The ZmWAKL–ZmWIK–ZmBLK1–ZmRBOH4 module provides quantitative resistance to gray leaf spot in maize

Zhong, Tao; Zhu, Mang; Zhang, Qianqian; Zhang, Yan; Deng, Suining; Guo, Chenyu; Xu, Ling; Liu, Tingting; Li, Yancong; Bi, Yaqi; Fan, Xingming; Balint-Kurti, Peter; Xu, Mingliang

doi:10.1038/s41588-023-01644-z

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Published: 18 January 2024

The ZmWAKL–ZmWIK–ZmBLK1–ZmRBOH4 module provides quantitative resistance to gray leaf spot in maize

Nature Genetics volume 56, pages 315–326 (2024)Cite this article

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Abstract

Gray leaf spot (GLS), caused by the fungal pathogens Cercospora zeae-maydis and Cercospora zeina, is a major foliar disease of maize worldwide (Zea mays L.). Here we demonstrate that ZmWAKL encoding cell-wall-associated receptor kinase-like protein is the causative gene at the major quantitative disease resistance locus against GLS. The ZmWAKL^Y protein, encoded by the resistance allele, can self-associate and interact with a leucine-rich repeat immune-related kinase ZmWIK on the plasma membrane. The ZmWAKL^Y/ZmWIK receptor complex interacts with and phosphorylates the receptor-like cytoplasmic kinase (RLCK) ZmBLK1, which in turn phosphorylates its downstream NADPH oxidase ZmRBOH4. Upon pathogen infection, ZmWAKL^Y phosphorylation activity is transiently increased, initiating immune signaling from ZmWAKL^Y, ZmWIK, ZmBLK1 to ZmRBOH4, ultimately triggering a reactive oxygen species burst. Our study thus uncovers the role of the maize ZmWAKL–ZmWIK–ZmBLK1–ZmRBOH4 receptor/signaling/executor module in perceiving the pathogen invasion, transducing immune signals, activating defense responses and conferring increased resistance to GLS.

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Main

Plants have evolved multiple, varied signal reception/transduction mechanisms to control cellular functions and coordinate defense responses at the cellular, tissue and organismal levels. The initial perception of pathogen infection is mediated by pattern-recognition receptors (PRRs) at the plasma membrane, which include receptor-like kinases (RLKs) and receptor-like proteins^1,2. The detection of pathogen-associated molecular patterns and damage-associated molecular patterns by cognate PRRs leads to pattern-triggered immunity (PTI), which includes rapid reactive oxygen species (ROS) production, calcium (Ca²⁺) influx, activation of calcium-dependent and mitogen-activated kinases, changes of immune-related gene expression and, in some cases, localized cell death^3,4,5. PTI is vital for preventing infection of most nonadapted microbes and restricting the growth of adapted microbes, termed basal resistance⁶.

Cell-wall-associated kinases (WAKs) and WAK-like kinases (WAKLs) represent a unique class of RLKs that are major regulators of fungal disease resistance in plant species. In Arabidopsis, WAKL22/RFO1 confers resistance to a broad spectrum of Fusarium races⁷. The maize gene ZmWAK is induced specifically upon Sporisorium reilianum infection in the mesocotyl to inhibit hyphal growth⁸. ZmWAK-RLK1 confers resistance to northern corn leaf blight caused by Exserohilum turcicum⁹. Other WAK genes, such as Xa4 in rice, TaWAKL4 and Snn1 in wheat, also confer resistance to fungal diseases^10,11,12.

Gray leaf spot (GLS) is a major foliar disease of maize caused by the fungal pathogens Cercospora zeae-maydis and Cercospora zeina¹³. Since its discovery in the United States in the 1920s, GLS has become a severe global maize disease. GLS was reported to cause substantial yield loss in susceptible cultivars¹⁴. In the northern United States and Ontario from 2016 to 2019, GLS caused the greatest estimated yield losses of any maize disease¹⁵. GLS resistance is inherited as a typical quantitative trait, and more than 100 quantitative trait loci (QTLs) have been identified across a range of studies^{14,16,17,18,19}. So far, ZmCCoAOMT2 (caffeoyl-CoA O-methyltransferase 2) and ZmMM1 (Mexicana lesion mimic 1) are the only two genes identified to be effective against GLS^20,21.

Here we report the map-based cloning of the GLS quantitative disease resistance (QDR) gene ZmWAKL and the elucidation of the signal transduction pathway, which activates defense responses against GLS in maize.

Fine-mapping of qRgls1 against GLS

We previously mapped the major QDR locus qRgls1 to the short arm of chromosome 8 in a segregating population derived from a cross between the highly GLS-resistant inbred line Y32 and GLS-susceptible inbred line Q11. qRgls1 was fine-mapped to a 1.4-Mb interval. The Y32 allele at qRgls1 enhanced resistance by 19.7–61.3% compared to the Q11 allele²². Here we identified further recombinants in the qRgls1 region with which we fine-mapped qRgls1 to a 122-kb interval between markers IDP2 and M2 (B73_RefGen_v4; Extended Data Fig. 1). We allowed five key BC₈F₇ recombinants to self-pollinate to develop homozygous lines with or without Y32 segments. The Y32 donor segments resulted in significantly reduced levels of GLS in recombinants II, III and IV, but not in recombinants I and V, further confirming the location of qRgls1 (Fig. 1a). The Y32 allele at qRgls1 reduced the GLS disease severity index (DSI) significantly by 14.7–21.6% (Extended Data Fig. 2a).

