Insect-derived long non-coding RNAs function as epigenetic effectors to reprogram plant immunity

Insect-derived long non-coding RNAs function as epigenetic effectors to reprogram plant immunity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Insect-derived long non-coding RNAs function as epigenetic effectors to reprogram plant immunity Yazhou Chen, Dong Wen, Shan Jiang, Zhuangzhuang Qiao, Chi Liu, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8297537/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Cross-kingdom RNAs are emerging as critical mediators of interspecies interactions, yet the functions of long RNAs such as mRNAs and long non-coding RNAs (lncRNAs) in recipient organisms remain largely unexplored. Here, we show that the brown planthopper ( Nilaparvata lugens , BPH), a major rice pest, translocates mRNAs and lncRNAs into rice plants, where they migrate systemically from feeding sites to distal tissues. Compared with BPH mRNAs, BPH Salivary gland Cross-kingdom LncRNA ( BSCL s) exhibit markedly higher stability in rice. Among them, mitochondrial-originated BSCL1 functions as a virulence factor that promotes BPH feeding and reproduction by suppressing host defense. Mechanistically, BSCL1 associates with the HIRA histone chaperone complex and displaces histone H3.3 from the promoters of transcription factors, including bHLH genes central to jasmonic acid signaling, thereby repressing transcriptional immunity. Our results identify BSCL s as systemic, RNA-based effectors that reprogram host defense at the epigenetic level, revealing a previously unrecognized mode of insect-mediated manipulation of plant immunity and highlighting lncRNAs as cross-kingdom regulators. Biological sciences/Zoology/Entomology Biological sciences/Plant sciences/Plant immunity/Effectors in plant pathology brown planthopper lncRNAs cross-kingdom RNAs BSCLs rice Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Cross-kingdom RNAs are RNA molecules of an organism that traffic and function in recipient organisms that belong to different biological kingdoms. Among these, cross-kingdom RNA interference (RNAi) phenomena that involve small RNAs (sRNAs) have been widely observed across various parasite-host interactions 1 – 5 . Those sRNAs traffic across the organisms and influence gene expression in the recipients, which reveals the critical roles of cross-kingdom RNAs in these interactions. In contrast, far less attention has been given to the functions of long RNA species such as mRNAs and long non-coding RNAs (lncRNAs) in recipient organisms, although they have been found to be intensively trafficked during parasite-host interactions. For example, plant phloem mRNAs have been found to traffic bidirectionally between parasite dodders and various host plants 6 , 7 . Arabidopsis mRNAs are delivered into the fungus and then translated by the fungal cells, leading to the reduction of fungal infection 8 . Cryptosporidium parvum lncRNAs were selectively delivered into the nuclei of human intestinal epithelial cells during infection 9 . Aphid lncRNAs are translocated into divergent host plants and systematically migrate in the plants and promote aphid colonization 10 . These studies suggest that long RNAs play essential roles in mediating parasite-host interactions; however, their function in the hosts remains largely unexplored. Sap-feeding insects such as aphids and planthoppers are excellent models for studying cross-kingdom RNAs. These insects suck the contents from the xylem and/or phloem tissues, the main components of plant vascular tissues 11 – 14 . Watery saliva secreted by insect salivary glands is injected into plant vascular tissues, where salivary molecules, including various RNAs, enter and traffic in the plants 5 , 10 , 13 , 15 , 16 . Moreover, these insects can be easily confined to specific areas of the plant, leaving the rest of the plant intact and uncontaminated, enabling tracking the migration of insect RNAs in the plant by various approaches such as qRT-PCR 5 , 10 , 16 . Additionally, insect-derived RNAs, as foreign RNA species, exhibit low sequence similarity to plant RNAs, making them highly distinguishable and ideal for detection using next-generation sequencing approaches 5 , 10 . Here, we used the brown planthopper ( Nilaparvata lugens Stål, BPH), a major rice pest worldwide 17 , to investigate the translocation of insect-derived RNAs into host plants. Our study identified both lncRNAs and mRNAs delivered into rice, with particular focus on salivary gland–derived lncRNAs, termed BSCL s ( BPH Salivary gland Cross-kingdom LncRNA s). These BSCL s were translocated into rice and migrated systemically from the feeding site to distal tissues, including leaves and roots. Compared with BPH mRNAs, BSCLs exhibited markedly higher stability within rice plants. Among them, mitochondrial-delivered BSCL1 promoted BPH feeding and reproduction by suppressing rice defense responses. BSCL1 displaced histone complexes from the promoters of transcription factor genes, leading to transcriptional repression of transcription factors such as bHLH, central to jasmonic acid (JA) signaling, thereby weakening host resistance to BPH. Results Identification and host-responsive expression of salivary lncRNAs in BPH To annotate lncRNAs in the BPH genome, we assembled strand-specific RNA-seq data from 11 libraries (Supplementary Table 1), identifying 49,435 transcripts from 47,350 genes (Supplementary Fig. 1A). After filtering, 24,433 putative lncRNAs (22,374 genes) were retained, including 10,268 previously reported and 12,106 newly identified (Supplementary Fig. 1A). Comparative analysis across 28 arthropod genomes showed that most lncRNA genes (57.9%, 12,957) are BPH-specific (Supplementary Fig. 1B, C), suggesting a recent evolutionary origin. To explore potential functions in BPH–rice interactions, we analyzed lncRNA expression in insects transferred from susceptible TN1 rice to resistant RHT plants. RNA-seq revealed 1,626 down-regulated and 1,291 up-regulated genes (Supplementary Fig. 1D, Supplementary data 1), including 932 lncRNAs enriched in salivary gland expression (Supplementary Fig. 1E). Notably, 606 differentially expressed lncRNAs were expressed in salivary glands, and most (78.7%, 477) were repressed by resistant plants (Supplementary Fig. 1F, G), implicating salivary lncRNAs in host colonization. BPH salivary gland lncRNAs are translocated into rice plants Previously, we reported that aphid salivary gland lncRNAs are translocated into plants, and lncRNA Ya1 acted as a virulence factor to promote aphid colonization 10 . Thus, we investigated whether BPH salivary gland lncRNAs are secreted into rice via feeding. We conducted cage experiments in which 30 male BPHs—chosen to prevent egg deposition on the plants—were confined to the leaf sheath (designated as feeding sites, FS) of 45-day-old rice plants for 2 days (Fig. 1 A). Controls were counterparts of untreated plants which the sheath was in an empty cage. Caged tissues were carefully washed to remove visible BPH tissues and were subsequently subjected to RNA-seq analysis. Bioinformatically, adaptor-removed RNA-seq reads were aligned to a merged genome file that the concatenated reference genomes of rice TN1 18 and BPH 19 (Supplementary Fig. 2A). Expectedly, the vast majority of reads in the replicates of controls and FS were mapped to the merged genome (Supplementary data 2). To gain more confidence, only uniquely aligned reads were used in the following analysis. The number of BPH reads in the FS varied largely among the replicates, resulting in different numbers of BPH lncRNAs identified (Supplementary data 2, Supplementary Fig. 2B). For instance, 3065 uniquely mapped reads from 951 transcripts were identified from one replicate, while fewer than 547 reads were identified in the other two. Such variation has been observed in other BPH experiments 5 . We chose the sample BPH-1-FS for further analysis since the highest number of BPH RNA reads were detected (244 transcripts with reads ≥ 4, Supplementary data 3). Among these 244 transcripts that are likely to be cross-kingdom RNAs (Supplementary data 3), 11 lncRNAs were expressed in the salivary glands and were also DE between BPH that fed on TN1 and RHT (Fig. 1 B, Supplementary data 1), which included 8 down-regulated and 3 up-regulated lncRNA genes (Fig. 1 C). We found that 10 out of 11 BPH lncRNAs were detected in the FS of rice plants in the repeated cage experiments but were absent from the same RNA samples that weren’t treated with reverse transcriptase and from the counterparts on the control plants (Supplementary Fig. 2C). Therefore, these lncRNAs were named BPH Salivary gland Cross-kingdom LncRNA ( BSCL ). Noticeably, the PCR bands of 7 BSCL s ( BSCL4-9 and BSCL11 ) were faint unless the PCR products were used as templates for another round of PCR amplification (Supplementary Fig. 2C). Three BSCLs ( BSCL1 , BSCL2 , BSCL3 ) were detected in more than two tested samples and absent from the controls (Supplementary Fig. 2C). In conclusion, BPH lncRNAs are translocated into rice plants via feeding, although there is some variation among different individuals. BPH BSCL s migrate systemically within rice plants Detection of BPH lncRNAs in the FS of rice sheath prompted us to assess whether these RNAs migrate inside rice plants. BPH feeding occurs mainly at the outer layer of rice stem sheaths, thus the FS tissues were dissected into the outer and inner layers (Fig. 1 D). The presence of BSCL1 , BSCL2 , and BSCL3 on each layer was examined (Fig. 1 D). PCR amplification with specific primers detected three BSCLs clearly in both layers, suggesting migration of these BSCL s in the rice plants. In addition to the FS, the tissues that were about 2 cm above and below the caged sheath, respectively termed up- and down-near feeding sites (UFS and DFS, Fig. 1 E), were also harvested. All three BSCLs were detected in the outer layers of FS and DFS (Fig. 1 E). Except for BSCL2 , both BSCL1 and BSCL3 were also found in the outer layers of UFS. Sequences of PCR products of these three lncRNAs obtained from rice sheath further confirmed the translocation of BPH lncRNAs into the rice plants (Supplementary Fig. 3A-C). BSCL1 was also found in the inner layers of UPS, FS, and DFS (Fig. 1 E), probably because of its relatively higher expression level. BSCL1 abundance in the outer layers ranked from the DFS, FS, then to UFS, which was similar as that in the inner layers (Fig. 1 E). It indicated that migration of BSCL s in the rice plants is likely from top to bottom in most outer sheaths where BPH actually feed on, and from bottom to top in the inner sheaths. The opposite may be achieved through the internodes, where nutrients and minerals are often redistributed throughout the entire plant. Indeed, we observed BSCL1 , BSCL2 , and BSCL3 as being noticeably abundant in the roots that were comparable to that in the feeding sites and less than in the top leaves (Fig. 1 F), when the feeding experiments were repeated with 14-day-old seedlings growing in hydroponics. In all experiments, the BSCL s were not detected in the control plants that were uninfested by BPH (Supplementary Fig. 2D, E). It strongly implies that the migration of BSCL s in rice plants is in alignment with the phloem streaming moving from the source to sink tissues. BPH BSCLs are more stable than BPH mRNAs in rice plants A substantial number of BPH mRNA reads were also found in the RNA-seq of FS (Supplementary Fig. 4A, Supplementary Table 2). This prompted us to investigate whether BPH mRNAs also migrated in the rice plants. Out of five selected BPH mRNAs (Supplementary Fig. 4A, B), two ( Nlug010593 and Nlug002054 ) were found in the FS by PCR (Fig. 2 A, Supplementary Fig. 4B). However, the presence of these mRNAs in rice plants can only be detected by primers targeting the shorter fragments (Supplementary Fig. 4C, D), indicating that BPH mRNAs in the rice plants have been fragmented. Fragmented RNAs are often less stable and more prone to degradation 20 , 21 . To assess the stability of BPH mRNAs and BSCL s in rice plants, we removed BPH from FS after 7 days of feeding and subsequently harvested samples at several time points (Fig. 2 B). On the 7th day of feeding (0 days post-BPH removal), BSCL s ( BSCL1 and BSCL2 ) and BPH mRNAs ( Nlug010593 and Nlug002054 ) were found in both the outer and inner layers of FS (Fig. 2 C-F). The abundance of BSCL1 and BSCL2 remained relatively higher than that of Nlug010593 and Nlug002054 (Fig. 2 C-F). Two days post BPH removal, the abundance of BPH mRNAs in the rice plants declined sharply. In contrast, the abundance of BSCL s in the rice plants remained high, even 8 days after the removal of BPH (Fig. 2 C-F), suggesting BPH BSCL s are more stable in rice plants than BPH mRNAs. BSCL1 is a mitochondrial-derived lncRNA To investigate the functions of BSCL s, we selected BSCL1 to conduct further experiments since BSCL1 is the most abundant in FS compared to other BSCL s. We determined the full-length BSCL1 sequence (708 nt) from brown planthopper (BPH) using 5′ and 3′ RACE (Supplementary Fig. 5A, B) and validated it by northern blotting (Supplementary Fig. 5C). The full-length transcript was detected in FS, NFS, and DFS tissues (Supplementary Fig. 5D). To determine the genomic origin of BSCL1 , we aligned its full-length sequence to the BPH reference genome. Although the sequence showed strong homology to a locus on Chromosome 9 (position 6540831–6541538), this nuclear copy contained a single-nucleotide mismatch (e.g., A→T at position 516) (Supplementary Fig. 6), indicating that it might be another source of the actively expressed BSCL1 transcript. To further resolve its origin, we performed a homology search against the NCBI database using the BSCL1 sequence. Strikingly, the reverse complement showed 100% identity to the BPH mitochondrial genome (position 14471–15178) (Supplementary Fig. 6). Notably, BSCL1 is reverse complementary to the mitochondrial 12S rRNA region, suggesting that it is transcribed from the opposite strand relative to the 12S rRNA gene. These results indicate that the Chromosome 9 locus corresponds to a nuclear-encoded mitochondrial sequence (NUMT), a class of mitochondrial DNA insertions typically considered transcriptionally inert 22 . Given the generally inactive nature of NUMTs, our findings strongly support the mitochondrial genome—rather than the nuclear NUMT copy—as the true source of the functional BSCL1 lncRNA. Visualization of BSCL1 localization in planta We examined the in-planta migration of BSCL1 using an RNA imaging approach, the RNA switch–controlled RNA-triggered fluorescence (RNA switch–RTF) system, which enables real-time visualization of RNA dynamics in living plants 23 . In this system, the RNA switch with a probe targeting BSCL1 can toggle between active and inactive states depending on the presence of the target RNA, thereby controlling GFP accumulation or degradation (Fig. 3 A). Based on the full-length sequence, we designed a dumbbell-shaped RNA switch with the probe (Supplementary Fig. 5E), which is essential for the RNA switch function. Agroinfiltration of N. benthamiana leaves with 35S::BSCL1 together with the RNA switch–RTF construct produced GFP signals in both the nucleus and cytoplasm (Fig. 3 B, C), confirming subcellular localization of BSCL1 (Fig. 3 C). No GFP fluorescence was observed when leaves were infiltrated with either BSCL1 or the RNA switch–RTF system alone (Fig. 3 C). Remarkably, GFP signals were also detected in tissues adjacent to the infiltration sites, where the RNA switch–RTF system had been introduced but not the BSCL1 plasmid (Fig. 3 C), demonstrating systemic movement of BSCL1 . By contrast, infiltration with 35S::GFP produced strong local fluorescence but no signals in neighboring tissues (Fig. 3 D). Together, these results demonstrate that BPH-derived BSCL s are not only localized within plant cells but also migrate systemically from the initial delivery sites to adjacent and distal tissues. BPH BSCL1 is a virulence factor that suppresses the plant defenses Although BSCL1 is derived from the mitochondrial genome, our findings indicated that the functional transcript is exported to the cytoplasm for secretion (Fig. 1 D, Fig. 3 C). Based on this cytoplasmic localization, we targeted the lncRNA using a canonical, cytoplasm-restricted RNA interference (RNAi) approach, employing two non-overlapping double-stranded RNA (dsRNA) sequences, rather than specialized mitochondrial gene silencing strategies 24 . The results showed that we had successfully inhibited the expression levels of BSCL1 in BPH (Fig. 4 A). The survival rate had no significant difference between dsBSCL1 and the dsGFP groups (Fig. 4 B), however, the honeydew production (Fig. 4 C, Supplementary Fig. 7A) and the total number of eggs per female (Fig. 4 D) were significantly reduced. To further assess the impact of BSCL1 on BPH performance, we generated stable transgenic plants in TN1 genetic background that produced 708-nt of BSCL1 (35S::BSCL1, lines 2 and 3) and 708-nt BSCL1 mutants in which two ATG start sites were mutated to stop codes (35S::BSCL1_mut, lines 8 and 11) (Fig. 4 E). BPH produces more honeydew (Fig. 4 F, Supplementary Fig. 7B) and eggs (Fig. 4 G) on both 35S::BSCL1 and 35S::BSCL1_mut compared to 35S::GFP and TN1 plants, suggesting BSCL1 in rice promote BPH colonization. These findings suggest that BPH BSCL1 is a virulence factor that plays a critical role in BPH-rice interactions. To assess whether plant defense pathways were affected by BSCL1 , we challenged TN1, 35S::GF plants, 35S::BSCL1_wt, and 35S::BSCL1_mut by M. oryzae . Six days posted the inoculation of M. oryzae , 35S::BSCL1_wt and 35S::BSCL1_mut plants exhibited a stronger hypersensitive response compared to TN1 and 35S::GFP plants (Fig. 4 H, I), suggesting that overall rice defense pathways were suppressed by BSCL1 . BSCL1 represses the expression of defense-related genes in rice To determine the host processes influenced by BSCL1 , we performed RNA-seq on rice plants (TN1, 35S::GFP, 35S::BSCL1_wt, and 35S::BSCL1_mut) with or without BPH feeding for 24 h. Principal component analysis (PCA) of transcriptomes revealed clear separation of sample groups (Supplementary Fig. 8A). Relative to non-feeding controls, BPH feeding altered the expression of 6,710 rice genes (Supplementary Fig. 8B, Supplementary data 4). We next examined whether BSCL1 modulates these plant responses. Coexpression analysis of differentially expressed (DE) genes grouped 6,404 genes into eight clusters based on expression patterns (Fig. 5 A, Supplementary data 5). TPM heatmaps confirmed these clusters (Supplementary Fig. 9). Expression profiles of 35S::BSCL1_mut plants closely resembled those of 35S::BSCL1_wt, indicating that BSCL1 functions as an RNA rather than through a peptide product (Supplementary Fig. 9). Four clusters (clusters 2, 3, 5, and 8) showed similar expression in 35S::BSCL1_wt, TN1, and 35S::GFP plants, suggesting that these groups are largely unrelated to BSCL1 activity (Fig. 5 A, Supplementary Fig. 9B, C, E, H). Cluster 4 genes were strongly induced in 35S::BSCL1_wt plants but suppressed after BPH feeding, a pattern absent in controls (Fig. 5 A, Supplementary Fig. 9D). By contrast, clusters 1, 6, and 7 exhibited distinct expression changes in 35S::BSCL1_wt plants (Fig. 5 A, Supplementary Fig. 9A, F, G). In clusters 1 and 7, many genes were strongly upregulated in TN1 and 35S::GFP plants after BPH feeding, but this induction was markedly repressed in 35S::BSCL1_wt plants (Fig. 5 A, Supplementary Fig. 9A, G). These clusters were significantly enriched for genes in the jasmonic acid (JA)-mediated defense pathway (Supplementary Fig. 10). Consistently, multiple JA pathway genes were repressed in both 35S::BSCL1_wt and 35S::BSCL1_mut compared to controls (Supplementary Fig. 11). Cluster 6 was of particular interest because its genes were downregulated upon BPH feeding and remained further suppressed in 35S::BSCL1_wt plants (Fig. 5 A, B). Notably, cluster 6 contained the highest number of transcription factor (TF) genes among all clusters (Fig. 5 B). Enriched families included bHLH, MYB-related, bZIP, Dof, Nin-like, ERF, G2-like, and TALE, with bHLH TFs most abundant (12/38; Fig. 5 B). Several members of this family—including bHLH96, bHLH6, and PTF1—are known regulators of JA- and SA-mediated defenses. Rice bHLH96 and bHLH6, in particular, have been implicated in resistance to BPH 25 , 26 . Their repression in both control plants and 35S::BSCL1_wt upon BPH feeding suggests that BSCL1 interferes with transcriptional regulation of key defense pathways (Fig. 5 C, D, Supplementary Table 3). BSCL1 targets the histone complex to repress transcription factor promoters In plants, lncRNAs have been reported to act through protein interactions 27 , 28 . To identify BSCL1 -binding partners in rice, we performed yeast three-hybrid (Y3H) screening using a rice cDNA library. A total of 197 yeast colonies grew on selective media, of which 104 exhibited strong X-β-gal activity (Supplementary Fig. 12, Supplementary data 6). These corresponded to 31 rice proteins, including enzymes, transcription factors, and components of the histone complex (Supplementary Table 4). Notably, vacuolar protein sorting-associated protein 2 homolog 2 (VPS), histone H3.3, transcription factor RF2a-like (RF2), ribonuclease J isoform X1, HIRA-interacting protein 3 (HIRIP3), and T-complex protein 1 subunit gamma were among the frequently recovered candidates. Given the central role of chromatin regulation in transcriptional control, we focused on histone H3.3 and HIRIP3. Interactions