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Article

WRKY36–PIL15 Transcription Factor Complex Negatively Regulates Sheath Blight Resistance and Seed Development in Rice

by
Siting Wang
1,†,
Qian Sun
1,†,
Shuo Yang
1,
Huan Chen
1,
Depeng Yuan
2,
Changxi Gan
3,
Haixia Chen
3,
Yongxi Zhi
3,
Hongyao Zhu
1,
Yue Gao
1,
Xiaofeng Zhu
1,* and
Yuanhu Xuan
2,*
1
College of Plant Protection, Shenyang Agricultural University, Shenyang 110866, China
2
State Key Laboratory of Elemento-Organic Chemistry and Department of Plant Protection, National Pesticide Engineering Research Center (Tianjin), Nankai University, Tianjin 300071, China
3
Zhengzhou Lvyeyuan Agricultural Technology Co., Ltd., Zhengzhou 450016, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Plants 2025, 14(4), 518; https://doi.org/10.3390/plants14040518
Submission received: 28 December 2024 / Revised: 2 February 2025 / Accepted: 5 February 2025 / Published: 8 February 2025
(This article belongs to the Special Issue The Mechanisms of Plant Resistance and Pathogenesis)
Browse Figures
Versions Notes

Abstract

:
Sheath blight (ShB) causes severe yield loss in rice. Previously, we demonstrated that the sugar will eventually be exported and the transporter 11 (SWEET11) mutation significantly improved rice resistance to ShB, but it caused severe defects in seed development. The present study found that WRKY36 and PIL15 directly activate SWEET11 to negatively regulate ShB. Interestingly, WRKY36 interacted with PIL15, WRKY36 and PIL15 directly activates miR530 to negatively regulate seed development. WRKY36 interacted with a key BR signaling transcription factor WRKY53. AOS2 is an effector protein from Rhizoctonia solani (R. solani) that interacts with WRKY53. Interestingly, AOS2 also interacts with WRKY36 and PIL15 to activate SWEET11 for sugar nutrition for R. solani. These data collectively suggest that WRKY36–PIL15 negatively regulates ShB resistance and seed development via the activation of SWEET11 and miR530, respectively. In addition, WRKY36 and PIL15 are the partners of the effector protein AOS2 by which R. solani hijacks sugar nutrition from rice.

Graphical Abstract

1. Introduction

Host plants have evolved disease resistance strategies to resist pathogens during their interaction. Sheath blight (ShB) in rice, caused by Rhizoctonia solani (R. solani), is the one of the most important diseases in rice, which can cause up to 50% yield loss [1]. Previous studies revealed that the resistance against ShB is controlled by multiple genes. At present, some of the quantitative trait locus (QTL)-linked genes have been cloned and functionally verified [2,3]. Phytohormone signals play an important role in the process of resistance of rice to ShB. Jasmonic acid (JA), salicylic acid (SA), and auxin signals regulate the resistance of rice to ShB [4,5]. Our previous study confirmed that ethylene and brassinosteroid (BR) signals positively and negatively regulate the resistance of rice to ShB, respectively [6]. WRKY transcription factors (TFs) are widely involved in the resistance of rice to ShB [7]. OsWRKY53 negatively regulates ShB resistance by activating SWEET2a [8]. In addition, SlWRKY36 and SlWRKY51 act as positive regulatory factors for salt stress tolerance, regulating tomato salt tolerance [9]. A recent study reported that the effector protein AOS2 from R. solani interacts with the rice TFs OsWRKY53 and OsGT1 and forms a TF complex to activate the expression of OsSWEET2a and OsSWEET3a, thereby promoting the pathogenesis of ShB [10].
The results of the genome-wide association study revealed that the natural variation of ZmFBL41 in maize affected the interaction between ZmFBL41 and ZmCAD, further affecting the host resistance [11]. Our previous study confirmed that OsLPA1 overexpression improves the resistance of rice to ShB [12]. A further study reported that OsLPA1 regulates the resistance of rice to ShB through interaction with OsKLP [13]. OsPhyB and OsLAZY1 negatively regulate the resistance of rice to ShB [14]. OsDEP1 interacts with OsLPA1 and inhibits the transcriptional activation of OsLPA1 downstream genes, thereby inhibiting the resistance of rice to ShB [15]. In addition, the protein phosphatase 2A catalytic subunit (PP2A-1) positively regulates the resistance of rice to ShB [16].
The interaction between plant pathogens and hosts is a complex and prolonged process. Extensive breeding practices have proven that improving host disease resistance often reduces crop yield, which is a trade-off between plant development and disease resistance [17,18]. Therefore, effective disease prevention and control, particularly exploring important genes that can simultaneously increase yield and disease resistance, is of great significance for ensuring national food security. The SA and JA pathways typically interact in an antagonistic manner and interact with the pathways of other plant hormones such as abscisic acid (ABA), gibberellic acid (GA), BR, and auxin to jointly regulate the trade-off between growth and immunity [19,20]. TFs are important factors that balance plant growth and immunity [21]. BZR1 blocks PTI signaling by interacting with different WRKYs or via transcriptional regulation with different WRKYs in Arabidopsis [18]. WRKY forms a transcription protein complex with MVQ (MPK3/6-targeted VQPs) protein, controlling the transcription of defense genes [22]. Our previous study reported that PIL15 negatively regulates rice resistance to ShB [23]. In addition, PIL15 activated miR530, a negative regulator of grain size to negatively regulate grain size [24]. PIL15 directly targets OsPUP7, affecting the transport of cytokinin, thereby affecting cell division and subsequent particle size [25]. The immune receptor NLR and cell-wall-associated kinase (WAK) play a dual role in balancing immunity and production. The two NLR genes at the Pigm locus not only confer resistance to Aspergillus oryzae but also increase yield [26]. Xa4 encodes a WAK and confers race-specific persistent resistance to Xoo without losing production potential [27].
SWEET is a family of sugar transporters. It has the function of bidirectional transport of sugar substances [28] and is involved in regulating plant–pathogen interactions [29]. Most studies have reported that activating the expression of the SWEETs is beneficial for pathogenic bacteria to hijack carbohydrate nutrients from plants to promote their growth [28,30]. In addition, some studies have reported that inhibiting the expression of SWEET can reduce the sugar content in the extracellular space and inhibit the growth of pathogenic bacteria [31]. OsSWEET11/Xa13/Os8N3, OsSWEET13/Xa25, and OsSWEET14/Os11N3 are important genes in rice regulating resistance to Xanthomonas oryzae pv. oryzae [32,33].
Our previous study reported that OsSWEET11 is significantly induced by R. solani infection, and sweet11 mutants are less susceptible to ShB [34]. However, in sweet11 mutants, the sucrose concentration in the embryo sacs significantly decreased and led to defective grain filling, which resulted in significantly lower 100-grain weight in sweet11 mutants than in wild-type plants [35]. In addition, we previously demonstrated Rubisco promoter expressing sucrose transport activity defected SWEET11 mutant protein increased ShB resistance and maintained normal grain filling [34]. Tissue-specific activation of DOF11 promoted rice resistance to ShB via the activation of SWEET14 without yield loss [31]. This suggested that tissue-specific regulation of SWEET expression might achieve the goal of improvement in ShB resistance without developmental defects in rice. Numerous studies have reported that WRKY TFs are widely involved in the resistance of rice to ShB [8,36]. In addition, our previous study reported that PIL15 repressors and overexpressors were less and more susceptible to ShB, respectively [23]. However, the regulation of SWEETs by TFs to modulate plant defense has not been studied in detail.
In this study, we observed that the WRKY36 and PIL15 activate SWEET11. Further, WRKY36 interacted with PIL15 to activate SWEET11, which negatively regulated rice resistance to ShB. In addition, WRKY36–PIL15 activated miR530 to negatively regulate grain size. R. solani effector protein AOS2 interacted with WRKY36 and PIL15 to activate SWEET11 for sugar efflux, via which R. solani activated SWEET11 expression as well as subsequent sugar efflux. Our study revealed the mechanism by which R. solani activated SWEET11 for sugar efflux and identified WRKY36 and PIL15 as the potential molecular targets in ShB-resistant breeding.

