Spatial regulation of rice leaf morphology by miRNA-targets complexes during viral infection

doi:10.21203/rs.3.rs-5045268/v1

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Spatial regulation of rice leaf morphology by miRNA-targets complexes during viral infection

https://doi.org/10.21203/rs.3.rs-5045268/v1

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Background: Leaf morphogenesis is essential for plant growth and development, yet the mechanisms by which plant viruses induce changes in leaf shape are not well understood. Rice ragged stunt virus (RRSV) infection induces distinct morphological abnormalities in rice leaves, including leaf tip curling and serrated margins, through unknown pathogenic mechanisms.

Results: This study reveals that key regulatory microRNAs (miR164, miR319, and miR156) and their target genes (CUC, TCP, and SPL) exhibit entirely opposite expression patterns in healthy and RRSV-infected leaves, indicating a profound impact on the leaf morphogenesis network. Significantly, the core protein OsCUC1, which typically functions by forming dimers, shows abnormal expression in the peripheral zone of the shoot apical meristem (SAM) under viral infection, leading to disruptions in leaf development. OsTCP1 was found to dynamically regulate OsCUC1 dimer formation by modifying its subcellular localization and interacting with OsSPL14 and OsSPL17, thereby influencing their regulatory functions. Genetic disruptions of OsCUC1, OsTCP1, and OsSPL14/OsSPL17 enhance the severity of RRSV infection, demonstrating their critical involvement in the viral pathogenic strategy.

Conclusions: The research uncovers a novel mechanism by which RRSV manipulates the expression and interactions of key regulatory factors, disrupting the delicate balance of the leaf morphogenesis network. These findings expand our understanding of viral manipulation of host development and provide a foundation for innovative strategies to enhance crop resilience.

RRSV

leaf morphogenesis

miRNA164

miR319

miR156

OsCUC1

OsTCP1

Leaves, key organs in plants, are responsible for photosynthesis and providing necessary energy for plant growth [1]. Leaf development is influenced by numerous factors and regulated by an extensive gene network [2, 3]. Plant hormones initiate the formation of leaf primordium [4-6]. As the leaf primordium differentiates from the periphery of the shoot apex meristem (SAM), a sequence of changes in gene expression occurs within the primordium cells. The silencing of the class I knotted 1-like homeobox (KNOX) gene is a particularly well-known change [7, 8]. The KNOX1 gene plays important roles in leaf development, maintaining SAM integrity and normal function, helping to control leaf edge morphology, and contributing to the formation of compound leaf morphology [9, 10]. During the establishment of adaxial–abaxial polarity [11], the class III homeodomain‐leucine zipper (HD-ZIP III) gene family (regulated by miR165 and miR166) [12] and ASYMMETRIC LEAVES 1 (AS1) and 2 (AS2) [13] act to promote adaxial identity. Simultaneously, the KANADI (KAN) gene family, YABBY gene family, and various AUXIN RESPONSE TRANSCRIPTION FACTORs (ARF3/4) participate in cell fate regulation on the abaxial side of the leaf [14, 15].

The CUP-SHAPED COTYLEDON (CUC) family also plays a key role in regulating leaf development. The CUC gene family, which includes CUC1, CUC2, and CUC3, is important in processes that determine organ boundary regions [16-18]. In Arabidopsis thaliana, the spatial distribution of the CUC2 gene and auxin at the leaf edge leads to serration at the leaf edge [19]. Additionally, the expression of CUC1 and CUC2 is negatively regulated by the miR164 family. The CUC gene family and KNOX1 protein exhibit specific expression at the organ boundary, mutually regulating each other. This interaction promotes standardization and development of the organ boundary [20-22].

In addition to miR164 and KNOX1, CUC is regulated by various factors. Genetic and expression analyses have revealed that CUC expression is negatively regulated by auxin maxima [23, 24]. Simultaneously, the TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) transcription factors regulate CUC expression [25-27]. In Arabidopsis, both CUC2 and CUC3 can interact with the miR319 target gene encoding the TCP protein [28]. The competitive binding of TCP and CUC leads to disruption of the CUC heterodimer structure, interfering with the complex CUC-mediated regulation of leaf morphology. As Arabidopsis progresses from seedlings to mature stages, the miR156 content gradually decreases. Consequently, expression of the miR156 target gene, encoding the protein SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL), increases. SPL and TCP then competitively recombine, disrupting the CUC-TCP dimer structure and releasing CUC to form additional heterodimers. This process also contributes to the regulation of leaf edge morphology, resulting in serration on the leaf edge that is characteristic of mature Arabidopsis [19, 28, 29].