of BSCL1 with both proteins were validated in repeated Y3H assays (Fig. 6 A). RNA-switch-RTF revealed that BSCL1 was colocalized with HIRIP3 in the nucleus and cytoplasm, with H3.3 in the nucleus, where H3.3 was mainly expressed (Fig. 6 B). These results suggest that BSCL1 associates with the HIRA histone chaperone complex, which normally deposits H3.3 into chromatin to maintain active transcription states. We next tested whether BSCL1 interferes with H3.3 deposition at promoters of defense-related transcription factors (e.g., bHLH6 in cluster 6). A luciferase (LUC) reporter driven by the bHLH6 promoter was co-expressed with combinations of H3.3, HIRIP3, and BSCL1 in rice. Neither H3.3 nor HIRIP3 alone altered promoter activity (Fig. 6 C), but co-expression with BSCL1 significantly suppressed LUC expression (Fig. 6 D, E). Importantly, simultaneous expression of H3.3 and HIRIP3 did not produce further repression beyond that observed with either factor alone (Fig. 6 F), indicating that BSCL1 disrupts the function of the H3.3–HIRIP3 complex at defense gene promoters (Fig. 6 G). Discussion We found that BPH actively translocated salivary gland RNAs into rice plants during feeding. These cross-kingdom RNAs move systemically from feeding sites on the sheath to distant tissues such as inner sheaths, upper leaves, and roots, likely through vascular transport. In rice, BPH mRNAs degraded relatively quickly—possibly via 5′-end decapping and processing as part of a conserved host defense strategy—whereas BPH lncRNAs persisted for extended periods, remaining detectable up to eight days after insect removal. Among these lncRNAs, BSCL1 emerged as a highly abundant and functionally important effector. Silencing BSCL1 in BPH reduced feeding and reproduction, while heterologous expression in rice enhanced BPH performance. Moreover, BSCL1 overexpression suppressed JA-induced defense transcription factors and compromised immunity against both BPH and the blast fungus M. oryzae , suggesting that it broadly disables rice plant defense. The discovery that the functional BSCL1 lncRNA originates from the mitochondrial genome introduces a fascinating layer of complexity to cross-kingdom communication. While NUMTs are widespread and typically represent transcriptionally silent relics of mtDNA transfer 29 , the active transcription, cytoplasmic export, and subsequent secretion of a mitochondrial-encoded RNA like BSCL1 is highly unusual. This finding strongly suggests a novel, specialized regulatory role for this subset of mitochondrial transcripts, distinct from the canonical nuclear-encoded effector molecules. In insects, mitochondrial transcription is fundamentally linked to essential metabolic processes and acute stress responses 30 , 31 ; its repurposing here for systemic cross-kingdom defense suppression hints at a highly evolved mechanism where fundamental cellular resources are co-opted for specialized pathogenicity. Further research is warranted to determine if other mitochondrial-derived RNAs are similarly utilized as mobile effectors in this or other insect-plant systems. Transcriptomic profiling revealed that BPH feeding orchestrates complex reprogramming of rice metabolism and defense. We observed repression of processes associated with cell walls, ion transport, and carbohydrate metabolism, but strong induction of jasmonic acid (JA)-related genes, RNA metabolism, circadian rhythm, and transporter activities (Supplementary Fig. 10). These patterns are consistent with previous findings: reinforcement of the cell wall enhances resistance 32 – 34 ; ion transport underpins defense signaling and metabolite biosynthesis 17 ; and carbohydrate metabolism, often manipulated by herbivores, is further exploited by BPH through hijacking SWEET13/14 sugar transporters 35 – 40 . Despite these suppressions, BPH feeding activates defense signaling, with strong induction of JA pathway genes and transcription factors such as WRKY, NAC, and MYB 25 , 41 – 45 and tissue-specific JA sectors including the MYC2–bHLH6 cascade 26 . Together, these findings highlight the dual nature of the interaction: rice perceives and counters insect attack, while BPH simultaneously manipulates host physiology to promote feeding. Our discovery of insect-derived lncRNAs reveals a previously unrecognized mechanism: BPH not only manipulates host physiology indirectly but also delivers its own genetic effectors to reprogram rice immunity at transcriptional and epigenetic levels. BSCL1 exemplifies this new class of mobile effectors. It suppresses JA-associated transcription factors such as OsbHLH6—key regulators of defense 25 , 26 —and interacts with the HIRA histone chaperone complex to block H3.3 deposition at their promoters (Fig. 6 G). Similar lncRNA-guided epigenetic mechanisms have been described in other systems 46 , 47 . These findings also force a reinterpretation of prior observations. The downregulation of defense TFs reported 41 and the apparent negative feedback in JA signaling 26 may partly result from direct suppression by insect lncRNAs. Likewise, sugar transport manipulations by BPH 40 likely occur in a defense-compromised context where RNA effectors such as BSCL1 have already silenced immune hubs. Thus, BPH deploys a multilayered strategy: altering host nutrition, modulating hormone signaling, and injecting RNA-based epigenetic suppressors that dismantle transcriptional control of immunity. BPH secretes a cocktail of salivary molecules to manipulate rice and promote feeding, including enzymatic proteins 48 , effector proteins 49 , 50 , and miRNAs 5 . In addition, numerous salivary gland–derived lncRNAs and mRNAs are translocated into rice, potentially trafficking systemically via the phloem. BPH mRNAs are rapidly degraded, consistent with a conserved host defense against foreign RNAs, as reflected by enrichment of RNA metabolism pathways in Cluster 2 upon BPH feeding (Supplementary Fig. 10). In contrast, a small subset of RNAs, including BSCL1 , remains stable as full-length transcripts, suggesting structural features that allow evasion of host RNA surveillance. While cross-kingdom transfer of long RNAs has been reported in parasitic plants, fungi, and aphids—including the aphid lncRNA Ya1 shown to function as an effector 6 – 8 , 10 — BSCL1 exemplifies these principles and raises key questions: can some insect mRNAs escape host surveillance and be translated in plant cells, as reported for fungal effectors 8 ? How do cross-kingdom lncRNAs such as BSCL1 and Ya1 evade detection, and how do resistant versus susceptible rice genotypes influence their stability, systemic movement, and function? Answering these questions will provide critical insight into the molecular strategies insects use to manipulate host immunity and may guide innovative approaches for pest control. In sum, the discovery of BPH BSCLs reveals lncRNAs as systemic epigenetic effectors that reprogram host immunity. This shifts the view of insect effectors from plant-centric to insect-centric and highlights lncRNAs as an overlooked class of cross-kingdom regulators. Beyond advancing basic understanding of plant–herbivore interactions, these findings suggest new strategies for pest control by targeting RNA effectors. Methods Insect rearing and plant growth Brown planthopper (BPH, N. lugens ) populations were originally collected from rice fields in Wuhan, China, and have been continuously maintained on the susceptible rice cultivar Taichung Native 1 (TN1) since 2007. Rice materials used in this study included TN1, the resistant cultivar Rathu Heenati (RHT) 51 , and transgenic lines (35S::GFP, 35S::BSCL1_wt, and 35S::BSCL1_mut). Rice seeds were sown in plastic pots (7.5 cm diameter, 10 cm height; one plant per pot) and placed in the fields of Huazhong Agricultural University. Plants were protected with insect-proof cages to prevent pest infestation. Tillering stage (30 ~ 45 day-old) plants were brought back to the laboratory for subsequent assays, and were grown under the controlled conditions of 27 ± 1°C, 70 ± 5% relative humidity, and a 14:10 h light/dark photoperiod. Hydroponically grown TN1 seedlings were germinated in Petri dishes (9 cm diameter, 2 cm height) containing water and incubated in a controlled growth chamber under the same controlled conditions mentioned above. Identification and evolutionary analysis of BPH lncRNAs To identify and analyze BPH lncRNAs, we employed a computational pipeline integrating Evolinc-I for lncRNA discovery and Evolinc-II for evolutionary conservation analysis 52 . RNA-seq datasets from BPH (NCBI project PRJNA514182) 53 were first quality-checked using FastQC (v0.11.5) 54 and low-quality bases reads were removed. RNA seq reads were mapped to the BPH genome using the RMTA (v2.6.3) pipeline 55 with default parameters. Transcript assembly was performed using StringTie (v2.2.3) 56 , and assembled GTF files were merged with Cuffmerge (v2.2.1.5) 57 to generate a comprehensive transcriptome. The Evolinc-I pipeline was then applied to identify candidate lncRNAs. Transcripts overlapping annotated protein-coding genes or transposable elements, or shorter than 200 nucleotides, were excluded. Coding potential was evaluated using CPC2 (v2.0) 58 , and transcripts with significant coding potential were removed. Remaining transcripts were further screened against the Rfam database (v15.0) 59 to exclude known structural RNAs, including tRNAs, rRNAs, and snoRNAs. The resulting set of transcripts was considered putative lncRNAs. To assess the evolutionary conservation of BPH lncRNAs, we utilized the Evolinc-II pipeline. This tool performs reciprocal BLAST analyses (Evalue cutoff, 1e-20) against 28 arthropod genomes (Supplementary Table 5). Homologous sequences were grouped into families based on sequence similarity. BPH feeding on different resistant rice lines At the tillering stage (~ 30 days), TN1 (susceptible) and RHT (resistant) rice plants were each infested with 30 third-instar BPH nymphs. After 48 h of feeding, surviving nymphs were collected and immediately snap-frozen in liquid nitrogen. Three biological replicates were performed per cultivar, and all samples were stored at -80°C for RNA extraction. BPH feeding experiments To test whether BPH secreted transcripts into rice, 30 male adults, to avoid egg deposition by females, were confined on stems of 45-day-old TN1 plants (tillering stage) using glass cylinders (10 cm length, 2.5 cm diameter). Uninfested plant stems with empty glass cylinders served as controls. After 48 h of feeding, feeding sites (FS) were excised, rinsed three times with deionized water, and snap-frozen in liquid nitrogen. Each treatment was conducted with two to three biological replicates. The experiment was repeated three times, achieving three replicates for controls and seven replicates for the FS. Samples were used for RNA-seq to identify BPH transcripts potentially translocated in the rice plants. RNA extraction, library preparation, and sequencing RNA-seq was performed by OE Biotech Co., Ltd. (Shanghai, China). Samples were frozen in liquid nitrogen, ground to a fine powder using sterilized stainless-steel beads in a TissueLyser II (Jingxin, Shanghai, China), and homogenized in TRIzol reagent (Invitrogen, 15596018CN, USA) following the manufacturer’s protocol. RNA concentration and purity were measured with a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA), and RNA integrity was assessed with an Agilent 2100 Bioanalyzer (Agilent Technologies, USA). Quantification was performed using a Qubit 2.0 Fluorometer (Thermo Scientific, USA). Only samples with RNA integrity number (RIN) > 7.0 were used for library construction. For each sample, 1 µg of high-quality total RNA was used to prepare strand-specific libraries with the VAHTS® Universal V6 RNA-seq Library Prep Kit (Vazyme, NR604, Nanjing, China). The libraries were assessed for quality and fragment size distribution using the Bioanalyzer 2100. Sequencing was performed on an NovaSeq 6000 platform (Illumina, USA), generating 150-bp paired-end reads (project No. PRJNA1314766, PRJNA1197768 (SRR31704824, SRR31704821, SRR31704820, SRR31704819, SRR31704818, SRR31704816), and PRJNA1330246). RNA-seq analysis RNA-seq datasets were analyzed from two sources: (i) publicly available data (PRJNA714229) covering diverse BPH tissues (antenna, fat body, gut, head, integument, ovipositor, ovary, and salivary gland) to investigate tissue-specific expression, and (ii) RNA-seq data generated in this study from BPH feeding on susceptible (TN1) and resistant (RHT) rice varieties to assess transcriptional responses to host resistance. The BPH reference genome 19 was used for read alignment, while feeding site samples were aligned against a merged reference of TN1 rice 18 and BPH 19 . Raw sequencing reads were processed with fastp (v0.20.1) 60 to remove adapter sequences and low-quality reads (phred score < 25). Reads shorter than 50 bp after trimming were discarded. The quality of clean reads was evaluated using FastQC (v0.11.5) 54 , and only reads passing quality control were retained for downstream analysis. Clean reads were aligned to the reference genome using RMTA (v2.6.3) 55 with the HISAT2 aligner. Parameters were set to trim 15 bases from the 5′ end of each read, and minimum and maximum intron lengths were specified as 20 and 500,000, respectively. The resulting BAM files were used for read quantification with HTSeq (v0.6.1) 61 , using the parameters -r, -i gene_id, -t exon, and library-specific strand settings (-s no for non-stranded or -s yes for stranded data). Only uniquely mapped reads overlapping annotated exons were counted. Differential gene expression was analyzed with edgeR (v3.0) 62 . Raw read counts from HTSeq were filtered to remove lowly expressed genes (counts per million, CPM < 1), normalized using the trimmed mean of M values (TMM) method, and tested for differential expression. P-values were corrected for multiple testing using the Benjamini–Hochberg method. Genes with false discovery rate (FDR)-adjusted p < 0.05 and |log2 fold change| ≥ 1 were considered significantly differentially expressed. Transcript abundance was also quantified as TPM (transcripts per million) using TPMCalculator (v0.0.3) 63 . BAM files generated by RMTA and the corresponding GTF annotation files were provided to TPMCalculator (-b for BAM, -g for annotation). Strand-specificity was set according to the RNA library type (--stranded no for non-stranded libraries). Migration of BPH-derived RNAs in rice plants To examine the systemic movement of BPH-delivered RNAs in rice, feeding experiments were performed at different developmental stages of TN1 plants. For tillering-stage plants (~ 30-day old), 50 fourth-instar BPH nymphs were confined on a stem using a glass cylinder cage (10 cm length, 2.5 cm diameter) and allowed to feed for 48 h. After feeding, tissues were collected from two additional positions relative to the feeding site (FS): 2 cm above the FS (UFS), 2 cm below the FS (DFS). To better resolve the spatial distribution of transcripts within stem tissues, FS, UFS, and DFS samples were dissected into two fractions: (i) outer layers (the two outermost sheaths) and (ii) inner layers (remaining tissues). All collected tissues were rinsed three times with sterile deionized water to remove surface contamination and immediately snap-frozen in liquid nitrogen. Control plants were treated with empty cages. Each treatment included three biological replicates, and the entire experiment was repeated three independent times. For hydroponic experiments, hydroponically grown TN1 plants at the three-leaf stage (~ 14-day old) were used. 20 fourth-instar nymphs were confined on the stems of four seedlings per cage for 48 h. After feeding, tissues were harvested from the leaf, FS, DFS, and root. Each tissue sample was thoroughly washed three times with sterile deionized water prior to freezing in liquid nitrogen. Seedlings caged without insects were used as controls. Each treatment was performed with three biological replicates, and the experiment was repeated three independent times. Experiments to analyze BPH RNA stability in rice plants To assess the stability of BPH-delivered mRNAs and lncRNAs in rice, a time-course feeding experiment was performed. Approximately 80 third-instar BPH nymphs were caged on stems of TN1 rice at the tillering stage (~ 30-day old) using glass cylinders (10 cm length, 2.5 cm diameter) and allowed to feed for seven days. After feeding, all insects were removed, and plant tissues were harvested at 0, 2, 4, 6, and 8 days post-removal. Four biological replicates were collected per time point. Tissues were dissected into outer and inner sheath layers, thoroughly washed with sterile deionized water, and snap-frozen in liquid nitrogen. The relative abundance of cross-kingdom RNAs was quantified by qRT-PCR. Two mRNAs ( Nlug010593 , Nlug002054) and two lncRNAs ( BSCL1 , BSCL2 ) were examined at multiple tissue sites (FS, UFS, DFS) across all time points. RNA extraction, cDNA synthesis, qRT-PCR Total RNA was extracted from collected tissues using TRIzol reagent (Invitrogen, 15596018CN, USA) and treated with RNase-free DNase I (Thermo Fisher Scientific, EN0521, USA) to remove residual genomic DNA. First-strand cDNA was synthesized from 1 µg of total RNA using a mixture of oligo (dT) and random primers with the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, K1622, USA), following the manufacturer’s instructions. Relative transcript abundance was measured by qRT-PCR. Two BPH mRNAs ( Nlug010593 , Nlug002054 ) and two lncRNAs ( BSCL1 , BSCL2 ) were analyzed at multiple tissue sites (FS, DFS, and UFS) across all time points. qRT-PCR reactions were performed in 20 µL volumes containing 10 µL SYBR Green (Takara, RR820A, Japan), 0.5 µL of each primer (10 µM), 1 µL of cDNA template, and 8 µL nuclease-free water, using the CFX Connect™ Real-Time System (Bio-Rad, USA). Cycling conditions were 95°C for 10 s, followed by 40 cycles of 95°C for 20 s, 60°C for 30 s, and 72°C for 30 s. Expression levels were normalized to O. sativa 18S rRNA ( Os18S ) and quantified using the 2^ –ΔCt method. Primer sequences are listed in Supplementary Table 6. Cloning and sequencing of BSCL1 To obtain the full-length cDNA of BSCL1 , 5’ and 3’ rapid amplification of cDNA ends (RACE) was performed. For 3’ RACE, 3 µg of BPH total RNA was added to an 80 µL ligation mixture containing 4 µL T4 RNA ligase, 8 µL T4 RNA ligase buffer, 8 µL BSA (Thermo Fisher Scientific, EL0021, USA), 8 µL ATP (Thermo Fisher Scientific, R0481, USA), and 10 pmol of 3’ RACE RNA adaptor (Supplementary Table 6). Ligation was carried out overnight at 16°C. The ligated RNA was reverse-transcribed into cDNA using an oligonucleotide complementary to the 3’ RACE adaptor with the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, K1622, USA). For 5’ RACE, 4.1 µL of BPH total RNA was mixed with 10 µL of ligation mixture containing 0.1 µL T4 RNA ligase, 1 µL T4 RNA ligase buffer, 1 µL BSA (Thermo Fisher Scientific, EL0021, USA), 1 µL ATP (Thermo Fisher Scientific, R0481, USA), and 20 pmol 5’ RACE RNA adaptor (Supplementary Table 6), followed by overnight ligation at 16°C. The ligated RNA was reverse-transcribed into cDNA using primers complementary to the 5’ RACE adaptor. RACE PCRs were performed with BSCL1 gene-specific forward or reverse primers together with the 3’ or 5’ RACE adaptor primers (Supplementary Table 6). Each 50 µL PCR reaction contained 1 µL Phanta® Max Super-Fidelity DNA Polymerase (Vazyme, P505, Nanjing, China), 25 µL Phanta Max Buffer, 1 µL dNTP mix, 1 µL cDNA template, 1 µL of each primer (10 µM), and 20 µL nuclease-free water. The cycling program was: 95°C for 3 min, followed by 36 cycles of 95°C for 15 s, 55°C for 15 s, and 72°C for 20 s, with a final extension at 72°C for 5 min. PCR products were purified, ligated into the pEASY®-Blunt Cloning vector (TransGen, CB101, Beijing, China), and sequenced to confirm the full-length BSCL1 cDNA. Northern blotting A 708-nt DNA fragment of BSCL1 was cloned under a T7 promoter. BSCL1 RNA transcript was synthesized using MAXIscript™ T7 Transcription Kit (Thermo Fisher Scientific, AM1314, USA). A Biotin-11-UTP (Thermo Fisher Scientific, AM8450, USA) labelled full-length of BSCL1 antisense was synthesized using the MAXIscript™ T7 Transcription Kit (Thermo Fisher Scientific, AM1314, USA). 