A hypersensitive response‐like lesion‐inducing protein modulates basal immunity against grey leaf spot in maize

Crop diseases cause substantial yield losses, posing a significant threat to global food security (Savary et al., 2019). In maize, grey leaf spot (GLS), caused by the fungal pathogens Cercospora zeina and Cercospora zeae-maydis, is a devastating foliar fungal disease capable of reducing yield by 11–69%, depending on the susceptibility of the hybrid cultivar and environmental conditions (Meisel et al., 2009; Mueller et al., 2020). Given the limitations of chemical control, genetic resistance is the most effective strategy for managing plant diseases, emphasizing the importance of elucidating the molecular mechanisms underlying pathogen resistance in maize. Hypersensitive response (HR)-like lesion-inducing proteins (HRLs) are a group of proteins containing a conserved HR-like lesion-inducing domain, yet their functions and mechanisms in plant–pathogen interactions remain largely unknow. In rice, OsHRL expression is induced by Xanthomonas oryzae pv. oryzae (Xoo) infection, and its overexpression enhances resistance to Xoo (Park et al., 2010). In soybean, GmHRL1 interacts with the Soybean mosaic virus (SMV) P3 protein, facilitating its nuclear translocation (Luan et al., 2019). Additionally, in tobacco, NbHRLI4 has been reported to induce cell death and negatively regulate turnip mosaic virus (TuMV) infection through a salicylic acid-mediated pathway (Wu et al., 2021). We recently cloned a major GLS resistance gene, ZmWAKL, encoding a cell wall-associated receptor kinase-like protein, and identified its co-receptor ZmWIK via immunoprecipitation-mass spectrometry (IP-MS) (Zhong et al., 2024). To further explore the ZmWAKL immune complex, the resistant ZmWAKLY was screened against the cDNA library of the disease-resistant inbred line Y32 using the split-ubiquitin yeast two-hybrid (Y2H) system. Fourteen proteins were identified as potential interactors, among which a HR-like lesion-inducing protein (ZmHRL, Zm00001d025020) was repeatedly detected (Table S1). Intriguingly, ZmHRL was not captured in our previous IP-MS analysis. Yeast assays confirmed that most candidates, except for Zm00001d051405, bound to ZmWAKLY but showed weak or no interaction with ZmWAKLQ (Figure 1a; Figure S1a). Transcriptome data (Yu et al., 2018) showed that ZmHRL was significantly induced by C. zeina infection (Figure S1b). Given the role of HRLs in plant disease resistance (Park et al., 2010; Wu et al., 2021), ZmHRL was selected for further investigation. The interaction between ZmWAKLY and ZmHRL was confirmed in N. benthamiana using split-luciferase complementation (SLC) and co-immunoprecipitation (Co-IP) assays, whereas ZmWAKLQ showed no interaction (Figure 1b,c). To further explore this interaction, ZmWAKL was divided into several segments based on its functional domains (Figure S2). SLC assays revealed that ZmHRL specifically binds to the transmembrane and intracellular regions of ZmWAKLY (ZmWAKLY/301–630) (Figure S2). Among eight HR-like lesion-inducing domain contained proteins in maize, ZmHRL is the most closely related to AtHRL, sharing 54.5% identity and conserved structure (Figure S3). AtHRL is predicted to localize to the endoplasmic reticulum (ER) (https://www.arabidopsis.org). Consistently, ZmHRL-GFP co-localizes with the ER marker AtHEDL-RFP in maize protoplasts, indicating ER membrane localization (Figure 1d). To evaluate the role of ZmHRL in GLS resistance, we generated seven independent ZmHRL-overexpressing (ZmHRL-OE) lines in the GLS-susceptible B73 background. These events were self-pollinated to generate homozygous transgenic lines and crossed with the susceptible line Q11 to produce T1BC1F1 populations. Transcript analysis confirmed higher ZmHRL expression in homozygous transgenic lines (Figure 1e). These lines showed enhanced GLS resistance, with smaller lesions and lower disease severity compared with B73 (Figure 1f,g). Similarly, ZmHRL-OE plants in the T1BC1F1 populations exhibited reduced GLS scale relative to their non-transgenic siblings (Figure S4). To further investigate its function, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to generate three Zmhrl knockout mutants with 2, 5, or 13 bp deletions in the B73 background (Figure S5a). All mutants exhibited significantly reduced GLS resistance