2. Results

2.1. WRKY36 and PIL15 Negatively Regulate Resistance of Rice to ShB

To isolate the transcription factor that might specifically regulate SWEET11, the yeast-one hybrid screening was performed using 2.0 kb of SWEET11 promoter as the bait. The results identified 15 putative transcriptional regulators including WRKY36 and PIL15. Previously, WRKYs and PIL15 were reported to regulate rice ShB resistance [7,23]; therefore, WRKY36 and PIL15 were further analyzed in relation to their regulation on SWEET11 transcription. The R. solani-dependent expression patterns revealed that PIL15 was significantly induced, whereas WRKY36 expression had no significant difference in R. solani infection (Figure S1). Therefore, the functions of WRKY36 and PIL15 in the resistance of rice to ShB were further investigated. Via genome editing, wrky36 and pil15 mutants were generated. wrky36-10 and wrky36-11 mutants have 1-bp insertion and 4-bp deletion at the 1st exon, respectively (Figure 1a). After R. solani inoculation, wrky36 mutants were less susceptible to ShB than the wild-type plants (Figure 1b,c). pil15-13 and pil15-14 mutants have 3-bp deletion and 1-bp insertion at the 2nd exon, respectively (Figure 1d). After R. solani inoculation, pil15 mutants were less susceptible to ShB than the wild-type plants (Figure 1e,f).
Since wrky36 and pil15 mutants were less susceptible to ShB, the mechanisms of WRKY36 and PIL15 in ShB resistance were further examined. To further evaluate the defense mechanism, plants overexpressing WRKY36 and PIL15 were tested. WRKY36 expression level was clearly higher in WRKY36 OX 1 and OX 2 than in wild-type plants (Figure 1g). After R. solani inoculation, WRKY36 OX plants were more susceptible to ShB than the wild-type plants (Figure 1h,i). PIL15 expression level was clearly higher in PIL15 eGFP OX 5 and OX 10 than in wild-type plants (Figure 1j). After R. solani inoculation, PIL15 eGFP OX plants were more susceptible to ShB than the wild-type plants (Figure 1k,l). Similarly, the PIL15 overexpression (PIL15 OX) and repressor (PIL15 RD) lines were examined [37]. PIL15 RDs were less susceptible to ShB than the wild-type plants, whereas PIL15 OXs were more susceptible to ShB than the wild-type plants (Figure 1m,n).

2.2. WRKY36 Interacts with PIL15 to Activate SWEET11

Yeast-one hybrid (Y1H) assay results revealed that WRKY36 or PIL15 activates the 2.0-kb SWEET11 promoter (Figure 2a). To verify the Y1H assay results, a transient assay was performed in rice protoplast cells. The results revealed that the co-expression of WRK36-Myc or PIL15-Myc with 2.0 kb of pSWEET11-GUS significantly activated the GUS expression level than the expression of pSWEET11-GUS alone. Western blot analysis was performed to detect the successful expression of WRK36-Myc or PIL15-Myc in the rice protoplast using an anti-Myc antibody (Figure 2b,c). Further, the SWEET11 promoter sequences were analyzed. The results indicated that the SWEET11 promoter region contains (T)TGAC(C/T) W-box (WRKY-binding) and (CACATG) PBE-box (PIL-binding) motifs (Figure 2d). Chromatin immunoprecipitation (ChIP) assay using WRKY36–GFP and PIL15–GFP plant calli as well as anti-GFP antibody revealed that WRKY36 bound to the P2 and P3 regions harboring W-box motif, whereas PIL15 bound to P4 with the enrichment of PBE-box motifs (Figure 2e).
Since both WRKY36 and PIL15 activate SWEET11, the interaction between WRKY36 and PIL15 was investigated. Yeast-two hybrid (Y2H) assay revealed that WRKY36 interacts with PIL15 (Figure 2f). Bimolecular fluorescence complementation (BiFC) assay with WRKY36 and PIL15 expression in Nicotiana benthamiana leaves revealed that WRKY36 interacts with PIL15 in the nucleus (Figure 2g). Co-immunoprecipitation (Co-IP) assay was performed by co-expressing WRKY36–GFP and PIL15-3xFlag or GFP and PIL15-3xFlag. Subsequent immunoprecipitation via anti-Flag antibody as well as Western blot analysis indicated that WRKY36–GFP interacts with PIL15-3xFlag (Figure 2h).
Furthermore, the function of WRKY36–PIL15 in SWEET11 activation was analyzed. The transient assay results revealed that WRKY36 or PIL15 expression activated pSWEET11, whereas co-expression of WRKY36 and PIL15 exhibited a stronger transcription activation activity than the expression of WRKY36 or PIL15 alone (Figure 2i). Further, wrky36/pil15 double mutants and SWEET11 OX/pil15 or SWEET11 OX/wrky36 plants were generated. After R. solani inoculation, pil15 and wrky36 single mutants were less and more susceptible to diseases than wild-type and wrky36/pil15 double mutants, respectively (Figure 2j,k). Inoculation of R. solani showed that SWEET11 OX and SWEET11 OX/pil15 plants exhibited similar lesion length, and SWEET11 OX and SWEET11 OX/pil15 plants were more susceptible than pil15 (Figure 2l,m). Similarly, inoculation of R. solani identified that SWEET11 OX and SWEET11 OX/wrky36 plants developed similar lesion length, and SWEET11 OX and SWEET11 OX/wrky36 plants were more susceptible than wrky36 (Figure 2n,o). In addition, the expression levels of SWEET11 in wrky36/pil15 double mutants and SWEET11 OX/pil15 or SWEET11 OX/wrky36 leaf sheaths were also detected. The results showed that the expression level of SWEET11 in pil15 and wrky36 single mutants were lower than that in the wild-type and higher than that in wrky36/pil15 double mutants (Figure S2a).
The expression levels of SWEET11 in SWEET11 OX and SWEET11 OX/pil15 leaf sheaths were similar, and SWEET11 OX and SWEET11 OX/pil15 plants were higher than pil15 and wild-type plants (Figure S2b). Similarly, the expression levels of SWEET11 in SWEET11 OX and SWEET11 OX/wrky36 leaf sheaths were similar, and SWEET11 OX and SWEET11 OX/wrky36 plants were higher than wrky36 and wild-type plants (Figure S2c). Meanwhile, the expression levels of SWEET11 in PIL15 eGFP OX and WRKY36 OX leaf sheaths were also detected. The results showed that the expression level of SWEET11 in PIL15 eGFP OX and WRKY36 OX plants was higher than that in wild-type plants (Figure S2d).

2.3. WRKY36 and PIL15 Negatively Regulate Seed Development

WRKY36–PIL15 activates SWEET11 to regulate resistance of rice to ShB, and sweet11 mutants exhibited defective seed development. Therefore, the seed development of WRKY36 and PIL15 mutants or overexpression plants was assessed. First, the plant height of wrky36 mutants and wild-type plants was similar (Figure 3a,b). Interestingly, wrky36 mutants developed relatively longer seeds than wild-type plants (Figure 3c,d); however, the width of seeds of wrky36 mutants and wild-type plants were similar (Figure 3e). Additionally, 1000-grain weight of wrky36 mutants was higher than that of wild-type plants (Figure 3f). However, there is no difference in the seed setting ration, panicle number per plant, and seed number per panicle between wrky36 mutants and wild-type plants (Figure 3g–i). The height of WRKY36 OX plants was significantly shorter than that of wild-type plants (Figure 3j,k). WRKY36 OX plants developed smaller seeds than wild-type plants. The seed length of WRKY36 OX plants was shorter, whereas the width was narrower than wild-type plants (Figure 3l–n). This developmental feature resulted in lower 1000-grain weight in WRKY36 OX than in wild-type plants (Figure 3o). WRKY36 OX plants have fewer seed numbers per panicle compared to wild-type plants (Figure 3p). However, there is no difference in the seed setting ration and panicle number per plant between WRKY36 OX and wild-type plants (Figure 3q,r).
First, the plant heights of pil15 RD mutants, PIL15 OX plants and wild-type plants were detected (Figure 4a,b). pil15 RD mutants have a shorter plant height than the wild-type plants, while PIL15 OX plants show no difference. Next, the function of PIL15 in rice seed development was analyzed. The seed length and width of pil15 mutants were relatively longer, whereas those of PIL15 eGFP OX plants were relatively shorter than those of wild-type plants (Figure 4c–e). These developmental differences resulted in higher 100-grain weight of pil15 mutants and lower 100-grain weight of PIL15 eGFP OX than that of wild-type plants (Figure 4f). In addition, compared with the wild-type plants, PIL15 eGFP OX plants have fewer seed number per panicle, while pil15 mutants has more seed number per panicle (Figure 4g). However, the seed setting ration and panicle number per plant of pil15 mutants, PIL15 eGFP OX and wild-type plants were similar (Figure 4h,i). Similarly, PIL15 OXs and PIL15 RDs were examined. PIL15 RDs developed larger seeds, whereas PIL15 OXs developed smaller seeds than wild-type plants (Figure 4j–l), and the 100-grain weight was higher in PIL15 RDs but lower in PIL15 OXs than in wild-type plants (Figure 4m). In addition, compared with wild-type plants, PIL15 OXs have fewer seed numbers per panicle, while PIL15 RDs have more seed numbers per panicle (Figure 4n). However, the seed setting ration and panicle numbers per plant of pil15 RDs, PIL15 OXs and wild-type plants were similar (Figure 4o,p).