10 ng of BSCL1 RNA and total BPH RNA were, respectively, mixed with RNA loading buffer (Takara, 9168, Japan), heated at 65°C for 10 min, and separated on a 10% TBE-Urea denaturing polyacrylamide gel (Beyotime, R0235S, Shanghai, China) in 1 × TBE buffer at 150 V for 90 min. RNAs were transferred to a nylon membrane (Beyotime, FFN10, Shanghai, China) by electroblotting at 60 V for 60 min in 0.5 × TBE buffer and cross-linked to the membrane using a UV crosslinker (Ultraviolet Products, CA, USA): twice on the gel-facing side and once on the opposite side. The membrane was pre-hybridized in 10 mL hybridization solution at 42°C for 1 h. The biotin-labeled BSCL1 antisense was denatured at 70°C for 10 min, cooled on ice for 5 min, and added to the hybridization solution for hybridization at 42°C overnight. Detection was carried out using the Chemiluminescent Nucleic Acid Detection Module Kit (Thermo Fisher Scientific, 89880, USA) following the manufacturer’s instructions. The membrane was blocked with 16 mL of Blocking Buffer for 15 min with gentle shaking. After decanting, 16 mL of conjugate/blocking solution was added, and the membrane was incubated for another 15 min. The membrane was washed four times for 5 min each with 20 mL 1 × wash solution under gentle shaking, then equilibrated in 30 mL Substrate Equilibration Buffer for 5 min. Chemiluminescent detection was performed by incubating the membrane in equal volumes of Luminol/Enhancer Solution and Stable Peroxide Solution for 5 min, followed by visualization using a CCD-equipped imaging system. Generating overexpression rice plants The BSCL1 sequence is 708 nt in length ( BSCL1_wt ) and contains two AUG codons at positions 164 and 253. To generate a mutant form, both AUGs were replaced with UAGs using overlapping PCR 64 , resulting in the BSCL1_mut sequence. For this, three overlapping fragments were amplified separately with primer pairs PC- BSCL1 -F– BSCL1 -ATG-164R (fragment 1), BSCL1 -ATG-164F– BSCL1 -ATG-253R (fragment 2), and BSCL1 -ATG-253F–PC- BSCL1 -R (fragment 3). Fragments 1 and 2 were first combined by overlap PCR with primers PC- BSCL1 -F and BSCL1 -ATG-253R. The resulting product was then fused with fragment 3 using primers PC- BSCL1 -F and PC- BSCL1 -R to obtain the 708-nt BSCL1_mut sequence. Primers were listed in the Supplementary Table 6. For rice transformation, the BSCL1_wt , BSCL1_mut and GFP sequences were cloned into the binary vector PC1300S under the control of the cauliflower mosaic virus 35S promoter. BSCL1_wt was amplified from BPH cDNA using primers carrying KpnI and XbaI restriction sites in the forward and reverse primers, respectively. BSCL1_wt , BSCL1_mut , and GFP were inserted into the PC1300S vector by homologous recombination using the ClonExpress Ultra One Step Cloning Kit (Vazyme, C115, Nanjing, China), yielding constructs PC1300S_35S::BSCL1_wt, PC1300S_35S::BSCL1_mut, PC1300S_35S::GFP. Plasmid constructs were first introduced into Agrobacterium tumefaciens strain EHA105 and verified by selection and molecular confirmation. The generation of transgenic rice was performed according to the method described by Hiei and Komari 65 , with minor modifications. Embryogenic calli were induced from surface-sterilized TN1 rice seeds and pre-cultured to increase transformation efficiency. The calli were then infected with the transformed Agrobacterium suspension and co-cultivated on medium containing acetosyringone to facilitate T-DNA transfer. Following co-cultivation, the calli were washed and placed on selective medium (N6 salts, N6 vitamins, 2,4-D 2 mg/L, Sucrose 30 g/L, Casein hydrolysate 0.3 g/L, Proline 0.5 g/L, Agar 8 g/L, Hygromycin B 50 mg/L, Cefotaxime 250 mg/L) containing the appropriate antibiotic to suppress bacterial growth and select for resistant transformants. Resistant calli were subsequently regenerated on hormone-supplemented medium (MS Basal Salt Mixture, MS Vitamin Stock, Sucrose 30 g/L, 6-BAP: 3.0 mg/L, NAA: 0.2 mg/L, Casein Hydrolysate 300 mg/L, Timentin 200 mg/L, Hygromycin 40 mg/L, Phytagel 3.2 g/L) to induce shoots, transferred to rooting medium (MS Basal Salt Mixture, MS Vitamin Stock, Sucrose 20 g/L, IBA 1.5 mg/L, Agar 7.0 g/L), and finally acclimatized in soil to obtain transgenic plants. Positive transformants were confirmed by PCR. The obtained positive plants of T2 generations were used for subsequent experiments. Rice blast fungal inoculation To evaluate the disease resistance of different rice lines, punch inoculation was carried out using the Magnaporthe oryzae Guy11 strain (was kindly provided by Professor Kabin Xie) as described previously 66 , with minor modifications. Conidia of M. oryzae were harvested from 10-day-old cultures grown on Oat-tomato agar medium (OTA, 30 g oatmeal, 200 mL tomato juice, 20 g agar, 1 L distilled water) under continuous light at 28°C. The spores were washed from the plate surface with sterile distilled water. The spore concentration was determined with hemocytometers and adjusted to 5 × 10 6 conidia/mL using sterile distilled water supplemented with 0.02% Tween-20 to promote even suspension. For inoculation, fully expanded leaves from 4- to 5-week-old rice plants (TN1, 35S::GFP, 35S::BSCL1_wt, and 35S::BSCL1_mut) were excised and placed on moist filter paper inside plastic boxes (30 × 20 × 10 cm). Two small wounds were made on each leaf using a sterile needle, and 5 µL of the spore suspension was carefully pipetted onto each wound site. Each plastic box contained leaves from a single rice line to avoid cross-contamination. After inoculation, the boxes were sealed with plastic bag to maintain high humidity and incubated in the dark at 28°C for 24 h. Subsequently, the boxes were transferred to growth chambers maintained at 28°C under a 14 h light / 10 h dark photoperiod. Disease progression was monitored daily. After 6 days, the leaves were photographed using a digital camera, and lesion development was recorded 67 , 68 . In total, 40 leaves per rice line were inoculated, serving as independent biological replicates for statistical analysis. Gene coexpression analysis and GO enrichment analysis To investigate the gene expression responses of different rice lines (TN1, 35S::GFP, 35S::BSCL1_wt, and 35S::BSCL1_mut) to BPH feeding, thirty fifth-instar BPH nymphs were confined on the stem of each rice plant at the tillering stage (~ 30 days old) using a cylindrical plastic cage. After 24 h of feeding, the outer layer of the feeding site (FS) was carefully excised, immediately snap-frozen in liquid nitrogen, and stored at − 80°C until RNA extraction. Three biological replicates were performed for each rice line, and non-infested plants were included as controls. RNA sequencing was performed by OE Biotech Co., Ltd. (Shanghai, China). RNA-seq analysis was discribed in the secton "RNA-seq analysis", which included RMTA for reads alignement, HTseq for reads count, and edgeR for identifying DEGs with |log 2 (fold change)| ≥ 1 and FDR-adjusted p value < 0.05. Co-expression analysis of DEGs was performed with the Mfuzz package in R (v2.6.1) 69 with default settings, and Gene Ontology (GO) enrichment analysis was carried out using the clusterProfiler package in R (v3.18.1) 70 . Localization of BSCL1 in planta For visualization of BSCL1 in plant cells, an RNA-triggered fluorescence (RTF) reporter system was employed 23 . The RTF system consists of two modules: (i) an RNA switch containing a probe sequence complementary to the target RNA, and (ii) a GFP expression cassette that is regulated by the RNA switch. In the absence of the target RNA, the RNA switch remains in an inactive conformation, leading to recruitment of the 26S proteasome and degradation of GFP, thereby preventing fluorescence. In contrast, binding of the target RNA to the probe region induces a conformational switch that prevents GFP degradation, allowing GFP accumulation and fluorescence detection. The RNA switch design method was adapted from Bai et al. 23 . Probes were designed using the Stellaris Probe Designer (Biosearch Technologies, https://www.biosearchtech.com/support/tools/design-software/stellaris-probe-designer ) with default settings, selecting sequences complementary to BSCL1 of approximately 21 nt in length and with annealing temperatures below 65°C. RNA switch secondary structures were predicted using mFold ( www.unafold.org/mfold/applications/rna-folding-form.php 71 ) with default parameters. Switches adopting a dumbbell-shaped structure, particularly those with shorter stems and larger bottom loops, were critical for functionality and guided the selection of optimal designs 23 . The fulll lenght of RNA switch sequeqnce including probe for BSCL1 was listed in Supplementary Table 6. The probe sequence specific for BSCL1 was cloned into the pcambia1300-AtU6::RNA-Switch–AtUbq10::RTF vector at the designated restriction sites, generating the RNA-switch- BSCL1 –RTF construct (Supplementary Fig. 4E). The RTF system comprises full-length GFP fused to a single-chain antibody (scFv) carrying an RRRG degron, together with 24 × GCN4 fused to Rev at its N-terminus. In this system, the scFv-GFP-RRRG fusion functions as the reporter, producing fluorescence only when stabilized, while the RRRG degron ensures that unbound GFP is rapidly degraded, minimizing background signal. The 24 × GCN4-Revd2 component binds to the RNA switch containing RRE sequences, recruiting multiple GFP-scFv molecules and thereby amplifying the fluorescence signal at the RNA site. Primers used for cloning are listed in Supplementary Table 6. The construct was sequence-verified by Sanger sequencing and introduced into A. tumefaciens GV3101 (pSoup) (Weidi, AC1002, Shanghai, China). The RNA-switch- BCSL1 –RTF construct was co-infiltrated with PC1300S-35S::BSCL1_wt into fully expanded leaves of 4-week-old N. benthamiana plants using the leaf infiltration method. A. tumefaciens cultures were grown overnight in LB medium containing appropriate antibiotics, harvested by centrifugation, and the bacterial pellet was washed once with infiltration buffer [10 mM MgCl 2 , 10 mM MES pH 5.6 (BioFroxx, 1086GR500, Germany), 100 µM acetosyringone (BioFroxx, 2279GR001, Germany)]. The pellet was then resuspended in infiltration buffer and adjusted to an OD 600 of 0.6. Equal volumes of GV3101 strains carrying the respective constructs were mixed and incubate in the dark for 2 h prior to infiltration. Infiltration was performed on the abaxial side of leaves using a 1-mL needleless syringe. Plants infiltrated with only the RTF construct or PC1300S-35S::BSCL1_wt served as negative controls, while those infiltrated with two constructs for GFP expression. PC1300S-35S::GFP alone was used as a positive control. Three days post-infiltration (dpi), infiltrated leaves were harvested, mounted in distilled water on glass slides, and imaged using a Leica SP8 confocal laser scanning microscope (Leica Microsystems, Germany). GFP fluorescence was excited at 488 nm and detected between 500–530 nm. Chlorophyll autofluorescence was collected at 650–700 nm to assist with subcellular localization. Yeast three-hybrid (Y3H) assay Y3H was adopted from the protocol developed by Professor Marvin Wickens’ lab at the University of Wisconsin-Madison 72 . The plasmid, p3HR2 for expressing BSCL1 , pIIIA/IRE-MS2 and pAD-IRP severed as positive controls, the yeast strain YBZ1, were kindly provided by Professor Marvin Wickens. The RNA expression plasmid was generated by inserting the 708-nt BSCL1 sequence into p3HR2 between XhoⅠ and XmaⅠ restriction sites. The recombinant plasmid p3HR2- BSCL1 was verified by restriction digestion and sequencing after amplification in E. coli (primers were listed in Supplementary Table 6). The Saccharomyces cerevisiae strain YBZ1 was used for yeast three-hybrid assays. Competent cells were prepared as follows: a single colony was inoculated from a YPDA (Coolaber, PM2011, Beijing, China) plate into 5 mL YPDA broth medium and grown overnight at 30°C with shaking (250 rpm). Cells were diluted to an OD600 of 1.2–1.5, pelleted at 1,000 g for 5 min, and sequentially washed with sterile water and TE/LiAc buffer [1 mL 10 × TE, 1 mL 10 × LiAc (Sigma, L4158, USA), 8 mL sterile water]. The final cell pellet was resuspended in 1× TE/LiAc to obtain competent cells. Transformation was performed by mixing competent cells with 40% PEG4000/TE/LiAc [1 mL 10 × TE, 1 mL 10 × LiAc (Sigma, L4158, USA), 8 mL 50% PEG4000 (Sigma, 95904, USA)], denatured salmon sperm DNA (Thermo Fisher Scientific, 15632011, USA), and p3HR2- BSCL1 , followed by incubation at 30°C for 30 min and heat shock at 42°C for 10 min. After recovery, cells were plated onto synthetic defined (SD) agar medium (Coolaber, PM2040, Beijing, China) lacking uracil (Coolaber, PM2270, Beijing, China) (SD-U) and incubated at 30°C for 2–3 days. Single colonies were verified by plasmid rescue, restriction digestion, and sequencing. Competent cells containing the p3HR2- BSCL1 were used for library transformation. To optimize 3-amino-1,2,4-triazole (3-AT) (Sigma, A8056, USA) concentration and test for autoactivation, yeast strains harboring RNA plasmids were co-transformed with empty pGAD-T7 vector. Transformants were spotted or plated onto SD agar medium (Coolaber, PM2040, Beijing, China) lacking uracil, leucine, and histidine (Coolaber, PM2170, Beijing, China) (SD-U-L-H), supplemented with increasing concentrations of 3-AT (0–100 mM). Growth was monitored after 3–5 days at 30°C, and the minimal concentration of 3-AT that suppressed background growth was selected for subsequent screening. Competent cells containing the p3HR2- BSCL1 were prepared as described above. For library transformation, cells were mixed with 40% PEG4000/TE/LiAc (1 mL 10 × TE, 1 mL 10 × LiAc, 8 mL 50% PEG4000), denatured salmon sperm DNA (Thermo Fisher Scientific, 15632011, USA), and 20 µg of cDNA library, kindly provided by Professor Yongjun Lin at Huazhong Agricultural University. After incubation at 30°C for 30 min with shaking, DMSO was added, and cells were heat shocked at 42°C for 15 min. Transformants were plated on SD-U-L-H plates supplemented with the optimized concentration of 3-AT. After 5–7 days at 30°C, colonies growing on selective plates were isolated as HIS + candidates. LacZ reporter assay and identificaiton of putative interactors The methods for LacZ screening and interaction validation were adapted from Bernstein et al. 73 . Briefly, HIS + colonies were replated onto SD agar medium (Coolaber, PM2040, Beijing, China) lacking uracil and leucine (Coolaber, PM2290, Beijing, China) (SD-U-L), overlaid with nitrocellulose membranes. After incubation at 30°C for 2–3 days, the membranes were briefly frozen in liquid nitrogen and then incubated at 37°C in buffer (Z-buffer supplemented with 65 µL β-mercaptoethanol and 200 µL of 25 mg/mL X-β-gal per 10 mL). Colonies that developed a blue color were scored as LacZ-positive. Plasmids were extracted from LacZ-positive yeast colonies, and the inserted sequences were amplified by PCR, verified by Sanger sequencing, and identified through BLAST searches against the NCBI database. To validate BSCL1 –protein interactions, full-length cDNAs of candidate genes were cloned into pGAD-T7 and co-transformed into YBZ1 cells together with either p3HR2-BSCL1 or the empty p3HR2 vector (negative control). The pIIIA/IRE-MS2 and pAD-IRP plasmids served as positive controls. Transformants were plated onto SD-U-L-H medium supplemented with the optimized concentration of 3-AT. Growth observed with the BSCL1 plasmid but absent with the empty vector was taken as evidence of a specific RNA–protein interaction. Colonies that turned blue on SD-U-L medium in the X-β-gal reporter assay were considered to represent positive interactions. Co-localization of BSCL1 with candidate target proteins The full-length CDSs of HIRIP3 and H3.3 were amplified by RT-PCR from rice cDNA and cloned into the pB7RWG between SpeⅠ and XhoⅠ restriction sites to generate C-terminal fusions with mCherry. Constructs were verified by Sanger sequencing and introduced into A. tumefaciens GV3101 (pSoup) (Weidi, AC1002, Shanghai, China) as described above. Agrobacterium strains carrying pB7RWG-HIRIP3-mCherry or pB7RWG-H3.3-mCherry were co-infiltrated with PC1300S-35S::BSCL1_wt and the RNA-switch- BSCL1 –RTF construct into N. benthamiana leaves, following the same infiltration protocol as above. Each protein was tested in independent infiltration assays. At 3 dpi, infiltrated leaves were examined by confocal microscopy Leica SP8 (Leica Microsystems, Germany). GFP signals ( BSCL1 detection via RNA-switch- BSCL1 –RTF system) were excited at 488 nm (emission 500–530 nm), while mCherry-tagged HIRIP3 and H3.3 were excited at 561 nm (emission 580–610 nm). Chlorophyll autofluorescence was collected at 650–700 nm to assist with subcellular localization. Images were collected in sequential scanning mode to prevent bleed-through between channels. Representative images were captured and processed using identical microscope settings for all samples. dsRNA synthesis, microinjection, and gene expression analysis in BPH Double-stranded RNA (dsRNA) targeting BSCL1 was synthesized by PCR amplification of two gene fragments ( dsBSCL1 -F1/ dsBSCL1 -R1: 315 bp, dsBSCL1 -F2/ dsBSCL1 -R2: 260 bp) with primers containing T7 RNA polymerase promoter sequences at both 5’ ends (Supplementary Table 6). The PCR products were used as templates for in vitro transcription with the T7 High Yield RNA Transcription Kit (Vazyme, TR101-01, Nanjing, China) following the manufacturer’s instructions. Synthesized dsRNAs were diluted to the appropriate concentration for microinjection. Third- to fourth-instar BPH nymphs were anesthetized with carbon dioxide for 20 s and injected with 200 ng dsRNA into the mesothorax using a Nanoliter 2010 microinjector (World Precision Instruments, USA). BPH injected with dsGFP served as the negative control. Each treatment was performed in three biological replicates. For RNAi efficiency assessment, ten nymphs per replicate were collected at 3 days post-injection. The remaining injected nymphs were maintained on three-leaf stage rice seedlings for subsequent experiments. Total RNA was isolated from microinjected nymphs or insects at different developmental stages using TRIzol reagent (Invitrogen, 15596018CN, USA). First-strand cDNA synthesis was performed with the PrimeScript RT Reagent Kit with gDNA Eraser (Takara, RR047A, Japan) following the manufacturer’s instructions. qRT-PCR was conducted as described above using gene-specific primers, with N. lugens Actin ( NlActin ) serving as the endogenous control (primers were listed in Supplementary Table 6). Relative expression levels were calculated using the 2^ –ΔΔCt method. BPH survival and fecundity assays To evaluate the effects of dsRNA-mediated gene silencing on BPH survival, approximately 100 third- to fourth-instar nymphs treated with gene-specific or control dsRNAs were transferred into 500-mL glass beakers containing fresh three-leaf stage TN1 seedlings 74 . Each beaker was treated as one biological replicate, with three replicates per treatment. Rice seedlings were replaced every 4 days to ensure a continuous food supply and minimize confounding effects from plant senescence. Beakers were maintained in a controlled growth chamber at 27 ± 1°C, 70 ± 5% relative humidity, and a 14:10 h light:dark photoperiod. The number of surviving nymphs was recorded every 24 h, and mortality was calculated relative to the initial number of insects. Monitoring continued until most individuals reached adulthood or exhibited mortality consistent with RNAi effects. Fecundity was assessed according to the methods described by Wen et al. 75 and Liu et al. 76 , with minor modifications. For BPH fecundity assays following microinjection, newly emerged dsRNA-treated adults (≤ 24 h old) were paired in glass tube (3 cm diameter, 25 cm high) supplied with five three-leaf stage TN1 rice seedlings for feeding and oviposition. Adults were transferred to a new glass tube with fresh rice seedlings every 4 days until all of the adults were dead. For fecundity assays on different rice lines, each rice plant was enclosed with a perforated plastic lid to allow plant growth and covered with an inverted transparent plastic cup (7.5 cm diameter) to confine insects. Five newly hatched nymphs (≤ 24 h old) were placed into each plastic cup containing a single rice plant. Upon adult emergence, one male–female pair was retained per cup for oviposition and removed after 10 days. Newly hatched nymphs were counted and removed daily until no further hatching occurred. A minimum of 15 biological replicates were performed for each dsRNA treatment or rice line to ensure robust statistical analysis. Honeydew secretion assay Honeydew secretion was measured following the method described by Guo et al. 32 . As described in fecundity assays, each rice plant was covered with a perforated plastic lid that allowed the plant to grow through. A 9-cm diameter filter paper was placed on the lid and covered with an inverted transparent plastic cup (7.5 cm diameter) to confine the insects. For assays after microinjection, two dsRNA-treated 5th instar nymphs were introduced into each cup and allowed to feed for 48 h, after which filter papers were collected. At least 15 biological replicates were performed per treatment. For assays comparing BPH feeding on different rice lines, five 5th instar nymphs were confined per cup and allowed to feed for 48 h, with at least 20 replicates per rice line. Collected filter papers were soaked in 0.1% (w/v) ninhydrin in acetone, dried in an oven at 60°C for 30 min, and honeydew spots visualized as violet or purple stains due to their amino acid content. Images were captured using a digital camera, and honeydew spot areas were quantified using ImageJ software. Dual-luciferase reporter assay in N. benthamiana Dual-luciferase reporter assays were performed as previously described 77 . The 1989-bp promoter region of bHLH6 was amplified from rice genomic DNA by PCR and cloned upstream of the firefly luciferase (LUC) coding sequence in the pGreenⅡ 0800-LUC vector, generating the Pro bHLH6 ::LUC reporter construct. In this vector, renilla luciferase (REN) driven by the CaMV 35S promoter served as an internal control for normalization of LUC activity. For effector constructs, the full-length sequence of BSCL1_wt , the full-length coding sequences (CDSs) of HIRIP3 and H3.3 , as well as a truncated GFP sequence (negative control for BSCL1 _wt), were amplified by PCR and cloned separately into pGreenⅡ 62-SK under the control of the CaMV 35S promoter. Primers used for all constructs are listed in SI Appendix , Table S6. All plasmids were verified by Sanger sequencing using specific primers and introduced into A. tumefaciens GV3101 (pSoup) (Weidi, AC1002, Shanghai, China). Agrobacterium strains carrying the reporter and effector constructs were cultured overnight in LB medium containing appropriate antibiotics at 28°C with