compared with B73 (Figure S5b,c). These findings indicate ZmHRL plays a critical role in GLS resistance in maize. RNA-seq analysis of ZmHRL-OE#12 and B73 identified 1086 upregulated and 1124 downregulated differentially expressed genes (DEGs) (Figure S6a). Gene ontology (GO) analysis revealed significant enrichment in oxidoreductase activity and calcium ion binding (Figure S6b). Notably, all nine genes within the oxidoreductase complex (GO:1990204) were downregulated, potentially leading to peroxide accumulation (Figure S6c). Plants generate reactive oxygen species (ROS) in response to environmental stimuli by activating oxidases and peroxidases. Consistent with this, two oxidases and two peroxidases were upregulated (Figure S6c). Furthermore, 31 calcium ion-binding genes were upregulated, including 12 encoding calcium-dependent protein kinases (CPKs), among which ZmCPK39 (Zm00001d015100) has been previously linked to multiple foliar disease resistance (Zhu et al., 2024) (Figure S6d). KEGG analysis showed enrichment in plant immunity-related pathways, including plant hormone signaling, phenylpropanoid biosynthesis, MAPK signalling, and plant–pathogen interactions (Figure S6e). Finally, RT-qPCR confirmed RNA-seq results for two peroxidase genes and the immune marker gene ZmNPR1 (Figure 1h,i). Transient expression of ZmHRL-GFP in N. benthamiana led to increased H2O2 accumulation (Figure S7a,b). Co-expression of ZmHRL-GFP with the resistant allele ZmWAKLY resulted in higher H2O2 levels than with the susceptible allele ZmWAKLQ (Figure 1j). Luminol-based assays showed higher flg22-induced ROS production in the resistant NIL-Y32 (ZmWAKLY) compared with the susceptible NIL-Q11 (ZmWAKLQ), while chitin-induced ROS levels were comparable (Figure S7c,d). This suggests that ZmWAKL is associated with the response to flg22 but not chitin. Furthermore, flg22-triggered ROS production was elevated in ZmHRL-OE lines but reduced in Zmhrl mutants compared with their recipient line B73 (Figure 1k,l). Sequence alignment revealed that ZmWAKLB (from B73) is highly similar to ZmWAKLY but contains multiple amino acid variations in both the extracellular and intracellular domains (Figure S8). These findings suggest that ZmWAKLB, like ZmWAKLY, may participate in immune signalling via ZmHRL to trigger ROS burst, albeit with differing efficiency. In conclusion, ZmHRL, an ER-localized protein, interacts with ZmWAKL to regulate ROS production, thereby enhancing GLS resistance. This study highlights the critical role of ER-localized proteins in plant defence, providing new insights for improving disease resistance in crops. Future research should elucidate how ZmHRL coordinates ER functions and immune responses to strengthen plant resilience. We thank the members of the Xu laboratory for their valuable discissions. We also appreciate the Center for Crop Functional Genomics and Molecular Breeding of China Agricultural University for providing transgenic technology support. This work was supported by the Biological Breeding-Major Projects (2023ZD04070) and the National Natural Science Foundation of China (32401852). The authors have declared no conflict of interest. M.X. and T.Z. designed the experiments; T.Z., C.G., and M.Z. performed the experiments; T.Z., M.Z., Q.Z., S.D., and M.X. analysed the data; T.Z. and M.X. wrote the manuscript; All authors discussed the results and commented on the manuscript. The data that supports the findings of this study are available in the Supporting Information of this article. Figure S1 Screening and identification of ZmHRL. Figure S2 Searching for ZmHRL binding region in ZmWAKLY and ZmWAKLQ. Figure S3 Phylogenetic analysis, conserved domain architecture, and structural prediction of HRLs. Figure S4 Overexpression of ZmHRL enhanced GLS resistance in backcross populations. Figure S5 Knockout of ZmHRL by CRISPR/Cas9 results in altered GLS resistance. Figure S6 RNA-seq analysis between ZmHRL-OE#12 and B73. Figure S7 ZmHRL-GFP ectopic expression in N. benthamiana enhances H2O2 accumulation and elicitors induced ROS burst in Q11 background. Figure S8 Sequence alignment of ZmWAKL from B73, Y32, and Q11. Table S1 Candidates ZmWAKL interacting with proteins and their putative functions. Table S2 Primers used in this study. Table S3 The source data for all figures. 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.