The miR164-CUC module is highly conserved in many plants, particularly concerning the control of organ boundary development and lateral branches [30, 31]. For example, in Arabidopsis, the miR164 target genes CUC1 and CUC2, and the non-target gene CUC3, exhibit redundant functions in terms of initiating the SAM and establishing organ boundaries [32]. In cotton, miR164 and its target gene CUC2 contribute to the development of lateral branches [33]. In monocotyledons, leaf morphology remains largely unaltered during development. Therefore, the regulatory function of miR164 and its target gene CUC may differ between monocotyledons and dicotyledons (e.g., Arabidopsis). In monocotyledonous plants, the expression domain of the miR164-CUC regulatory module is altered, and the spatial expression pattern differs between monocotyledonous meristem differs from the pattern in dicotyledonous meristem; however, miR164, its target gene CUC2, and the non-target gene CUC3 share similar protein functions [34]. For instance, in maize, NAM/CUC3 is associated with boundary specification around the SAM and the development of boundaries in lateral organs [35].

In rice, the role of miR164-regulated CUC in leaf development was recently recognized [36, 37]. OsCUC1 and OsCUC3 form heterodimers in rice, with key regulatory roles concerning organ boundary specification in the meristem. Rice plants lacking OsCUC1 or overexpressing miR164 exhibit abnormal leaf development phenotypes with wrinkled and curled leaves; the resulting plants are short and stunted [36, 37]. miR319 and its target genes also have been implicated in the regulation of leaf morphology in rice. Overexpression of miR319a and miR319b in rice leads to increased leaf width, whereas downregulation of the target gene OsPCF1 enhances cold tolerance in rice [38]. Additionally, overexpression of miR319a and miR319b, as well as interference with the target gene OsTCP21, exacerbates symptoms in rice ragged stunt virus (RRSV)-infected rice leaves, suggesting that miR319 is involved in regulating virus-induced changes to leaf morphology [39]. As a monocotyledonous plant, rice does not undergo substantial changes in leaf shape during the transition from seedlings to mature plants, unlike Arabidopsis [40, 41]. There is evidence indicate that the transition from vegetative reproduction to reproductive development in monocotyledonous plants (e.g., rice) is characterized by morphological changes in lateral branches. The SPLs-NL1-PLA1 pathway, regulated by miR156/529, controls bract growth in rice, influencing the transition between vegetative and reproductive branches; this influence is illustrated by morphological changes in leaves [42]. Recent research concerning miR156 in rice has revealed that its expression gradually increases during leaf development but decreases during shoot development. Additionally, the expression patterns of miR159, miR164, and miR172 are altered in miR156-overexpressing plants [43]. In summary, although the expression pattern of miR156 in rice differs from the expression pattern in Arabidopsis, the conserved function of miR156 in regulating the plant's transition from seedlings to maturity persists. Therefore, it remains unknown whether the CUC-TCP-SPL regulatory network, extensively studied in Arabidopsis and responsible for morphological changes during leaf development, exists in rice.

A common phenomenon observed after plant virus infection is the viral regulation of leaf morphological development, but the underlying mechanism is currently unclear. RRSV, a member of the genus Oryzavirus [44] that causes severe decline in rice yield [45, 46], can induce unique symptoms such as leaf tip curl and serration on the leaf edge; the mechanism of pathogenicity has not been elucidated [47]. Previous studies have demonstrated that RRSV infection in rice leads to the upregulation of miR319, thereby inhibiting the jasmonic acid (JA)-mediated antiviral defense pathway; this enables viral infection and leads to symptom onset. Additionally, miR164, miR156, and their target genes respond to RRSV infection [39], suggesting a widespread response of miRNAs during the RRSV infection process. However, it remains unclear how regulatory networks, formed by multiple miRNAs, respond to RRSV and participate in the pathogenic mechanisms. In this study, we observed that the regulatory network formed by miR164, miR319, miR156, and their target genes CUC, TCP, and SPL dynamically responds to RRSV infection, actively participating in the pathogenic regulation of leaf dysplasia. This study provides significant new insights into the molecular interactions governing leaf morphogenesis and their manipulation by viral pathogens. By elucidating the intricate network of miRNAs and their target proteins in the context of viral infection, it advances our understanding of how viruses hijack host developmental processes to facilitate their own propagation. These findings have broad implications for developing novel strategies to mitigate the impact of viral diseases on crops, potentially leading to more resilient agricultural systems.