**Fig. 1: Map-based cloning of the quantitative GLS-resistance gene at the *qRgls1* locus.**

We selected two homozygous lines derived from recombinant IV, which shared a similar genetic background, but differed at qRgls1, named these near-isogenic lines NIL-Y32 (with the Y32 allele) and NIL-Q11 (with the Q11 allele; Fig. 1a). NIL-Y32 was more resistant to C. zeina than NIL-Q11 at 44 d postinoculation (dpi; Extended Data Fig. 2b,c). C. zeae-maydis and C. zeina conidiophores erupt through the stomata during infection^13,23. Here scanning electron microscopy showed that there was much more profuse conidiation on NIL-Q11 compared to NIL-Y32 (Extended Data Fig. 2d). In several previous studies, GLS resistance has been associated with increased time to flowering^14,24. These two NILs showed no significant differences in three flowering-related traits when grown under long-day or short-day conditions (Extended Data Fig. 2e,f).

We identified, sequenced and annotated two overlapping bacterial artificial chromosome (BAC) clones from the resistant parental line Y32 encompassing the qRgls1 region (Supplementary Fig. 1a,b). The qRgls1 interval was ~122 kb in B73 (B73_RefGen_v4) but only ~60 kb in Y32. The three predicted genes within the Y32 qRgls1 region were all shared with B73, encoded two cell-wall-associated receptor kinase-like proteins (Zm00001d008457, hereinafter referred to as ZmPR5L; Zm00001d008458, hereinafter referred to as ZmWAKL) and a hypothetical protein (Zm00001d008459). The B73 qRgls1 region additionally harbored a gene encoding a leucine-rich repeat RLK (Zm00001d008460; Fig. 1a and Supplementary Fig. 1b). We can’t detect expressions of either Zm00001d008459 or Zm00001d008460 by RT–qPCR; however, expressions of both ZmPR5L and ZmWAKL were detected, and these genes were considered candidates for qRgls1 (Supplementary Fig. 1c).

We determined the full-size complementary DNA (cDNA) sequence of ZmWAKL by rapid amplification of cDNA ends and amplified the full-size cDNA of ZmPR5L by RT–PCR (Supplementary Fig. 1d,e). We designated the Y32 alleles ZmWAKL^Y and ZmPR5L^Y and the Q11 alleles ZmWAKL^Q and ZmPR5L^Q. ZmWAKL^Y differs from ZmWAKL^Q predominantly in the extracellular domain (ECD) with only 62.8% sequence identity. The intracellular domain (ICD) is more conserved with 92.9% sequence identity. Both ZmWAKL isoforms are non-arginine-aspartate (non-RD) kinases comprising the extracellular galacturonan-binding domain (GUB), transmembrane (TM) and cytoplasmic serine/threonine kinase (STK) domains (Extended Data Fig. 3a and Supplementary Fig. 2a). By contrast, ZmPR5L^Y and ZmPR5L^Q shared 98.4% sequence identity (Extended Data Fig. 3b and Supplementary Fig. 2b).

ZmWAKL is the causal gene underlying qRgls1

We isolated a 9.2-kb Y32 genomic fragment comprising the native ZmWAKL^Y allele for the complementation test (Fig. 1b). We independently obtained five transgenic events in the background of the GLS-susceptible line B73. We self-pollinated three of them to develop homozygous transgenic lines expressing ZmWAKL^Y that had significantly higher GLS resistance than B73 (Fig. 1c–e). Notably, the enhanced GLS resistance did not cause substantial changes in plant architecture or flowering time (Supplementary Fig. 3a,b). We backcrossed another two transgenic events to Q11 to produce T₁BC₁F₁ and T₁BC₃F₁ populations. Transgenic plants consistently exhibited significantly lower DSI than their nontransgenic siblings (Fig. 1f–h). These results indicate that the ZmWAKL^Y allele confers resistance to GLS.

We also generated ten and three independent transgenic events overexpressing ZmWAKL^Y-GFP (GFP, green fluorescent protein) and ZmWAKL^Q-GFP in the B73 background, respectively (Fig. 1i and Extended Data Fig. 4a). ZmWAKL-GFP was detectable by immunoblotting with α-GFP antibody (Fig. 1j,m and Extended Data Fig. 4b). We self-pollinated five of the ZmWAKL^Y-GFP transgenic events to generate homozygous transgenic lines. All these lines exhibited fewer lesions and significantly reduced GLS scales, but similar morphology and flowering time, compared to B73 (Fig. 1k,l and Supplementary Fig. 3c,d). We backcrossed the remaining five ZmWAKL^Y-GFP transgenic events to Q11 to generate T₁BC₁F₁ and T₁BC₃F₁ populations. Transgenic plants displayed significantly lower DSI than their nontransgenic siblings (Fig. 1n,o). No obvious difference in GLS resistance between overexpression and native ZmWAKL^Y alleles suggested that the ZmWAKL^Y sequence, rather than its expression level, underpinned the variation in GLS resistance. By contrast, plants overexpressing ZmWAKL^Q-GFP showed no differences in DSI relative to their nontransgenic siblings (Extended Data Fig. 4c,d).

We generated seven independent transgenic events in B73 overexpressing ZmWAKL^C, a chimeric gene encoding a protein consisting of the ZmWAKL^Y ECD/TM and ZmWAKL^Q ICD (Extended Data Fig. 4e). We backcrossed these events to Q11 to produce T₁BC₂F₁ and T₁BC₃F₁ populations. Overall, transgenic plants displayed higher ZmWAKL expression levels and lower DSI values compared to their nontransgenic siblings (Extended Data Fig. 4f,g). The chimeric gene ZmWAKL^C lowered DSI in Q11 by 4.5–15.6%, a little less than the ZmWAKL^Y (7.9–19.5%; Extended Data Fig. 4h), suggesting that the ZmWAKL^Y ECD has a key role in GLS resistance.

For the other candidate, ZmPR5L, we obtained three overexpression transgenic events and two clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) knocked out Zmpr5l null mutants. All transgenic plants showed similar disease severity to B73 (Extended Data Fig. 5), suggesting that ZmPR5L is not involved in GLS resistance.