2.4. WRKY36–PIL15 Directly Activates miR530

WRKY36 and PIL15 activate SWEET11 to regulate the resistance of rice to ShB, and sweet11 mutants exhibited defective seed development; therefore, the expression levels of SWEET11 in the seeds of WRKY36 and PIL15 mutants were examined. The results showed that there was no difference in the expression level of SWEET11 between WRKY36 and PIL15 mutants compared to wild-type plants (Figure S3). It is proved that WRKY36 and PIL15 might regulate the expression of SWEET11 in leaf sheaths, but not in the seeds. A previous study revealed that PIL15 activates miR530 to negatively regulate grain size [24]. To analyze whether WRKY36 also activates miR530, miR530 promoter sequences were analyzed. The W-box motif was observed within 2.0 kb of miR530 promoter region [38,39] (Figure 5a). The Y1H assay results indicated that both WRKY36 and PIL15 activate pmiR530 (Figure 5b). To verify the results of Y1H assay, the transient assay was performed. In the protoplast cells, WRKY36 or PIL15 activated pmiR530 but not W-box (mwpmiR530)- or G-box (mgpmiR530)-mutated pmiR530. Additionally, co-expression of WRKY36 and PIL15 exhibited an additive effect on the activation of pmiR530 (Figure 5c). ChIP-qPCR results further confirmed that WRKY36 and PIL15 bound to the miR530 promoter (Figure 5d,e). To analyze miR530 expression levels in wkry36 mutants and WRKY36 OX plants, the miR530 expression levels were assessed in the developing caryopsis of wrky36 mutants, WRKY36 OX, and wild-type plants. The RT-qPCR results revealed that the miR530 expression level was significantly lower in wrky36 mutants but higher in WRKY36 OX plants than in wild-type plants (Figure 5f).

2.5. AOS2 Interacts with WRKY36 and PIL15 to Activate SWEET11

Plants overexpressing WRKY36 exhibited an enlarged lamina joint angle (Figure 3g), a typical BR signaling phenotype in rice [40]. Further, Y2H assay revealed that WRKY36 interacts with WRKY53 (Figure 6a), the key BR signaling TF [41]. BiFC assay results confirmed that WRKY36 and WRKY53 interact in the nucleus of tobacco cells (Figure 6b). In addition, Co-IP assay results indicated that 3xFlag-WRKY53 interacts with WRKY36–GFP but not with GFP, indicating that WRKY36 interacts with WRKY53 (Figure 6c). After R. solani inoculation, wrky53 mutants were less susceptible to the ShB than wild-type plants (Figure 6d,e). However, wrky53 mutant seeds were smaller, and WRKY53 OX seeds were bigger than wild-type seeds, which was different from the results of wrky36 mutants (Figure 6f–i) [41]. In addition, the interaction between PIL15 and WRKY53 was assessed. The results of the Y2H assay revealed that WRKY53 interacts with PIL15 (Figure 6j). The BiFC results confirmed that WRKY53 interacts with PIL15 in the nucleus of tobacco cells (Figure 6k). However, unlike WRKY36 and PIL15, WRKY53 did not directly activate the SWEET11 promoter (Figure S4). The results indicated that WRKY53 might play a similar function as that of WRKY36 and PIL15 in the resistance of rice to ShB but not in grain development.
Recently, we identified that the R. solani effector protein AOS2 interacts with WRKY53 to activate SWEET2a and SWEET3a for sugar nutrition in pathogens [10]. Since WRKY36 and PIL15 interact with WRKY53, the interaction between AOS2 and WRKY36 as well as AOS2 and PIL15 was assessed. The results of the Y2H assay revealed that AOS2 interacts with WRKY36 and PIL15 (Figure 7a). The BiFC results confirmed that AOS2 interacts with WRKY36 and PIL15 in the nucleus of tobacco cells (Figure 7b). In addition, the Co-IP assay results indicated that 3xFlag-WRKY36 interacts with AOS2-GFP but not with GFP, indicating that WRKY36 interacts with AOS2 (Figure 7c). Spray-induced gene silencing (SIGS) of AOS2 inhibited R. solani-induced SWEET11 expression [10,42] (Figure 7d,e). To investigate whether AOS2–WRKY36–PIL15 form a transcriptional complex to activate SWEET11, the transient assay was performed. The results indicated that AOS2, WRKY36, and PIL15 activate pSWEET11, and the co-expression of AOS2–WRKY36–PIL15 revealed higher activation against pSWEET11 than the expression of AOS2, WRKY36, or PIL15 alone (Figure 7f).

3. Discussion

In any infection, the goal of pathogens is to hijack nutrients from the host. Extensive studies revealed that SWEET sugar transporters are the targets of various pathogens [43,44,45]. Pathogenic infection activates SWEET expression during the early infection stage to obtain sugars from host plants; therefore, inhibition of SWEET activation during pathogen infection could successfully control disease [10,34]. However, SWEET also plays key roles in grain filling. The mutation of SWEET genes exhibited defects in grain development [46]. Some studies reported that the mutation of TAL-binding sequences in SWEET gene promoters successfully improved the resistance of rice to bacterial blight without any growth defect [47]. However, the TFs that regulate SWEET in a tissue-specific manner to balance rice resistance and normal growth have not been studied in detail.

3.1. WRKY36–PIL15–AOS2 Activates SWEET11 to Efflux Sugar

SWEET11 negatively regulated the resistance of rice to ShB, suggesting that SWEET11 is a susceptible gene to ShB; however, it positively controlled the grain filling. To assess whether SWEET11 expression is tissue specific, the Y1H assay was conducted between SWEET11 and the TFs WRKY36 and PIL15 that regulate resistance to R. solani. The Y1H assay revealed that WRKY36 and PIL15 bind to the promoter to activate SWEET11. After R. solani inoculation, wrky36 and pil15 mutants were less susceptible whereas WRKY36 OXs and PIL15 OXs were more susceptible to ShB. Further genetic combinations revealed that wrky36/pil15 was less susceptible to ShB than wrky36 and pil15. In addition, overexpression of SWEET11 in wrky36 or pil15 background increased the wrky36 or pil15 ShB-resistant phenotype, and the lesion lengths on SWEET11 OX/wrky36 and SWEET11 OX/pil15 were similar with on SWEET11 OX. These data suggested that WRKY36 and PIL15 might activate SWEET11 to increase susceptibility of ShB. Furthermore, WRKY36 interacted with PIL15 to form a transcriptional complex, and WRKY36 and PIL15 had synergistic effects on the activation of SWEET11. SWEET11 and PIL15 expressions are sensitive to R. solani inoculation, while WRKY36 expression level is not altered by R. solani infection. However, WRKY36 activates SWEET11 expression, suggesting that WRKY36 protein might be post-translationally modified upon R. solani infection to increase its activity or stability to form more stable transcriptional complex harboring WRKY36–PIL15. WRKY36 OX plants exhibited an enlarged lamina joint angle, a typical BR signaling phenotype. Further screening revealed that WRKY36 interacts with WRKY53, a key BR signaling TF. However, wrky36 mutant is different from wrky53 mutant [41,48]; it developed normal plant height, suggesting that WRKY36 might has functional redundancy with other WRKY genes for regulating BR signaling. Furthermore, WRKY53 interacted with PIL15; however, pil15 mutants and PIL15 OX plants did not exhibit BR signaling phenotype. However, WRKY36, PIL15, and WRKY53 negatively regulated the resistance of rice to ShB. These data suggested that WRKY36–PIL15–WRKY53 might form a transcriptional complex to regulate defense of rice to ShB. Recently, we demonstrated that AOS2, an effector protein secreted from R. solani, interacts with WKRY53 to activate SWEET2a and SWEET3a to hijack sugar nutrition from rice [10]. Interestingly, AOS2 also interacts with WRKY36 and PIL15 in the nucleus to activate SWEET11, and suppression of AOS2 expression using the SIGS approach inhibited the R. solani-infection-mediated induction of SWEET11. These data suggested that R. solani secrets effector protein AOS2 into the nucleus of a plant cell; further, AOS2–WRKY36–PIL15 transcriptional complex activates sucrose transporter SWEET11 for obtaining sugars from rice plants (Figure 8a). Since transcription factor complex involving AOS2 activates multiple SWEETs, the expression levels of SWEET2a and SWEET3a in PIL15 and WRKY36 mutants and overexpressing plants were detected. The RT-qPCR results showed that the expression of SWEET2a and SWEET3a in PIL15 and WRKY36 mutants and overexpressing plants did not exhibit a certain pattern. In addition, the expression levels of SWEET11 in WRKY53 mutants, overexpressing plants, and gt1 mutant plants were also detected. RT-qPCR results showed that the expression level of SWEET11 in WRKY53 overexpressing plants was higher than that in wild-type plants, but there was no difference between wrky53 mutant and wild-type plants. Meanwhile, there was no difference in the expression level of SWEET11 between the gt1 mutant and wild-type plants (Figure S5), implying that R. solani effector AOS2 might interact with different rice transcription factors to activate different sets of SWEETs for sugar nutrition. These findings further implied the complex regulation of R. solani and rice interaction, and surprisingly, a single effector AOS2 could control glucose and sucrose efflux from rice by hijacking different transcriptional complexes.