RRSV infection regulates the spatial expression patterns of leaf phenotype-related miRNAs and their target genes

Leaf complexity in Arabidopsis is regulated by a conserved miRNA transcription factor regulatory network [28, 48]. To investigate the function of this network during rice virus infection, we identified key genes involved in Arabidopsis leaf morphogenesis (AtCUC2, AtCUC3, AtTCP4, and AtSPL9) and their corresponding rice homologs. Phylogenetic analysis demonstrated that OsCUC1 and OsCUC3 are closely related to AtCUC2 and AtCUC3, respectively (Fig. S1a; Table S1). Additionally, OsTCP1 aligns with AtTCP4 (Fig. S1b; Table S1). and OsSPL14 and OsSPL17 correspond to AtSPL9 (Fig. S1c; Table S1). We performed transcriptome and small RNA sequencing on RRSV-infected and control rice (Zhonghua 11, ZH11) at 7-, 14-, and 21-days post-inoculation (dpi). Our results revealed changes in miRNA expression: miR164 decreased at 7 and 21 dpi, miR319 exhibited dynamic expression patterns over time, and miR156 increased at 14 and 21 dpi (Fig. S2a, c; Table S2). Target gene analysis indicated that OsOMTN3 (OsCUC1 was not detected) expression increased progressively in infected rice, whereas OsTCP1 expression decreased at 14 and 21 dpi. OsSPL17 expression remained stable throughout the experiment (Fig. S2b, c; Table S3). To understand the long-term expression of miRNAs regulating leaf morphology, miR164, miR156, miR319, and their target genes were analyzed in mock and RRSV-infected rice from 7 to 56 dpi (Fig. S3a-c). Viral infection led to plant stunting and intensified leaf wrinkling (Fig. S3a, b). miR164 was downregulated during the symptomatic phase (Fig. S3d), while its target gene OsCUC1 was upregulated (Fig. S3e). miR319 and miR156 showed altered expression in infected rice, with miR319 upregulated and miR156 significantly higher during the symptomatic phase (Fig. S3f, h). The target genes OsTCP1 and OsSPL17 exhibited opposite and increasing expression patterns, respectively, in infected rice (Fig. S3g, i). These results indicated that RRSV infection causes dynamic temporal alterations in the expression patterns of miR164, miR319, miR156, and their target genes.

To explore the regulation of spatial expression patterns concerning miRNAs and their target genes during RRSV infection, rice leaves from mock and RRSV-infected samples were selected at 42 dpi. They were divided into four segments: C1, C2, C3, and C4 (for mock samples), as well as R1, R2, R3, and R4 (for RRSV-infected samples). Among these, C4 and R4 represent the respective proximal axis of the two types of leaves. The expression levels of the three miRNAs and their target genes in all segments were compared with the base C4 segment in mock leaves (Fig. 1a). In mock leaves, from the proximal to distal axis (C4 to C1), the expression patterns of miR164, miR319, and miR156 exhibited an initial decrease, followed by an increase (Fig. 1b-d). In contrast, in RRSV-infected leaves, the expression patterns of these three miRNAs demonstrated an initial increase, followed by a decrease (Fig. 1b-d). Intriguingly, compared with mock leaves, the expression levels of these three miRNAs were significantly increased in the middle segments of RRSV-infected leaves (Fig. 1b-d). The target genes regulated by these three miRNAs were also influenced by viral infection. In mock leaves, the three target genes OsCUC1, OsTCP1 and OsSPL17 showed a gradual decrease in expression from the proximal to distal axis (Fig. 1e-g), whereas. In RRSV-infected leaves, the expression patterns of OsCUC1 and OsSPL17 exhibited the opposite trend, gradually increasing from the proximal to distal axis (Fig. 1e, g), whereas OsTCP1 displayed a zig-zag expression pattern (Fig. 1f). Notably, compared with mock leaves, the expression levels of OsCUC1 and OsSPL17 were significantly increased in the visibly affected R2 and R1 segments of infected leaves (Fig. 1e, g), whereas OsTCP1 showed a significant increase in the R2 segment and a significant decrease in the R1 segment (Fig. 1f). Our results indicated that RRSV infection alters the spatial expression patterns of the three miRNAs and their target genes, causing opposite expression patterns in normal and RRSV-infected leaves. The overall changes among OsTCP1, OsCUC1, and OsSPL17 suggested an antagonistic relationship, similar to the antagonistic interaction observed among AtTCP4, AtCUC2, and AtSPL9 in Arabidopsis. These findings implied that OsTCP1, OsCUC1, and OsSPL17 regulate rice leaf development during infection in a manner similar to Arabidopsis, supporting the notion of a conserved regulatory mechanism in leaf morphogenesis before and after viral infection in rice.