Molecular characterization of ZmWAKL

The ZmWAKL^Q in NIL-Q11 exhibited higher expression levels than the ZmWAKL^Y in NIL-Y32 in all tissues except the mesocotyl and root at the seedling stage and the top leaf at the maturity stage (Extended Data Fig. 6a). In plants spray-inoculated at the two-leaf stage in the greenhouse, expression levels of both ZmWAKL alleles were rapidly induced by C. zeina infection, peaking at 6 h postinoculation (hpi), with ZmWAKL^Y expression being higher than ZmWAKL^Q (Extended Data Fig. 6b). In field grown plants infusion-inoculated with inocula into the whorl at the 10-leaf stage, the expression levels of both ZmWAKL^Y and ZmWAKL^Q alleles gradually increased after ~20 dpi and peaked at 29 dpi, with ZmWAKL^Y expression higher at the peak (Extended Data Fig. 6c).

We transiently transformed onion epidermal cells with constructs ZmWAKL^Y-EGFP (EGFP, enhanced GFP) and ZmWAKL^Q-EGFP for localization studies, finding that both ZmWAKL isoforms localized to the plasma membrane (Fig. 2a and Extended Data Fig. 6d). Using both ZmWAKL^Y-GFP and ZmWAKL^Q-GFP transgenic maize lines, we observed that GFP signal colocalizes with the membrane-impermeable dye FM4-64 (Extended Data Fig. 6e).

**Fig. 2: Molecular characterization of ZmWAKL.**

Phylogenetic analysis of WAKs/WAKLs reported in rice^10,25,26,27, tomato²⁸, Arabidopsis^{7,29,30,31,32}, wheat^11,12,33,34 and maize^8,9 showed them clustered into three clades with a clear distinction between arginine-aspartate (RD) and non-RD kinases. Both ZmWAKL^Y and ZmWAKL^Q are closer to ZmWAK-RLK1 (ref. ⁹) than to ZmWAK (ref. ⁸; Supplementary Fig. 4). We surveyed ZmWAKL sequences in 98 maize inbred lines (Supplementary Table 2) and identified 48 ZmWAKL haplotypes differentiated by numerous SNPs and insertions and deletions (InDels), especially in the extracellular region (Supplementary Fig. 5), which exhibited a nucleotide diversity (π) >3.4-fold higher than the intracellular region (Supplementary Table 3).

The self-association and kinase activity of ZmWAKL

Homodimerization occurs as part of the activation process of some PRRs³⁵. In a split-ubiquitin-based membrane yeast two-hybrid assay, we observed that the GLS-resistant ZmWAKL^Y, but not the GLS-susceptible ZmWAKL^Q, was able to associate with itself (Fig. 2b). We confirmed these results by split-luciferase complementation (SLC) and co-immunoprecipitation (Co-IP) assays (Fig. 2c,d). Furthermore, we determined that the GUB domain in the ZmWAKL^Y ECD (ZmWAKL^Y/GUB), but not the STK domain in its ICD (ZmWAKL^Y/STK), could self-associate in both yeast two-hybrid and SLC assays, while neither ZmWAKL^Q/GUB nor ZmWAKL^Q/STK was able to do so (Fig. 2e,f).

The maltose-binding protein (MBP)-tagged ZmWAKL^Y ICD (ZmWAKL^Y/ICD-MBP), but not its kinase-inactive variant ZmWAKL^Y/ICD,K391E-MBP, exhibited autophosphorylation and phosphorylated the universal kinase substrate myelin basic protein (MBP^a) in vitro, indicating that ZmWAKL^Y/ICD-MBP has kinase activity (Fig. 2g). By contrast, neither ZmWAK^Q/ICD-MBP nor its kinase-inactive variant ZmWAKL^Q/ICD,K430E-MBP had kinase activity (Fig. 2g). We also performed a phosphorylation assay by immunoprecipitating ZmWAKL^Y-GFP and ZmWAKL^Q-GFP from their respective overexpressed transgenic plants. We detected kinase activities from immunoprecipitates of both ZmWAKL-GFP isoforms, as indicated by phosphorylated MBP^a (Fig. 2h). Given that ZmWAKL^Q has no kinase activity in vitro, we speculate that a protein(s) coprecipitated with ZmWAKL^Q-GFP may phosphorylate MBP^a.

ZmWIK is a coreceptor of ZmWAKL

Plant PRRs usually interact with a coreceptor to recognize the substrate and transduce signal³⁶. To identify a possible ZmWAKL coreceptor, we performed repeated IP using extracts from transgenic plants overexpressing ZmWAKL^Y-GFP followed by mass spectrometry (MS). Of six identified plasma membrane-tethered kinases (Supplementary Table 4), only a leucine-rich repeat receptor-like serine/threonine-protein kinase (Zm00001d028560, hereinafter referred to as ZmWIK) and a protein kinase (Zm00001d011700, hereinafter referred to as ZmPK2) can interact with both ZmWAKL isoforms (Fig. 3a and Extended Data Fig. 7a–d). ZmPK2, lacking an extracellular receptor-like domain and showing weaker fluorescence and lower gene expression in leaves, is unlikely to be a coreceptor of ZmWAKL (Extended Data Fig. 7a–e).

