shaking at 200 rpm under dark conditions for 4 d.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eProtoplast isolation\u003c/h3\u003e\n\u003cp\u003eThe fungal cultures were divided into batches and transferred to sterilized 50 mL centrifuge tubes. The tubes were then centrifuged at 4\u0026deg;C and 5,000 \u0026times; g for 10 min. The pellet was washed three times with 0.7 mol/L NaCl, followed by final centrifugation at 4\u0026deg;C and 5,000 \u0026times; g for 5 min, after which the supernatant was discarded. In case of enzymatic digestion, 0.2 g of fungal biomass was suspended in 1 mL of enzyme solution (prepared in 0.7 mol/L NaCl: 2.0% lysing enzymes, 1.5% Driselase basidiomycetes, 1.0% snailase, 1.0% cellulase, and 3 mg/ml Bovine serum albumin). The enzyme solution was filter-sterilized using a 0.45 \u0026micro;m filter column. The mixture was incubated in a water bath at 32\u0026deg;C for 4\u0026ndash;5 h.\u003c/p\u003e \u003cp\u003eThe digested suspension was filtered through two layers of lens cleaning paper glass funnel (pre-wetted with 0.7 mol/L NaCl) into a 50 mL centrifuge tube. To the filtrate, 1 mL of STC buffer (1.2 M sorbitol, 10 mM Tris-HCl, 50 mM CaCl₂, pH 7.5) was added, and the mixture was centrifuged at 4\u0026deg;C and 3,000 \u0026times; g for 20 min. The supernatant was discarded, and the pellet was resuspended in 1 mL of STC buffer, followed by centrifugation at 4\u0026deg;C and 2,000 \u0026times;g for 10 min. This washing step was repeated once. The final protoplasts pellet was gently resuspended in 500 \u0026micro;L of STC buffer. Protoplast concentration was quantified using a hemocytometer under a microscope, and the suspension was diluted with STC buffer to achieve a final density of \u0026ge;\u0026thinsp;5 \u0026times; 10⁶ protoplasts per mL.\u003c/p\u003e\n\u003ch3\u003eKnockout and overexpression vector construction\u003c/h3\u003e\n\u003cp\u003eThe overexpression vector was constructed using the plasmid backbone pPCT74. To facilitate detection, an HA epitope tag was incorporated into the construct. The target gene was placed under the control of the \u003cem\u003eToxA\u003c/em\u003e promoter, a strong constitutive promoter in fungi, to ensure high expression levels. The native termination codon of the gene was removed, and an HA tag was fused to the C-terminal end of the protein, enabling efficient detection via PCR methods.\u003c/p\u003e \u003cp\u003eThe strategy based on the split-marker approach was used to obtain \u003cem\u003eE. gansuensis\u003c/em\u003e secreted protein essential (\u003cem\u003eEgSPE\u003c/em\u003e) gene knockout strains. In the obtained target gene sequence, an upstream primer was designed approximately 1,000 bp before the start codon (excluding the start codon), with the 5' end of the upstream primer incorporating a hygromycin linker homologous arm. A downstream primer was designed approximately 1,000 bp after the stop codon (excluding the stop codon), with the 3' end of the downstream primer incorporating a hygromycin linker homologous arm. The fused product was then purified and used as a template for amplification to obtain the knockout fragment. Additionally, we employed a strategy using hygromycin as the homologous arm, amplifying a fragment from approximately 1,000 bp before the start codon to the first two-thirds of the hygromycin sequence, and another fragment from the last two-thirds of the hygromycin sequence to approximately 1,000 bp after the stop codon. The overlap between the two truncated \u003cem\u003ehph\u003c/em\u003e gene fragments was 500\u0026ndash;700 bp.\u003c/p\u003e\n\u003ch3\u003ePEG-mediated protoplast transformation\u003c/h3\u003e\n\u003cp\u003eTransformation using 80 \u0026micro;L of protoplasts was mixed with 1 \u0026micro;g of linearized plasmid (overexpression and knockout fragments), then added 5 \u0026micro;L of 50 mM spermidine and stirred thoroughly. Subsequently, added 90 \u0026micro;L of 40% PEG 4,000 and incubated at room temperature for 10 min. Then added 2 volumes of STC (360 \u0026micro;L), centrifuged at 4\u0026deg;C and 2,000\u0026times;g for 5 min, and repeated the centrifugation step three times. Finally, retained 100 \u0026micro;L of STC and gently resuspended the pellet by flicking.