3.2. WRKY36–PIL15–miR530 Signaling Negatively Regulates Grain Size

The sweet11 mutant exhibited improved resistance to ShB but defective grain filling. WRKY36 and PIL15 activate SWEET11 to negatively regulate the resistance of rice to ShB; therefore, seed development was examined in the mutants. Unlike sweet11 mutant, wrky36 and pil15 mutants developed larger seeds than wild-type plants. The seed length of wrky36 mutant was longer but the width was the same compared with wild-type plants. The seed length and width of pil15 mutant were longer than those of wild-type plants. The seeds of WRKY36 OX and PIL15 OX were smaller than the wild-type seeds. These developmental features resulted in higher 1000-grain weight of wrky36 mutants, higher 100-grain weight of pil15 mutants, lower 1000-grain weight of WRKY36 OX and lower 100-grain weight of PIL15 OX than that of their corresponding wild-types. The seed setting ration, panicle numbers per plant, and seed numbers per panicle of wrky36 mutants were similar to the wild-type plants. WRKY36 OX plants have fewer seed numbers per panicle compared to the wild-type plants, but the seed setting ration and panicle numbers per plant of wrky36 mutants were similar to the wild-type plants. The seed setting ration and panicle number per plant of pil15 mutants or pil15 RDs and PIL15 eGFP OX or PIL15 OXs were similar than wild-type plants. Compared with wild-type plants, PIL15 eGFP OX or PIL15 OXs have fewer seed numbers per panicle, while pil15 mutants or pil15 RDs have more seed numbers per panicle, suggesting that WRKY36 and PIL15 are specifically inhibit seed size in rice.
WRKY53 positively regulates seed size by activating BR signaling, suggesting the differential regulation of seed development by WRKY53 and WRKY36. MicroRNA (miRNA) is a type of non-coding small RNA that regulates gene expression by altering the translation efficiency or stability of targeted mRNA. It plays an important role in regulating plant growth, development, and response to pathogens [49,50]. Previously, PIL15 was reported to activate miR530, a negative regulator of seed development [24]. Promoter sequence analysis indicated that W-box and G-box appear in the miR530 promoter. Y1H, transient, and ChIP assays confirmed that WRKY36 bound to miR530 promoter to activate its transcription. In addition, miR530 expression level was significantly lower in the developing caryopsis of wrky36 mutants but higher in WRKY36 OX plants, which further confirmed the transcriptional activation of WRKY36 to miR530 in plants. These data suggested that WRKY36–PIL15–SWEET11 in the green tissues negatively regulated the resistance of rice to ShB, and WRKY36–PIL15–miR530 in the seeds negatively regulated grain size (Figure 8b).

3.3. The Potential Application of WRKY36–PIL15 in ShB-Resistant Breeding

Increasing yield and resistance are some of the main goals of crop breeding. However, the signaling pathways of resistance and yield are often antagonistically regulated [18]. Our study demonstrated that WRKY36 and PIL15 activate SWEET11 and miR530 in the green tissue and seeds, respectively. wrky36 and pil15 mutants exhibited increased resistance to ShB and yield by controlling grain size, respectively. This suggested that WRKY36 and PIL15 might be the molecular targets for ShB-resistant rice breeding. wrky36 and pil15 mutants exhibited increased yield under the normal condition; however, we believe they will have advantage under R. solai infection. Currently, the genome sequence of rice core resources is available, and Crispr/Cas9-mediated genome editing technique is improving fast. Therefore, identification of useful gene sources for resistance and yield trade-off will be helpful for the precise design of molecular breeding. During domestication and improvement, rice evolved genetically to adapt the environmental changes as well as improve yield. In the rice population, SNP and indel (insertion and deletion) occurred in a high frequency, which makes different traits of rice cultivars [51]. In this study, wrky36 and pil15 mutants exhibited resistance to ShB and developed large grain; therefore, investigation of elite haplotypes of WRKY36 and PIL15 with relatively lower expression levels will provide useful gene source for rice breeding. Integration of those minor effect genes could generate elite cultivars by cross, a general breeding approach in rice. Further, maize sheath blight causes severe yield effects, and it is also caused by R. solani AG1-IA, the same pathogen causing rice ShB; and some mechanisms are close between rice and maize [11]. Therefore, the results we identified in rice might be able to be phenocopied in maize, which could help maize breeders to increase efficiency. Lastly, our study provides a useful approach for simultaneously improving rice yield and ShB resistance, and the mechanism could be useful for crop breeding.

4. Materials and Methods

4.1. Plant Materials and Growth Conditions

Wild-type (WT) (Oryza sativa L. Nipponbare, Zhonghua 11, and Longjing 11), sweet11 mutants (Nip), PIL15 OXs (Nip), pil15 RDs (Nip), wrky36 mutants (ZH11), WRKY36 OXs (ZH11), pil15 mutants (ZH11), PIL15 eGFP OXs (ZH11), wrky53 (LJ11), and WRKY53 OXs (LJ11) were used in this study. The plants were inoculated with R. solani AGI-1A and grown for approximately 2 months in a greenhouse at 30–24 °C (day and night). The tobacco used for injection (N. benthamiana) was grown for approximately 1 month in a 22 °C light incubator (16 h day/8 h night).

4.2. Pathogens

R. solani AG1-IA was cultured on potato dextrose agar (PDA) medium. The 0.4 × 0.8 cm2 of wood bark was placed on the edge of a 90 mm PDA medium. Further, 1 cm2 of fungal mass was placed at the center. Fungal mass is used for leaf inoculation, while wood bark is used for leaf sheath inoculation. The Petri dish was incubated in dark at 30 °C for approximately 2 days. The infected bark was placed at the angle between the penultimate leaf sheath of rice. Image J 1.53e software was used to measure the typical lesion length and lesion area after 1–7 days of inoculation (https://imagej.nih.gov/ij/, accessed on 8 September 2023). Six leaf sheaths or leaves from each line were analyzed, and the experiments were repeated three times. The data in disease spot statistical chart are presented as the mean ± SE (n > 10).

4.3. Generation of Transgenic Rice Plants

To generate overexpression lines, Ubi: WRKY36 and 35S: PIL15 were transformed into ZH11 plant calli. CRISPR/Cas9-generated mutants (sweet11, wrky36, and pil15) were used. PIL15 OXs (Nip), pil15 RD (Nip), wrky53 (LJ11), and WRKY53 OX (LJ11) were generated as described previously [37,52]. A specific 300-bp fragment from WRKY36 was cloned into the pH7GWIWG2(II) vector and then transformed into pil15 to construct wrky36/pil15 double mutants. SWEET11 was cloned into the pCambia1381-Ubi vector and then transformed into the pil15 to construct SWEET11 OX/pil15. SWEET11 was cloned into the pCambia1381-Ubi vector and then transformed into the wrky36 to construct SWEET11 OX/wrky36.

4.4. Yeast-One Hybrid Assay

In the study, all pHISi-1 carriers were linearized using XhoI before transformation. The 2.0 kb of SWEET11 promoter sequences (XhoI site was mutated) and rice cDNA TF library (Ouyibio, Shanghai, China) were cloned into pHISi-1 and pGAD424 to obtain pSWEET11-pHISi-1 and pGAD424-TF yeast vectors, respectively. The pGAD424-TF plasmid was used to transform YM4271 yeast strain carrying the linearized pSWEET11-pHISi-1. Two DNA fragments containing two adjacent G-box and one normal G-box element in the 2.0 kb of miR530 promoter were inserted into pHISi-1 to obtain the pmiR530 A-pHISi-1 and pmiR530 B-pHISi-1 plasmids, respectively. Similarly, four fragments predicted on the 2.0 kb of miR530 promoter, each containing one W-box, were inserted into pHISi-1 to obtain the pmiR530 a/b/c/d -pHISi-1 plasmids, respectively. The coding sequences of PIL15 and WRKY36 were cloned into the pGAD424 vector. The recombinant pGAD424-WRKY36/PIL15 plasmid was used to transform YM4271 yeast strain carrying the linearized pmiR530 A/B/a/b/c/d-pHISi-1. pGAD424 empty vector was used as the control. The analysis of the interaction between proteins and DNA was conducted on synthetic dropout (SD) -Leu or -His media containing various concentrations of 3-aminotriazole (3-AT). The relevant primer information is given in Table S1.

4.5. Yeast-Two Hybrid Assay

The coding sequences of WRKY36, PIL15, WRKY53, and AOS2 were cloned into the yeast expression vector pGAD424 or pGBT9. The recombinant pGAD424-PIL15/WRKY36/WRKY53 plasmid was used to transform AH109 yeast strain. The pGBT9-WRKY36/WRKY53/AOS2 plasmid was used to transform Y187 yeast strain. The diploid yeast cells were grown on SD/-Leu-Trp, -His, or -Leu-Trp-His media containing various concentrations of 3-AT to screen for interactions between proteins after mating. The relevant primer information is given in Table S1.

4.6. Co-IP

The coding sequences of PIL15, WRKY36, AOS2, and WRKY53 were cloned into the pGD3G3Flag and pGD3GGM vectors, respectively, to generate the PIL15-3xFlag, WRKY36–GFP, AOS2-GFP, 3xFlag-WRKY36 and 3xFlag-WRKY53 plasmids. All plasmids were used to transform Agrobacterium GV3101 and expressed in N. benthamiana leaves by co-infiltration. The injected leaves were placed in dark for 2 days. Co-IP experiment was conducted as described previously [23]. The relevant primer information is given in Table S1.