The localization of a ZmWIK-GFP fusion in onion epidermal cells and Nicotiana benthamiana leaves was consistent with the plasma membrane location (Extended Data Fig. 7f,g). In a SLC assay, stronger luminescence was seen in the interaction of ZmWIK-nLUC with cLUC-ZmWAKL^Y than with cLUC-ZmWAKL^Q (Fig. 3b). In a Co-IP assay, ZmWIK-Myc could be coprecipitated with ZmWAKL^Y-GFP and ZmWAKL^Q-GFP (Fig. 3c). To identify the domains of ZmWIK and ZmWAKL responsible for the interaction, we divided ZmWIK and ZmWAKL each into two parts, ECD/TM and ICD. In a SLC assay, ZmWIK^ECD,TM-nLUC interacted with cLUC-ZmWAKL^Y/ECD,TM, but not with cLUC-ZmWAKL^Q/ECD,TM, while ZmWIK^ICD-nLUC interacted relatively weakly with cLUC-ZmWAKL^Y/ICD or cLUC-ZmWAKL^Q/ICD (Fig. 3d). In a pull-down assay, more His-ZmWAKL^Y/ICD was pulled down by ZmWIK^ICD-MBP than His-ZmWAKL^Q/ICD (His, histidine; Fig. 3e). To pinpoint the difference between the two ZmWAKL^ICD isoforms in interacting with ZmWIK, we performed multiple SLC assays. Although cLUC-ZmWAKL^Y/ICD and cLUC-ZmWAKL^Q/ICD showed similar protein abundance, luminescence intensity was significantly higher when ZmWIK-nLUC co-expressed with cLUC-ZmWAKL^Y/ICD than with cLUC-ZmWAKL^Q/ICD (Supplementary Fig. 6). These findings suggest that both the ECD and ICD of ZmWAKL^Y contribute to its preferential interaction with ZmWIK compared to ZmWAKL^Q.

In vitro, the purified His-ZmWIK^ICD exhibited autophosphorylation and strong transphosphorylation of MBP^a, whereas the kinase-inactive variant His-ZmWIK^ICD,K339E did not (Fig. 3f). Although His-ZmWIK^ICD had strong kinase activity, it only weakly phosphorylated the two kinase-inactive ZmWAKL^ICD-MBP variants (Fig. 3g). ZmWAKL^Y/ICD-MBP also showed autophosphorylation but weak transphosphorylation of His-ZmWIK^ICD,K339E, while ZmWAKL^Q/ICD-MBP displayed neither autophosphorylation nor transphosphorylation (Fig. 3h). We speculate that the use of kinase-inactive variants as substrates may underlie the relatively weak reciprocal phosphorylation, like what has been observed in the previous report³⁷. When ZmWAKL^Y/ICD-MBP and His-ZmWIK^ICD were mixed in vitro, they enhanced each other’s phosphorylation levels, with His-ZmWIK^ICD being more strongly phosphorylated than ZmWAKL^Y/ICD-MBP. Reduced phosphorylation levels were observed when using ZmWAKL^Q/ICD-MBP or two kinase-inactive ZmWAKL^ICD-MBP variants in place of ZmWAKL^Y/ICD-MBP (Fig. 3i). His-ZmWIK^ICD strongly phosphorylated MBP^a and adding ZmWAKL^Y/ICD-MBP further increased the phosphorylation levels of both His-ZmWIK^ICD and MBP^a. Intriguingly, ZmWAKL^Q/ICD-MBP had similar effects, although to a lesser extent (Fig. 3j,k).

Two transgenic lines overexpressing ZmWIK showed significantly fewer GLS symptoms compared to B73 (Extended Data Fig. 7h–j). An ethyl methanesulfonate (EMS) induced null mutant Zmwik, harboring a premature translation termination codon, was backcrossed into NIL-Y32. The plants with Zmwik were more susceptible to GLS than wild-type counterparts in the field trials (Fig. 3l–n), suggesting that ZmWIK may act downstream of ZmWAKL^Y in GLS resistance. Overall, these data indicate that ZmWIK binds to ZmWAKL^Y at the plasma membrane and lack of either protein reduces GLS resistance. Moreover, they mutually phosphorylate each other, with more frequent phosphorylation made by ZmWAKL^Y to ZmWIK. We thus speculate that ZmWIK may be a coreceptor of ZmWAKL.

ZmWAKL/ZmWIK interacts with ZmBLK1 for immune signaling

RLCKs, Botrytis-induced kinase 1 (BIK1) and PBL1 (avrPphB susceptible 1-like 1), have emerged as core components linking PRRs to downstream defenses³⁸. We hypothesized that a similar RLCK might contribute to ZmWAKL-mediated immunity. Thus, two maize BIK1/PBL1 homologs, ZmBLK1 (Zm00001d034662) and ZmBLK1-1 (Zm00001d012958), sharing 61.1%/69.0% and 60.4%/69.0% identical sequences to AtBIK1 and AtPBL1, respectively, were identified (Supplementary Fig. 7a). Phylogenetic analysis indicated that ZmBLK1/ZmBLK1-1 clustered in the same clade as OsRLCK176 (refs. ^39,40), OsRLCK57 (ref. ⁴¹), SlTPK1b (tomato protein kinase 1)⁴², AtBIK1 (ref. ⁴³) and AtPBL1 (ref. ⁴⁴), all of which are involved in ROS production via the NADPH oxidase respiratory burst oxidase homolog D (RBOHD). Other RLCKs, OsRLCK185 (ref. ⁴⁵), AtPBL27 (PBS1-like 27)⁴⁶, AtPCRK1/2 (PTI compromised RLCK 1/2)⁴⁷ and AtBSK1 (brassinosteroid-signaling kinase 1)⁴⁸, are involved in triggering a mitogen-activated protein kinase signaling cascade, clustered in a separate clade (Supplementary Fig. 7b). These findings suggest that ZmBLK1 and ZmBLK1-1 may be involved in ROS-mediated defense responses.

In SLC assays, cLUC-ZmBLK1, but not cLUC-ZmBLK1-1, interacted with ZmWIK-nLUC and ZmWAKL^Y-nLUC, and to a lesser extent with ZmWAKL^Q-nLUC (Fig. 4a and Extended Data Fig. 8a). Using Co-IP assays, we observed that ZmBLK1-Myc was associated with ZmWIK-GFP (Fig. 4b), and specifically with ZmWIK^ICD-GFP, but not ZmWIK^ECD,TM-GFP (Fig. 4c). ZmBLK1-Myc was also associated with both ZmWAKL-GFP isoforms in a Co-IP assay (Fig. 4d). ZmBLK1 was reported to contribute to disease resistance and tethered to the plasma membrane through a post-translational N-terminal myristoylation motif⁴⁹. This plasma membrane localization of ZmBLK1 would facilitate its interaction with ZmWIK and ZmWAKL. With all three proteins present, we detected both ZmBLK1-Myc and cLUC-ZmWIK in the complexes immunoprecipitated by either ZmWAKL^Y-GFP or ZmWAKL^Q-GFP in Co-IP assays (Fig. 4e). Thus, ZmBLK1, ZmWAKL and ZmWIK may form an immune signaling complex at the plasma membrane.