\u003c/p\u003e\n\u003ch3\u003eProtoplast regeneration and screening for stable transformants\u003c/h3\u003e\n\u003cp\u003eA 100 \u0026micro;L of the transformation product was spread onto regeneration medium (PDB, 0.6 M sucrose, and 1.0% agar). Hygromycin (100 \u0026micro;g/mL) (Solarbio: H8080) was used to screen the overexpression transformants and knockout transformants. Untransformed protoplasts were spread onto regeneration plates with and without antibiotics, followed by incubation at 22\u0026deg;C in the dark for 7\u0026ndash;10 d. Then, the grown transformants were transferred to fresh antibiotic-resistant medium and DNA was extracted for identification (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA). The \u003cem\u003eHyg\u003c/em\u003e-F/R primer pairs were employed to detect the introduction of the hygromycin gene, \u003cem\u003eEgSPE\u003c/em\u003e-F/R to verify gene knockout, and \u003cem\u003eEgSPE\u003c/em\u003e-outF/R to confirm whether the target gene was replaced by hygromycin (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB). The PCR band sizes were used to determine the results (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC, D, E).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of fungal growth rate\u003c/h2\u003e \u003cp\u003eAn inoculation pipette of 0.5 mm was used to inoculate the wild-type, overexpressed and knockout strains onto PDA medium. The wild-type \u003cem\u003eE. gansuensis\u003c/em\u003e was inoculated onto an antibiotic-free medium, and the overexpressed and knockout strains were inoculated onto a medium containing hygromycin (100 \u0026micro;g/mL) (Solarbio: H8080). The colony diameter was recorded every three days using a vernier caliper, and measured continuously for 30 d. Each strain had three replicates, and the experiment was repeated three times. The growth rate was fastest on the 18th day.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRT-qPCR analysis\u003c/h3\u003e\n\u003cp\u003eReverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) was used to determine the expression level of the target gene of the overexpressed strain. RNA was extracted from a single (transformed) mycelium colony, reverse transcribed into cDNA, diluted to an appropriate concentration, and designed specific primers for the target gene with extension factor as the internal reference gene. ABclonal SYBR Green (RT-qPCR reagent No. RK21203) was used to perform RT-qPCR for the overexpressed transformed strain \u003cem\u003eE. gansuensis\u003c/em\u003e, and the procedure was repeated three times. The RT-qPCR instrument automatically generated amplification and melting curve, through which the Ct value of target gene and internal reference gene in each sample was determined (Ct, cycle threshold, the number of cycles when the fluorescence signal reached the set threshold).\u003c/p\u003e\n\u003ch3\u003eDetection of endophytic fungal colonization rate\u003c/h3\u003e\n\u003cp\u003eTwo months post-inoculation, the colonization rate of endophytic fungi in drunken horse grass was assessed for each transgenic strain treatment. Cut off a tiller and trim about 2 cm of the stem from the root upwards, and extract DNA for subsequent experiments. Fungal colonization was detected by PCR amplification of the indole alkaloid biosynthesis gene \u003cem\u003eidtG\u003c/em\u003e. A total of 60 samples were analyzed per treatment.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTranscriptome sequencing and data analysis\u003c/h2\u003e \u003cp\u003eThe total RNA from inoculated stems with different time points inoculated and control was individually extracted to construct the RNA-seq library. Sequencing was performed using Illumina NovaSeq6,000 platform. DESeq2 (V1.22.2) /edgeR (V3.6.8) was used for differential expression significance analysis, and the screening threshold was FDR (false discovery rate)\u0026thinsp;\u0026lt;\u0026thinsp;0.05, log\u003csub\u003e2\u003c/sub\u003eFC (fold change)\u0026thinsp;\u0026gt;\u0026thinsp;1 or \u0026lt; -1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of drought resistance of transgenic strains in plants\u003c/h2\u003e \u003cp\u003eThe genetic screen used the firefly luciferase reporter gene driven by the drought responsive \u003cem\u003eRD29A\u003c/em\u003e promoter (\u003cem\u003eRD29A::LUC\u003c/em\u003e). In \u003cem\u003eN. benthamiana\u003c/em\u003e, each leaf was divided into two halves, with \u003cem\u003eproRD29A::LUC\u003c/em\u003e co-expressed alongside EV on one side and EgSPE on the other. After 24 h of expression, the experimental group was treated with 20% PEG6,000 for 8 h, water treatment as a control. Leaves were cut and sprayed with D-Luciferin (GlpBio: GC11860) on the underside of the leaves, then observe and photographed using the Living Imaging system (PerkinElmer IVIS Lumina III).