shaking. Cells were harvested by centrifugation, washed, and resuspended in infiltration buffer [10 mM MgCl 2 , 10 mM MES pH 5.6 (BioFroxx, 1086GR500, Germany), 100 µM acetosyringone (BioFroxx, 2279GR001, Germany)] to an OD 600 of 0.6. For co-infiltration, equal volumes of Agrobacterium cultures carrying reporter and effector constructs were mixed. Fully expanded leaves of 4-week-old N. benthamiana plants were infiltrated on the abaxial side using a 1-mL needleless syringe. Each construct combination was infiltrated into at least three independent leaves from different plants as biological replicates. Infiltrated plants were maintained under standard growth conditions (22–25°C, 14 h light/10 h dark) for 3 days to allow transient expression. Firefly (LUC) and renilla (REN) luciferase signals were measured using the Dual-Luciferase Reporter Assay Kit (Vazyme, DL101-01, Nanjing, China) according to the manufacturer’s instructions. Leaf discs (approximately 1.0 cm diameter) were collected from infiltrated zones, homogenized in passive lysis buffer, and luminescence was measured with a multimode reader Spark (TECAN, Zurich, Switzerland). Relative LUC activity was calculated as the ratio of LUC:REN signals for each sample. For live imaging, D-Luciferin potassium salt (1 mM, Yeasen, 40902ES01, Shanghai, China) was evenly applied to the abaxial surface of infiltrated leaves. Luminescence was visualized using the NightSHADE L985 live imaging system (Berthold, Germany), and images were captured under standardized exposure conditions. Data analysis Statistical analysis was performed with SPSS 20.0 (IBM, Armonk City, NY, USA). Paired Student’s t test or one-way ANOVA test followed by Tukey’s multiple comparisons test was used to analyze the results of survival rate, honeydew measurement, fecundity measurement, and qRT-PCR. Correlation analysis and Fisher’s Exact test were performed in R. P < 0.05 was considered as statistically significant. All results are presented as means ± SE. Declarations Reporting summary Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article. Data availability The raw data used to generate all figures in this study are available in the Supplementary Information/Source Data file. Transcriptomics data have been deposited in the SRA database under project numbers PRJNA1197768, PRJNA1314766, and PRJNA1330246. Source data are provided as a Source Data file. Source data are provided with this paper. Competing interests The authors declare no competing interests. Author contributions Y.C. and H.H. conceived and designed the study, supervised the project, and finalized the manuscript. D.W. carried out the majority of the experimental work. S.J. and Z.Q. contributed to the experimental setup and bioassays. Bioinformatic analyses were conducted jointly by Y.C. and D.W. C.L., J.W., R.H., and Y.Z. contributed to the performed research. Y.H. and W.M. contributed to analyzed data and experiment design. The manuscript was drafted by Y.C. and D.W., with all authors contributing to its revision. All authors reviewed the manuscript, provided feedback, and approved the final version. Acknowledgments This project is funded by the National Key Research and Development Program of China (project No. 2024YFD1400700 to H.H.), the Hubei Grain Yield Improvement Program (project No. 2662025YJ017 to H.H.), the National Key Research and Development Program of China (project No. 2023YFF1000703 to Y.C.), the National Natural Science Foundation of China (project No. 32172392 to Y.C.), the Hubei Hongshan Laboratory (project No. 2022hszd026 to Y.C.), the Startup Foundation for Advanced Talents at HZAU to Y.C., the Fundamental Research Funds for the Central Universities (Program No. 2022ZKPY003 to Y.C.), and the Wuhan Yingcai Talent Program to Y.C. 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Curr Biol 35:2614–2629 Additional Declarations There is NO Competing Interest. Supplementary Files Supplementarydata1.xlsx dataset 1 Supplementarydata2.xlsx dataset 2 Supplementarydata3.xlsx dataset 3 Supplementarydata4.xlsx dataset 4 Supplementarydata5.xlsx dataset 5 Supplementarydata6.xlsx dataset 6 SupplementaryInformation.docx Insect-derived long non-coding RNAs function as epigenetic effectors to reprogram plant immunity Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-8297537","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":558622663,"identity":"69185ecb-0871-46cb-acad-ee9d45d1940d","order_by":0,"name":"Yazhou 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07:31:19","extension":"xml","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":189333,"visible":true,"origin":"","legend":"","description":"","filename":"NCOMMS250990060structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8297537/v1/e98fa496a2f4be3a30208f78.xml"},{"id":98042411,"identity":"d65630ce-115b-4d1f-abd3-371a58c5fd7a","added_by":"auto","created_at":"2025-12-12 07:31:20","extension":"html","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":209447,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8297537/v1/ac68b440fccae08dbd07eecf.html"},{"id":98042380,"identity":"2b831215-b60e-47e0-8255-00ad9a790f87","added_by":"auto","created_at":"2025-12-12 07:31:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":534892,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBPH-delivered lncRNAs systematically migrate in rice. \u003c/strong\u003e(A) Schematic overview of the feeding experiment. FS, the leaf sheath segment directly exposed to BPH feeding. (B) Venn diagram showing the overlap of lncRNAs identified in FS, DE in response to RHT, and expressed in BPH salivary glands. (C) Heatmap of \u003cem\u003eBSCLs \u003c/em\u003etranscript abundance (TPM values). The bar plot shows the sequencing reads of \u003cem\u003eBSCLs\u003c/em\u003edetected in FS. (D) Migration of \u003cem\u003eBSCL1\u003c/em\u003e, \u003cem\u003eBSCL2\u003c/em\u003e, and \u003cem\u003eBSCL3\u003c/em\u003e from the outer to inner layers of the FS. A schematic of inner and outer layers is shown on the left. (E) Migration of \u003cem\u003eBSCL1\u003c/em\u003e, \u003cem\u003eBSCL2\u003c/em\u003e, and \u003cem\u003eBSCL3\u003c/em\u003e from FS to the UFS, DFS regions of the sheath, and the leaf blade. A schematic of FS, UFS, and DFS is shown on the left. Box plots indicate the relative abundance of \u003cem\u003eBSCL1\u003c/em\u003e, \u003cem\u003eBSCL2\u003c/em\u003e, and \u003cem\u003eBSCL3\u003c/em\u003e at different sampling sites, determined by qRT-PCR. (F) Migration of \u003cem\u003eBSCL1\u003c/em\u003e, \u003cem\u003eBSCL2\u003c/em\u003e, and \u003cem\u003eBSCL3\u003c/em\u003e to the rice roots. A schematic of leaf blade, FS, DFS and root is shown on the left. Box plots indicate the relative abundance of \u003cem\u003eBSCL1\u003c/em\u003e, \u003cem\u003eBSCL2\u003c/em\u003e, and \u003cem\u003eBSCL3\u003c/em\u003e at different sampling sites, determined by qRT-PCR. Numbers under the boxpot show the detection ratio of \u003cem\u003eBSCL1, BSCL2\u003c/em\u003e and \u003cem\u003eBSCL3 \u003c/em\u003eat each site. Different lowercase letters indicate statistically significant differences at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 according to one-way ANOVA followed by Tukey’s multiple comparisons test.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8297537/v1/066858348964158542fed8b0.png"},{"id":98425597,"identity":"142ff2b5-09aa-43e7-a8ed-eee9f2fedc29","added_by":"auto","created_at":"2025-12-17 16:34:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":450628,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eBSCL\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003es persist in rice plants whereas BPH mRNAs are rapidly degraded. \u003c/strong\u003e(A) BPH mRNAs were translocated into the FS of rice plants. (B) Schematic of sampling strategy showing collection at different time points post-BPH infestation. (C, D) The relative abundance of \u003cem\u003emRNA-Nlug010593\u003c/em\u003e and \u003cem\u003emRNA-Nlug002054\u003c/em\u003etranscripts declined rapidly in both the outer (C) and inner (D) layers of UFS, FS, and DFS across time points. (E, F) In contrast, \u003cem\u003eBSCL1\u003c/em\u003e and \u003cem\u003eBSCL2 \u003c/em\u003etranscripts persisted for extended periods in both the outer (E) and inner (F) layers of UFS, FS, and DFS. A schematic of the inner and outer layers is provided in Fig. 2D. Data are shown as mean ± SE (n = 4 biological replicates).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8297537/v1/a5d748754f3eb93504c1afb3.png"},{"id":98042383,"identity":"21136559-f8f1-4fae-921a-28ad1b1d7319","added_by":"auto","created_at":"2025-12-12 07:31:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":491674,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVisualization of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBSCL1 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003elocalization and movement.\u003c/strong\u003e (A) Schematic of the RNA-switch–RTF reporter system. The red sequence in RNA-switch represents the probe for \u003cem\u003eBSCL1\u003c/em\u003e. In the absence of target RNA, the RNA-switch remains inactive due to base pairing between the linkers and their complementary sequences in the probe, leading to GFP degradation via 26S proteasome. Upon target RNA binding, the probe hybridizes with the target, triggering the switch to an unfolding active state, recruiting in GFP accumulation at the RNA site (For details, please refer to the Materials and Methods section). (B) Schematic of infiltration injection site in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. (C) Visualization of \u003cem\u003eBSCL1\u003c/em\u003e localization and movement in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves using the RNA-switch RTF system. The infiltration site was injected with either a mixture of the \u003cem\u003eBSCL1\u003c/em\u003e expression construct and the RNA-switch–RTF construct, or with the RNA-switch–RTF construct alone, while the adjacent site was treated with the RNA-switch–RTF construct accordingly. (D) GFP expressed alone is unable to move in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. None indicated no construct infiltrated.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8297537/v1/878f8ceaa5808a867d0c6ca6.png"},{"id":98427217,"identity":"9c385bfa-bc3f-4383-9620-ed9d3da50756","added_by":"auto","created_at":"2025-12-17 16:39:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":580889,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eBSCL1 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003epromotes BPH colonization by suppressing rice defense responses. \u003c/strong\u003e(A) Silencing of \u003cem\u003eBSCL1\u003c/em\u003eby dsRNA injection. \u003cem\u003edsGFP\u003c/em\u003e served as a negative control. Data are means ± SE from three independent biological replicates. (B) Survival rates were unaffected by dsRNA treatment (90–100 insects per replicate, three replicates per treatment). (C, D) Honeydew excretion (C) and fecundity (D) were reduced after \u003cem\u003eBSCL1 \u003c/em\u003esilencing (n = 15 and n = 12–28 biological replicates, respectively). (E) Schematic of transgenic rice construction. (F, G) Honeydew excretion (F) and fecundity (G) were increased in BPH feeding on rice stably expressing \u003cem\u003eBSCL1_wt\u003c/em\u003e or \u003cem\u003eBSCL1_mut\u003c/em\u003e (n = 20–24 and n = 15–20 biological replicates, respectively). (H, I) Transgenic expression of \u003cem\u003eBSCL1_wt\u003c/em\u003e or \u003cem\u003eBSCL1_mut\u003c/em\u003e increased rice susceptibility to \u003cem\u003eM. oryzae\u003c/em\u003e. (H) Disease symptoms caused by \u003cem\u003eM. oryzae\u003c/em\u003e strain Guy11 at 7 dpi. (I) Hypersensitive response proportion of each rice line in (H). Stacked bars are color-coded to show the proportions (in percentage) of each hypersensitive response scale (0–5) out of total infiltrated leaves scored (For details, please refer to the Materials and Methods section). Total of 40 leaves are scored for each stacked column. Statistical significance was determined by paired Student’s \u003cem\u003et \u003c/em\u003etest: ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ns = not significant.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8297537/v1/5470ef0c724eaf59c17fc65a.png"},{"id":98425776,"identity":"ae5c3693-51c0-4ed9-8718-8a8bc2823e3d","added_by":"auto","created_at":"2025-12-17 16:35:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":842429,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eBSCL1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-induced DE genes are enriched for defense-related TFs\u003c/strong\u003e. (A) Expression trends of DE genes grouped into eight clusters. CK, uninfested plants; BPH, plants infested by BPH. The colors indicate the membership belonging to the clusters. Red, yellowish, and greenish rank from high, medium, and low. (B) Enrichment of TFs across clusters. Bar plots on the right and top indicate the total number of TFs per family and per cluster, respectively. Red marker bars indicate the highest values in each plots. (C, D) Heatmaps of TPM for enriched \u003cem\u003eWRKY\u003c/em\u003e genes in clusters 1 and 7 (C) and \u003cem\u003ebHLH\u003c/em\u003e gene families in clusters 3 and 6 (D). Genes marked with red dots have been previously reported to be associated with rice defense against BPH [\u003cem\u003eWRKY72\u003c/em\u003e\u003csup\u003e42\u003c/sup\u003e, \u003cem\u003eWRKY24\u003c/em\u003e\u003csup\u003e43\u003c/sup\u003e, \u003cem\u003eWRKY71\u003c/em\u003e\u003csup\u003e44\u003c/sup\u003e, \u003cem\u003eWRKY45\u003c/em\u003e\u003csup\u003e45\u003c/sup\u003e, \u003cem\u003ebHLH96\u003c/em\u003e\u003csup\u003e25\u003c/sup\u003e, \u003cem\u003ebHLH6\u003c/em\u003e\u003csup\u003e26\u003c/sup\u003e].\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8297537/v1/67fb5d87f5caf00eee18cb49.png"},{"id":98427170,"identity":"ac0d1c21-875d-4852-9bfc-02a67be00556","added_by":"auto","created_at":"2025-12-17 16:39:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":687601,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eBSCL1 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eevicts the histone complex from the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebHLH6\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e promoter and represses its transcription.\u003c/strong\u003e (A) Yeast three-hybrid screening identified H3.3 and HIRIP3 as \u003cem\u003eBSCL1\u003c/em\u003e-interacting proteins. MS2 sequences fused to \u003cem\u003eBSCL1\u003c/em\u003eserved as bait, and a rice cDNA library fused to the Gal4 activation domain served as prey. Interaction activated reporter genes (\u003cem\u003eHIS3\u003c/em\u003e and \u003cem\u003elacZ\u003c/em\u003e). (B) Co-localization of \u003cem\u003eBSCL1\u003c/em\u003e with H3.3 and HIRIP3 in the nucleus and cytoplasm of \u003cem\u003eN. benthamiana\u003c/em\u003e epidermal cells. \u003cem\u003eBSCL1 \u003c/em\u003elocalization was visualized with the RNA-switch–RTF system, and H3.3/HIRIP3 were fused to mCherry. (C) Expression of H3.3 or HIRIP3 alone did not affect luciferase activity driven by the \u003cem\u003ebHLH6\u003c/em\u003e promoter. (D–F) Co-expression of \u003cem\u003eBSCL1 \u003c/em\u003ewith H3.3 (D) or HIRIP3 (E), or both (F) suppressed \u003cem\u003ebHLH6\u003c/em\u003e promoter activity. Boxplots show luciferase activity (n = 12). Bioluminescence images of representative treatments are shown below. Different lowercase letters indicate significant differences (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, one-way ANOVA with Tukey’s test). (G) Proposed model of \u003cem\u003eBSCL1 \u003c/em\u003efunction. In uninfested plants, the histone complex regulates \u003cem\u003ebHLH6\u003c/em\u003e transcription, contributing to rice resistance to BPH. During infestation, \u003cem\u003eBSCL1\u003c/em\u003eis secreted into rice, where it disrupts histone–DNA interactions at the \u003cem\u003ebHLH6\u003c/em\u003e promoter, repressing transcription and promoting BPH colonization.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8297537/v1/8a602b53eb4e59343785dd82.png"},{"id":101751436,"identity":"a47af726-4128-4666-9f00-67c3379134ce","added_by":"auto","created_at":"2026-02-03 10:20:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5178043,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8297537/v1/f4595e4f-17bb-45a8-93b5-29c51f7f7bf7.pdf"},{"id":98042386,"identity":"1c69c270-96ef-4af9-9f29-205354ed8d5f","added_by":"auto","created_at":"2025-12-12 07:31:19","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":253661,"visible":true,"origin":"","legend":"dataset 1","description":"","filename":"Supplementarydata1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8297537/v1/590db0d44e8c9b3a7e38841b.xlsx"},{"id":98042387,"identity":"8e8d12a0-824b-4a33-8221-9316055a25fc","added_by":"auto","created_at":"2025-12-12 07:31:19","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":11913,"visible":true,"origin":"","legend":"dataset 2","description":"","filename":"Supplementarydata2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8297537/v1/0da442ca3dbace47e60b8d6a.xlsx"},{"id":98042389,"identity":"a720b01c-5071-48d9-9c26-3d415774d515","added_by":"auto","created_at":"2025-12-12 07:31:19","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":32154,"visible":true,"origin":"","legend":"dataset 3","description":"","filename":"Supplementarydata3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8297537/v1/ac3c63f89bf55c097f1511d4.xlsx"},{"id":98426964,"identity":"5b3d9ddd-220f-4652-b627-31202c8937c7","added_by":"auto","created_at":"2025-12-17 16:39:07","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1771730,"visible":true,"origin":"","legend":"\u003cp\u003edataset 4\u003c/p\u003e","description":"","filename":"Supplementarydata4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8297537/v1/cd3c412636e8861b7e09d0f1.xlsx"},{"id":98425702,"identity":"b218f400-da00-41c6-a7d5-9ed873433d3c","added_by":"auto","created_at":"2025-12-17 16:35:06","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":577550,"visible":true,"origin":"","legend":"\u003cp\u003edataset 5\u003c/p\u003e","description":"","filename":"Supplementarydata5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8297537/v1/d961fb4b191156ecabb43429.xlsx"},{"id":98427965,"identity":"b3e84428-d50b-4276-880e-bac0f4374a8d","added_by":"auto","created_at":"2025-12-17 16:41:26","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":13577,"visible":true,"origin":"","legend":"\u003cp\u003edataset 6\u003c/p\u003e","description":"","filename":"Supplementarydata6.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8297537/v1/e7441b42940bc609d054b365.xlsx"},{"id":98427446,"identity":"7ed43425-5bf6-489b-87df-ea80adee1224","added_by":"auto","created_at":"2025-12-17 16:40:26","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":9108723,"visible":true,"origin":"","legend":"Insect-derived long non-coding RNAs function as epigenetic effectors to reprogram plant immunity","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-8297537/v1/c819f4b00b9d00cc92638d72.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Insect-derived long non-coding RNAs function as epigenetic effectors to reprogram plant immunity","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCross-kingdom RNAs are RNA molecules of an organism that traffic and function in recipient organisms that belong to different biological kingdoms. Among these, cross-kingdom RNA interference (RNAi) phenomena that involve small RNAs (sRNAs) have been widely observed across various parasite-host interactions\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Those sRNAs traffic across the organisms and influence gene expression in the recipients, which reveals the critical roles of cross-kingdom RNAs in these interactions.\u003c/p\u003e \u003cp\u003eIn contrast, far less attention has been given to the functions of long RNA species such as mRNAs and long non-coding RNAs (lncRNAs) in recipient organisms, although they have been found to be intensively trafficked during parasite-host interactions. For example, plant phloem mRNAs have been found to traffic bidirectionally between parasite dodders and various host plants\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Arabidopsis mRNAs are delivered into the fungus and then translated by the fungal cells, leading to the reduction of fungal infection\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eCryptosporidium parvum\u003c/em\u003e lncRNAs were selectively delivered into the nuclei of human intestinal epithelial cells during infection\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Aphid lncRNAs are translocated into divergent host plants and systematically migrate in the plants and promote aphid colonization\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. These studies suggest that long RNAs play essential roles in mediating parasite-host interactions; however, their function in the hosts remains largely unexplored.