OsCUC1 expression is downregulated in response to RRSV infection, contributing to viral pathogenesis

In the model plant Arabidopsis, CUC2 plays a key role in controlling leaf morphology through an antagonistic interaction with auxin, directly causing serration to form on leaf edges [19]. Previous studies have demonstrated that the absence of OsCUC1 in rice results in severe dwarfism, along with curled and shrunken leaves [36, 37]; this phenotype mimics the results of RRSV infection. To examine the regulatory effects of RRSV infection on OsCUC1, we infected pOsCUC1::rOsCUC1-GFP transgenic plants [37] with RRSV and compared them with mock samples. The results showed distinct fluorescence signals in mock samples across the peripheral, margin, and boundary regions of SAM; after RRSV infection, the fluorescence signals in these areas were weak or absent (Fig. 2a). To investigate the role of OsCUC1 in the RRSV infection process, we obtained OsCUC1 deletion mutant rice plants [37]. We initially assessed the expression levels of miR164, miR319, and miR156, along with their target genes OsCUC1, OsCUC3, OsTCP1, OsSPL14, and OsSPL17 in the OsCUC1 mutant lines. The results indicated a significant downregulation of miR164 expression, while the expression of OsCUC3 remains unchanged. Both miR319 and its target gene OsTCP1 exhibited notable downregulation in expression. Conversely, miR156 showed significant upregulation in expression, with its target genes OsSPL14 and OsSPL17 displaying marked downregulation (Fig. 2b, c). These findings suggest a regulatory role of OsCUC1 in modulating leaf phenotype-associated miRNAs and their target genes. We then conducted RRSV inoculation experiments on OsCUC1 deletion plants to assess the involvement of OsCUC1 in viral pathogenesis. The findings revealed that the OsCUC1 mutant rice exhibited greater disease severity compared with wild-type (WT) rice after RRSV inoculation (Fig. 2d). Statistical analyses of plant height showed that in both mock and RRSV-infected samples, the heights of OsCUC1 mutant rice plants were shorter than the height of WT rice plants (Fig. 2e). Virus accumulation in OsCUC1 mutant rice plants was greater than virus accumulation in WT rice (Fig. 2f), indicating that OsCUC1 mutation positively regulated RRSV pathogenesis. Given that OsCUC1 is a target of miR164, we further investigated the role of miR164 in viral pathogenesis (Fig. 2g; Fig. S4a). Our findings demonstrated that miR164 overexpression (OE) lines exhibited increased susceptibility to RRSV, whereas miR164 short tandem target mimic (STTM) transgenic lines (mimic) displayed enhanced resistance (Fig. 2h, i). These findings suggested that RRSV infection alters OsCUC1 expression in the SAM, potentially influencing leaf development. Accordingly, RRSV infection can attenuate and suppress the expression of OsCUC1 in the peripheral zone of the SAM, thereby affecting leaf development; genetic disruption of OsCUC1 expression contributes to viral pathogenesis.

Interaction network of OsTCP1, OsCUC1, and OsSPL14/ OsSPL17 proteins in rice

In Arabidopsis, the miR319 target gene TCP4 and miR156 target gene SPL9 jointly regulate the CUC protein complex, thereby dynamically regulates leaf margin serration [28]. To validate the existence of this target gene interaction network in rice, we investigated the interactions of the rice homolog OsTCP1 with CUC and SPL. Through yeast two-hybrid experiments, we confirmed that OsCUC1 can form homodimers，OsTCP1 interacts with OsCUC1, OsSPL14, and OsSPL17 (Fig. 3a). AlphaFold software was used to predict the protein structures of OsCUC1, OsTCP1, OsSPL14, and OsSPL17; rigid and flexible docking analyses were performed to identify interaction interfaces among these proteins. The results indicated that there were hydrogen bonds involving R28, H30, P31, Y40, E35, P25, and G26 was formed between OsCUC1 itself (Fig. 3b). The hydrogen bonds and salt bridges were present between D47, G48, D86, S160, S162 and T163 in OsCUC1 and R108, D119, D122 and R123 in OsTCP1 (Fig. 3c). There were hydrogen bonds involving K131, Q179, and L417 in OsSPL14, as well as R89, A99, and R108 in OsTCP1 (Fig. 3d). OsSPL17 residues involved in hydrogen bonds were T259, D260, S278, and N299, which interacted with A365, R372, Q321, and S262 in OsTCP1; a salt bridge was formed between S399 and L400 in OsSPL17 and D119 and S130 in OsTCP1 (Fig. 3e). Bimolecular fluorescence complementation (BiFC) experiments also confirmed that OsCUC1 can form homodimers, OsTCP1 interacts with OsCUC1, OsSPL14, and OsSPL17 (Fig. 3f). As transcription factors, OsCUC1, OsSPL14, and OsSPL17 are exclusively localized in the cell nucleus; conversely, OsTCP1 is expressed in both the nucleus and cytoplasm, exhibiting substantial regions of cytoplasmic expression (Fig. S5). The interactions of OsTCP1 with OsCUC1, OsSPL14, and OsSPL17 were not limited to the nucleus; fluorescence signals were also observed in the cytoplasm (Fig. 3f). These results indicated that OsTCP1 in rice may also function as an intermediate bridge, interacting with OsCUC1, OsSPL14, and OsSPL17; such interactions influence the nuclear localization and expression patterns of OsCUC1, OsSPL14, and OsSPL17.