**Fig. 4: ZmBLK1 is a cytoplasmic kinase downstream of the ZmWAKL/ZmWIK complex.**

ZmBLK1 is phosphorylated by ZmWAKL/ZmWIK

ZmBLK1-GST (GST, glutathione S-transferase) exhibited autophosphorylation and transphosphorylation of MBP^a, but the kinase-inactive variant ZmBLK1^K114E-GST did not (Fig. 4f). His-ZmWIK^ICD had self-phosphorylated and strongly phosphorylated ZmBLK1-GST and, to a lesser extent, ZmBLK1^K114E-GST. In contrast, ZmBLK1-GST, although showing significant autophosphorylation, hardly phosphorylated His-ZmWIK^ICD,K339E (Fig. 4g). With respect to reciprocal phosphorylation between ZmBLK1 and two ZmWAKL^ICD isoforms, ZmWAKL^Y/ICD-MBP phosphorylated ZmBLK1^K114E-GST, whereas ZmWAKL^Q/ICD-MBP didn’t (Fig. 4h). In turn, ZmBLK1-GST phosphorylated ZmWAKL^Y/ICD,K391E-MBP moderately and ZmWAKL^Q/ICD,K430E-MBP more weakly (Fig. 4i). Collectively, these data suggest that reciprocal phosphorylation between the three proteins is not equivalent, with the phosphorelay being primarily directed from ZmWAKL^Y/ZmWIK to ZmBLK1.

The autophosphorylation and transphosphorylation relationships of ZmWAKL, ZmWIK and ZmBLK1 are likely to be more complex when all three proteins are present. Based on the presumptive phosphorelay pathway, we investigated the phosphorylation level of ZmBLK1 by the ZmWAKL/ZmWIK receptor complex. In the presence of the two kinase-inactive variants His-ZmWIK^ICD,K339E and ZmBLK1^K114E-GST, neither autophosphorylation nor transphosphorylation could be detected for ZmWAKL^Y/ICD-MBP (Fig. 4j(I)). The phosphorylation of the kinase-inactive variant ZmBLK1^K114E-GST by His-ZmWIK^ICD did not change appreciably upon the addition of ZmWAKL^Y/ICD-MBP, ZmWAKL^Q/ICD-MBP or two kinase-inactive ZmWAKL^ICD-MBP variants (Fig. 4j (II)). In the presence of the kinase-inactive variant His-ZmWIK^ICD,K339E, the addition of ZmWAKL^Y/ICD-MBP, ZmWAKL^Q/ICD-MBP or two kinase-inactive ZmWAKL^ICD-MBP variants did not appear to alter the phosphorylation level of ZmBLK1-GST in any way (Fig. 4j (III)). Notably, His-ZmWIK^ICD could strongly phosphorylate ZmBLK1-GST, and this transphosphorylation was further increased upon the addition of ZmWAKL^Y/ICD-MBP, but not by the addition of ZmWAKL^Q/ICD-MBP or two kinase-inactive ZmWAKL^ICD-MBP variants (Fig. 4j (IV)).

In N. benthamiana, we found ZmWAKL^Y-GFP caused more phosphorylation of ZmBLK1-Myc than ZmWAKL^Q-GFP when co-expressed with cLUC-ZmWIK (Extended Data Fig. 8b). The cLUC-ZmWIK and ZmBLK1-Myc constructs were cotransfected into protoplasts isolated from transgenic plants overexpressing ZmWAKL^Y-GFP or ZmWAKL^Q-GFP. In immunoprecipitates obtained using α-Myc magnetic beads, ZmBLK1-Myc was more phosphorylated in the presence of ZmWAKL^Y-GFP compared to ZmWAKL^Q-GFP (Extended Data Fig. 8c). The more ZmBLK1-Myc phosphorylated, the higher its kinase activity (Extended Data Fig. 8d).

We speculate that in the ZmWAKL/ZmWIK/ZmBLK1 complex, ZmWIK directly phosphorylates ZmBLK1, with ZmWAKL^Y enhancing this, while ZmWAKL^Q does not. These findings support an immune phosphorelay from ZmWAKL^Y to ZmWIK to ZmBLK1 in GLS resistance.

ZmBLK1 interacts with and phosphorylates ZmRBOH4

In Arabidopsis, BIK1 directly binds to and phosphorylates the N-terminal domain of RBOHD, leading to a ROS burst that mediates plant immunity^43,50. In maize genome, six RBOHs were identified, of which ZmRBOH4 was the most closely related to AtRBOHD with 63.3% sequence identity (Extended Data Fig. 9a). In SLC assays, ZmRBOH4 interacted with ZmBLK1 but not ZmBLK1-1; furthermore, the N-terminal domain of ZmRBOH4 directly interacted with ZmBLK1, but not with two ZmWAKL isoforms or ZmWIK (Fig. 5a and Extended Data Fig. 9b,c). We confirmed these results by pull-down assays (Fig. 5b and Extended Data Fig. 9d). For the other five ZmRBOHs, ZmRBOH1/ZmRBOH5/ZmRBOH6 but not ZmRBOH2/ZmRBOH3 interacted with ZmBLK1 in SLC assays (Extended Data Fig. 9e). However, unlike ZmRBOH4, which has high and pathogen-inducible gene expression in leaves (Extended Data Fig. 9f), ZmRBOH1 showed relatively low gene expression, which could not be induced by pathogen inoculation, and ZmRBOH5/ZmRBOH6 were exclusively expressed in the anthers (Supplementary Fig. 8a,b). Hence, only ZmRBOH4 is likely involved in the immune signaling against GLS.