\u003c/p\u003e \u003cp\u003eSap-feeding insects such as aphids and planthoppers are excellent models for studying cross-kingdom RNAs. These insects suck the contents from the xylem and/or phloem tissues, the main components of plant vascular tissues\u003csup\u003e\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Watery saliva secreted by insect salivary glands is injected into plant vascular tissues, where salivary molecules, including various RNAs, enter and traffic in the plants\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Moreover, these insects can be easily confined to specific areas of the plant, leaving the rest of the plant intact and uncontaminated, enabling tracking the migration of insect RNAs in the plant by various approaches such as qRT-PCR\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Additionally, insect-derived RNAs, as foreign RNA species, exhibit low sequence similarity to plant RNAs, making them highly distinguishable and ideal for detection using next-generation sequencing approaches\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHere, we used the brown planthopper (\u003cem\u003eNilaparvata lugens\u003c/em\u003e St\u0026aring;l, BPH), a major rice pest worldwide\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, to investigate the translocation of insect-derived RNAs into host plants. Our study identified both lncRNAs and mRNAs delivered into rice, with particular focus on salivary gland\u0026ndash;derived lncRNAs, termed \u003cem\u003eBSCL\u003c/em\u003es (\u003cem\u003eBPH Salivary gland Cross-kingdom LncRNA\u003c/em\u003es). These \u003cem\u003eBSCL\u003c/em\u003es were translocated into rice and migrated systemically from the feeding site to distal tissues, including leaves and roots. Compared with BPH mRNAs, \u003cem\u003eBSCLs\u003c/em\u003e exhibited markedly higher stability within rice plants. Among them, mitochondrial-delivered \u003cem\u003eBSCL1\u003c/em\u003e promoted BPH feeding and reproduction by suppressing rice defense responses. \u003cem\u003eBSCL1\u003c/em\u003e displaced histone complexes from the promoters of transcription factor genes, leading to transcriptional repression of transcription factors such as bHLH, central to jasmonic acid (JA) signaling, thereby weakening host resistance to BPH.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIdentification and host-responsive expression of salivary lncRNAs in BPH\u003c/h2\u003e \u003cp\u003eTo annotate lncRNAs in the BPH genome, we assembled strand-specific RNA-seq data from 11 libraries (Supplementary Table\u0026nbsp;1), identifying 49,435 transcripts from 47,350 genes (Supplementary Fig.\u0026nbsp;1A). After filtering, 24,433 putative lncRNAs (22,374 genes) were retained, including 10,268 previously reported and 12,106 newly identified (Supplementary Fig.\u0026nbsp;1A). Comparative analysis across 28 arthropod genomes showed that most lncRNA genes (57.9%, 12,957) are BPH-specific (Supplementary Fig.\u0026nbsp;1B, C), suggesting a recent evolutionary origin.\u003c/p\u003e \u003cp\u003eTo explore potential functions in BPH\u0026ndash;rice interactions, we analyzed lncRNA expression in insects transferred from susceptible TN1 rice to resistant RHT plants. RNA-seq revealed 1,626 down-regulated and 1,291 up-regulated genes (Supplementary Fig.\u0026nbsp;1D, Supplementary data 1), including 932 lncRNAs enriched in salivary gland expression (Supplementary Fig.\u0026nbsp;1E). Notably, 606 differentially expressed lncRNAs were expressed in salivary glands, and most (78.7%, 477) were repressed by resistant plants (Supplementary Fig.\u0026nbsp;1F, G), implicating salivary lncRNAs in host colonization.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBPH salivary gland lncRNAs are translocated into rice plants\u003c/h3\u003e\n\u003cp\u003ePreviously, we reported that aphid salivary gland lncRNAs are translocated into plants, and lncRNA \u003cem\u003eYa1\u003c/em\u003e acted as a virulence factor to promote aphid colonization\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Thus, we investigated whether BPH salivary gland lncRNAs are secreted into rice via feeding. We conducted cage experiments in which 30 male BPHs\u0026mdash;chosen to prevent egg deposition on the plants\u0026mdash;were confined to the leaf sheath (designated as feeding sites, FS) of 45-day-old rice plants for 2 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Controls were counterparts of untreated plants which the sheath was in an empty cage. Caged tissues were carefully washed to remove visible BPH tissues and were subsequently subjected to RNA-seq analysis. Bioinformatically, adaptor-removed RNA-seq reads were aligned to a merged genome file that the concatenated reference genomes of rice TN1\u003csup\u003e18\u003c/sup\u003e and BPH\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e (Supplementary Fig.\u0026nbsp;2A). Expectedly, the vast majority of reads in the replicates of controls and FS were mapped to the merged genome (Supplementary data 2). To gain more confidence, only uniquely aligned reads were used in the following analysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe number of BPH reads in the FS varied largely among the replicates, resulting in different numbers of BPH lncRNAs identified (Supplementary data 2, Supplementary Fig.\u0026nbsp;2B). For instance, 3065 uniquely mapped reads from 951 transcripts were identified from one replicate, while fewer than 547 reads were identified in the other two. Such variation has been observed in other BPH experiments\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. We chose the sample BPH-1-FS for further analysis since the highest number of BPH RNA reads were detected (244 transcripts with reads\u0026thinsp;\u0026ge;\u0026thinsp;4, Supplementary data 3).\u003c/p\u003e \u003cp\u003eAmong these 244 transcripts that are likely to be cross-kingdom RNAs (Supplementary data 3), 11 lncRNAs were expressed in the salivary glands and were also DE between BPH that fed on TN1 and RHT (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, Supplementary data 1), which included 8 down-regulated and 3 up-regulated lncRNA genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). We found that 10 out of 11 BPH lncRNAs were detected in the FS of rice plants in the repeated cage experiments but were absent from the same RNA samples that weren\u0026rsquo;t treated with reverse transcriptase and from the counterparts on the control plants (Supplementary Fig.\u0026nbsp;2C). Therefore, these lncRNAs were named \u003cem\u003eBPH Salivary gland Cross-kingdom LncRNA\u003c/em\u003e (\u003cem\u003eBSCL\u003c/em\u003e). Noticeably, the PCR bands of 7 \u003cem\u003eBSCL\u003c/em\u003es (\u003cem\u003eBSCL4-9\u003c/em\u003e and \u003cem\u003eBSCL11\u003c/em\u003e) were faint unless the PCR products were used as templates for another round of PCR amplification (Supplementary Fig.\u0026nbsp;2C). Three \u003cem\u003eBSCLs\u003c/em\u003e (\u003cem\u003eBSCL1\u003c/em\u003e, \u003cem\u003eBSCL2\u003c/em\u003e, \u003cem\u003eBSCL3\u003c/em\u003e) were detected in more than two tested samples and absent from the controls (Supplementary Fig.\u0026nbsp;2C).\u003c/p\u003e \u003cp\u003eIn conclusion, BPH lncRNAs are translocated into rice plants via feeding, although there is some variation among different individuals.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBPH\u003c/b\u003e \u003cb\u003eBSCL\u003c/b\u003e\u003cb\u003es migrate systemically within rice plants\u003c/b\u003e\u003c/p\u003e \u003cp\u003eDetection of BPH lncRNAs in the FS of rice sheath prompted us to assess whether these RNAs migrate inside rice plants. BPH feeding occurs mainly at the outer layer of rice stem sheaths, thus the FS tissues were dissected into the outer and inner layers (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). The presence of \u003cem\u003eBSCL1\u003c/em\u003e, \u003cem\u003eBSCL2\u003c/em\u003e, and \u003cem\u003eBSCL3\u003c/em\u003e on each layer was examined (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). PCR amplification with specific primers detected three \u003cem\u003eBSCLs\u003c/em\u003e clearly in both layers, suggesting migration of these \u003cem\u003eBSCL\u003c/em\u003es in the rice plants.\u003c/p\u003e \u003cp\u003eIn addition to the FS, the tissues that were about 2 cm above and below the caged sheath, respectively termed up- and down-near feeding sites (UFS and DFS, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), were also harvested. All three \u003cem\u003eBSCLs\u003c/em\u003e were detected in the outer layers of FS and DFS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Except for \u003cem\u003eBSCL2\u003c/em\u003e, both \u003cem\u003eBSCL1\u003c/em\u003e and \u003cem\u003eBSCL3\u003c/em\u003e were also found in the outer layers of UFS. Sequences of PCR products of these three lncRNAs obtained from rice sheath further confirmed the translocation of BPH lncRNAs into the rice plants (Supplementary Fig.\u0026nbsp;3A-C). \u003cem\u003eBSCL1\u003c/em\u003e was also found in the inner layers of UPS, FS, and DFS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), probably because of its relatively higher expression level.\u003c/p\u003e \u003cp\u003e \u003cem\u003eBSCL1\u003c/em\u003e abundance in the outer layers ranked from the DFS, FS, then to UFS, which was similar as that in the inner layers (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). It indicated that migration of \u003cem\u003eBSCL\u003c/em\u003es in the rice plants is likely from top to bottom in most outer sheaths where BPH actually feed on, and from bottom to top in the inner sheaths. The opposite may be achieved through the internodes, where nutrients and minerals are often redistributed throughout the entire plant. Indeed, we observed \u003cem\u003eBSCL1\u003c/em\u003e, \u003cem\u003eBSCL2\u003c/em\u003e, and \u003cem\u003eBSCL3\u003c/em\u003e as being noticeably abundant in the roots that were comparable to that in the feeding sites and less than in the top leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF), when the feeding experiments were repeated with 14-day-old seedlings growing in hydroponics. In all experiments, the \u003cem\u003eBSCL\u003c/em\u003es were not detected in the control plants that were uninfested by BPH (Supplementary Fig.\u0026nbsp;2D, E). It strongly implies that the migration of \u003cem\u003eBSCL\u003c/em\u003es in rice plants is in alignment with the phloem streaming moving from the source to sink tissues.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBPH\u003c/b\u003e \u003cb\u003eBSCLs\u003c/b\u003e \u003cb\u003eare more stable than BPH mRNAs in rice plants\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA substantial number of BPH mRNA reads were also found in the RNA-seq of FS (Supplementary Fig.\u0026nbsp;4A, Supplementary Table\u0026nbsp;2). This prompted us to investigate whether BPH mRNAs also migrated in the rice plants. Out of five selected BPH mRNAs (Supplementary Fig.\u0026nbsp;4A, B), two (\u003cem\u003eNlug010593\u003c/em\u003e and \u003cem\u003eNlug002054\u003c/em\u003e) were found in the FS by PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, Supplementary Fig.\u0026nbsp;4B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHowever, the presence of these mRNAs in rice plants can only be detected by primers targeting the shorter fragments (Supplementary Fig.\u0026nbsp;4C, D), indicating that BPH mRNAs in the rice plants have been fragmented. Fragmented RNAs are often less stable and more prone to degradation\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. To assess the stability of BPH mRNAs and \u003cem\u003eBSCL\u003c/em\u003es in rice plants, we removed BPH from FS after 7 days of feeding and subsequently harvested samples at several time points (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). On the 7th day of feeding (0 days post-BPH removal), \u003cem\u003eBSCL\u003c/em\u003es (\u003cem\u003eBSCL1\u003c/em\u003e and \u003cem\u003eBSCL2\u003c/em\u003e) and BPH mRNAs (\u003cem\u003eNlug010593\u003c/em\u003e and \u003cem\u003eNlug002054\u003c/em\u003e) were found in both the outer and inner layers of FS (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-F). The abundance of \u003cem\u003eBSCL1\u003c/em\u003e and \u003cem\u003eBSCL2\u003c/em\u003e remained relatively higher than that of \u003cem\u003eNlug010593\u003c/em\u003e and \u003cem\u003eNlug002054\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-F). Two days post BPH removal, the abundance of BPH mRNAs in the rice plants declined sharply. In contrast, the abundance of \u003cem\u003eBSCL\u003c/em\u003es in the rice plants remained high, even 8 days after the removal of BPH (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-F), suggesting BPH \u003cem\u003eBSCL\u003c/em\u003es are more stable in rice plants than BPH mRNAs.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBSCL1\u003c/b\u003e \u003cb\u003eis a mitochondrial-derived lncRNA\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the functions of \u003cem\u003eBSCL\u003c/em\u003es, we selected \u003cem\u003eBSCL1\u003c/em\u003e to conduct further experiments since \u003cem\u003eBSCL1\u003c/em\u003e is the most abundant in FS compared to other \u003cem\u003eBSCL\u003c/em\u003es. We determined the full-length \u003cem\u003eBSCL1\u003c/em\u003e sequence (708 nt) from brown planthopper (BPH) using 5\u0026prime; and 3\u0026prime; RACE (Supplementary Fig.\u0026nbsp;5A, B) and validated it by northern blotting (Supplementary Fig.\u0026nbsp;5C). The full-length transcript was detected in FS, NFS, and DFS tissues (Supplementary Fig.\u0026nbsp;5D).\u003c/p\u003e \u003cp\u003eTo determine the genomic origin of \u003cem\u003eBSCL1\u003c/em\u003e, we aligned its full-length sequence to the BPH reference genome. Although the sequence showed strong homology to a locus on Chromosome 9 (position 6540831\u0026ndash;6541538), this nuclear copy contained a single-nucleotide mismatch (e.g., A\u0026rarr;T at position 516) (Supplementary Fig.\u0026nbsp;6), indicating that it might be another source of the actively expressed \u003cem\u003eBSCL1\u003c/em\u003e transcript. To further resolve its origin, we performed a homology search against the NCBI database using the \u003cem\u003eBSCL1\u003c/em\u003e sequence. Strikingly, the reverse complement showed 100% identity to the BPH mitochondrial genome (position 14471\u0026ndash;15178) (Supplementary Fig.\u0026nbsp;6). Notably, \u003cem\u003eBSCL1\u003c/em\u003e is reverse complementary to the mitochondrial 12S rRNA region, suggesting that it is transcribed from the opposite strand relative to the 12S rRNA gene. These results indicate that the Chromosome 9 locus corresponds to a nuclear-encoded mitochondrial sequence (NUMT), a class of mitochondrial DNA insertions typically considered transcriptionally inert\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Given the generally inactive nature of NUMTs, our findings strongly support the mitochondrial genome\u0026mdash;rather than the nuclear NUMT copy\u0026mdash;as the true source of the functional \u003cem\u003eBSCL1\u003c/em\u003e lncRNA.\u003c/p\u003e \u003cp\u003e \u003cb\u003eVisualization of\u003c/b\u003e \u003cb\u003eBSCL1\u003c/b\u003e \u003cb\u003elocalization in planta\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe examined the in-planta migration of \u003cem\u003eBSCL1\u003c/em\u003e using an RNA imaging approach, the RNA switch\u0026ndash;controlled RNA-triggered fluorescence (RNA switch\u0026ndash;RTF) system, which enables real-time visualization of RNA dynamics in living plants\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. In this system, the RNA switch with a probe targeting \u003cem\u003eBSCL1\u003c/em\u003e can toggle between active and inactive states depending on the presence of the target RNA, thereby controlling GFP accumulation or degradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on the full-length sequence, we designed a dumbbell-shaped RNA switch with the probe (Supplementary Fig.\u0026nbsp;5E), which is essential for the RNA switch function. Agroinfiltration of \u003cem\u003eN. benthamiana\u003c/em\u003e leaves with \u003cem\u003e35S::BSCL1\u003c/em\u003e together with the RNA switch\u0026ndash;RTF construct produced GFP signals in both the nucleus and cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C), confirming subcellular localization of \u003cem\u003eBSCL1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). No GFP fluorescence was observed when leaves were infiltrated with either \u003cem\u003eBSCL1\u003c/em\u003e or the RNA switch\u0026ndash;RTF system alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Remarkably, GFP signals were also detected in tissues adjacent to the infiltration sites, where the RNA switch\u0026ndash;RTF system had been introduced but not the \u003cem\u003eBSCL1\u003c/em\u003e plasmid (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), demonstrating systemic movement of \u003cem\u003eBSCL1\u003c/em\u003e. By contrast, infiltration with \u003cem\u003e35S::GFP\u003c/em\u003e produced strong local fluorescence but no signals in neighboring tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eTogether, these results demonstrate that BPH-derived \u003cem\u003eBSCL\u003c/em\u003es are not only localized within plant cells but also migrate systemically from the initial delivery sites to adjacent and distal tissues.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBPH\u003c/b\u003e \u003cb\u003eBSCL1\u003c/b\u003e \u003cb\u003eis a virulence factor that suppresses the plant defenses\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAlthough \u003cem\u003eBSCL1\u003c/em\u003e is derived from the mitochondrial genome, our findings indicated that the functional transcript is exported to the cytoplasm for secretion (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Based on this cytoplasmic localization, we targeted the lncRNA using a canonical, cytoplasm-restricted RNA interference (RNAi) approach, employing two non-overlapping double-stranded RNA (dsRNA) sequences, rather than specialized mitochondrial gene silencing strategies\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. The results showed that we had successfully inhibited the expression levels of \u003cem\u003eBSCL1\u003c/em\u003e in BPH (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The survival rate had no significant difference between \u003cem\u003edsBSCL1\u003c/em\u003e and the \u003cem\u003edsGFP\u003c/em\u003e groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), however, the honeydew production (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, Supplementary Fig.\u0026nbsp;7A) and the total number of eggs per female (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD) were significantly reduced.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further assess the impact of \u003cem\u003eBSCL1\u003c/em\u003e on BPH performance, we generated stable transgenic plants in TN1 genetic background that produced 708-nt of \u003cem\u003eBSCL1\u003c/em\u003e (35S::BSCL1, lines 2 and 3) and 708-nt \u003cem\u003eBSCL1\u003c/em\u003e mutants in which two ATG start sites were mutated to stop codes (35S::BSCL1_mut, lines 8 and 11) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). BPH produces more honeydew (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, Supplementary Fig.\u0026nbsp;7B) and eggs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG) on both 35S::BSCL1 and 35S::BSCL1_mut compared to 35S::GFP and TN1 plants, suggesting \u003cem\u003eBSCL1\u003c/em\u003e in rice promote BPH colonization. These findings suggest that BPH \u003cem\u003eBSCL1\u003c/em\u003e is a virulence factor that plays a critical role in BPH-rice interactions.\u003c/p\u003e \u003cp\u003eTo assess whether plant defense pathways were affected by \u003cem\u003eBSCL1\u003c/em\u003e, we challenged TN1, 35S::GF plants, 35S::BSCL1_wt, and 35S::BSCL1_mut by \u003cem\u003eM. oryzae\u003c/em\u003e. Six days posted the inoculation of \u003cem\u003eM. oryzae\u003c/em\u003e, 35S::BSCL1_wt and 35S::BSCL1_mut plants exhibited a stronger hypersensitive response compared to TN1 and 35S::GFP plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH, I), suggesting that overall rice defense pathways were suppressed by \u003cem\u003eBSCL1\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBSCL1\u003c/b\u003e \u003cb\u003erepresses the expression of defense-related genes in rice\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo determine the host processes influenced by \u003cem\u003eBSCL1\u003c/em\u003e, we performed RNA-seq on rice plants (TN1, 35S::GFP, 35S::BSCL1_wt, and 35S::BSCL1_mut) with or without BPH feeding for 24 h. Principal component analysis (PCA) of transcriptomes revealed clear separation of sample groups (Supplementary Fig.\u0026nbsp;8A). Relative to non-feeding controls, BPH feeding altered the expression of 6,710 rice genes (Supplementary Fig.\u0026nbsp;8B, Supplementary data 4).\u003c/p\u003e \u003cp\u003eWe next examined whether \u003cem\u003eBSCL1\u003c/em\u003e modulates these plant responses. Coexpression analysis of differentially expressed (DE) genes grouped 6,404 genes into eight clusters based on expression patterns (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, Supplementary data 5). TPM heatmaps confirmed these clusters (Supplementary Fig.\u0026nbsp;9). Expression profiles of 35S::BSCL1_mut plants closely resembled those of 35S::BSCL1_wt, indicating that \u003cem\u003eBSCL1\u003c/em\u003e functions as an RNA rather than through a peptide product (Supplementary Fig.\u0026nbsp;9).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFour clusters (clusters 2, 3, 5, and 8) showed similar expression in 35S::BSCL1_wt, TN1, and 35S::GFP plants, suggesting that these groups are largely unrelated to \u003cem\u003eBSCL1\u003c/em\u003e activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, Supplementary Fig.\u0026nbsp;9B, C, E, H). Cluster 4 genes were strongly induced in 35S::BSCL1_wt plants but suppressed after BPH feeding, a pattern absent in controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, Supplementary Fig.