In Arabidopsis, both homotypic and heterotypic dimers can form between CUC2 and CUC3, jointly regulating leaf margin serration [28]. To explore the potential conserved functions of CUC in rice, we conducted yeast two-hybrid experiments, which revealed that OsCUC1 and OsCUC3 can and interact with each other to form heterotypic dimers (Fig. S6a-d). As OsCUC1 expression contributes to viral pathogenesis. To explore whether OsCUC3 is involved in RRSV pathogenesis, we obtained OsCUC3 knockout mutant plants (Fig. S6e) and conducted virus inoculation experiments. In both mock and RRSV-infected samples, the height of OsCUC3 mutant plants were shorter than the height of WT rice plants (Fig. S6f, g). Virus accumulation was higher in OsCUC3 mutant rice than in WT rice plants (Fig. S6h), indicating that the absence of OsCUC3 has positive regulatory effects on RRSV pathogenesis. These results collectively demonstrated that OsCUC1 and OsCUC3 can form heterotypic dimers in rice, and both are involved in viral pathogenesis.

OsTCP1 regulates OsCUC1 self-complex formation through cellular localization and interaction with OsSPL14 and OsSPL17

Our previous research has shown that there is complex pairwise interactions among OsCUC1, OsTCP1, OsSPL14/ OsSPL17 proteins (Fig. 3). To further explore these interactions, we first co-expressed OsCUC1 and OsTCP1 proteins. The co-localization of OsCUC1 with OsTCP1 suggested that the signal of OsTCP1 was similar to its original nuclear localization (Fig. S5); the signal of OsCUC1, originally present solely in the nucleus (Fig. S5), was also found in the cytoplasm in a pattern that partially overlapped with OsTCP1 during co-localization (Fig. 4a). These observations suggested a potential role for OsTCP1 in regulating OsCUC1 self-interaction. We then added OsTCP1 to the OsCUC1 interaction system, with the unrelated protein GUS as a control. The results showed that compared to the control, the fluorescence of OsCUC1 self-interaction was weakened after adding OsTCP1 (Fig. 4b). Statistical results also showed that OsTCP1 significantly reduced the fluorescence signal intensity of OsCUC1 self-interaction (Fig. 4c), indicating that OsTCP1 could disrupt OsCUC1 self-interaction. To address the effect of OsSPL14 and OsSPL17 on the interaction between OsTCP1 and OsCUC1, we added OsSPL14 and OsSPL17 to the BiFC system of OsTCP1 and OsCUC1, respectively, with GUS protein as an unrelated control. Both fluorescence observation and statistical data showed that OsSPL14 and OsSPL17 could significantly disrupt the interaction between OsTCP1 and OsCUC1 (Fig. 4d, e). These results suggested that OsTCP1 may regulate OsCUC1 self-interaction in rice by altering subcellular localization or interacting with OsSPL14 and OsSPL17.

MiR319 overexpression and OsTCP1 knockout exacerbate viral infection

To investigate whether miR319 and OsTCP1 participates in viral pathogenesis, we established miR319 OE, mimic lines and OsTCP1 knockout mutant via CRISPR/Cas9 (Fig. S4b; Fig. S7a-c). Phenotypic observations and statistical analyses revealed that the Ostcp1 mutant displayed severe dwarfing and increased tillering compared with WT rice plants (Fig. S7a, c). We used quantitative reverse transcription polymerase chain reaction RT-qPCR) to assess the expression patterns of miR164, miR319, and miR156 in OsTCP1 mutants. The results indicated significant downregulation of the expression levels of these three miRNAs in Ostcp1 mutants compared with WT rice plants (Fig. 5d). The corresponding target genes—OsCUC1, OsCUC3, and OsSPL14—were significantly upregulated in Ostcp1 mutants (Fig. 5e), suggesting that OsTCP1 had negative regulatory effects on OsCUC1, OsCUC3, and OsSPL14. Virus inoculation experiments revealed that, in both mock and RRSV-infected samples, miR319 OE and Ostcp1 mutant plants exhibited more severe dwarfing compared with WT rice plants (Fig. 5a, b, f, g). Viral genome detection showed that virus accumulation levels were higher in miR319 OE and Ostcp1 mutant rice than in WT rice (Fig. 5c, h), indicating that the absence of OsTCP1 has positive regulatory effects on RRSV pathogenesis. These results demonstrated that OsTCP1 negatively regulates OsCUC1 and OsCUC3 in rice, and OsTCP1 loss leads to more severe viral pathogenesis.