**Fig. 5: ZmRBOH4 receives immune signal from ZmBLK1 and regulates GLS resistance in maize.**

Both ZmWAKL^Y/ICD-MBP and His-ZmWIK^ICD showed autophosphorylation, but phosphorylated His-N-ZmRBOH4 not at all or weakly, respectively (Fig. 5c,d). This is consistent with the lack of direct interaction between ZmRBOH4 and ZmWAKL^Y (or ZmWIK; Extended Data Fig. 9b–d). By contrast, ZmBLK1-GST strongly phosphorylated His-N-ZmRBOH4 (Fig. 5e). When all four proteins, ZmWAKL, ZmWIK, ZmBLK1 and ZmRBOH4, were mixed in vitro, their phosphorylation levels varied substantially, from strongly phosphorylated ZmBLK1-GST and His-N-ZmRBOH4 to barely phosphorylated His-ZmWIK^ICD to undetectably phosphorylated ZmWAKL^Y/ICD-MBP. Phosphorylation of His-N-ZmRBOH4 was somewhat enhanced in the presence of ZmWAKL^Y/ICD-MBP compared to ZmWAKL^Q/ICD-MBP and the two kinase-inactive ZmWAKL^ICD-MBP variants (Fig. 5f). In the presence of kinase-active ZmWAKL^Y/ICD-MBP and ZmBLK1-GST, His-N-ZmRBOH4 displayed weaker phosphorylation when His-ZmWIK^ICD was replaced by its kinase-inactive variant His-ZmWIK^ICD,K339E. However, the substitution of ZmBLK1-GST by its kinase-inactive variant ZmBLK1^K114E-GST almost eliminated His-N-ZmRBOH4 phosphorylation. When both kinase-inactive His-ZmWIK^ICD,K339E and ZmBLK1^K114E-GST variants were present, no His-N-ZmRBOH4 phosphorylation could be detected (Fig. 5g). We interpreted these results as evidence that ZmBLK1 is the major upstream kinase phosphorylating N-ZmRBOH4-GST in this assay, and ZmRBOH4 may join the ZmWAKL^Y/ZmWIK/ZmBLK1 complex to form an immune module.

To verify the function of ZmRBOH4, we knocked out ZmRBOH4 in B73 using CRISPR/Cas9. Notably, these two knocked out lines exhibited significantly reduced plant height and increased GLS susceptibility compared with wild-type plants under artificial inoculation (Fig. 5h,i and Extended Data Fig. 10), indicating ZmRBOH4 is involved in regulating GLS resistance.

ZmWAKL-mediated immunity against GLS in vivo

As noted above, ZmWAKL could be induced by C. zeina infection with a peak at 6 hpi (Extended Data Fig. 6b). Here we wanted to know what would happen to ZmWAKL^Y and ZmWAKL^Q proteins in response to C. zeina infection. Transgenic plants overexpressing ZmWAKL^Y-GFP inoculated with C. zeina showed increased levels of phosphorylated ZmWAKL^Y, peaking at 6 hpi, with stronger kinase activity (Fig. 6a). However, in a parallel experiment, ZmWAKL^Q-GFP appeared to be unaffected by C. zeina infection, exhibiting only basal phosphorylation level and kinase activity (Fig. 6b). Like what was previously observed (Fig. 2h), we speculate that coprecipitated protein(s) in the ZmWAKL^Q-GFP immunoprecipitate, rather than ZmWAKL^Q-GFP itself, may be responsible for this basal activity. To gain insight into the phosphorylation changes of the other proteins, we performed proteomic and phosphoproteomic analyses on transgenic plants overexpressing ZmWAKL^Y-GFP and ZmWAKL^Q-GFP collected at 6 hpi. We detected that the phosphorylation levels of ZmBLK1 and ZmRBOH4 showed significant increases in ZmWAKL^Y-GFP plants compared to those in ZmWAKL^Q-GFP plants (Fig. 6c and Supplementary Table 5).

**Fig. 6: The ZmWAKL-triggered immune response to *C. zeina* and proposed working model.**

Considering our evidence that immune signaling, initiated by ZmWAKL^Y in response to C. zeina attack, eventually converges to ZmRBOH4 via ZmWIK and ZmBLK1, it appears that GLS resistance may be associated with a ROS burst. We observed that NIL-Y32 exhibited higher C. zeina-induced ROS levels compared to NIL-Q11 (Fig. 6d) and Zmwik and Zmrboh4 null mutants showed lower C. zeina-induced ROS levels than wild-type plants (Fig. 6e,f).

Working model of maize WAKL–WIK–BLK1–RBOH4 immune module

We characterized the role of the cell-wall-associated RLK ZmWAKL in QDR to GLS in maize. Our investigations suggest the following model: ZmWAKL^Y could self-associate and interact with the coreceptor ZmWIK to form the ZmWAKL^Y/ZmWIK receptor complex at the plasma membrane. Upon challenge by the pathogen C. zeina, ZmWAKL^Y is activated and transiently increases its phosphorylation activity, and together with ZmWIK, transmits immune signals to ZmBLK1 and then to ZmRBOH4, which triggers a ROS burst to incur innate immunity (Fig. 6g). In contrast to ZmWAKL^Y, ZmWAKL^Q is unable to associate with itself, shows no autophosphorylation or kinase activity and interacts weakly with ZmWIK. Upon pathogen challenge, ZmWAKL^Q shows no detectable increase in its phosphorylation activity, thus failing to trigger immune responses, leading to GLS susceptibility (Fig. 6g). The maize ZmWAKL–ZmWIK–ZmBLK1–ZmRBOH4 immune module perceives pathogen invasion, transduces immune signals and activates defense responses, eventually conferring quantitative resistance to GLS.