4.7. BiFC Assay

The PIL15, WRKY36, and AOS2 were fused with N-terminus of YFP in PXNGW, PIL15, WRKY36, WRKY53 were fused with C-terminus of CFP in PXCGW. PIL15–nYFP, WRKY36–nYFP, AOS2-nYFP, WRKY36–cYFP, WRKY53–cYFP [10], and PIL15–cYFP were generated. The corresponding vector was transferred into Agrobacterium tumefaciens strain GV3101. The strain was grown overnight on LB medium containing appropriate antibiotics at 28 °C. Collect bacteria, resuspend them in buffer (10 mM MES, pH 5.6, 10 mM MgCl2, and 150 µM acetosyringone), and then incubate at room temperature for 3 h before inoculation. The tobacco leaves were manually injected. Incubate plants at room temperature under continuous low light for 2 to 4 days [53]. The relevant primer information is given in Table S1.

4.8. Microscopic Observation

A. tumefaciens-infected N. benthamiana leaves were analyzed at 48 h after injection. Leaf discs were placed on a slide and visualized using a X36 objective lens on an inverted microscope (TOKYO Corporation, Tokyo, Japan). GFP and CFP fluorescence were observed using an excitation wavelength of 488 nm, while RFP was observed using an excitation wavelength of 561 nm. When observed under a microscope, both GFP and RFP fluorescence channels are opened. The appearance of green fluorescence indicates the presence of interactions between proteins. H2B-RFP is used as a nuclear localization marker. If GFP and RFP undergo fluorescence merging, it indicates that the protein interaction is in the nucleus.

4.9. Transactivation Assay

Effectors 35S: PIL15, 35S: WRKY36, reporter pSWEET11 GUS, and internal control 35S: LUC were transformed into protoplasts [54]. The β-glucuronidase (GUS) activity was detected using the GUS assay kit (Promega, Madison, WI, USA). The expression of luciferase (LUC) was detected using a luciferase assay kit (Promega, Madison, WI, USA) [55,56]. The relevant primer information is given in Table S1.

4.10. RNA Isolation and RT-qPCR

Total RNA was extracted from rice seedlings or inoculated leaves using RNAiso Plus reagent (Takara, Dalian, China), and genomic DNA was removed. cDNA was synthesized using the PrimeScript RT reagent kit with gDNA Eraser (Takara, Dalian, China) as per the manufacturer’s instructions. RT-qPCR was performed using SYBR qPCR Master Mix (Vazyme, Nanjing, China). The relative expression levels were calculated using the 2−ΔΔCT method [57]. The relevant primer information is given in Table S1. Ubiquitin was used as the internal control for RT-qPCR. The data are presented as the mean ± SE (n = 3).

4.11. Chromatin Immunoprecipitation (ChIP)-qPCR Assay

According to the reference paper, three-week-old overexpressing rice plants with GFP tags were subjected to chromatin immunoprecipitation. DNA was used for ChIP-qPCR analysis [58]. The relevant primer information is given in Table S1.

4.12. SIGS Assay

One-month-old rice was sprayed with enzyme-free water and dsAOS2, and inoculated with R. solani. Sample and extract RNA from 0 to 72 h after inoculation to detect the expression levels of AOS2 and SWEET11. The relevant primer information is given in Table S1.

4.13. Lamina Joint Assay

The 1 cm of the flag leaf, the lamina joint, and 1 cm of the leaf sheath were selected with good growth conditions at the rice heading stage. The angles of lamina joint bending were measured using Image J 1.53e software (https://imagej.nih.gov/ij/, accessed on 8 September 2023) [59].

4.14. Production Shape Assay

For the statistical analyses of seed length and width, more than 100 seeds from WRKY36, PIL15, and WRKY53 mutants and overexpressions were calculated. For the statistical analyses of plant height, number of effective grains per main panicle, panicle number per plant, and seed number per panicle, the number of statistical samples for WRKY36 and PIL15 mutants and overexpressions were 20 plants.

4.15. Statistical Analysis

Prism 8 was used for statistical analysis (GraphPad, San Diego, CA, USA). All data were expressed as mean ± standard error. The comparison between different groups was conducted through one-way analysis of variance. Significance was determined using Student’s t-test. p < 0.05 was considered significant. Significant differences: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 (Student’s t-test). Different letters above the bars denote statistically significant differences (p < 0.05).

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants14040518/s1, Figure S1. Screening of WRKY36 and PIL15. The expression levels of the 15 putative transcription factors were analysed after 0, 12, 24 and 48 h of R. solani infection. Ten leaves were sampled at each time point for RNA extraction. Figure S2. SWEET11 expression levels in wrky36 and pil15 single mutants, wrky36/pil15 double mutants, SWEET11 OX, SWEET11 OX/pil15 or SWEET11 OX/wrky36, and PIL15 OX or WRKY36 OX plant leaf sheaths by RT-qPCR. (a) The expression of SWEET11 in WT, pil15, wrky36, and wrky36/pil15 was examined using RT-qPCR. (b) The expression of SWEET11 in WT, pil15, SWEET11 OX, and SWEET11 OX/pil15 was examined using RT-qPCR. (c) The expression of SWEET11 in WT, wrky36, SWEET11 OX, and SWEET11 OX/wrky36 was examined using RT-qPCR. (d) The expression of SWEET11 in WT, PIL15 eGFP OXs, and WRKY36 OXs was examined using RT-qPCR. Statistical differences between groups were analyzed using the Student’s t-test (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns indicates nonsignificant). Different letters indicate significant differences between groups at p < 0.05. Figure S3. SWEET11 expression level in wrky36 and pil15 single mutant seeds by RT-qPCR. (a) The expression of SWEET11 in WT, wrky36-10, and wrky36-11 was examined using RT-qPCR. (b) The expression of SWEET11 in WT, pil15 RD 2, and pil15 RD 22 was examined using RT-qPCR. Statistical differences between groups were analyzed using the Student’s t-test (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns indicates nonsignificant). Different letters indicate significant differences between groups at p < 0.05. Figure S4. Y1H assay of the binding of WRKY53 to the SWEET11 promoter. The 2.0-kb region of SWEET11 promoter sequences (XhoI site was mutated) and the WRKY53 coding sequences were cloned into pHISi-1 and pGAD424 to obtain pSWEET11-pHISi-1 and pGAD424-WRKY53 yeast vectors, respectively. The pGAD424-WRKY53 plasmid was used to transform the Y2M4271 yeast strain carrying the linearized pSWEET11-pHISi-1. pGAD424 empty vector was used as control. The analysis of the interaction between proteins and DNA was conducted on synthetic dropout (SD) -Leu or -His media containing different concentrations of 3-aminotriazole (3-AT). The relevant primer information is shown in Table S1. Figure S5. SWEET11 expression levels in WRKY53 mutants, overexpressed plants, and gt1 mutants. SWEET2a and SWEET3a expression levels in WRKY36 and PIL15 mutants and overexpressed plant leaf sheaths. (a) The expression of SWEET11 in LJ11, WRKY53 OX, and wrky53 was examined using RT-qPCR. (b) The expression of SWEET2a in WT, WRKY36 OXs, and wrky36 was examined using RT-qPCR. (c) The expression of SWEET3a in WT, WRKY36 OXs, and wrky36 was examined using RT-qPCR. (d) The expression of SWEET2a in WT, PIL15 eGFP OXs, and pil15s was examined using RT-qPCR. (e) The expression of SWEET3a in WT, PIL15 eGFP OXs, and pil15s was examined using RT-qPCR. (f) The expression of SWEET3a in WT, PIL15 OXs, and pil15 RDs was examined using RT-qPCR. (g) The expression of SWEET2a in WT, PIL15 OXs, and pil15 RDs was examined using RT-qPCR. (h) The expression of SWEET11 in WT and gt1 was examined using RT-qPCR. Statistical differences between groups were analyzed using the Student’s t-test (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns indicates nonsignificant). Different letters indicate significant differences between groups at p < 0.05. Table S1. Primers used in this study.

Author Contributions

S.W.: Investigation, Data curation, Writing—original draft. Q.S.: Investigation, Validation, Formal analysis. S.Y.: Data curation, Writing—review and editing. H.C. (Huan Chen): Data curation, Writing—review and editing. D.Y.: Data curation, Writing—review and editing. C.G.: Data curation, Writing—review and editing. H.C. (Haixia Chen): Validation, Formal analysis. Y.Z.: Validation, Formal analysis. H.Z.: Validation, Formal analysis. S.W.: Writing—review and editing. Y.G.: Methodology, Investigation, Validation. Y.X. and X.Z.: Funding acquisition, Conceptualization, Supervision, Project administration, Writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the National Natural Science Foundation of China (32372482, 32072406, 32272499 and 32302379); the Natural Science Foundation of Liaoning Province (2021-MS-223); the Natural Science Foundation of the Education Department of Liaoning Province (LJKZ0641).