MiR156 overexpression and OsSPL14/OsSPL17 knockout exacerbate viral infection

Mutants of OsSPL14 and OsSPL17 were also generated in rice (Fig. S7d-i), both of which displayed a dwarf phenotype (Fig. S7d, g). Initially, the expression levels of miR164, miR319, and miR156, along with their target genes OsCUC1, OsCUC3, OsTCP1, OsSPL14, and OsSPL17, were assessed in the rice mutants of OsSPL14 and OsSPL17. Significant downregulation of miR164 and its target genes OsCUC1 and OsCUC3 was observed in both rice mutants of OsSPL14 and OsSPL17. In the Osspl14 mutant, miR319 was significantly downregulated, with no change observed in the expression level of its target gene OsTCP1. Conversely, in the Osspl17 mutant, miR319 expression remained unchanged, while its target gene OsTCP1 was significantly upregulated. miR156 was significantly downregulated in both mutants of OsSPL14 and OsSPL17, accompanied by significant downregulation of their target genes OsSPL14 and OsSPL17 in their respective mutants (Fig. 6d, e). These findings suggest a potential regulatory role of OsSPL14 and OsSPL17 in leaf phenotypic-associated miRNAs and their target genes.

To access the role of miR156 and targets OsSPL14 and OsSPL17 in RRSV infection, we generated miR156 OE and mimic rice lines (Fig. S4c). Virus inoculation experiments revealed that, in both mock and RRSV-infected samples, miR164 OE and Osspl14/17 mutant plants exhibited more severe dwarfing compared with WT rice plants (Fig. 6a, b, f, g, I, j). Viral genome detection showed that virus levels were higher in miR319 OE and Osspl14/17 mutant rice than in WT rice (Fig. 6c, h, k). Conversely, miR156 mimic rice lines exhibited enhanced virus resistance relative to WT rice plants, characterized by milder symptoms and reduced virus accumulation (Fig. 6a-c), indicating that overexpression of miR156 or the absence of OsTCP1 has positive regulatory effects on RRSV pathogenesis.

This study confirmed that RRSV infection can dynamically regulate the expression patterns of miR164, miR319, and miR156, along with their target genes (OsCUC1, OsTCP1, and OsSPL14/OsSPL17), across different leaf positions (Fig. 1). Furthermore, it induces aberrant expression of OsCUC1 in the peripheral zone of the SAM, thereby disrupting leaf development (Fig. 2). A complex protein interaction network exists among the target proteins OsCUC1, OsTCP1, and OsSPL14/OsSPL17 (Fig. 3), with OsTCP1 serving as a bridge. It dynamically regulates the formation of OsCUC1 dimers by altering its subcellular localization and interactions, thus participating in the formation of leaf margin phenotypes (Fig. 4). Moreover, miRNA164, miRNA319, and miRNA156, along with their target genes OsCUC1, OsTCP1, and OsSPL14/OsSPL17, are all involved in regulating the interaction between rice and the virus (Fig. 2; Fig. 5; Fig. 6). Genetic mutations in OsCUC1, OsTCP1, and OsSPL14/OsSPL17 facilitate RRSV pathogenesis (Fig. 2; Fig. 5; Fig. 6).

Abnormal leaf phenotype development is a typical symptom of viral infection in plant hosts [49-51]. For example, tomato leaf curl New Delhi virus (ToLCNDV) infection in tomato leaves leads to upward leaf curling, a hallmark of disease. There is evidence that miR159/319 and miR172 are associated with leaf curl disease and could potentially be used in viral resistance strategies [52]. Additionally, Arabidopsis expressing turnip mosaic virus (TuMV) HC-Pro reportedly exhibited a series of developmental abnormalities, including serrated leaves. Further analysis suggested that the ethylene-responsive transcription factor RAV2 contributes to the observed defects in leaf shape [53]. However, the regulatory mechanisms underlying virus-induced leaf phenotype abnormalities have been unclear. The present study validated the classical miRNA target network that regulates leaf phenotype development in Arabidopsis; this network dynamically responds to viral infection in rice and contributes to the formation of virus-induced pathogenic leaf phenotypes (Fig. 1). Accordingly, we propose the following working model. Viral infection directly downregulates expression of the core gene OsCUC1 in the SAM peripheral zone. Additionally, miR164, miR319, and miR156, as well as their target genes (OsCUC1, OsTCP1, and OsSPL14) exhibit opposite expression patterns in healthy and diseased leaves, indicating that viral infection disrupts leaf development via regulation of the relevant miRNA target network (Fig. 7). The present findings expand the broader understanding of mechanisms by which viral infection influences abnormal leaf phenotype development.