Discussion

Identifying the causative gene underlying QDR is challenging due to the difficulties with the genetic dissection and verification of this quantitative trait. Here we adopted a sequential fine-mapping strategy and multiple transgenic approaches to demonstrate that ZmWAKL is the causative gene for qRgls1 (Fig. 1). Characterizing ZmWAKL allowed us to identify its coreceptor ZmWIK, a plasma-membrane-localized RLK (Figs. 2 and 3). Thereafter, partly based on analogies to systems found in Arabidopsis, we identified the ZmBLK1 and ZmRBOH4 as downstream components of the ZmWAKL^Y/ZmWIK receptor complex (Figs. 4 and 5).

Extracellular ROS are produced primarily through activation of RBOHs at the plasma membrane and act as a pivotal early component of PTI responses⁵¹. Besides, RBOHs are crucial for growth and development^51,52, like knocking out ZmRBOH4 results in weaker plants (Extended Data Fig. 10). The reduced vitality of ZmRBOH4 mutants might also contribute to the increased GLS susceptibility (Fig. 5h,i).

In cereal crops, the molecular mechanism of ROS production and its contribution to QDR are not completely understood. Therefore, the discovery of the maize ZmWAKL–ZmWIK–ZmBLK1–ZmRBOH4 immune module will have a significant influence on the conceptual underpinnings of QDR in plants.

Methods

Plant materials

In our previous study, the major QTL-qRgls1 is restricted to a ~1.4-Mb region flanked by makers GZ204 and IDP5 (ref. ²²). To fine-map qRgls1, the recombinants were self-pollinated and backcrossed to Q11 to develop BC₂F₅ (1,865 plants), BC₅F₇ (1,055 plants) and BC₇F₇ (1,162 plants) populations. The five BC₇F₇ recombinants were subsequently backcrossed to Q11 to generate BC₈F₇ generations, which were then self-pollinated to develop BC₈F₈ homozygous lines with/without Y32 segments. Comparison of Y32 donor regions with GLS resistance allowed us to confirm the final qRgls1 region. From the recombinant IV, we obtained a pair of near-isogenic lines differing at qRgls1, namely NIL-Y32 (with qRgls1) and NIL-Q11 (without qRgls1). The two NILs had 96.12% genetic identity (as assessed with the Axiom_M6H60k genotyping chip, Thermo Fisher Scientific) and were used for exploring the molecular mechanism underlying ZmWAKL-mediated innate immunity. In addition, a collection of 98 maize inbred lines (Supplementary Table 2) was used for examining allelic variations of the ZmWAKL locus.

The EMS mutant line Zmwik was obtained from the Maize EMS-induced Mutant Database (http://www.elabcaas.cn/memd/public/index.html#/; mutant ID: EMS4-0073f9). The mutant was generated in the B73 background and contained the heterozygous ZmWIK locus. The mutant was backcrossed twice to NIL-Y32, followed by self-pollination, to introduce ZmWAKL^Y into the ZmWIK wild-type and its homozygous Zmwik mutant to evaluate GLS resistance.

Field assays

The fine-mapping populations, maize inbred lines and ZmWIK mutants were planted in Baoshan (Yunnan, China) and naturally infected with the pathogenic fungus C. zeina. Baoshan is the ideal place for GLS development due to sufficient natural disease stress. The seeds were sown at 5 m in length with 0.5 m between rows and 0.25 m between plants within a row.

In Beijing, transgenic plants and NILs were planted in the same way as in Baoshan, except for using the 4-m long plot and being artificially inoculated with C. zeina. The GLS inoculum was prepared using the pathogenic fungus C. zeina. The fungus was cultured on potato dextrose agar medium, and the spores were washed off with ddH₂O to prepare a spore inoculum at a concentration of 5 × 10³ spores per ml. Plants were inoculated at the 10-leaf stage by pouring 5-ml inoculum into the leaf whorl. Samples were collected at different time points after inoculation, as specified in each experiment. Each sample contains leaf tissues collected from at least three individual plants. For gene expression analysis, one leaf punch per plant was collected from the middle of the inoculated leaf, and each genotype/treatment sample contained three punches, which were pooled and stored at −80 °C. For scanning electron microscope (SEM) analysis, four punches across the center of each leaf were collected.

The GLS symptoms are scored three times at 1-week intervals, starting 2 weeks after pollination. The number and size of lesions on the leaves of the entire plant were used to assess the GLS severity. GLS scale was rated using the following scale: 1 (highly resistant), 3 (resistant), 5 (intermediate resistant/susceptible), 7 (susceptible) and 9 (highly susceptible). To convert the scale to a DSI, scales 1, 3, 5, 7 to 9 were assigned to a distinct numerical value of 0, 0.25, 0.5, 0.75 and 1, respectively.

$${\rm{DSI}}( \% )=\frac{\begin{array}{l}{\rm{Assigned}}\,{\rm{disease}}\,{\rm{severity}}\,{\rm{value}}\\\,\times\, {\rm{number}}\,{\rm{of}}\,{\rm{plants}}\,{\rm{in}}\,{\rm{corresponding}}\,{\rm{grade}}\end{array}}{1\,\times\, {\rm{total}}\,{\rm{number}}\,{\rm{of}}\,{\rm{plants}}}\times 100$$

Days to heading, silking and anthesis were recorded for each plant from NILs and homozygous transgenic lines.

Sequential fine-mapping of qRgls1

Newly developed markers in the qRgls1 region were used to genotype recombinants (Supplementary Table 1). In each generation, new recombinants within the mapped qRgls1 region were selected and backcrossed to Q11 to produce progeny for fine-mapping. The qRgls1 locus was fine-mapped by comparing the genotypes of the recombinants with their deduced phenotypes based on the resistant performance of their progeny. A significant difference in the DSI between the heterozygous and homozygous genotypes of the offspring indicates the presence of the resistance allele at qRgls1 in the heterozygous region of their parental recombinant; otherwise, there is no resistance allele at qRgls1.