\u0026nbsp;9D). By contrast, clusters 1, 6, and 7 exhibited distinct expression changes in 35S::BSCL1_wt plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, Supplementary Fig.\u0026nbsp;9A, F, G). In clusters 1 and 7, many genes were strongly upregulated in TN1 and 35S::GFP plants after BPH feeding, but this induction was markedly repressed in 35S::BSCL1_wt plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, Supplementary Fig.\u0026nbsp;9A, G). These clusters were significantly enriched for genes in the jasmonic acid (JA)-mediated defense pathway (Supplementary Fig.\u0026nbsp;10). Consistently, multiple JA pathway genes were repressed in both 35S::BSCL1_wt and 35S::BSCL1_mut compared to controls (Supplementary Fig.\u0026nbsp;11).\u003c/p\u003e \u003cp\u003eCluster 6 was of particular interest because its genes were downregulated upon BPH feeding and remained further suppressed in 35S::BSCL1_wt plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). Notably, cluster 6 contained the highest number of transcription factor (TF) genes among all clusters (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Enriched families included bHLH, MYB-related, bZIP, Dof, Nin-like, ERF, G2-like, and TALE, with bHLH TFs most abundant (12/38; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Several members of this family\u0026mdash;including bHLH96, bHLH6, and PTF1\u0026mdash;are known regulators of JA- and SA-mediated defenses. Rice bHLH96 and bHLH6, in particular, have been implicated in resistance to BPH\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Their repression in both control plants and 35S::BSCL1_wt upon BPH feeding suggests that \u003cem\u003eBSCL1\u003c/em\u003e interferes with transcriptional regulation of key defense pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D, Supplementary Table\u0026nbsp;3).\u003c/p\u003e \u003cp\u003e \u003cb\u003eBSCL1\u003c/b\u003e \u003cb\u003etargets the histone complex to repress transcription factor promoters\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn plants, lncRNAs have been reported to act through protein interactions\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. To identify \u003cem\u003eBSCL1\u003c/em\u003e-binding partners in rice, we performed yeast three-hybrid (Y3H) screening using a rice cDNA library. A total of 197 yeast colonies grew on selective media, of which 104 exhibited strong X-β-gal activity (Supplementary Fig.\u0026nbsp;12, Supplementary data 6). These corresponded to 31 rice proteins, including enzymes, transcription factors, and components of the histone complex (Supplementary Table\u0026nbsp;4). Notably, vacuolar protein sorting-associated protein 2 homolog 2 (VPS), histone H3.3, transcription factor RF2a-like (RF2), ribonuclease J isoform X1, HIRA-interacting protein 3 (HIRIP3), and T-complex protein 1 subunit gamma were among the frequently recovered candidates.\u003c/p\u003e \u003cp\u003eGiven the central role of chromatin regulation in transcriptional control, we focused on histone H3.3 and HIRIP3. Interactions of \u003cem\u003eBSCL1\u003c/em\u003e with both proteins were validated in repeated Y3H assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). RNA-switch-RTF revealed that \u003cem\u003eBSCL1\u003c/em\u003e was colocalized with HIRIP3 in the nucleus and cytoplasm, with H3.3 in the nucleus, where H3.3 was mainly expressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). These results suggest that \u003cem\u003eBSCL1\u003c/em\u003e associates with the HIRA histone chaperone complex, which normally deposits H3.3 into chromatin to maintain active transcription states.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe next tested whether \u003cem\u003eBSCL1\u003c/em\u003e interferes with H3.3 deposition at promoters of defense-related transcription factors (e.g., bHLH6 in cluster 6). A luciferase (LUC) reporter driven by the bHLH6 promoter was co-expressed with combinations of H3.3, HIRIP3, and \u003cem\u003eBSCL1\u003c/em\u003e in rice. Neither H3.3 nor HIRIP3 alone altered promoter activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), but co-expression with \u003cem\u003eBSCL1\u003c/em\u003e significantly suppressed LUC expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, E). Importantly, simultaneous expression of H3.3 and HIRIP3 did not produce further repression beyond that observed with either factor alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF), indicating that \u003cem\u003eBSCL1\u003c/em\u003e disrupts the function of the H3.3\u0026ndash;HIRIP3 complex at defense gene promoters (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe found that BPH actively translocated salivary gland RNAs into rice plants during feeding. These cross-kingdom RNAs move systemically from feeding sites on the sheath to distant tissues such as inner sheaths, upper leaves, and roots, likely through vascular transport. In rice, BPH mRNAs degraded relatively quickly\u0026mdash;possibly via 5\u0026prime;-end decapping and processing as part of a conserved host defense strategy\u0026mdash;whereas BPH lncRNAs persisted for extended periods, remaining detectable up to eight days after insect removal. Among these lncRNAs, \u003cem\u003eBSCL1\u003c/em\u003e emerged as a highly abundant and functionally important effector. Silencing \u003cem\u003eBSCL1\u003c/em\u003e in BPH reduced feeding and reproduction, while heterologous expression in rice enhanced BPH performance. Moreover, \u003cem\u003eBSCL1\u003c/em\u003e overexpression suppressed JA-induced defense transcription factors and compromised immunity against both BPH and the blast fungus \u003cem\u003eM. oryzae\u003c/em\u003e, suggesting that it broadly disables rice plant defense.\u003c/p\u003e \u003cp\u003eThe discovery that the functional \u003cem\u003eBSCL1\u003c/em\u003e lncRNA originates from the mitochondrial genome introduces a fascinating layer of complexity to cross-kingdom communication. While NUMTs are widespread and typically represent transcriptionally silent relics of mtDNA transfer\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, the active transcription, cytoplasmic export, and subsequent secretion of a mitochondrial-encoded RNA like \u003cem\u003eBSCL1\u003c/em\u003e is highly unusual. This finding strongly suggests a novel, specialized regulatory role for this subset of mitochondrial transcripts, distinct from the canonical nuclear-encoded effector molecules. In insects, mitochondrial transcription is fundamentally linked to essential metabolic processes and acute stress responses\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e; its repurposing here for systemic cross-kingdom defense suppression hints at a highly evolved mechanism where fundamental cellular resources are co-opted for specialized pathogenicity. Further research is warranted to determine if other mitochondrial-derived RNAs are similarly utilized as mobile effectors in this or other insect-plant systems.\u003c/p\u003e \u003cp\u003eTranscriptomic profiling revealed that BPH feeding orchestrates complex reprogramming of rice metabolism and defense. We observed repression of processes associated with cell walls, ion transport, and carbohydrate metabolism, but strong induction of jasmonic acid (JA)-related genes, RNA metabolism, circadian rhythm, and transporter activities (Supplementary Fig.\u0026nbsp;10). These patterns are consistent with previous findings: reinforcement of the cell wall enhances resistance\u003csup\u003e\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e; ion transport underpins defense signaling and metabolite biosynthesis\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e; and carbohydrate metabolism, often manipulated by herbivores, is further exploited by BPH through hijacking SWEET13/14 sugar transporters\u003csup\u003e\u003cspan additionalcitationids=\"CR36 CR37 CR38 CR39\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Despite these suppressions, BPH feeding activates defense signaling, with strong induction of JA pathway genes and transcription factors such as WRKY, NAC, and MYB\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan additionalcitationids=\"CR42 CR43 CR44\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e and tissue-specific JA sectors including the MYC2\u0026ndash;bHLH6 cascade\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Together, these findings highlight the dual nature of the interaction: rice perceives and counters insect attack, while BPH simultaneously manipulates host physiology to promote feeding.\u003c/p\u003e \u003cp\u003eOur discovery of insect-derived lncRNAs reveals a previously unrecognized mechanism: BPH not only manipulates host physiology indirectly but also delivers its own genetic effectors to reprogram rice immunity at transcriptional and epigenetic levels. \u003cem\u003eBSCL1\u003c/em\u003e exemplifies this new class of mobile effectors. It suppresses JA-associated transcription factors such as OsbHLH6\u0026mdash;key regulators of defense\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e\u0026mdash;and interacts with the HIRA histone chaperone complex to block H3.3 deposition at their promoters (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). Similar lncRNA-guided epigenetic mechanisms have been described in other systems\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. These findings also force a reinterpretation of prior observations. The downregulation of defense TFs reported\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e and the apparent negative feedback in JA signaling\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e may partly result from direct suppression by insect lncRNAs. Likewise, sugar transport manipulations by BPH\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e likely occur in a defense-compromised context where RNA effectors such as \u003cem\u003eBSCL1\u003c/em\u003e have already silenced immune hubs. Thus, BPH deploys a multilayered strategy: altering host nutrition, modulating hormone signaling, and injecting RNA-based epigenetic suppressors that dismantle transcriptional control of immunity.\u003c/p\u003e \u003cp\u003eBPH secretes a cocktail of salivary molecules to manipulate rice and promote feeding, including enzymatic proteins\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, effector proteins\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e, and miRNAs\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. In addition, numerous salivary gland\u0026ndash;derived lncRNAs and mRNAs are translocated into rice, potentially trafficking systemically via the phloem. BPH mRNAs are rapidly degraded, consistent with a conserved host defense against foreign RNAs, as reflected by enrichment of RNA metabolism pathways in Cluster 2 upon BPH feeding (Supplementary Fig.\u0026nbsp;10). In contrast, a small subset of RNAs, including \u003cem\u003eBSCL1\u003c/em\u003e, remains stable as full-length transcripts, suggesting structural features that allow evasion of host RNA surveillance. While cross-kingdom transfer of long RNAs has been reported in parasitic plants, fungi, and aphids\u0026mdash;including the aphid lncRNA \u003cem\u003eYa1\u003c/em\u003e shown to function as an effector\u003csup\u003e\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e\u0026mdash;\u003cem\u003eBSCL1\u003c/em\u003e exemplifies these principles and raises key questions: can some insect mRNAs escape host surveillance and be translated in plant cells, as reported for fungal effectors\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e? How do cross-kingdom lncRNAs such as \u003cem\u003eBSCL1\u003c/em\u003e and \u003cem\u003eYa1\u003c/em\u003e evade detection, and how do resistant versus susceptible rice genotypes influence their stability, systemic movement, and function? Answering these questions will provide critical insight into the molecular strategies insects use to manipulate host immunity and may guide innovative approaches for pest control.\u003c/p\u003e \u003cp\u003eIn sum, the discovery of BPH \u003cem\u003eBSCLs\u003c/em\u003e reveals lncRNAs as systemic epigenetic effectors that reprogram host immunity. This shifts the view of insect effectors from plant-centric to insect-centric and highlights lncRNAs as an overlooked class of cross-kingdom regulators. Beyond advancing basic understanding of plant\u0026ndash;herbivore interactions, these findings suggest new strategies for pest control by targeting RNA effectors.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eInsect rearing and plant growth\u003c/h2\u003e \u003cp\u003eBrown planthopper (BPH, \u003cem\u003eN. lugens\u003c/em\u003e) populations were originally collected from rice fields in Wuhan, China, and have been continuously maintained on the susceptible rice cultivar Taichung Native 1 (TN1) since 2007.\u003c/p\u003e \u003cp\u003eRice materials used in this study included TN1, the resistant cultivar Rathu Heenati (RHT)\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e, and transgenic lines (35S::GFP, 35S::BSCL1_wt, and 35S::BSCL1_mut). Rice seeds were sown in plastic pots (7.5 cm diameter, 10 cm height; one plant per pot) and placed in the fields of Huazhong Agricultural University. Plants were protected with insect-proof cages to prevent pest infestation. Tillering stage (30\u0026thinsp;~\u0026thinsp;45 day-old) plants were brought back to the laboratory for subsequent assays, and were grown under the controlled conditions of 27\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, 70\u0026thinsp;\u0026plusmn;\u0026thinsp;5% relative humidity, and a 14:10 h light/dark photoperiod.\u003c/p\u003e \u003cp\u003eHydroponically grown TN1 seedlings were germinated in Petri dishes (9 cm diameter, 2 cm height) containing water and incubated in a controlled growth chamber under the same controlled conditions mentioned above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIdentification and evolutionary analysis of BPH lncRNAs\u003c/h2\u003e \u003cp\u003eTo identify and analyze BPH lncRNAs, we employed a computational pipeline integrating Evolinc-I for lncRNA discovery and Evolinc-II for evolutionary conservation analysis\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. RNA-seq datasets from BPH (NCBI project PRJNA514182)\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e were first quality-checked using FastQC (v0.11.5)\u003csup\u003e54\u003c/sup\u003e and low-quality bases reads were removed. RNA seq reads were mapped to the BPH genome using the RMTA (v2.6.3) pipeline\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e with default parameters. Transcript assembly was performed using StringTie (v2.2.3)\u003csup\u003e56\u003c/sup\u003e, and assembled GTF files were merged with Cuffmerge (v2.2.1.5)\u003csup\u003e57\u003c/sup\u003e to generate a comprehensive transcriptome. The Evolinc-I pipeline was then applied to identify candidate lncRNAs. Transcripts overlapping annotated protein-coding genes or transposable elements, or shorter than 200 nucleotides, were excluded. Coding potential was evaluated using CPC2 (v2.0)\u003csup\u003e58\u003c/sup\u003e, and transcripts with significant coding potential were removed. Remaining transcripts were further screened against the Rfam database (v15.0)\u003csup\u003e59\u003c/sup\u003e to exclude known structural RNAs, including tRNAs, rRNAs, and snoRNAs. The resulting set of transcripts was considered putative lncRNAs.\u003c/p\u003e \u003cp\u003eTo assess the evolutionary conservation of BPH lncRNAs, we utilized the Evolinc-II pipeline. This tool performs reciprocal BLAST analyses (Evalue cutoff, 1e-20) against 28 arthropod genomes (Supplementary Table\u0026nbsp;5). Homologous sequences were grouped into families based on sequence similarity.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBPH feeding on different resistant rice lines\u003c/h3\u003e\n\u003cp\u003eAt the tillering stage (~\u0026thinsp;30 days), TN1 (susceptible) and RHT (resistant) rice plants were each infested with 30 third-instar BPH nymphs. After 48 h of feeding, surviving nymphs were collected and immediately snap-frozen in liquid nitrogen. Three biological replicates were performed per cultivar, and all samples were stored at -80\u0026deg;C for RNA extraction.\u003c/p\u003e\n\u003ch3\u003eBPH feeding experiments\u003c/h3\u003e\n\u003cp\u003eTo test whether BPH secreted transcripts into rice, 30 male adults, to avoid egg deposition by females, were confined on stems of 45-day-old TN1 plants (tillering stage) using glass cylinders (10 cm length, 2.5 cm diameter). Uninfested plant stems with empty glass cylinders served as controls. After 48 h of feeding, feeding sites (FS) were excised, rinsed three times with deionized water, and snap-frozen in liquid nitrogen. Each treatment was conducted with two to three biological replicates. The experiment was repeated three times, achieving three replicates for controls and seven replicates for the FS. Samples were used for RNA-seq to identify BPH transcripts potentially translocated in the rice plants.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction, library preparation, and sequencing\u003c/h2\u003e \u003cp\u003eRNA-seq was performed by OE Biotech Co., Ltd. (Shanghai, China). Samples were frozen in liquid nitrogen, ground to a fine powder using sterilized stainless-steel beads in a TissueLyser II (Jingxin, Shanghai, China), and homogenized in TRIzol reagent (Invitrogen, 15596018CN, USA) following the manufacturer\u0026rsquo;s protocol. RNA concentration and purity were measured with a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA), and RNA integrity was assessed with an Agilent 2100 Bioanalyzer (Agilent Technologies, USA). Quantification was performed using a Qubit 2.0 Fluorometer (Thermo Scientific, USA). Only samples with RNA integrity number (RIN)\u0026thinsp;\u0026gt;\u0026thinsp;7.0 were used for library construction.\u003c/p\u003e \u003cp\u003eFor each sample, 1 \u0026micro;g of high-quality total RNA was used to prepare strand-specific libraries with the VAHTS\u0026reg; Universal V6 RNA-seq Library Prep Kit (Vazyme, NR604, Nanjing, China). The libraries were assessed for quality and fragment size distribution using the Bioanalyzer 2100. Sequencing was performed on an NovaSeq 6000 platform (Illumina, USA), generating 150-bp paired-end reads (project No. PRJNA1314766, PRJNA1197768 (SRR31704824, SRR31704821, SRR31704820, SRR31704819, SRR31704818, SRR31704816), and PRJNA1330246).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eRNA-seq analysis\u003c/h2\u003e \u003cp\u003eRNA-seq datasets were analyzed from two sources: (i) publicly available data (PRJNA714229) covering diverse BPH tissues (antenna, fat body, gut, head, integument, ovipositor, ovary, and salivary gland) to investigate tissue-specific expression, and (ii) RNA-seq data generated in this study from BPH feeding on susceptible (TN1) and resistant (RHT) rice varieties to assess transcriptional responses to host resistance. The BPH reference genome\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e was used for read alignment, while feeding site samples were aligned against a merged reference of TN1 rice\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e and BPH\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRaw sequencing reads were processed with fastp (v0.20.1)\u003csup\u003e60\u003c/sup\u003e to remove adapter sequences and low-quality reads (phred score\u0026thinsp;\u0026lt;\u0026thinsp;25). Reads shorter than 50 bp after trimming were discarded. The quality of clean reads was evaluated using FastQC (v0.11.5)\u003csup\u003e54\u003c/sup\u003e, and only reads passing quality control were retained for downstream analysis.\u003c/p\u003e \u003cp\u003eClean reads were aligned to the reference genome using RMTA (v2.6.3)\u003csup\u003e55\u003c/sup\u003e with the HISAT2 aligner. Parameters were set to trim 15 bases from the 5\u0026prime; end of each read, and minimum and maximum intron lengths were specified as 20 and 500,000, respectively. The resulting BAM files were used for read quantification with HTSeq (v0.6.1)\u003csup\u003e61\u003c/sup\u003e, using the parameters -r, -i gene_id, -t exon, and library-specific strand settings (-s no for non-stranded or -s yes for stranded data). Only uniquely mapped reads overlapping annotated exons were counted.\u003c/p\u003e \u003cp\u003eDifferential gene expression was analyzed with edgeR (v3.0)\u003csup\u003e62\u003c/sup\u003e. Raw read counts from HTSeq were filtered to remove lowly expressed genes (counts per million, CPM\u0026thinsp;\u0026lt;\u0026thinsp;1), normalized using the trimmed mean of M values (TMM) method, and tested for differential expression. P-values were corrected for multiple testing using the Benjamini\u0026ndash;Hochberg method. Genes with false discovery rate (FDR)-adjusted \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and |log2 fold change| \u0026ge; 1 were considered significantly differentially expressed.\u003c/p\u003e \u003cp\u003eTranscript abundance was also quantified as TPM (transcripts per million) using TPMCalculator (v0.0.3)\u003csup\u003e63\u003c/sup\u003e. BAM files generated by RMTA and the corresponding GTF annotation files were provided to TPMCalculator (-b for BAM, -g for annotation). Strand-specificity was set according to the RNA library type (--stranded no for non-stranded libraries).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMigration of BPH-derived RNAs in rice plants\u003c/h2\u003e \u003cp\u003eTo examine the systemic movement of BPH-delivered RNAs in rice, feeding experiments were performed at different developmental stages of TN1 plants. For tillering-stage plants (~\u0026thinsp;30-day old), 50 fourth-instar BPH nymphs were confined on a stem using a glass cylinder cage (10 cm length, 2.5 cm diameter) and allowed to feed for 48 h. After feeding, tissues were collected from two additional positions relative to the feeding site (FS): 2 cm above the FS (UFS), 2 cm below the FS (DFS).