Previous studies have shown that miR164-CUC [36, 37], miR319-TCP [38, 54], and miR156-SPL [43, 55] are involved in the regulation of leaf morphology. OsCUC1, the rice homolog of AtCUC2 in Arabidopsis, is also involved in regulating leaf shape, demonstrating functional conservation. However, whereas AtCUC2 in Arabidopsis is involved in regulating serration at the leaf margin, rice leaves do not exhibit serration under normal growth conditions. The loss of OsCUC1 in rice hinders normal leaf development, resulting in abnormalities such as deformation and wrinkling [36, 37]. Although miR319-TCP in rice is also involved in the regulation of leaf morphology, its impact differs from the impact of TCP in Arabidopsis, which affects serration on leaf edges [25]. Overexpression of miR319 in rice leads to the downregulation of target genes such as OsPCF5 (OsTCP1), resulting in broader leaves [38]. There is evidence that miR156 expression gradually increases during leaf development and decreases during shoot development; it influences tillering, initiation rate, and early leaf maturity [43]. The present study showed that miR156 content at a given leaf position gradually decreased over time (Fig. S3h). This finding suggests that the regulatory pattern of miR156-SPL during leaf development in rice may differ from the corresponding pattern in Arabidopsis. Although the three miRNAs and their target genes display varying degrees of involvement in the regulation of rice leaf development, their relationships and responses to viral stress are unclear. The present study explored the working model of this regulatory network in rice and its underlying effects on biotic stress.

In Arabidopsis, TCP dynamically regulates the formation of CUC dimers by binding to CUC and SPL, thereby controlling leaf margin morphology [28]. Our results indicate that in rice, OsTCP1 interacts with OsCUC1, OsCUC3, OsSPL14, and OsSPL17 (Fig. 5). The interactions of OsTCP1 with CUC and SPL regulate OsCUC1 dimers and alter the subcellular localizations of these proteins (Fig. 3; Fig. 4). Therefore, we speculate that OsTCP1 modulates the transcription factor functionality of CUC and SPL through interaction-induced changes in protein localization. Analyses of TCP subcellular localization revealed that OsTCP1 exhibits rapid shuttling between the nucleus and cytoplasm (Fig. S8a), possibly exporting or importing proteins with which it interacts. Furthermore, we found that OsTCP1 has a large disordered sequence region at its 5' end (Fig. S8d). We also observed that OsTCP1 exhibits weak phase separation (Fig. S8b, c). These characteristics may help to elucidate the biological implications of protein interactions in rice.

Leaf margin serration development is governed by a complex interplay of plant hormones, miRNAs, transcription factors (including NAM/CUC, TCP, and SPL), the KNOX gene family, and other regulatory factors. These regulatory mechanisms demonstrate distinct patterns in simple and compound leaves. Auxin, a critical phytohormone, plays a pivotal role in initiating leaf margin serrations. The regulatory function of auxin in serration development is primarily dependent on its concentration gradient along the leaf margin. Moreover, several key components of the auxin signaling pathway, including PINFORMED 1 (PIN1)[56, 57], AUXIN RESISTANT 1 (AUX1), LIKE AUXIN RESISTANT 1/2/3 (LAX 1/2/3)[58], and the AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA)[59, 60] gene family, are involved in modulating serration development. RNA-Seq analysis of auxin-related genes (Table S4) revealed that RRSV infection significantly regulates the expression of genes associated with the auxin pathway (Fig. S9a-c). Employing DR5-GUS transgenic materials, we observed that auxin levels in RRSV-infected serrated leaves were significantly reduced compared to mock leaves, and the spatial distribution of auxin was altered (Fig. S9d). These findings imply that auxin may play a role in modulating the pathogenic leaf phenotype associated with RRSV infection.

In conclusion, our findings suggest that miR164, miR319, and miR156, as well as their target genes, are involved in responses to viral infection, influencing the regulation of virus-induced abnormal leaf development phenotypes. These results provide insights into the regulatory mechanisms of leaf development in response to biotic stress. Because leaves are vital developmental organs in plants, and viral infection often influences leaf morphogenesis, a comprehensive understanding of this mechanism will support the cultivation of disease-resistant varieties.

Plant materials and growth conditions

Plant growth and virus inoculation were carried out as previously describe [39, 61]. Rice seedlings were cultivated in a greenhouse at 28–30˚C with a photoperiod of 16 hours of light and 8 hours of darkness or under normal field conditions.

Virus infection and virus strain and vector insect

The 7-day-old rice seedlings were used for virus inoculation with 1–2 viruliferous brown planthoppers (BPH) per plant for 2 days. Subsequently, the insects were removed, and the rice seedlings were maintained under the same growing conditions. Non-viruliferous BPH-inoculated rice plants served as mock controls. At 7, 14, 21, 28, 35, 42, 49, and 56 dpi, the seedlings were photographed and harvested for detection. For inoculation and virus infection statistics, rice seedlings at 4 weeks post-inoculation (wpi) were photographed and harvested to detect viral genomic segments. A minimum of 30 rice seedlings were used for each sample.

Phylogenetic Analysis

The genes of the rice CUC, TCP, SPL gene family, and the Arabidopsis CUC2, CUC3, TCP4, SPL9 were chosen as objects for evolutionary tree analysis. Protein sequences were obtained from the Rice Genome Annotation Project (http://rice.uga.edu/index.shtml) and TAIR (https://www.arabidopsis.org/index.jsp) based on their accession numbers. Sequence alignment was performed, and the phylogenetic tree was constructed using MEGA10 with the maximum likelihood method and bootstrap analysis. The accession numbers are provided in Table S1.