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eWGA-AF488 staining assay\u003c/h2\u003e \u003cp\u003eAt 6 dpi, tissue segments (extending 1 cm above the inoculation site) were collected for assessment of fungal colonization. The samples were processed for fluorescence imaging according to the staining protocol described by [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], Wheat Germ Agglutinin-Alexa Fluor 488 (WGA-AF488) was used to stain fungal hyphae. Observation was carried out on a Leica SP8 laser scanning confocal microscope, with excitation/emission settings of 488/500\u0026ndash;530 nm for eGFP (WGA-AF488).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eValidation of the secretion function of the predicted signal peptide of EgSPE\u003c/h2\u003e \u003cp\u003eThe yeast signal sequence trap assay used the pSUC2T7M13Ori (pSUC2) vector, which carries a truncated SUC2 gene lacking the initiating methionine and the native signal peptide [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The secretion activity of the EgSPE signal peptide was verified. DNA fragments of the signal peptide were synthesized by SynbioB and inserted into pSUC2 through EcoRI and XhoI restriction sites, fused in-frame with the invertase gene [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Using the lithium acetate method, 20 ng of the plasmid was transformed into the enzyme-deficient yeast strain YTK12.\u003c/p\u003e \u003cp\u003eAfter transformation, yeast cells were plated on CMD-W (minus-tryptophan) plates and transferred to fresh CMD-W plates, then stored at 30\u0026deg;C. PCR with vector-specific primers was used to confirm the transformation. To assess the secretion of the transformant enzyme, colonies were replicated onto YPRAA plates containing raffinose and lacking glucose (1% yeast extract, 2% peptone, 2% raffinose, and 2 \u0026micro;g/mL antibiotic A). The activity of the transformant enzyme was measured by TTC reduction to insoluble red triphenylformazan. Yeast cultures were inoculated into sucrose medium and incubated at 30\u0026deg;C for 24 h. The pellets were collected, washed, and resuspended in distilled water. The cultures were incubated with 0.1% TTC at 35\u0026deg;C for 35 min, followed by an additional 10 min at room temperature, and color change was monitored.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe Statistical Product and Service Solutions (SPSS) software was used to conduct the statistical analyzes. All experimental data were tested by Student\u0026rsquo;s t-test and Tukey post-test. Prism 10.1.2 software (GraphPad, San Diego, CA, USA) was used to generate figures.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eExpression of the secreted protein EgSPE in\u003c/b\u003e \u003cb\u003eE. gansuensis\u003c/b\u003e \u003cb\u003eis highly induced during initial host colonization\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn order to explore the molecular mechanism of \u003cem\u003eE. gansuensis\u003c/em\u003e colonization in \u003cem\u003eA. inebrians\u003c/em\u003e, we performed transcriptomic analysis of the \u003cem\u003eA. inebrians\u003c/em\u003e-\u003cem\u003eE. gansuensis\u003c/em\u003e interaction at 0 and 96 hours post-inoculation (hpi). Differential expression analysis revealed 276 significantly upregulated and 1,468 downregulated fungal genes during host infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Functional enrichment analysis showed that upregulated genes were primarily associated with transmembrane export, immune regulation, DNA repair, and tubulin assembly (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Among these, a gene encoding a secreted protein, designated \u003cem\u003eEgSPE\u003c/em\u003e (\u003cem\u003eE. gansuensis\u003c/em\u003e essential secreted protein), exhibited the most significant induction following host inoculation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eWe further validated EgSPE expression under colonization. \u003cem\u003eEgSPE\u003c/em\u003e transcript levels were markedly elevated during fungal inoculation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Structural analysis predicted that EgSPE contains an N-terminal signal peptide and a carbohydrate-binding module (CBM) family 19 domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). We then validated the functional secretion of the predicted signal peptide via a yeast signal sequence trap assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). These results demonstrate that the N-terminal signal peptide effectively directs protein secretion \u003cem\u003ein vivo\u003c/em\u003e, supporting the classification of EgSPE as a functional effector during the initial infection phase.