\u003c/p\u003e \u003cp\u003eTo better resolve the spatial distribution of transcripts within stem tissues, FS, UFS, and DFS samples were dissected into two fractions: (i) outer layers (the two outermost sheaths) and (ii) inner layers (remaining tissues). All collected tissues were rinsed three times with sterile deionized water to remove surface contamination and immediately snap-frozen in liquid nitrogen. Control plants were treated with empty cages. Each treatment included three biological replicates, and the entire experiment was repeated three independent times.\u003c/p\u003e \u003cp\u003eFor hydroponic experiments, hydroponically grown TN1 plants at the three-leaf stage (~\u0026thinsp;14-day old) were used. 20 fourth-instar nymphs were confined on the stems of four seedlings per cage for 48 h. After feeding, tissues were harvested from the leaf, FS, DFS, and root. Each tissue sample was thoroughly washed three times with sterile deionized water prior to freezing in liquid nitrogen. Seedlings caged without insects were used as controls. Each treatment was performed with three biological replicates, and the experiment was repeated three independent times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eExperiments to analyze BPH RNA stability in rice plants\u003c/h2\u003e \u003cp\u003eTo assess the stability of BPH-delivered mRNAs and lncRNAs in rice, a time-course feeding experiment was performed. Approximately 80 third-instar BPH nymphs were caged on stems of TN1 rice at the tillering stage (~\u0026thinsp;30-day old) using glass cylinders (10 cm length, 2.5 cm diameter) and allowed to feed for seven days. After feeding, all insects were removed, and plant tissues were harvested at 0, 2, 4, 6, and 8 days post-removal. Four biological replicates were collected per time point. Tissues were dissected into outer and inner sheath layers, thoroughly washed with sterile deionized water, and snap-frozen in liquid nitrogen.\u003c/p\u003e \u003cp\u003eThe relative abundance of cross-kingdom RNAs was quantified by qRT-PCR. Two mRNAs (\u003cem\u003eNlug010593\u003c/em\u003e, \u003cem\u003eNlug002054)\u003c/em\u003e and two lncRNAs (\u003cem\u003eBSCL1\u003c/em\u003e, \u003cem\u003eBSCL2\u003c/em\u003e) were examined at multiple tissue sites (FS, UFS, DFS) across all time points.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction, cDNA synthesis, qRT-PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from collected tissues using TRIzol reagent (Invitrogen, 15596018CN, USA) and treated with RNase-free DNase I (Thermo Fisher Scientific, EN0521, USA) to remove residual genomic DNA. First-strand cDNA was synthesized from 1 \u0026micro;g of total RNA using a mixture of oligo (dT) and random primers with the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, K1622, USA), following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003cp\u003eRelative transcript abundance was measured by qRT-PCR. Two BPH mRNAs (\u003cem\u003eNlug010593\u003c/em\u003e, \u003cem\u003eNlug002054\u003c/em\u003e) and two lncRNAs (\u003cem\u003eBSCL1\u003c/em\u003e, \u003cem\u003eBSCL2\u003c/em\u003e) were analyzed at multiple tissue sites (FS, DFS, and UFS) across all time points. qRT-PCR reactions were performed in 20 \u0026micro;L volumes containing 10 \u0026micro;L SYBR Green (Takara, RR820A, Japan), 0.5 \u0026micro;L of each primer (10 \u0026micro;M), 1 \u0026micro;L of cDNA template, and 8 \u0026micro;L nuclease-free water, using the CFX Connect\u0026trade; Real-Time System (Bio-Rad, USA). Cycling conditions were 95\u0026deg;C for 10 s, followed by 40 cycles of 95\u0026deg;C for 20 s, 60\u0026deg;C for 30 s, and 72\u0026deg;C for 30 s. Expression levels were normalized to \u003cem\u003eO. sativa\u003c/em\u003e 18S rRNA (\u003cem\u003eOs18S\u003c/em\u003e) and quantified using the 2^\u003csup\u003e\u0026ndash;ΔCt\u003c/sup\u003e method. Primer sequences are listed in Supplementary Table\u0026nbsp;6.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCloning and sequencing of\u003c/b\u003e \u003cb\u003eBSCL1\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo obtain the full-length cDNA of \u003cem\u003eBSCL1\u003c/em\u003e, 5\u0026rsquo; and 3\u0026rsquo; rapid amplification of cDNA ends (RACE) was performed. For 3\u0026rsquo; RACE, 3 \u0026micro;g of BPH total RNA was added to an 80 \u0026micro;L ligation mixture containing 4 \u0026micro;L T4 RNA ligase, 8 \u0026micro;L T4 RNA ligase buffer, 8 \u0026micro;L BSA (Thermo Fisher Scientific, EL0021, USA), 8 \u0026micro;L ATP (Thermo Fisher Scientific, R0481, USA), and 10 pmol of 3\u0026rsquo; RACE RNA adaptor (Supplementary Table\u0026nbsp;6). Ligation was carried out overnight at 16\u0026deg;C. The ligated RNA was reverse-transcribed into cDNA using an oligonucleotide complementary to the 3\u0026rsquo; RACE adaptor with the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, K1622, USA). For 5\u0026rsquo; RACE, 4.1 \u0026micro;L of BPH total RNA was mixed with 10 \u0026micro;L of ligation mixture containing 0.1 \u0026micro;L T4 RNA ligase, 1 \u0026micro;L T4 RNA ligase buffer, 1 \u0026micro;L BSA (Thermo Fisher Scientific, EL0021, USA), 1 \u0026micro;L ATP (Thermo Fisher Scientific, R0481, USA), and 20 pmol 5\u0026rsquo; RACE RNA adaptor (Supplementary Table\u0026nbsp;6), followed by overnight ligation at 16\u0026deg;C. The ligated RNA was reverse-transcribed into cDNA using primers complementary to the 5\u0026rsquo; RACE adaptor.\u003c/p\u003e \u003cp\u003eRACE PCRs were performed with \u003cem\u003eBSCL1\u003c/em\u003e gene-specific forward or reverse primers together with the 3\u0026rsquo; or 5\u0026rsquo; RACE adaptor primers (Supplementary Table\u0026nbsp;6). Each 50 \u0026micro;L PCR reaction contained 1 \u0026micro;L Phanta\u0026reg; Max Super-Fidelity DNA Polymerase (Vazyme, P505, Nanjing, China), 25 \u0026micro;L Phanta Max Buffer, 1 \u0026micro;L dNTP mix, 1 \u0026micro;L cDNA template, 1 \u0026micro;L of each primer (10 \u0026micro;M), and 20 \u0026micro;L nuclease-free water. The cycling program was: 95\u0026deg;C for 3 min, followed by 36 cycles of 95\u0026deg;C for 15 s, 55\u0026deg;C for 15 s, and 72\u0026deg;C for 20 s, with a final extension at 72\u0026deg;C for 5 min. PCR products were purified, ligated into the pEASY\u0026reg;-Blunt Cloning vector (TransGen, CB101, Beijing, China), and sequenced to confirm the full-length \u003cem\u003eBSCL1\u003c/em\u003e cDNA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eNorthern blotting\u003c/h2\u003e \u003cp\u003eA 708-nt DNA fragment of \u003cem\u003eBSCL1\u003c/em\u003e was cloned under a T7 promoter. \u003cem\u003eBSCL1\u003c/em\u003e RNA transcript was synthesized using MAXIscript\u0026trade; T7 Transcription Kit (Thermo Fisher Scientific, AM1314, USA). A Biotin-11-UTP (Thermo Fisher Scientific, AM8450, USA) labelled full-length of \u003cem\u003eBSCL1\u003c/em\u003e antisense was synthesized using the MAXIscript\u0026trade; T7 Transcription Kit (Thermo Fisher Scientific, AM1314, USA). 10 ng of \u003cem\u003eBSCL1\u003c/em\u003e RNA and total BPH RNA were, respectively, mixed with RNA loading buffer (Takara, 9168, Japan), heated at 65\u0026deg;C for 10 min, and separated on a 10% TBE-Urea denaturing polyacrylamide gel (Beyotime, R0235S, Shanghai, China) in 1 \u0026times; TBE buffer at 150 V for 90 min. RNAs were transferred to a nylon membrane (Beyotime, FFN10, Shanghai, China) by electroblotting at 60 V for 60 min in 0.5 \u0026times; TBE buffer and cross-linked to the membrane using a UV crosslinker (Ultraviolet Products, CA, USA): twice on the gel-facing side and once on the opposite side. The membrane was pre-hybridized in 10 mL hybridization solution at 42\u0026deg;C for 1 h. The biotin-labeled \u003cem\u003eBSCL1\u003c/em\u003e antisense was denatured at 70\u0026deg;C for 10 min, cooled on ice for 5 min, and added to the hybridization solution for hybridization at 42\u0026deg;C overnight.\u003c/p\u003e \u003cp\u003eDetection was carried out using the Chemiluminescent Nucleic Acid Detection Module Kit (Thermo Fisher Scientific, 89880, USA) following the manufacturer\u0026rsquo;s instructions. The membrane was blocked with 16 mL of Blocking Buffer for 15 min with gentle shaking. After decanting, 16 mL of conjugate/blocking solution was added, and the membrane was incubated for another 15 min. The membrane was washed four times for 5 min each with 20 mL 1 \u0026times; wash solution under gentle shaking, then equilibrated in 30 mL Substrate Equilibration Buffer for 5 min. Chemiluminescent detection was performed by incubating the membrane in equal volumes of Luminol/Enhancer Solution and Stable Peroxide Solution for 5 min, followed by visualization using a CCD-equipped imaging system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eGenerating overexpression rice plants\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eBSCL1\u003c/em\u003e sequence is 708 nt in length (\u003cem\u003eBSCL1_wt\u003c/em\u003e) and contains two AUG codons at positions 164 and 253. To generate a mutant form, both AUGs were replaced with UAGs using overlapping PCR\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e, resulting in the \u003cem\u003eBSCL1_mut\u003c/em\u003e sequence. For this, three overlapping fragments were amplified separately with primer pairs PC-\u003cem\u003eBSCL1\u003c/em\u003e-F\u0026ndash;\u003cem\u003eBSCL1\u003c/em\u003e-ATG-164R (fragment 1), \u003cem\u003eBSCL1\u003c/em\u003e-ATG-164F\u0026ndash;\u003cem\u003eBSCL1\u003c/em\u003e-ATG-253R (fragment 2), and \u003cem\u003eBSCL1\u003c/em\u003e-ATG-253F\u0026ndash;PC-\u003cem\u003eBSCL1\u003c/em\u003e-R (fragment 3). Fragments 1 and 2 were first combined by overlap PCR with primers PC-\u003cem\u003eBSCL1\u003c/em\u003e-F and \u003cem\u003eBSCL1\u003c/em\u003e-ATG-253R. The resulting product was then fused with fragment 3 using primers PC-\u003cem\u003eBSCL1\u003c/em\u003e-F and PC-\u003cem\u003eBSCL1\u003c/em\u003e-R to obtain the 708-nt \u003cem\u003eBSCL1_mut\u003c/em\u003e sequence. Primers were listed in the Supplementary Table\u0026nbsp;6.\u003c/p\u003e \u003cp\u003eFor rice transformation, the \u003cem\u003eBSCL1_wt\u003c/em\u003e, \u003cem\u003eBSCL1_mut\u003c/em\u003e and \u003cem\u003eGFP\u003c/em\u003e sequences were cloned into the binary vector PC1300S under the control of the cauliflower mosaic virus 35S promoter. \u003cem\u003eBSCL1_wt\u003c/em\u003e was amplified from BPH cDNA using primers carrying KpnI and XbaI restriction sites in the forward and reverse primers, respectively. \u003cem\u003eBSCL1_wt\u003c/em\u003e, \u003cem\u003eBSCL1_mut\u003c/em\u003e, and \u003cem\u003eGFP\u003c/em\u003e were inserted into the PC1300S vector by homologous recombination using the ClonExpress Ultra One Step Cloning Kit (Vazyme, C115, Nanjing, China), yielding constructs PC1300S_35S::BSCL1_wt, PC1300S_35S::BSCL1_mut, PC1300S_35S::GFP. Plasmid constructs were first introduced into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain EHA105 and verified by selection and molecular confirmation.\u003c/p\u003e \u003cp\u003eThe generation of transgenic rice was performed according to the method described by Hiei and Komari\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e, with minor modifications. Embryogenic calli were induced from surface-sterilized TN1 rice seeds and pre-cultured to increase transformation efficiency. The calli were then infected with the transformed \u003cem\u003eAgrobacterium\u003c/em\u003e suspension and co-cultivated on medium containing acetosyringone to facilitate T-DNA transfer. Following co-cultivation, the calli were washed and placed on selective medium (N6 salts, N6 vitamins, 2,4-D 2 mg/L, Sucrose 30 g/L, Casein hydrolysate 0.3 g/L, Proline 0.5 g/L, Agar 8 g/L, Hygromycin B 50 mg/L, Cefotaxime 250 mg/L) containing the appropriate antibiotic to suppress bacterial growth and select for resistant transformants. Resistant calli were subsequently regenerated on hormone-supplemented medium (MS Basal Salt Mixture, MS Vitamin Stock, Sucrose 30 g/L, 6-BAP: 3.0 mg/L, NAA: 0.2 mg/L, Casein Hydrolysate 300 mg/L, Timentin 200 mg/L, Hygromycin 40 mg/L, Phytagel 3.2 g/L) to induce shoots, transferred to rooting medium (MS Basal Salt Mixture, MS Vitamin Stock, Sucrose 20 g/L, IBA 1.5 mg/L, Agar 7.0 g/L), and finally acclimatized in soil to obtain transgenic plants. Positive transformants were confirmed by PCR. The obtained positive plants of T2 generations were used for subsequent experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eRice blast fungal inoculation\u003c/h2\u003e \u003cp\u003eTo evaluate the disease resistance of different rice lines, punch inoculation was carried out using the \u003cem\u003eMagnaporthe oryzae\u003c/em\u003e Guy11 strain (was kindly provided by Professor Kabin Xie) as described previously\u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e, with minor modifications. Conidia of \u003cem\u003eM. oryzae\u003c/em\u003e were harvested from 10-day-old cultures grown on Oat-tomato agar medium (OTA, 30 g oatmeal, 200 mL tomato juice, 20 g agar, 1 L distilled water) under continuous light at 28\u0026deg;C. The spores were washed from the plate surface with sterile distilled water. The spore concentration was determined with hemocytometers and adjusted to 5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e conidia/mL using sterile distilled water supplemented with 0.02% Tween-20 to promote even suspension.\u003c/p\u003e \u003cp\u003eFor inoculation, fully expanded leaves from 4- to 5-week-old rice plants (TN1, 35S::GFP, 35S::BSCL1_wt, and 35S::BSCL1_mut) were excised and placed on moist filter paper inside plastic boxes (30 \u0026times; 20 \u0026times; 10 cm). Two small wounds were made on each leaf using a sterile needle, and 5 \u0026micro;L of the spore suspension was carefully pipetted onto each wound site. Each plastic box contained leaves from a single rice line to avoid cross-contamination. After inoculation, the boxes were sealed with plastic bag to maintain high humidity and incubated in the dark at 28\u0026deg;C for 24 h. Subsequently, the boxes were transferred to growth chambers maintained at 28\u0026deg;C under a 14 h light / 10 h dark photoperiod. Disease progression was monitored daily. After 6 days, the leaves were photographed using a digital camera, and lesion development was recorded\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e,\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. In total, 40 leaves per rice line were inoculated, serving as independent biological replicates for statistical analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eGene coexpression analysis and GO enrichment analysis\u003c/h2\u003e \u003cp\u003eTo investigate the gene expression responses of different rice lines (TN1, 35S::GFP, 35S::BSCL1_wt, and 35S::BSCL1_mut) to BPH feeding, thirty fifth-instar BPH nymphs were confined on the stem of each rice plant at the tillering stage (~\u0026thinsp;30 days old) using a cylindrical plastic cage. After 24 h of feeding, the outer layer of the feeding site (FS) was carefully excised, immediately snap-frozen in liquid nitrogen, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until RNA extraction. Three biological replicates were performed for each rice line, and non-infested plants were included as controls.\u003c/p\u003e \u003cp\u003eRNA sequencing was performed by OE Biotech Co., Ltd. (Shanghai, China). RNA-seq analysis was discribed in the secton \"RNA-seq analysis\", which included RMTA for reads alignement, HTseq for reads count, and edgeR for identifying DEGs with |log\u003csub\u003e2\u003c/sub\u003e(fold change)| \u0026ge; 1 and FDR-adjusted \u003cem\u003ep\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Co-expression analysis of DEGs was performed with the Mfuzz package in R (v2.6.1)\u003csup\u003e69\u003c/sup\u003e with default settings, and Gene Ontology (GO) enrichment analysis was carried out using the clusterProfiler package in R (v3.18.1)\u003csup\u003e70\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLocalization of\u003c/b\u003e \u003cb\u003eBSCL1\u003c/b\u003e \u003cb\u003ein planta\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFor visualization of \u003cem\u003eBSCL1\u003c/em\u003e in plant cells, an RNA-triggered fluorescence (RTF) reporter system was employed\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The RTF system consists of two modules: (i) an RNA switch containing a probe sequence complementary to the target RNA, and (ii) a GFP expression cassette that is regulated by the RNA switch. In the absence of the target RNA, the RNA switch remains in an inactive conformation, leading to recruitment of the 26S proteasome and degradation of GFP, thereby preventing fluorescence. In contrast, binding of the target RNA to the probe region induces a conformational switch that prevents GFP degradation, allowing GFP accumulation and fluorescence detection.\u003c/p\u003e \u003cp\u003eThe RNA switch design method was adapted from Bai et al.\u003csup\u003e23\u003c/sup\u003e. Probes were designed using the Stellaris Probe Designer (Biosearch Technologies, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.biosearchtech.com/support/tools/design-software/stellaris-probe-designer\u003c/span\u003e\u003cspan address=\"https://www.biosearchtech.com/support/tools/design-software/stellaris-probe-designer\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with default settings, selecting sequences complementary to \u003cem\u003eBSCL1\u003c/em\u003e of approximately 21 nt in length and with annealing temperatures below 65\u0026deg;C. RNA switch secondary structures were predicted using mFold (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"https://www.biosearchtech.com/support/tools/design-software/stellaris-probe-designer\" target=\"_blank\"\u003ewww.unafold.org/mfold/applications/rna-folding-form.php\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.unafold.org/mfold/applications/rna-folding-form.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003csup\u003e71\u003c/sup\u003e) with default parameters. Switches adopting a dumbbell-shaped structure, particularly those with shorter stems and larger bottom loops, were critical for functionality and guided the selection of optimal designs\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The fulll lenght of RNA switch sequeqnce including probe for \u003cem\u003eBSCL1\u003c/em\u003e was listed in Supplementary Table\u0026nbsp;6.\u003c/p\u003e \u003cp\u003eThe probe sequence specific for \u003cem\u003eBSCL1\u003c/em\u003e was cloned into the pcambia1300-AtU6::RNA-Switch\u0026ndash;AtUbq10::RTF vector at the designated restriction sites, generating the RNA-switch-\u003cem\u003eBSCL1\u003c/em\u003e\u0026ndash;RTF construct (Supplementary Fig.\u0026nbsp;4E). The RTF system comprises full-length GFP fused to a single-chain antibody (scFv) carrying an RRRG degron, together with 24 \u0026times; GCN4 fused to Rev at its N-terminus. In this system, the scFv-GFP-RRRG fusion functions as the reporter, producing fluorescence only when stabilized, while the RRRG degron ensures that unbound GFP is rapidly degraded, minimizing background signal. The 24 \u0026times; GCN4-Revd2 component binds to the RNA switch containing RRE sequences, recruiting multiple GFP-scFv molecules and thereby amplifying the fluorescence signal at the RNA site. Primers used for cloning are listed in Supplementary Table\u0026nbsp;6. The construct was sequence-verified by Sanger sequencing and introduced into \u003cem\u003eA. tumefaciens\u003c/em\u003e GV3101 (pSoup) (Weidi, AC1002, Shanghai, China).\u003c/p\u003e \u003cp\u003eThe RNA-switch-\u003cem\u003eBCSL1\u003c/em\u003e\u0026ndash;RTF construct was co-infiltrated with PC1300S-35S::BSCL1_wt into fully expanded leaves of 4-week-old \u003cem\u003eN. benthamiana\u003c/em\u003e plants using the leaf infiltration method. \u003cem\u003eA. tumefaciens\u003c/em\u003e cultures were grown overnight in LB medium containing appropriate antibiotics, harvested by centrifugation, and the bacterial pellet was washed once with infiltration buffer [10 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 10 mM MES pH 5.6 (BioFroxx, 1086GR500, Germany), 100 \u0026micro;M acetosyringone (BioFroxx, 2279GR001, Germany)]. The pellet was then resuspended in infiltration buffer and adjusted to an OD\u003csub\u003e600\u003c/sub\u003e of 0.6. Equal volumes of GV3101 strains carrying the respective constructs were mixed and incubate in the dark for 2 h prior to infiltration. Infiltration was performed on the abaxial side of leaves using a 1-mL needleless syringe. Plants infiltrated with only the RTF construct or PC1300S-35S::BSCL1_wt served as negative controls, while those infiltrated with two constructs for GFP expression. PC1300S-35S::GFP alone was used as a positive control.\u003c/p\u003e \u003cp\u003eThree days post-infiltration (dpi), infiltrated leaves were harvested, mounted in distilled water on glass slides, and imaged using a Leica SP8 confocal laser scanning microscope (Leica Microsystems, Germany). GFP fluorescence was excited at 488 nm and detected between 500\u0026ndash;530 nm. Chlorophyll autofluorescence was collected at 650\u0026ndash;700 nm to assist with subcellular localization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eYeast three-hybrid (Y3H) assay\u003c/h2\u003e \u003cp\u003eY3H was adopted from the protocol developed by Professor Marvin Wickens\u0026rsquo; lab at the University of Wisconsin-Madison\u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. The plasmid, p3HR2 for expressing \u003cem\u003eBSCL1\u003c/em\u003e, pIIIA/IRE-MS2 and pAD-IRP severed as positive controls, the yeast strain YBZ1, were kindly provided by Professor Marvin Wickens.