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

Conflicts of Interest

Authors Changxi Gan, Haixia Chen and Yongxi Zhi were employed by Zhengzhou Lvyeyuan Agricultural Technology Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Figure 1. Response of WRKY36 and PIL15 to Rhizoctonia solani AG1-1A. (a,d) Mutation site sequence information of WRKY36 and PIL15 genome-editing mutants generated using CRISPR/Cas9. (b) WT and wrky36 mutants were inoculated with R. solani. (c) The length of lesions on the leaf sheath surface was measured. (e) WT and pil15 mutants were inoculated with R. solani. (f) The length of lesions on the leaf sheath surface was measured. (g) The expression of WRKY36 in WT and WRKY36 OXs was examined using RT-qPCR. (h) WT and WRKY36 OXs were inoculated with R. solani. (i) The length of lesions on the leaf sheath surface was measured. (j) The expression of PIL15 in WT and PIL15 eGFP OXs was examined using RT-qPCR. (k) WT and PIL15 eGFP OXs were inoculated with R. solani. (l) The length of lesions on the leaf sheath surface was measured. (m) WT, PIL15 OXs and pil15 RDs were inoculated with R. solani. (n) The length of lesion on the leaf sheath surface was measured. Statistical differences between groups were analyzed using the Student’s t-test (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).
Figure 1. Response of WRKY36 and PIL15 to Rhizoctonia solani AG1-1A. (a,d) Mutation site sequence information of WRKY36 and PIL15 genome-editing mutants generated using CRISPR/Cas9. (b) WT and wrky36 mutants were inoculated with R. solani. (c) The length of lesions on the leaf sheath surface was measured. (e) WT and pil15 mutants were inoculated with R. solani. (f) The length of lesions on the leaf sheath surface was measured. (g) The expression of WRKY36 in WT and WRKY36 OXs was examined using RT-qPCR. (h) WT and WRKY36 OXs were inoculated with R. solani. (i) The length of lesions on the leaf sheath surface was measured. (j) The expression of PIL15 in WT and PIL15 eGFP OXs was examined using RT-qPCR. (k) WT and PIL15 eGFP OXs were inoculated with R. solani. (l) The length of lesions on the leaf sheath surface was measured. (m) WT, PIL15 OXs and pil15 RDs were inoculated with R. solani. (n) The length of lesion on the leaf sheath surface was measured. Statistical differences between groups were analyzed using the Student’s t-test (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).
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Figure 2. Confirmation of activation of SWEET11 by WRKY36 or PIL15, and the interaction between WRKY36 and PIL15. (a) Yeast-one hybrid assay of the binding of PIL15 or WRKY36 to the SWEET11 promoter. (b) Reporter, effector, and internal control vector diagram. (c) Transactivation assays verified that WRKY36 and PIL15 activated SWEET11 expression. (d) The putative W-box and PBE-box elements in the 2.0-kb region of the SWEET11 promoter. (e) ChIP-qPCR assay showing that WRKY36 and PIL15 directly bind to the W-box and PBE-box motif in the SWEET11 promoter, respectively. The promoter fragment of SWEET11 containing the W-box motif (P2, P3) was enriched in the ChIP-qPCR analysis rather than in the negative control (P1). Similarly, the promoter fragment of SWEET11 containing the PBE-box motif (P4) was enriched in the ChIP-qPCR analysis rather than in the negative control (P1). (f) Yeast-two hybrid assay verified the interaction between PIL15 and WRKY36. (g) BiFC assay verified the interaction between PIL15 and WRKY36 in N. benthamiana leaves. Co-transformation of PIL15-nYFP and WRKY36–cCFP led to the reconstitution of GFP signal, whereas no signal was detected when WRKY36–cCFP and nYFP were co-expressed. Co-transformation of cCFP and nYFP was used as negative control. (h) Co-IP assays indicated the interaction between PIL15 and WRKY36 in plants. PIL15-3xFlag and WRKY36–GFP were co-expressed in N. benthamiana leaves. The co-expression of PIL15-3xFlag and GFP was used as a negative control. (i) The transient assay showed that WRKY36 and PIL15 both activated SWEET11 expression in an additive manner. The promoter of the SWEET11-driven reporter gene was co-expressed with WRKY36 or PIL15 individually or together. (j) WT, pil15, wrky36, wrky36/pil15 plants were inoculated with R. solani. (k) The length of lesions on the leaf sheath was measured. (l) WT, pil15, SWEET11 OX, SWEET11 OX/pil15 plants were inoculated with R. solani. (m) The length of lesions on the leaf sheath was measured. (n) WT, wrky36, SWEET11 OX, SWEET11 OX/wrky36 plants were inoculated with R. solani. (o) The length of lesions on the leaf sheath was measured. Statistical differences between groups were analyzed using the Student’s t-test (* p < 0.05; **** p < 0.0001). Different letters above the bars indicate significant differences at p < 0.05.
Figure 2. Confirmation of activation of SWEET11 by WRKY36 or PIL15, and the interaction between WRKY36 and PIL15. (a) Yeast-one hybrid assay of the binding of PIL15 or WRKY36 to the SWEET11 promoter. (b) Reporter, effector, and internal control vector diagram. (c) Transactivation assays verified that WRKY36 and PIL15 activated SWEET11 expression. (d) The putative W-box and PBE-box elements in the 2.0-kb region of the SWEET11 promoter. (e) ChIP-qPCR assay showing that WRKY36 and PIL15 directly bind to the W-box and PBE-box motif in the SWEET11 promoter, respectively. The promoter fragment of SWEET11 containing the W-box motif (P2, P3) was enriched in the ChIP-qPCR analysis rather than in the negative control (P1). Similarly, the promoter fragment of SWEET11 containing the PBE-box motif (P4) was enriched in the ChIP-qPCR analysis rather than in the negative control (P1). (f) Yeast-two hybrid assay verified the interaction between PIL15 and WRKY36. (g) BiFC assay verified the interaction between PIL15 and WRKY36 in N. benthamiana leaves. Co-transformation of PIL15-nYFP and WRKY36–cCFP led to the reconstitution of GFP signal, whereas no signal was detected when WRKY36–cCFP and nYFP were co-expressed. Co-transformation of cCFP and nYFP was used as negative control. (h) Co-IP assays indicated the interaction between PIL15 and WRKY36 in plants. PIL15-3xFlag and WRKY36–GFP were co-expressed in N. benthamiana leaves. The co-expression of PIL15-3xFlag and GFP was used as a negative control. (i) The transient assay showed that WRKY36 and PIL15 both activated SWEET11 expression in an additive manner. The promoter of the SWEET11-driven reporter gene was co-expressed with WRKY36 or PIL15 individually or together. (j) WT, pil15, wrky36, wrky36/pil15 plants were inoculated with R. solani. (k) The length of lesions on the leaf sheath was measured. (l) WT, pil15, SWEET11 OX, SWEET11 OX/pil15 plants were inoculated with R. solani. (m) The length of lesions on the leaf sheath was measured. (n) WT, wrky36, SWEET11 OX, SWEET11 OX/wrky36 plants were inoculated with R. solani. (o) The length of lesions on the leaf sheath was measured. Statistical differences between groups were analyzed using the Student’s t-test (* p < 0.05; **** p < 0.0001). Different letters above the bars indicate significant differences at p < 0.05.
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Figure 3. WRKY36 regulates seed development. (a) Phenotypes of 3-month-old wrky36 mutants and WT plants. Scale bar = 20 cm. (b) The height of plants was measured. (c) Grains with hulls from wrky36 and WT. Scale bar = 1 cm. (d) Seed length of wrky36 and WT. (e) Seed width of wrky36 and WT. (f) The 1000-grain weight of wrky36 and WT. (g) Number of effective grains per panicle in wrky36 and WT. (h) Panicle number per plant of wrky36 and WT. (i) The seed setting rate (%) of wrky36 and WT. (j) Phenotypes of 3-month-old WRKY36 OXs and WT plants. Scale bar = 20 cm. (k) The height of plants was measured. (l) Grains with hulls from WRKY36 OXs and WT. Scale bar = 1 cm. (m) Seed length of WRKY36 OXs and WT. (n) Seed width of WRKY36 OXs and WT. (o) The 1000-grain weight of WRKY36 OXs and WT. (p) Number of effective grains per panicle in WRKY36 OX and WT. (q) Panicle number per plant of WRKY36 OX and WT. (r) The seed setting rate (%) of WRKY36 OX and WT. The data in (b,d–i,k,m–r) are presented as the mean ± SE (b,g–i,k,p–r, n = 20 plants; c–f and l–o, n > 100 seeds; f,o, n = 3 replicates). Statistical differences between groups were analyzed using the Student’s t-test (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns indicates nonsignificant).
Figure 3. WRKY36 regulates seed development. (a) Phenotypes of 3-month-old wrky36 mutants and WT plants. Scale bar = 20 cm. (b) The height of plants was measured. (c) Grains with hulls from wrky36 and WT. Scale bar = 1 cm. (d) Seed length of wrky36 and WT. (e) Seed width of wrky36 and WT. (f) The 1000-grain weight of wrky36 and WT. (g) Number of effective grains per panicle in wrky36 and WT. (h) Panicle number per plant of wrky36 and WT. (i) The seed setting rate (%) of wrky36 and WT. (j) Phenotypes of 3-month-old WRKY36 OXs and WT plants. Scale bar = 20 cm. (k) The height of plants was measured. (l) Grains with hulls from WRKY36 OXs and WT. Scale bar = 1 cm. (m) Seed length of WRKY36 OXs and WT. (n) Seed width of WRKY36 OXs and WT. (o) The 1000-grain weight of WRKY36 OXs and WT. (p) Number of effective grains per panicle in WRKY36 OX and WT. (q) Panicle number per plant of WRKY36 OX and WT. (r) The seed setting rate (%) of WRKY36 OX and WT. The data in (b,d–i,k,m–r) are presented as the mean ± SE (b,g–i,k,p–r, n = 20 plants; c–f and l–o, n > 100 seeds; f,o, n = 3 replicates). Statistical differences between groups were analyzed using the Student’s t-test (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns indicates nonsignificant).
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Figure 4. PIL15 regulates seed development. (a) Phenotypes of 3-month-old PIL15 RD mutants, PIL15 OXs and WT plants. Scale bar = 20 cm. (b) The height of plants was measured. (c) Grains with hulls from pil15 mutants, PIL15 eGFP OXs, and WT. Scale bar = 1 cm. (d) Seed length of pil15 mutants, PIL15 eGFP OXs, and WT. (e) Seed width of pil15 mutants, PIL15 eGFP OXs, and WT. (f) The 100-grain weight of pil15 mutants, PIL15 eGFP OXs, and WT. (g) Number of effective grains per panicle in pil15 mutants, PIL15 eGFP OXs, and WT. (h) Panicle number per plant of pil15 mutants, PIL15 eGFP OXs, and WT. (i) The seed setting rate (%) of pil15 mutants, PIL15 eGFP OXs, and WT. (j) Grains with hulls from pil15 RDs, PIL15 OXs, and WT. Scale bar = 1 cm. (k) Seed length of pil15 RDs, PIL15 OXs, and WT. (l) Seed width of pil15 RDs, PIL15 OXs, and WT. (m) The 100-grain weight of pil15 RDs, PIL15 OXs, and WT. (n) Number of effective grains per panicle in pil15 RDs, PIL15 OXs, and WT. (o) Panicle number per plant of pil15 RDs, PIL15 OXs, and WT. (p) The seed setting rate (%) of pil15 RDs, PIL15 OXs, and WT. The data in (b–i,k–p) are presented as the mean ± SE (b,g–i,n–p, n = 20 plants; c–f,j–m, n > 100 seeds; f,m, n = 3 replicates). Statistical differences between groups were analyzed using the Student’s t-test (* p < 0.05; ** p < 0.01; *** p < 0.001; ns indicates nonsignificant).
Figure 4. PIL15 regulates seed development. (a) Phenotypes of 3-month-old PIL15 RD mutants, PIL15 OXs and WT plants. Scale bar = 20 cm. (b) The height of plants was measured. (c) Grains with hulls from pil15 mutants, PIL15 eGFP OXs, and WT. Scale bar = 1 cm. (d) Seed length of pil15 mutants, PIL15 eGFP OXs, and WT. (e) Seed width of pil15 mutants, PIL15 eGFP OXs, and WT. (f) The 100-grain weight of pil15 mutants, PIL15 eGFP OXs, and WT. (g) Number of effective grains per panicle in pil15 mutants, PIL15 eGFP OXs, and WT. (h) Panicle number per plant of pil15 mutants, PIL15 eGFP OXs, and WT. (i) The seed setting rate (%) of pil15 mutants, PIL15 eGFP OXs, and WT. (j) Grains with hulls from pil15 RDs, PIL15 OXs, and WT. Scale bar = 1 cm. (k) Seed length of pil15 RDs, PIL15 OXs, and WT. (l) Seed width of pil15 RDs, PIL15 OXs, and WT. (m) The 100-grain weight of pil15 RDs, PIL15 OXs, and WT. (n) Number of effective grains per panicle in pil15 RDs, PIL15 OXs, and WT. (o) Panicle number per plant of pil15 RDs, PIL15 OXs, and WT. (p) The seed setting rate (%) of pil15 RDs, PIL15 OXs, and WT. The data in (b–i,k–p) are presented as the mean ± SE (b,g–i,n–p, n = 20 plants; c–f,j–m, n > 100 seeds; f,m, n = 3 replicates). Statistical differences between groups were analyzed using the Student’s t-test (* p < 0.05; ** p < 0.01; *** p < 0.001; ns indicates nonsignificant).
