<scp>ScWRKY6</scp> Interacts With <scp>ScSAG39</scp> to Regulate Immune Homeostasis by Transcriptional Control of <i>ScPR1</i>

Sugarcane (Saccharum spp.) is essential for global sugar and bioenergy production, but its yield and quality are severely threatened by fungal diseases (Ling et al. 2025). Plant defence against pathogens is primarily regulated by transcription factors (TFs) (Buscaill and Rivas 2014), among which WRKYs can act as positive or negative immune regulators (Huang et al. 2022). We previously reported that ScWRKY4 interacts with ScJAZ13 to suppress JA signalling and immune gene expression, increasing susceptibility to pathogens (Wang et al. 2024). More recently, we found that ScWRKY2 reduces resistance to smut disease by interacting with the chloroplast protein ScPsbP and inducing ROS scavenging genes (Wang et al. 2025). These findings indicate that WRKY TFs play diverse roles in sugarcane immunity. However, their contribution to immune homeostasis during fungal infection remains unclear. Here, we identified the sugarcane ScWRKY6, a class II-d WRKY TF, which contains two nuclear localization signals (NLSs), two nuclear export signals (NESs), a conserved zinc finger motif, and a WRKY domain (Figure 1A,B; Figure S1A–C; Table S1). Its expression is markedly induced by smut, pokkah boeng, and brown stripe diseases, suggesting its potential role in the broad-spectrum antifungal response of sugarcane (Figure 1C). Notably, ScWRKY6 was revealed as a nuclear protein that promotes intracellular ROS accumulation, as indicated by elevated ROS-scavenging gene expression and stronger H2DCF-DA (2′, 7′-dichlorodihydrofluorescein diacetate) fluorescence (Figure 1D,E; Figure S1D). These results suggest that ScWRKY6 may function as a regulator in sugarcane response to fungal pathogens. Subsequently, a rice (Oryza sativa)–Magnaporthe oryzae system was established to investigate the regulatory role of ScWRKY6 in fungal disease resistance using genetic and biochemical approaches (Figure S1E–G). Interestingly, ScWRKY6-OE transgenic plants exhibited fewer disease lesions and significantly higher H2O2 accumulation than wild-type (WT) plants after inoculation with the M. oryzae strain Guy11 (Figure 1F,G). In punch inoculation assays, lesion lengths in ScWRKY6-OE plants were significantly shorter than those in WT (Figure 1G,H). Besides, the expression level of the defence-related gene OsPR1 was markedly higher in ScWRKY6-OE plants at 48 h post-inoculation (Figure 1I), indicating that ScWRKY6 could positively regulate rice resistance to M. oryzae. To elucidate the role of ScWRKY6 in rice blast resistance, we performed RNA-seq on 12 cDNA libraries from WT and ScWRKY6-OE1 plants inoculated with (T) or without M. oryzae (CK) (Figure S2A; Table S2). A total of 4947 and 1838 DEGs were identified in the WT-CK_vs_WT-T and ScWRKY6-CK_vs_ScWRKY6-T groups, respectively (Figure S2B–E). Among them, the up-regulated genes in WT-CK_vs_WT-T were primarily associated with fundamental metabolic and biosynthetic pathways. In contrast, the up-regulated genes in ScWRKY6-CK_vs_ScWRKY6-T were predominantly enriched in pathways related to plant immune responses (Figure S2F,G; Table S3). Interestingly, ScWRKY6 overexpression activates the phenylpropanoid biosynthesis pathway, with most related DEGs up-regulated, including key enzymes like PAL, C4H, and 4CL (Figure S3). Surprisingly, but reasonably, numerous TFs and resistance (R) genes, such as NLR-, RLK- and WAK-type genes, were specifically up-regulated in ScWRKY6-OE lines (Figure S4; Tables S4 and S5), indicating that ScWRKY6 enhances rice defence against M. oryzae by promoting secondary metabolism and R gene expression. Furthermore, we showed that ScWRKY6 could up-regulate the expression of OsPR1 to mediate plant immune responses (Figure 1I). To determine whether it directly regulates PR1, the promoter of the homologous gene ScPR1, which was transcriptionally active and inducible upon Sporisorium scitamineum infection, was cloned from sugarcane (Figure S5A–C). This promoter contained two W-box motifs, one of which (probe1) was specifically bound by ScWRKY6 (Figure 1J,K; Figure S5D). Dual-luciferase and ChIP-qPCR assays further demonstrated that ScWRKY6 could activate ScPR1 transcription (Figure 1L; Figure S6). These results indicate that ScWRKY6 directly binds to the ScPR1 promoter, most probably contributing to the pathogen-induced expression of ScPR1. To identify the potential ScWRKY6-interacting proteins involved in disease responses, a yeast two-hybrid (Y2H) screen was conducted using a sugarcane cDNA library from smut-infected buds (Figure S7A). They were mainly involved in arabinose metabolism, protein degradation, and cysteine protease activity (Figure S7B). Among them, ScSAG39, a cysteine protease gene down-regulated after S. scitamineum infection (Figure S8A–C), was selected for further study. Overexpression of ScSAG39 in Nicotiana benthamiana aggravated disease symptoms and reduced H2O2 accumulation, implying a negative role in plant defence. This was further supported by altered expression of ROS- and HR-related genes (Figure S8D–F). We also found that ScSAG39 was an endoplasmic reticulum–associated protein (Figure S8G). Its interaction with ScWRKY6 was confirmed by Y2H, BiFC, and Co-IP assays (Figure S9). Notably, co-expression assays showed that changes in ScSAG39 abundance altered the nuclear localization of ScWRKY6 (Figure 1M–O). ScSAG39 restricted the nuclear import of ScWRKY6, and reduced ScSAG39 levels allowed more ScWRKY6 to accumulate in the nucleus (Figure 1N; Figure S9A). In addition, ScSAG39 also inhibited the activation of ScPR1 by competitively blocking ScWRKY6 binding to the W-box element (Figure 1P), and its co-expression with ScWRKY6 led to a marked decrease in luciferase activity driven by the ScPR1 promoter (Figure 1Q; Figure S10). Furthermore, both ScSAG39 and the W-box element interacted with the same key residue, lysine 310 of ScWRKY6 (Figure S11), which informed us that ScSAG39 competitively occupied the site required for transcriptional activation of ScPR1. Overall, this study demonstrates the positive role of sugarcane ScWRKY6 in enhancing fungal disease resistance. ScWRKY6 interacts with ScSAG39. Reduced ScSAG39 levels lead to greater nuclear accumulation of ScWRKY6 and activation of ScPR1, whereas high ScSAG39 levels inhibit ScPR1 expression by interfering with ScWRKY6 binding to the W-box element, thereby modulating plant immune responses (Figure 1R). These findings provide novel insights into the regulation of plant immune homeostasis. Y.Q., Q.W., and Y.Z. conceived and designed the project. S.Z., D.W., L.Q., S.C., and Q.H. analyzed the data. S.Z., G.W., K.L., Q.D., and T.S. performed the experiments. S.Z. and Q.W. wrote the manuscript draft. Y.Q., Q.W., Y.Z., and Y.S. revised the manuscript. The RNA-seq data have been deposited at Beijing Institute of Genomics Data Center (http://bigd.big.ac.cn), accession number is PRJCA036085. Tables S1–S6. pbi70444-sup-0001-TableS1–S6.xlsx. Figures S1–S11. pbi70444-sup-0002-FigureS1–S11.docx. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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