\u003c/p\u003e \u003cp\u003eThe RNA expression plasmid was generated by inserting the 708-nt \u003cem\u003eBSCL1\u003c/em\u003e sequence into p3HR2 between XhoⅠ and XmaⅠ restriction sites. The recombinant plasmid p3HR2-\u003cem\u003eBSCL1\u003c/em\u003e was verified by restriction digestion and sequencing after amplification in \u003cem\u003eE. coli\u003c/em\u003e (primers were listed in Supplementary Table\u0026nbsp;6). The \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e strain YBZ1 was used for yeast three-hybrid assays. Competent cells were prepared as follows: a single colony was inoculated from a YPDA (Coolaber, PM2011, Beijing, China) plate into 5 mL YPDA broth medium and grown overnight at 30\u0026deg;C with shaking (250 rpm). Cells were diluted to an OD600 of 1.2\u0026ndash;1.5, pelleted at 1,000 g for 5 min, and sequentially washed with sterile water and TE/LiAc buffer [1 mL 10 \u0026times; TE, 1 mL 10 \u0026times; LiAc (Sigma, L4158, USA), 8 mL sterile water]. The final cell pellet was resuspended in 1\u0026times; TE/LiAc to obtain competent cells.\u003c/p\u003e \u003cp\u003eTransformation was performed by mixing competent cells with 40% PEG4000/TE/LiAc [1 mL 10 \u0026times; TE, 1 mL 10 \u0026times; LiAc (Sigma, L4158, USA), 8 mL 50% PEG4000 (Sigma, 95904, USA)], denatured salmon sperm DNA (Thermo Fisher Scientific, 15632011, USA), and p3HR2-\u003cem\u003eBSCL1\u003c/em\u003e, followed by incubation at 30\u0026deg;C for 30 min and heat shock at 42\u0026deg;C for 10 min. After recovery, cells were plated onto synthetic defined (SD) agar medium (Coolaber, PM2040, Beijing, China) lacking uracil (Coolaber, PM2270, Beijing, China) (SD-U) and incubated at 30\u0026deg;C for 2\u0026ndash;3 days. Single colonies were verified by plasmid rescue, restriction digestion, and sequencing. Competent cells containing the p3HR2-\u003cem\u003eBSCL1\u003c/em\u003e were used for library transformation.\u003c/p\u003e \u003cp\u003eTo optimize 3-amino-1,2,4-triazole (3-AT) (Sigma, A8056, USA) concentration and test for autoactivation, yeast strains harboring RNA plasmids were co-transformed with empty pGAD-T7 vector. Transformants were spotted or plated onto SD agar medium (Coolaber, PM2040, Beijing, China) lacking uracil, leucine, and histidine (Coolaber, PM2170, Beijing, China) (SD-U-L-H), supplemented with increasing concentrations of 3-AT (0\u0026ndash;100 mM). Growth was monitored after 3\u0026ndash;5 days at 30\u0026deg;C, and the minimal concentration of 3-AT that suppressed background growth was selected for subsequent screening.\u003c/p\u003e \u003cp\u003eCompetent cells containing the p3HR2-\u003cem\u003eBSCL1\u003c/em\u003e were prepared as described above. For library transformation, cells were mixed with 40% PEG4000/TE/LiAc (1 mL 10 \u0026times; TE, 1 mL 10 \u0026times; LiAc, 8 mL 50% PEG4000), denatured salmon sperm DNA (Thermo Fisher Scientific, 15632011, USA), and 20 \u0026micro;g of cDNA library, kindly provided by Professor Yongjun Lin at Huazhong Agricultural University. After incubation at 30\u0026deg;C for 30 min with shaking, DMSO was added, and cells were heat shocked at 42\u0026deg;C for 15 min. Transformants were plated on SD-U-L-H plates supplemented with the optimized concentration of 3-AT. After 5\u0026ndash;7 days at 30\u0026deg;C, colonies growing on selective plates were isolated as HIS\u003csup\u003e+\u003c/sup\u003e candidates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eLacZ reporter assay and identificaiton of putative interactors\u003c/h2\u003e \u003cp\u003eThe methods for LacZ screening and interaction validation were adapted from Bernstein et al.\u003csup\u003e73\u003c/sup\u003e. Briefly, HIS\u003csup\u003e+\u003c/sup\u003e colonies were replated onto SD agar medium (Coolaber, PM2040, Beijing, China) lacking uracil and leucine (Coolaber, PM2290, Beijing, China) (SD-U-L), overlaid with nitrocellulose membranes. After incubation at 30\u0026deg;C for 2\u0026ndash;3 days, the membranes were briefly frozen in liquid nitrogen and then incubated at 37\u0026deg;C in buffer (Z-buffer supplemented with 65 \u0026micro;L β-mercaptoethanol and 200 \u0026micro;L of 25 mg/mL X-β-gal per 10 mL). Colonies that developed a blue color were scored as LacZ-positive.\u003c/p\u003e \u003cp\u003ePlasmids were extracted from LacZ-positive yeast colonies, and the inserted sequences were amplified by PCR, verified by Sanger sequencing, and identified through BLAST searches against the NCBI database. To validate \u003cem\u003eBSCL1\u003c/em\u003e\u0026ndash;protein interactions, full-length cDNAs of candidate genes were cloned into pGAD-T7 and co-transformed into YBZ1 cells together with either p3HR2-BSCL1 or the empty p3HR2 vector (negative control). The pIIIA/IRE-MS2 and pAD-IRP plasmids served as positive controls. Transformants were plated onto SD-U-L-H medium supplemented with the optimized concentration of 3-AT. Growth observed with the \u003cem\u003eBSCL1\u003c/em\u003e plasmid but absent with the empty vector was taken as evidence of a specific RNA\u0026ndash;protein interaction. Colonies that turned blue on SD-U-L medium in the X-β-gal reporter assay were considered to represent positive interactions.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCo-localization of\u003c/b\u003e \u003cb\u003eBSCL1\u003c/b\u003e \u003cb\u003ewith candidate target proteins\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe full-length CDSs of HIRIP3 and H3.3 were amplified by RT-PCR from rice cDNA and cloned into the pB7RWG between SpeⅠ and XhoⅠ restriction sites to generate C-terminal fusions with mCherry. Constructs were verified by Sanger sequencing and introduced into \u003cem\u003eA. tumefaciens\u003c/em\u003e GV3101 (pSoup) (Weidi, AC1002, Shanghai, China) as described above.\u003c/p\u003e \u003cp\u003e \u003cem\u003eAgrobacterium\u003c/em\u003e strains carrying pB7RWG-HIRIP3-mCherry or pB7RWG-H3.3-mCherry were co-infiltrated with PC1300S-35S::BSCL1_wt and the RNA-switch-\u003cem\u003eBSCL1\u003c/em\u003e\u0026ndash;RTF construct into \u003cem\u003eN. benthamiana\u003c/em\u003e leaves, following the same infiltration protocol as above. Each protein was tested in independent infiltration assays.\u003c/p\u003e \u003cp\u003eAt 3 dpi, infiltrated leaves were examined by confocal microscopy Leica SP8 (Leica Microsystems, Germany). GFP signals (\u003cem\u003eBSCL1\u003c/em\u003e detection via RNA-switch-\u003cem\u003eBSCL1\u003c/em\u003e\u0026ndash;RTF system) were excited at 488 nm (emission 500\u0026ndash;530 nm), while mCherry-tagged HIRIP3 and H3.3 were excited at 561 nm (emission 580\u0026ndash;610 nm). Chlorophyll autofluorescence was collected at 650\u0026ndash;700 nm to assist with subcellular localization. Images were collected in sequential scanning mode to prevent bleed-through between channels. Representative images were captured and processed using identical microscope settings for all samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003edsRNA synthesis, microinjection, and gene expression analysis in BPH\u003c/h2\u003e \u003cp\u003eDouble-stranded RNA (dsRNA) targeting \u003cem\u003eBSCL1\u003c/em\u003e was synthesized by PCR amplification of two gene fragments (\u003cem\u003edsBSCL1\u003c/em\u003e-F1/\u003cem\u003edsBSCL1\u003c/em\u003e-R1: 315 bp, \u003cem\u003edsBSCL1\u003c/em\u003e-F2/\u003cem\u003edsBSCL1\u003c/em\u003e-R2: 260 bp) with primers containing T7 RNA polymerase promoter sequences at both 5\u0026rsquo; ends (Supplementary Table\u0026nbsp;6). The PCR products were used as templates for in vitro transcription with the T7 High Yield RNA Transcription Kit (Vazyme, TR101-01, Nanjing, China) following the manufacturer\u0026rsquo;s instructions. Synthesized dsRNAs were diluted to the appropriate concentration for microinjection.\u003c/p\u003e \u003cp\u003eThird- to fourth-instar BPH nymphs were anesthetized with carbon dioxide for 20 s and injected with 200 ng dsRNA into the mesothorax using a Nanoliter 2010 microinjector (World Precision Instruments, USA). BPH injected with \u003cem\u003edsGFP\u003c/em\u003e served as the negative control. Each treatment was performed in three biological replicates. For RNAi efficiency assessment, ten nymphs per replicate were collected at 3 days post-injection. The remaining injected nymphs were maintained on three-leaf stage rice seedlings for subsequent experiments.\u003c/p\u003e \u003cp\u003eTotal RNA was isolated from microinjected nymphs or insects at different developmental stages using TRIzol reagent (Invitrogen, 15596018CN, USA). First-strand cDNA synthesis was performed with the PrimeScript RT Reagent Kit with gDNA Eraser (Takara, RR047A, Japan) following the manufacturer\u0026rsquo;s instructions. qRT-PCR was conducted as described above using gene-specific primers, with \u003cem\u003eN. lugens Actin\u003c/em\u003e (\u003cem\u003eNlActin\u003c/em\u003e) serving as the endogenous control (primers were listed in Supplementary Table\u0026nbsp;6). Relative expression levels were calculated using the 2^\u003csup\u003e\u0026ndash;ΔΔCt\u003c/sup\u003e method.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eBPH survival and fecundity assays\u003c/h2\u003e \u003cp\u003eTo evaluate the effects of dsRNA-mediated gene silencing on BPH survival, approximately 100 third- to fourth-instar nymphs treated with gene-specific or control dsRNAs were transferred into 500-mL glass beakers containing fresh three-leaf stage TN1 seedlings\u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e. Each beaker was treated as one biological replicate, with three replicates per treatment. Rice seedlings were replaced every 4 days to ensure a continuous food supply and minimize confounding effects from plant senescence. Beakers were maintained in a controlled growth chamber at 27\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, 70\u0026thinsp;\u0026plusmn;\u0026thinsp;5% relative humidity, and a 14:10 h light:dark photoperiod. The number of surviving nymphs was recorded every 24 h, and mortality was calculated relative to the initial number of insects. Monitoring continued until most individuals reached adulthood or exhibited mortality consistent with RNAi effects.\u003c/p\u003e \u003cp\u003eFecundity was assessed according to the methods described by Wen et al.\u003csup\u003e75\u003c/sup\u003e and Liu et al.\u003csup\u003e76\u003c/sup\u003e, with minor modifications. For BPH fecundity assays following microinjection, newly emerged dsRNA-treated adults (\u0026le;\u0026thinsp;24 h old) were paired in glass tube (3 cm diameter, 25 cm high) supplied with five three-leaf stage TN1 rice seedlings for feeding and oviposition. Adults were transferred to a new glass tube with fresh rice seedlings every 4 days until all of the adults were dead. For fecundity assays on different rice lines, each rice plant was enclosed with a perforated plastic lid to allow plant growth and covered with an inverted transparent plastic cup (7.5 cm diameter) to confine insects. Five newly hatched nymphs (\u0026le;\u0026thinsp;24 h old) were placed into each plastic cup containing a single rice plant. Upon adult emergence, one male\u0026ndash;female pair was retained per cup for oviposition and removed after 10 days. Newly hatched nymphs were counted and removed daily until no further hatching occurred. A minimum of 15 biological replicates were performed for each dsRNA treatment or rice line to ensure robust statistical analysis.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eHoneydew secretion assay\u003c/h2\u003e \u003cp\u003eHoneydew secretion was measured following the method described by Guo et al.\u003csup\u003e32\u003c/sup\u003e. As described in fecundity assays, each rice plant was covered with a perforated plastic lid that allowed the plant to grow through. A 9-cm diameter filter paper was placed on the lid and covered with an inverted transparent plastic cup (7.5 cm diameter) to confine the insects.\u003c/p\u003e \u003cp\u003eFor assays after microinjection, two dsRNA-treated 5th instar nymphs were introduced into each cup and allowed to feed for 48 h, after which filter papers were collected. At least 15 biological replicates were performed per treatment. For assays comparing BPH feeding on different rice lines, five 5th instar nymphs were confined per cup and allowed to feed for 48 h, with at least 20 replicates per rice line.\u003c/p\u003e \u003cp\u003eCollected filter papers were soaked in 0.1% (w/v) ninhydrin in acetone, dried in an oven at 60\u0026deg;C for 30 min, and honeydew spots visualized as violet or purple stains due to their amino acid content. Images were captured using a digital camera, and honeydew spot areas were quantified using ImageJ software.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDual-luciferase reporter assay in\u003c/b\u003e \u003cb\u003eN. benthamiana\u003c/b\u003e\u003c/p\u003e \u003cp\u003eDual-luciferase reporter assays were performed as previously described\u003csup\u003e\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e. The 1989-bp promoter region of bHLH6 was amplified from rice genomic DNA by PCR and cloned upstream of the firefly luciferase (LUC) coding sequence in the pGreenⅡ 0800-LUC vector, generating the Pro\u003csub\u003ebHLH6\u003c/sub\u003e::LUC reporter construct. In this vector, renilla luciferase (REN) driven by the CaMV 35S promoter served as an internal control for normalization of LUC activity.\u003c/p\u003e \u003cp\u003eFor effector constructs, the full-length sequence of \u003cem\u003eBSCL1_wt\u003c/em\u003e, the full-length coding sequences (CDSs) of \u003cem\u003eHIRIP3\u003c/em\u003e and \u003cem\u003eH3.3\u003c/em\u003e, as well as a truncated \u003cem\u003eGFP\u003c/em\u003e sequence (negative control for \u003cem\u003eBSCL1\u003c/em\u003e_wt), were amplified by PCR and cloned separately into pGreenⅡ 62-SK under the control of the CaMV 35S promoter. Primers used for all constructs are listed in \u003cem\u003eSI Appendix\u003c/em\u003e, Table S6. All plasmids were verified by Sanger sequencing using specific primers and introduced into \u003cem\u003eA. tumefaciens\u003c/em\u003e GV3101 (pSoup) (Weidi, AC1002, Shanghai, China).\u003c/p\u003e \u003cp\u003e \u003cem\u003eAgrobacterium\u003c/em\u003e strains carrying the reporter and effector constructs were cultured overnight in LB medium containing appropriate antibiotics at 28\u0026deg;C with shaking. Cells were harvested by centrifugation, washed, and resuspended in infiltration buffer [10 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 10 mM MES pH 5.6 (BioFroxx, 1086GR500, Germany), 100 \u0026micro;M acetosyringone (BioFroxx, 2279GR001, Germany)] to an OD\u003csub\u003e600\u003c/sub\u003e of 0.6. For co-infiltration, equal volumes of \u003cem\u003eAgrobacterium\u003c/em\u003e cultures carrying reporter and effector constructs were mixed. Fully expanded leaves of 4-week-old \u003cem\u003eN. benthamiana\u003c/em\u003e plants were infiltrated on the abaxial side using a 1-mL needleless syringe. Each construct combination was infiltrated into at least three independent leaves from different plants as biological replicates.\u003c/p\u003e \u003cp\u003eInfiltrated plants were maintained under standard growth conditions (22\u0026ndash;25\u0026deg;C, 14 h light/10 h dark) for 3 days to allow transient expression. Firefly (LUC) and renilla (REN) luciferase signals were measured using the Dual-Luciferase Reporter Assay Kit (Vazyme, DL101-01, Nanjing, China) according to the manufacturer\u0026rsquo;s instructions. Leaf discs (approximately 1.0 cm diameter) were collected from infiltrated zones, homogenized in passive lysis buffer, and luminescence was measured with a multimode reader Spark (TECAN, Zurich, Switzerland). Relative LUC activity was calculated as the ratio of LUC:REN signals for each sample.\u003c/p\u003e \u003cp\u003eFor live imaging, D-Luciferin potassium salt (1 mM, Yeasen, 40902ES01, Shanghai, China) was evenly applied to the abaxial surface of infiltrated leaves. Luminescence was visualized using the NightSHADE L985 live imaging system (Berthold, Germany), and images were captured under standardized exposure conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed with SPSS 20.0 (IBM, Armonk City, NY, USA). Paired Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test or one-way ANOVA test followed by Tukey\u0026rsquo;s multiple comparisons test was used to analyze the results of survival rate, honeydew measurement, fecundity measurement, and qRT-PCR. Correlation analysis and Fisher\u0026rsquo;s Exact test were performed in R. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered as statistically significant. All results are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SE.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eReporting summary\u003c/h2\u003e \u003cp\u003eFurther information on research design is available in the Nature Portfolio Reporting Summary linked to this article.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe raw data used to generate all figures in this study are available in the Supplementary Information/Source Data file. Transcriptomics data have been deposited in the SRA database under project numbers PRJNA1197768, PRJNA1314766, and PRJNA1330246. Source data are provided as a Source Data file. Source data are provided with this paper.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eY.C. and H.H. conceived and designed the study, supervised the project, and finalized the manuscript. D.W. carried out the majority of the experimental work. S.J. and Z.Q. contributed to the experimental setup and bioassays. Bioinformatic analyses were conducted jointly by Y.C. and D.W. C.L., J.W., R.H., and Y.Z. contributed to the performed research. Y.H. and W.M. contributed to analyzed data and experiment design. The manuscript was drafted by Y.C. and D.W., with all authors contributing to its revision. All authors reviewed the manuscript, provided feedback, and approved the final version.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThis project is funded by the National Key Research and Development Program of China (project No. 2024YFD1400700 to H.H.), the Hubei Grain Yield Improvement Program (project No. 2662025YJ017 to H.H.), the National Key Research and Development Program of China (project No. 2023YFF1000703 to Y.C.), the National Natural Science Foundation of China (project No. 32172392 to Y.C.), the Hubei Hongshan Laboratory (project No. 2022hszd026 to Y.C.), the Startup Foundation for Advanced Talents at HZAU to Y.C., the Fundamental Research Funds for the Central Universities (Program No. 2022ZKPY003 to Y.C.), and the Wuhan Yingcai Talent Program to Y.C.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhu K et al (2017) Plant microRNAs in larval food regulate honeybee caste development. 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Curr Biol 35:2614\u0026ndash;2629\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"brown planthopper, lncRNAs, cross-kingdom RNAs, BSCLs, rice","lastPublishedDoi":"10.21203/rs.3.rs-8297537/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8297537/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCross-kingdom RNAs are emerging as critical mediators of interspecies interactions, yet the functions of long RNAs such as mRNAs and long non-coding RNAs (lncRNAs) in recipient organisms remain largely unexplored. Here, we show that the brown planthopper (\u003cem\u003eNilaparvata lugens\u003c/em\u003e, BPH), a major rice pest, translocates mRNAs and lncRNAs into rice plants, where they migrate systemically from feeding sites to distal tissues. Compared with BPH mRNAs, \u003cem\u003eBPH Salivary gland Cross-kingdom LncRNA\u003c/em\u003e (\u003cem\u003eBSCL\u003c/em\u003es) exhibit markedly higher stability in rice. Among them, mitochondrial-originated \u003cem\u003eBSCL1\u003c/em\u003e functions as a virulence factor that promotes BPH feeding and reproduction by suppressing host defense. Mechanistically, \u003cem\u003eBSCL1\u003c/em\u003e associates with the HIRA histone chaperone complex and displaces histone H3.3 from the promoters of transcription factors, including bHLH genes central to jasmonic acid signaling, thereby repressing transcriptional immunity. Our results identify \u003cem\u003eBSCL\u003c/em\u003es as systemic, RNA-based effectors that reprogram host defense at the epigenetic level, revealing a previously unrecognized mode of insect-mediated manipulation of plant immunity and highlighting lncRNAs as cross-kingdom regulators.\u003c/p\u003e","manuscriptTitle":"Insect-derived long non-coding RNAs function as epigenetic effectors to reprogram plant immunity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-12 07:31:14","doi":"10.21203/rs.3.rs-8297537/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8563d943-a24b-4f9d-a646-92686491d69c","owner":[],"postedDate":"December 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":59464972,"name":"Biological sciences/Zoology/Entomology"},{"id":59464973,"name":"Biological sciences/Plant sciences/Plant immunity/Effectors in plant pathology"}],"tags":[],"updatedAt":"2026-01-23T09:12:38+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-12 07:31:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8297537","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8297537","identity":"rs-8297537","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}