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eSubstantially improved transformation system enables functional analysis of EgSPE\u003c/h2\u003e \u003cp\u003eTo functionally characterize EgSPE, efficient genetic manipulation of \u003cem\u003eE. gansuensis\u003c/em\u003e was required. However, conventional transformation approaches for filamentous fungi are largely ineffective in \u003cem\u003eEpichlo\u0026euml;\u003c/em\u003e species, primarily due to their slow growth and limited protoplast yield. In particular, hyphal maturation leads to extensive chitin-glucan cross-linking in the cell wall, which markedly reduces enzymatic digestibility [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Consistent with this, 15-day-old mycelia grown on PDA-the standard condition for biomass accumulation-yielded only\u0026thinsp;~\u0026thinsp;1.8 \u0026times; 10⁶ protoplasts g⁻\u0026sup1; after enzymatic treatment, with incomplete cell wall digestion (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA, B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo overcome this bottleneck, we redesigned the hyphal culture strategy to obtain younger, more enzymatically susceptible mycelia. Briefly, colony fragments were homogenized and spread on cellophane-overlaid PDA to generate synchronized micro-colonies for 4 d, which were subsequently subcultured in YPD liquid medium for another 4 d (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). This optimized approach dramatically improved cell wall digestibility, increasing protoplast yield to 6.9 \u0026times; 10⁸ g⁻\u0026sup1; with complete digestion (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D). Consequently, we observed a 9-fold increase in transformation efficiency, yielding over 175 transformants \u0026micro;g⁻\u0026sup1; DNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eUsing this substantially improved system, we successfully generated both \u003cem\u003eEgSPE\u003c/em\u003e overexpression and knockout mutants (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA-F). One overexpression line exhibiting\u0026thinsp;~\u0026thinsp;70-fold elevated transcript levels was selected for further analyzes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). The application of a split-marker homologous recombination strategy further ensured precise gene targeting, achieving a knockout frequency of 12% for the Δ\u003cem\u003eegspe\u003c/em\u003e mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC-F). Collectively, this optimized platform enables rapid and efficient genetic manipulation of \u003cem\u003eE. gansuensis\u003c/em\u003e, thereby providing a robust foundation for dissecting the molecular mechanisms underlying \u003cem\u003eEpichlo\u0026euml;\u003c/em\u003e\u0026ndash;grass symbiosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEgSPE is required for mycelial growth of\u003c/b\u003e \u003cb\u003eE. gansuensis\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eTo determine whether EgSPE contributes to fungal development, we examined colony morphology and radial growth of overexpression and knockout strains in comparison with the wild type on PDA medium. While overexpression lines (OE-\u003cem\u003eEgSPE\u003c/em\u003e) displayed growth characteristics indistinguishable from the wild type, knockout mutants (Δ\u003cem\u003eegspe\u003c/em\u003e) exhibited reduced colony size, altered pigmentation and significantly slower radial expansion (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). These results indicate that EgSPE is essential for maintaining normal vegetative growth and mycelial morphogenesis in \u003cem\u003eE. gansuensis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eEgSPE is essential for early fungal proliferation and systemic host colonization\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eTo determine the role of EgSPE in host-fungus interactions, we monitored the progression of fungal invasion in \u003cem\u003eA. inebrians\u003c/em\u003e. At 6 days post-inoculation (dpi), the capacity for early hyphal expansion was assessed in tissues approximately 1 cm above the inoculation site (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Quantitative biomass analysis and WGA-AF488 fluorescence staining revealed that while OE-\u003cem\u003eEgSPE\u003c/em\u003e strains exhibited proliferation levels comparable to the wild type, the Δ\u003cem\u003eegspe\u003c/em\u003e mutants were significantly impaired in their ability to invade host tissues, showing a marked reduction in fungal biomass (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C).