Constructs and transformation

The generation of CRISPR/Cas9 knock out lines and Agrobacterium tumefaciens-mediated rice transformation were carried out in BioRun Co., Ltd. (Wuhan, China). The primers for generating the constructs were listed in Table S5.

RT-qPCR analysis

For RT-qPCR, total RNA was extracted and then reverse transcribed using the StarScript II RT Mix with gDNA Remover (Genstar). The cDNA was used as the template for RT-qPCR. RT-qPCR was performed using the 2×RealStar Fast SYBR qPCR Mix (Genstar) following the manufacturer’s instructions. The rice OsEF1α gene was used as an internal control. Primer sequences in this assay are listed in Table S5.

Subcellular localization and BiFC

We constructed the recombinant eukaryotic expression plasmids p35S::OsCUC1-GFP, p35S::OsCUC3-GFP, p35S::OsTCP1-GFP, p35S::OsSPL14-GFP, p35S::OsSPL17-GFP , and the coding sequences of OsCUC1, OsCUC3, OsTCP1, OsSPL14, OsSPL17 were correspondingly cloned into p35S-Vn, p35S-Vc. The plasmids were co-expressed in rice protoplasts or Nicotiana benthamiana leaf. The subcellular localization and the protein-protein interaction was evaluated using a confocal laser scanning microscope (LSM880, Carl Zeiss, Oberkochen, Germany). Primer sequences for the constructions are listed in Table S5.

Tissue expression analysis

For fresh sections, samples were embedded in 5% agar and cut with a microslicer (50 lm thick). The samples were photographed using a microscope (DS-Ri2, Nikon, Japan).

Yeast two-hybrid assay

The CDS of OsCUC1, OsCUC3, OsTCP1, OsSPL14, OsSPL17 were cloned into pGADT7 and pGBKT7 vector for Y2H. Yeast transformation was carried out according to the instructions (Clontech, Mountain View, California, USA). Yeast AH109 cells were co-transformed with OsCUC1, OsCUC3, OsTCP1, OsSPL14, OsSPL17. All yeast transformants were grown on an SD/-Leu/-Trp and then transferred to SD/-Leu/-Trp/-His/-Ade medium for interaction test.

RNA-sequencing

The total RNA from single-cell sequencing samples was extracted and sent to NovelBio Bio-Pharm Technology Company for RNA sequencing. The original data underwent FastQC testing, followed by the utilization of Trimmomatic to eliminate joints and poor-quality sequences. The minimum reads length was maintained at 75 bp, and a second FastQC assessment was conducted to obtain clean data. The Nipponbare genome (IRGSP 1.0) (http://rapdb.dna.affrc.go.jp/) was selected as the reference genome. Using the HISAT2 software, the data were aligned to the reference genome, producing SAM files, which were then sorted by location and compressed into BAM files using Samtools. Utilizing the annotation file of the reference genome and featureCounts, the exon region of each gene was tallied with reads to derive the expression matrix for each gene. Subsequently, StringTie was employed to normalize gene expression levels, and FPKM was used for quantification.

Protein interaction prediction analysis

After Protein sequences of OsCUC1, OsCUC3, OsTCP1, OsSPL14, OsSPL17 were collected, AlphaFold3 software was used for 3D structure prediction[62]. The obtained cif result files were analyzed using the PDBePISA (https://www.ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver) interaction interface, Finally, all structure drawings are drawn by pymol software (The PyMOL Molecular Graphics System, Version 3.0 Schrödinger, LLC; https://pymol.org).

Acknowledgements

We thank all the members in Wu lab for helpful discussions.

Authors’ contributions

S.Z., and J.W. conceived the idea and designed the projects. L.W., J.Z., S.L. and J.R. performed the yeast two-hybrid related experiments. L.W., J.Z., S.L., and L.Y. performed the BiFC related experiments. L.W., J.R., and W.Y. performed the virus inoculation related experiments. L.W., J.Z., X.Y., X.L. and Salem performed the detection related experiments. L.W., J.L., and C.D. performed the tissue expression analysis related experiments. L.W., and S.Z. analyzed RNA-seq raw data, S.Z., and J.W. wrote the manuscript with input from L.W., Z.W., and Salem. All authors worked collaboratively to edit and revise the paper.

Funding

This work was supported by grants from the National Key R&D Program of China (2021YFD1400500), National Natural Science Foundation of China (32025031, 32170167), National Key R&D Program of China (2023YFF1000500), Hainan Seed Industry Laboratory and China National Seed Group (B23YQ1514, B23CQ15EP).

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Spatial regulation of rice leaf morphology by miRNA-targets complexes during viral infection

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