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Figure 5. Confirmation of activation of miR530 by WRKY36–PIL15. (a) Putative W-box and G-box elements in the 2.0-kb region of the miR530 promoter. (b) Yeast-one hybrid assay of the binding of PIL15 and WRKY36 to the miR530 promoter. (c) The transient assay showed that WRKY36 and PIL15 both activated miR530 expression in an additive manner. WRKY36 and PIL15 cannot activate miR530 transcription with W-box and G-box site mutations in the miR530 promoter (mwpmiR530 and mgpmiR530). (d) ChIP-qPCR assay showing that WRKY36 directly bind to the W-box motif in the miR530 promoter. The promoter fragment of miR530 containing the W-box motif (P1, P2, and P4) was enriched in the ChIP-qPCR analysis rather than in the negative control (P3). (e) ChIP-qPCR assay showing that PIL15 directly bind to the G-box motif in the miR530 promoter. The promoter fragment of miR530 containing the G-box motif (P3, P4) was enriched in the ChIP-qPCR analysis. (f) Expression of miR530 (miR530 precursor) in wkry36, WRKY36 OX and WT plants. Ubiquitin was used as the internal control for RT-qPCR. The data in (c–f) are presented as the mean ± SE (n = 3). Statistical differences between groups were analyzed using the Student’s t-test (* p < 0.05; ** p < 0.01; *** p < 0.001). Different letters above the bars indicate significant differences at p < 0.05.
Figure 5. Confirmation of activation of miR530 by WRKY36–PIL15. (a) Putative W-box and G-box elements in the 2.0-kb region of the miR530 promoter. (b) Yeast-one hybrid assay of the binding of PIL15 and WRKY36 to the miR530 promoter. (c) The transient assay showed that WRKY36 and PIL15 both activated miR530 expression in an additive manner. WRKY36 and PIL15 cannot activate miR530 transcription with W-box and G-box site mutations in the miR530 promoter (mwpmiR530 and mgpmiR530). (d) ChIP-qPCR assay showing that WRKY36 directly bind to the W-box motif in the miR530 promoter. The promoter fragment of miR530 containing the W-box motif (P1, P2, and P4) was enriched in the ChIP-qPCR analysis rather than in the negative control (P3). (e) ChIP-qPCR assay showing that PIL15 directly bind to the G-box motif in the miR530 promoter. The promoter fragment of miR530 containing the G-box motif (P3, P4) was enriched in the ChIP-qPCR analysis. (f) Expression of miR530 (miR530 precursor) in wkry36, WRKY36 OX and WT plants. Ubiquitin was used as the internal control for RT-qPCR. The data in (c–f) are presented as the mean ± SE (n = 3). Statistical differences between groups were analyzed using the Student’s t-test (* p < 0.05; ** p < 0.01; *** p < 0.001). Different letters above the bars indicate significant differences at p < 0.05.
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Figure 6. Confirmation of the interaction between WRKY36 and WRKY53. (a) Yeast-two hybrid assay verified the interaction between WRKY53 and WRKY36. (b) BiFC assay indicated the interaction between WRKY53 and WRKY36 in N. benthamiana leaves. Co-transformation of WRKY36–nYFP and WRKY53–cCFP led to the reconstitution of GFP signal, whereas no signal was detected when WRKY53–cCFP and nYFP were co-expressed. Co-transformation of cCFP and nYFP was used as negative control. (c) Co-IP assays indicated the interaction between WRKY53 and WRKY36 in plants. 3xFlag-WRKY53 and WRKY36–GFP were co-expressed in N. benthamiana leaves. The co-expression of 3xFlag-WRKY53 and GFP was used as a negative control. (d) WT and wrky53 mutants were inoculated with R. solani. (e) The length of lesions on the leaf sheath surface was measured. (d) Six leaf sheaths from each line were analyzed, and the experiments were repeated three times. The data in (e) are presented as the mean ± SE (n > 10). (f) Grains with hulls from wrky53, WRKY53 OX, and WT. Scale bar = 1 cm. (g) Seed length of wrky53, WRKY53 OX, and WT. (h) Seed width of wrky53, WRKY53 OX, and WT. (i) The 100-grain weight of wrky53, WRKY53 OX, and WT. The data in (g–i) are presented as the mean ± SE. (f–h, n > 100 seeds; i, n = 3 replicates). (j) Yeast-two hybrid assay verified the interaction between WRKY53 and PIL15. (k) BiFC assay indicated the interaction between WRKY53 and PIL15 in N. benthamiana leaves. Co-transformation of PIL15-nYFP and WRKY53-cCFP led to the reconstitution of GFP signal, whereas no signal was detected when WRKY53-cCFP and nYFP were co-expressed. Co-transformation of cCFP and nYFP was used as negative control. Statistical differences between groups were analyzed using the Student’s t-test (* p < 0.05; ** p < 0.01; *** p < 0.001).
Figure 6. Confirmation of the interaction between WRKY36 and WRKY53. (a) Yeast-two hybrid assay verified the interaction between WRKY53 and WRKY36. (b) BiFC assay indicated the interaction between WRKY53 and WRKY36 in N. benthamiana leaves. Co-transformation of WRKY36–nYFP and WRKY53–cCFP led to the reconstitution of GFP signal, whereas no signal was detected when WRKY53–cCFP and nYFP were co-expressed. Co-transformation of cCFP and nYFP was used as negative control. (c) Co-IP assays indicated the interaction between WRKY53 and WRKY36 in plants. 3xFlag-WRKY53 and WRKY36–GFP were co-expressed in N. benthamiana leaves. The co-expression of 3xFlag-WRKY53 and GFP was used as a negative control. (d) WT and wrky53 mutants were inoculated with R. solani. (e) The length of lesions on the leaf sheath surface was measured. (d) Six leaf sheaths from each line were analyzed, and the experiments were repeated three times. The data in (e) are presented as the mean ± SE (n > 10). (f) Grains with hulls from wrky53, WRKY53 OX, and WT. Scale bar = 1 cm. (g) Seed length of wrky53, WRKY53 OX, and WT. (h) Seed width of wrky53, WRKY53 OX, and WT. (i) The 100-grain weight of wrky53, WRKY53 OX, and WT. The data in (g–i) are presented as the mean ± SE. (f–h, n > 100 seeds; i, n = 3 replicates). (j) Yeast-two hybrid assay verified the interaction between WRKY53 and PIL15. (k) BiFC assay indicated the interaction between WRKY53 and PIL15 in N. benthamiana leaves. Co-transformation of PIL15-nYFP and WRKY53-cCFP led to the reconstitution of GFP signal, whereas no signal was detected when WRKY53-cCFP and nYFP were co-expressed. Co-transformation of cCFP and nYFP was used as negative control. Statistical differences between groups were analyzed using the Student’s t-test (* p < 0.05; ** p < 0.01; *** p < 0.001).
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Figure 7. AOS2–WRKY36–PIL15 transcriptional complex activates SWEET11. (a) Yeast-two hybrid assay verified the interaction of WRKY53, PIL15, and WRKY36 with AOS2. (b) BiFC assay indicated the interaction of WRKY36 and PIL15 with AOS2 in N. benthamiana leaves. Co-transformation of AOS2-nYFP and WRKY36–cCFP or AOS2-nYFP and PIL15-cCFP led to the reconstitution of GFP signal, whereas no signal was detected when AOS2-nYFP and cCFP were co-expressed. Co-transformation of cCFP and nYFP was used as negative control. (c) Co-IP assays indicated the interaction between WRKY36 and AOS2 in plants. 3xFlag-WRKY36 and AOS2-GFP were co-expressed in N. benthamiana leaves. The co-expression of 3xFlag-WRKY36 and GFP was used as a negative control. (d) RT-qPCR analysis of AOS2 gene expression levels 0, 24, 48, and 72 h after R. solani inoculation. Mock was treated with RNase-free water. Rs-α-tubulin was used as the internal control for RT-qPCR. (e) RT-qPCR analysis of SWEET11 gene expression levels 0, 24, 48, and 72 h after R. solani inoculation. Mock was treated with RNase-free water. Ubiquitin was used as the internal control for RT-qPCR. (f) The incidence of disease in leaves treated with mock (RNase free water) or dsAOS2 after inoculation with R. solani at 24, 48, and 72 h. (g) The statistical results of % of lesion area covered on the leaves shown were calculated. (h) The transient assay showed that WRKY36, PIL15, and AOS2 both activated SWEET11 expression in an additive manner. The data in (d–f) are presented as the mean ± SE (n = 3). Statistical differences between groups were analyzed using the Student’s t-test (* p < 0.05; ** p < 0.01; **** p < 0.0001; ns indicates nonsignificant). Different letters above the bars indicate significant differences at p < 0.05.
Figure 7. AOS2–WRKY36–PIL15 transcriptional complex activates SWEET11. (a) Yeast-two hybrid assay verified the interaction of WRKY53, PIL15, and WRKY36 with AOS2. (b) BiFC assay indicated the interaction of WRKY36 and PIL15 with AOS2 in N. benthamiana leaves. Co-transformation of AOS2-nYFP and WRKY36–cCFP or AOS2-nYFP and PIL15-cCFP led to the reconstitution of GFP signal, whereas no signal was detected when AOS2-nYFP and cCFP were co-expressed. Co-transformation of cCFP and nYFP was used as negative control. (c) Co-IP assays indicated the interaction between WRKY36 and AOS2 in plants. 3xFlag-WRKY36 and AOS2-GFP were co-expressed in N. benthamiana leaves. The co-expression of 3xFlag-WRKY36 and GFP was used as a negative control. (d) RT-qPCR analysis of AOS2 gene expression levels 0, 24, 48, and 72 h after R. solani inoculation. Mock was treated with RNase-free water. Rs-α-tubulin was used as the internal control for RT-qPCR. (e) RT-qPCR analysis of SWEET11 gene expression levels 0, 24, 48, and 72 h after R. solani inoculation. Mock was treated with RNase-free water. Ubiquitin was used as the internal control for RT-qPCR. (f) The incidence of disease in leaves treated with mock (RNase free water) or dsAOS2 after inoculation with R. solani at 24, 48, and 72 h. (g) The statistical results of % of lesion area covered on the leaves shown were calculated. (h) The transient assay showed that WRKY36, PIL15, and AOS2 both activated SWEET11 expression in an additive manner. The data in (d–f) are presented as the mean ± SE (n = 3). Statistical differences between groups were analyzed using the Student’s t-test (* p < 0.05; ** p < 0.01; **** p < 0.0001; ns indicates nonsignificant). Different letters above the bars indicate significant differences at p < 0.05.
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Figure 8. Speculative model shows the PIL15-WRKY36 transcription complex regulates ShB resistance and seed development in rice (Extension from [10]). (a) The AOS2 secreted by Rhizoctonia solani and WRKY36–PIL15 form a transcription factor complex, activating SWEET11 in the nucleus. Sucrose is exported from the cytoplasm to the extracellular vesicles through SWEET11, and is metabolized into glucose and fructose through cell wall invertase, which are utilized by Rhizoctonia solani. (b) The PIL15 and WRKY36 transcription factor complexes negatively regulate rice grain size and resistance to ShB by activating miR530 and SWEET11. If the activations of miR530 and SWEET11 by PIL15 and WRKY36 are inhibited, rice yield and ShB resistance are improved.
Figure 8. Speculative model shows the PIL15-WRKY36 transcription complex regulates ShB resistance and seed development in rice (Extension from [10]). (a) The AOS2 secreted by Rhizoctonia solani and WRKY36–PIL15 form a transcription factor complex, activating SWEET11 in the nucleus. Sucrose is exported from the cytoplasm to the extracellular vesicles through SWEET11, and is metabolized into glucose and fructose through cell wall invertase, which are utilized by Rhizoctonia solani. (b) The PIL15 and WRKY36 transcription factor complexes negatively regulate rice grain size and resistance to ShB by activating miR530 and SWEET11. If the activations of miR530 and SWEET11 by PIL15 and WRKY36 are inhibited, rice yield and ShB resistance are improved.
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MDPI and ACS Style