\u003c/p\u003e \u003cp\u003eWe further evaluated whether this early invasion defect impacts the long-term establishment of symbiosis. Analysis of two-month-old plants showed that OE-\u003cem\u003eEgSPE\u003c/em\u003e did not significantly increase the colonization rate, whereas Δ\u003cem\u003eegspe\u003c/em\u003e mutants resulted in a pronounced reduction compared with the wild type (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Collectively, these results demonstrate that EgSPE promotes both early fungal proliferation in host tissues and subsequent systemic colonization in the \u003cem\u003eE. gansuensis\u003c/em\u003e-\u003cem\u003eA. inebrians\u003c/em\u003e symbiosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEgSPE activates host drought responses and enhances drought tolerance in\u003c/b\u003e \u003cb\u003eA. inebrians\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eE. gansuensis-A. inebrians\u003c/em\u003e symbiosis is known to enhance host tolerance to multiple environmental stresses, particularly drought [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Consistent with this role, \u003cem\u003eEgSPE\u003c/em\u003e expression was not only induced during endophyte-host interaction but further upregulated under drought stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Heterologous expression of EgSPE in \u003cem\u003eN. benthamiana\u003c/em\u003e significantly enhanced the expression of the drought-responsive marker gene \u003cem\u003eRD29A\u003c/em\u003e under drought treatment, suggesting a specialized role of EgSPE in endophyte-mediated drought tolerance (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C).\u003c/p\u003e \u003cp\u003eTo further confirm the functional role of EgSPE in the native host, we assessed drought responses of \u003cem\u003eA. inebrians\u003c/em\u003e colonized by different \u003cem\u003eE. gansuensis\u003c/em\u003e strains after verifying successful colonization via fungal marker detection (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003eS2\u003c/span\u003eG). Under drought treatment, plants inoculated with the wild-type and OE-\u003cem\u003eEgSPE\u003c/em\u003e strains exhibited significantly enhanced drought tolerance compared with endophyte-free plants (Eg-free). In contrast, plants colonized by Δ\u003cem\u003eegspe\u003c/em\u003e mutants failed to confer any significant drought resistance (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eWe further analyzed the expression of downstream drought-responsive genes including \u003cem\u003eAiRD22\u003c/em\u003e, \u003cem\u003eAiNAC5\u003c/em\u003e, and \u003cem\u003eAiABA1\u003c/em\u003e. At 48 and 96 hpi, inoculation with Eg-wt, OE-\u003cem\u003eEgSPE\u003c/em\u003e, and Δ\u003cem\u003eegspe\u003c/em\u003e mutants all induced the expression of these downstream drought-responsive genes compared with Eg-free. Notably, the upregulation of these genes was more pronounced in plants colonized by the OE-\u003cem\u003eEgSPE\u003c/em\u003e strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). After 48 h of inoculation, treated with PEG6,000 for 48 h, OE-\u003cem\u003eEgSPE\u003c/em\u003e colonization significantly enhanced the expression of \u003cem\u003eAiRD22\u003c/em\u003e, \u003cem\u003eAiNAC5\u003c/em\u003e, and \u003cem\u003eAiABA1\u003c/em\u003e relative to Eg-free. By contrast, only slight increases in the expression of these genes were observed in plants colonized by the Δ\u003cem\u003eegspe\u003c/em\u003e mutants, and no significant induction was detected under PEG6,000 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eF, G). Together, these results indicate that EgSPE plays a critical role in \u003cem\u003eE. gansuensis\u003c/em\u003e-mediated enhancement of drought tolerance in \u003cem\u003eA. inebrians\u003c/em\u003e, likely through activation of host drought-responsive pathways.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003e \u003cem\u003eEpichlo\u0026euml;\u003c/em\u003e endophytic fungal symbionts are widely recognized for conferring enhanced stress tolerance to their host plants [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Notably, these endophytes improve drought resistance in grasses without compromising host growth, a trait of considerable value for developing more resilient crops and forages under the pressures of climate change. Understanding the molecular mechanisms underlying \u003cem\u003eEpichlo\u0026euml;\u003c/em\u003e\u0026ndash;grass interactions is therefore critical for the efficient and widespread application of this endophyte genus. In this study, we identified a protein effector, EgSPE, which plays multiple essential roles in \u003cem\u003eE. gansuensis\u003c/em\u003e and its interaction with host grass, including the regulation of fungal morphology and growth rate, the establishment of systemic symbiosis, and the mediation of host drought tolerance. This effector may serve as a key entry point for elucidating the molecular crosstalk mechanisms in \u003cem\u003eEpichlo\u0026euml;\u003c/em\u003e\u0026ndash;grass interactions.\u003c/p\u003e \u003cp\u003e \u003cem\u003eEgSPE\u003c/em\u003e encodes a small protein of 81 amino acids, contains a single carbohydrate-binding module (CBM) domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). CBMs are non-catalytic domains that typically enhance polysaccharide-degrading efficiency by targeting enzymes to their substrates and disrupting substrate structure; they also participate in diverse biological processes-including pathogen defense and biosynthesis-through specific carbohydrate recognition [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. For instance, LysM (CBM50) effectors such as Ecp6, Slp1, and RsLysM sequester chitin oligomers in the apoplast, competing with plant chitin receptors like CEBiP to prevent chitin perception and thereby dampening ROS bursts and defense-related gene expression [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Certain CBM containing proteins can enhance reactive oxygen species (ROS) bursts and contribute to drought-stress tolerance [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEgSPE contains a CBM 19 domain previously characterized as a chitin-binding module in fungal chitinases, suggesting a potential role in chitin-associated processes [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The phenotypic alterations observed in the Δ\u003cem\u003eegspe\u003c/em\u003e mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e) suggest that EgSPE contributes to fungal morphogenesis and symbiotic performance. One possibility is that EgSPE directly participates in cell wall organization through chitin binding, as proper chitin distribution is essential for hyphal growth and colony architecture [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Disruption of this process could alter cell wall structure and consequently affect colony morphology and pigment deposition, potentially explaining the white-to-pink transition observed in the mutants. Alternatively, EgSPE may function as a non-catalytic accessory factor that facilitates the localization or activity of cell wall-modifying enzymes such as chitinases or glucanases. Consistent with this view, CBMs often enhance polysaccharide remodeling by coordinating enzyme\u0026ndash;substrate interactions within insoluble cell wall matrices [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Together, these observations support a role for EgSPE in chitin-associated cell wall processes that influence fungal development and host interaction.\u003c/p\u003e \u003cp\u003eIn addition, EgSPE appears to exert a cross-kingdom function, as its secretion induces the expression of drought-responsive genes in plants. This observation suggests that EgSPE may act as a fungal-derived elicitor or microbe-associated molecular pattern (MAMP) that is perceived by plant pattern recognition receptors (PRRs), such as LysM receptor-like kinases involved in the recognition of fungal carbohydrate signals [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Activation of PRR-mediated signaling typically triggers early immune responses, including reactive oxygen species (ROS) accumulation, Ca\u0026sup2;⁺ influx, and MAPK cascade activation, which subsequently regulate downstream transcription factors such as WRKY and DREB, leading to the induction of ABA-dependent and drought-responsive genes [\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Consistent with this hypothesis, increasing evidence indicates that \u003cem\u003eEpichlo\u0026euml;\u003c/em\u003e endophytes enhance host tolerance to drought stress by modulating antioxidant systems and phytohormone signaling pathways. In particular, abscisic acid (ABA) plays a central role in coordinating stress-responsive gene expression and mitigating oxidative damage in endophyte-associated grasses [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe identification and functional characterization of EgSPE highlight the importance of secreted proteins in regulating the growth and development of \u003cem\u003eEpichlo\u0026euml;\u003c/em\u003e endophytes and their symbiotic interaction with host grasses. Our findings provide direct evidence that secreted proteins contribute to multiple aspects of the \u003cem\u003eEpichlo\u0026euml;\u003c/em\u003e\u0026ndash;grass association, an area that has remained poorly understood. These results suggest that \u003cem\u003eEpichlo\u0026euml;\u003c/em\u003e-derived secreted proteins may represent valuable molecular tools for dissecting the complex molecular dialogue between endophytes and their host plants. Future studies should aim to identify additional effector or secreted proteins in \u003cem\u003eEpichlo\u0026euml;\u003c/em\u003e and to elucidate the molecular mechanisms underlying their secretion, perception and downstream signaling in host plants. The optimized fungal transformation system developed in this study will facilitate functional genetic analyzes and accelerate the discovery of symbiosis-related effectors, thereby advancing our understanding of the molecular basis of \u003cem\u003eEpichlo\u0026euml;\u003c/em\u003e-grass symbiosis.