Wang, S.; Sun, Q.; Yang, S.; Chen, H.; Yuan, D.; Gan, C.; Chen, H.; Zhi, Y.; Zhu, H.; Gao, Y.; et al. WRKY36–PIL15 Transcription Factor Complex Negatively Regulates Sheath Blight Resistance and Seed Development in Rice. Plants 2025, 14, 518. https://doi.org/10.3390/plants14040518

AMA Style

Wang S, Sun Q, Yang S, Chen H, Yuan D, Gan C, Chen H, Zhi Y, Zhu H, Gao Y, et al. WRKY36–PIL15 Transcription Factor Complex Negatively Regulates Sheath Blight Resistance and Seed Development in Rice. Plants. 2025; 14(4):518. https://doi.org/10.3390/plants14040518

Chicago/Turabian Style

Wang, Siting, Qian Sun, Shuo Yang, Huan Chen, Depeng Yuan, Changxi Gan, Haixia Chen, Yongxi Zhi, Hongyao Zhu, Yue Gao, and et al. 2025. "WRKY36–PIL15 Transcription Factor Complex Negatively Regulates Sheath Blight Resistance and Seed Development in Rice" Plants 14, no. 4: 518. https://doi.org/10.3390/plants14040518

APA Style

Wang, S., Sun, Q., Yang, S., Chen, H., Yuan, D., Gan, C., Chen, H., Zhi, Y., Zhu, H., Gao, Y., Zhu, X., & Xuan, Y. (2025). WRKY36–PIL15 Transcription Factor Complex Negatively Regulates Sheath Blight Resistance and Seed Development in Rice. Plants, 14(4), 518. https://doi.org/10.3390/plants14040518

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