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eIn this study, we identify EgSPE as a secreted effector from \u003cem\u003eE. gansuensis\u003c/em\u003e that contributes to symbiotic establishment and host drought tolerance in \u003cem\u003eA. inebrians\u003c/em\u003e. Our results show that EgSPE is essential for normal fungal growth and efficient host colonization, and its absence abolishes the endophyte's ability to enhance drought tolerance in the host. Furthermore, EgSPE is associated with the activation of drought-responsive gene expression in both heterologous and native host systems. Together, these findings reveal a role for EgSPE in mediating mutualistic benefits in this grass\u0026ndash;endophyte association and provide a mechanistic basis for further investigation of stress adaptation in symbiotic systems.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eEgSPE \u003cem\u003eEpichlo\u0026euml; gansuensis\u003c/em\u003e essential secreted protein\u003c/p\u003e \u003cp\u003eOE-\u003cem\u003eEgSPE EgSPE\u003c/em\u003e-overexpressed \u003cem\u003eEpichlo\u0026euml; gansuensis\u003c/em\u003e strain\u003c/p\u003e \u003cp\u003eΔ\u003cem\u003eegspe EgSPE\u003c/em\u003e knockout \u003cem\u003eEpichlo\u0026euml; gansuensis\u003c/em\u003e strain\u003c/p\u003e \u003cp\u003e \u003cem\u003eRD29A Responsive to Desiccation 29A\u003c/em\u003e \u003c/p\u003e \u003cp\u003eSPs secreted proteins\u003c/p\u003e \u003cp\u003ehpi hours post-inoculation\u003c/p\u003e \u003cp\u003edpi days post-inoculation\u003c/p\u003e \u003cp\u003eCBM carbohydrate-binding module\u003c/p\u003e \u003cp\u003eGO Gene Ontology\u003c/p\u003e \u003cp\u003eROS reactive oxygen species\u003c/p\u003e \u003cp\u003eMAMP microbe-associated molecular pattern\u003c/p\u003e \u003cp\u003ePRRs plant pattern recognition receptors\u003c/p\u003e \u003cp\u003eABA abscisic acid\u003c/p\u003e \u003cp\u003eNCBI National Center for Biotechnology Information\u003c/p\u003e \u003cp\u003ePDA potato dextrose agar\u003c/p\u003e \u003cp\u003eYPD yeast peptone dextrose medium\u003c/p\u003e \u003cp\u003eWGA-AF488 Wheat Germ Agglutinin-Alexa Fluor 488\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research was performed in accordance with Chinese law and international guidelines. The collection of the plant species was permitted by the University Ethical Committee. Lei Lei and Haijuan Zhang identified the studied plant using the WFO Plant List (https://www.wfoplantlist.org/) and deposited the voucher specimen in the Lanzhou University Herbarium (ID:00098860).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets analysed during the current study are available from the corresponding author ([email protected]) on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the National Natural Science Foundation of China (32300241, U21A20239); Gansu Province Intellectual Property Program (22ZSCQD01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL.L. and C.J.L. conceived the study. L.L., H.J.Z. and H.T.S. designed the experiments and wrote the manuscript. L.L. and C.J.L. revised the manuscript to the present form. The final manuscript was confirmed by all authors and approved for submission.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Dr. Jingtao Li (Qingdao Agricultural University) for provided a fungal overexpression vector.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLee K, Missaoui A, Mahmud K, Presley H, Lonnee M. Interaction between grasses and \u003cem\u003eEpichlo\u0026euml;\u003c/em\u003e endophytes and its significance to biotic and abiotic stress tolerance and the rhizosphere. Microorganisms. 2021;9(11):2186\u0026ndash;213.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFardella PA, Clarke BB, Belanger FC. 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Transcriptomic analyses giving insights into molecular regulation mechanisms involved in cold tolerance by \u003cem\u003eEpichlo\u0026euml;\u003c/em\u003e endophyte in seed germination of \u003cem\u003eAchnatherum inebrians\u003c/em\u003e. Plant Growth Regul. 2016;80:367\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng C, Wang JF, Hou WP, Malik K, Zhao CZ, Niu XL, Liu YL, Huang R, Li CJ, Nan ZB. Elucidating the molecular mechanisms by which seed-borne endophytic fungi, \u003cem\u003eEpichlo\u0026euml; gansuensis\u003c/em\u003e, increases the tolerance of \u003cem\u003eAchnatherum inebrians\u003c/em\u003e to NaCl stress. Int J Mol Sci. 2021;22(24):13191.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHao MS, Mazurkewich S, Li H, Kvammen A, Saha S, Koskela S, Inman AR, Nakajima M, Tanaka N, Nakai H, Br\u0026auml;nd\u0026eacute;n G, Bulone V, Larsbrink J, McKee LS. 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Functional evaluation of the signal peptides of secreted proteins. Bio-Protocol. 2018;8(9):e2839.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Epichloë endophyte, transformation system, secreted protein, drought tolerance","lastPublishedDoi":"10.21203/rs.3.rs-9167048/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9167048/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003e \u003cem\u003eEpichlo\u0026euml;\u003c/em\u003e endophytes form beneficial symbioses with cool-season grasses, enhancing host tolerance to abiotic stresses such as drought while maintaining normal plant growth. However, the molecular mechanisms underpinning this symbiosis, particularly the role of fungal-secreted effectors, remain largely unexplored.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn this study, we identify EgSPE, a secreted effector from \u003cem\u003eEpichlo\u0026euml; gansuensis\u003c/em\u003e, as a key regulator of symbiotic establishment and host drought tolerance in drunken horse grass (\u003cem\u003eAchnatherum inebrians\u003c/em\u003e). Transcriptome profiling during host colonization revealed \u003cem\u003eEgSPE\u003c/em\u003e as a strongly induced gene encoding a secreted protein. Functional characterization facilitated by a substantially improved transformation system demonstrates that EgSPE is indispensable for fungal growth and efficient host colonization, as its deletion severely disrupted symbiotic establishment. Notably, EgSPE activates the host drought-responsive signaling by inducing the marker gene \u003cem\u003eRD29A\u003c/em\u003e in a heterologous system (\u003cem\u003eNicotiana benthamiana\u003c/em\u003e) and upregulating stress-related genes (\u003cem\u003eAiRD22\u003c/em\u003e, \u003cem\u003eAiNAC5\u003c/em\u003e, and \u003cem\u003eAiABA1\u003c/em\u003e) in its native host (\u003cem\u003eA. inebrians\u003c/em\u003e). Consistently, only the \u003cem\u003eE. gansuensis\u003c/em\u003e wild-type and OE-\u003cem\u003eEgSPE\u003c/em\u003e strains enhanced host drought tolerance, whereas the Δ\u003cem\u003eegspe\u003c/em\u003e mutants failed to confer this benefit.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eIn summary, our research findings identify EgSPE as a fungal effector that plays an important role in the establishment of symbiosis and in the host's drought response, providing strong evidence for how \u003cem\u003eE. gansuensis\u003c/em\u003e promotes abiotic stress tolerance in grasses.\u003c/p\u003e","manuscriptTitle":"EgSPE, a secreted effector from Epichloë gansuensis, modulates symbiotic establishment and host drought tolerance","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-06 13:10:50","doi":"10.21203/rs.3.rs-9167048/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-17T08:00:32+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-13T20:41:28+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-12T03:47:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"197686866391403061982342703124725297911","date":"2026-04-06T19:22:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"14451032544702901805920303502191831710","date":"2026-04-06T15:30:35+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-01T09:14:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-01T09:05:02+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-03-30T06:46:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-28T14:26:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2026-03-28T14:21:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6f864abf-fe96-4d76-9fae-6dcf5ec735ef","owner":[],"postedDate":"April 6th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-13T00:53:19+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-06 13:10:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9167048","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9167048","identity":"rs-9167048","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}