Genetic transformation

From the positive Y32 BAC clone 57-9-1-93, a 9.2 kb genomic fragment, containing the 2.1-kb promoter, 2.2-kb ZmWAKL^Y coding and 4.9-kb 3′-UTRs, was subcloned into pCAMBIA3301 according to the method described previously⁸. To construct overexpression vector, the fused gene of ZmWAKL coding sequence (CDS) and EGFP was cloned into the express vector pBCXUN-Myc driven by the maize ubiquitin promoter as described earlier⁵³. In addition, we constructed overexpression vectors for the chimeric genes ZmWAKL^C, ZmPR5L and ZmWIK. To knockout the endogenous genes ZmPR5L and ZmRBOH4, CRISPR/Cas9 vectors were constructed following the protocol described previously⁵⁴. These vectors were transferred to Agrobacterium strain EHA105 and then transformed into the maize inbred line B73.

The genotypes were confirmed by PCR or sequencing standard PCR products. All T₁-positive or edited plants from each transgenic event were self-pollinated to produce homozygous transgenic plants or backcrossed to the susceptible parent Q11 or NIL-Y32 to produce backcross populations for functional verification.

Plant protein extraction and immunoblot analysis

Plant total protein was extracted from maize or tobacco leaves. The tissue sample was ground in liquid nitrogen and resuspended in an equal volume (1:1 fresh wt/vol) of the extraction buffer (50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF) and 0.2 % Triton X-100) on ice for at least 30 min and then centrifuged at 12,000g for 30 min at 4 °C to obtain the supernatant. The remaining supernatant was incubated with ~5 μl magnetic beads for 2 h at 4 °C. The beads were magnetically separated, washed twice and then heated in 100 μl of 1× SDS sample buffer at 95 °C for 5 min. The protein sample was subjected to electrophoresis on a 10% (wt/vol) polyacrylamide gel and transferred to a polyvinylidene fluoride blotting membrane (GE HealthCare, A10116498). The membrane was incubated for 1 h at room temperature with 5% nonfat milk in tris-buffered saline (20 mM Tris–HCl (pH 7.6) and 150 mM NaCl) containing 0.05% Tween 20 (TBST). All antibody incubations were performed in 1× phosphate-buffered saline (1× PBS; 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, 1.4 mM KH₂PO₄, pH 7.4). The fused protein was detected with the corresponding tag antibody at a dilution of 1:2,000 (vol/vol) overnight at 4 °C. α-Actin (dilution of 1:2,000 (vol/vol)) was used as an endogenous control (EASYBIO, BE0028). The membrane was incubated for 1 h with an immunoglobulin horseradish peroxidase-conjugated secondary antibody at a dilution of 1:5,000 (vol/vol) to visualize the signal.

Subcellular localization

The CDS of ZmWAKL^Y, ZmWAKL^Q and ZmWIK were inserted into the vector pEZS-NL to create EGFP-fused expression constructs driven by the 35S promoter. Similarly, the ZmWIK CDS was cloned into pSuper1300-GFP to generate the fusion construct. The ZmWIK-GFP constructs were transformed into tobacco leaves using Agrobacterium-mediated method. The ZmWAKL^Y-EGFP, ZmWAKL^Q-EGFP and ZmWIK-EGFP constructs were also mixed with nano-grade gold particles and bombarded into onion epidermal cells, which were then cultured at 28 °C in the dark for 8–10 h. The GFP signals were visualized under a confocal microscope (Zeiss). Plasmolysis was achieved by incubating onion epidermal cells in 0.3 g ml⁻¹ sucrose for 5 min.

Fluorescence was examined in the root epidermal tissues of 1-week-old ZmWAKL^Y-GFP or ZmWAKL^Q-GFP overexpression transgenic plants and B73 under a confocal microscope. The membrane-impermeable dye FM4-64 (MedchemExpress, HY-103466) was used as the plant membrane marker.

Phylogenetic analysis

The amino acid sequences of reported immune-related WAKs/WAKLs and RLCKs were downloaded from the National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/). The ZmRBOH1-6 amino acid sequences were retrieved from the Gramene database (http://www.gramene.org/). The full-length sequences were used for the phylogenetic analysis, and the phylogenetic trees were constructed using the neighbor-joining method in MEGA 7.0 (http://www.megasoftware.net). Bootstrap values from 1,000 pseudo-replicates were used to provide support for the nodes in the phylogenetic tree. The conserved domains were predicted using the NCBI Conserved Domains Database (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi).

Yeast two-hybrid assay

The DUAL membrane system was used to verify the self-association of ZmWAKL. The signal peptide sequences of ZmWAKL^Y and ZmWAKL^Q were removed, and the remaining sequences were cloned into the pBT3-SUC vector. The full-length CDS of ZmWAKL^Y and ZmWAKL^Q were cloned into the pPR3-N vector. These vectors were cotransformed into the yeast strain NMY51 to detect protein–protein interactions according to the manufacturer’s user guide (Clontech). Then, the GUB domain (amino acids 46–155 in ZmWAKL^Y and amino acids 37–182 in ZmWAKL^Q) and the intracellular kinase domain (amino acids 362–630 in ZmWAKL^Y and amino acids 404–666 in ZmWAKL^Q) were cloned into pGADT7 and pGBKT7 vectors, respectively, to verify the interaction domains. These vectors were cotransformed into the Y2HGold strain, and the protein interactions were tested on a selective medium.

SLC assay

The CDS of ZmWAKL^Y, ZmWAKL^Q, ZmBLK1, ZmWIK, ZmRBOH4 and their gene segments were cloned into the JW771-35S-CLuc vector (cLUC) and JW772-35S-NLuc vector (nLUC), respectively, to generate fusion proteins with the C-terminal or N-terminal fragment of the luciferase gene. ZmWIK and ZmWAKL were divided into their ECD/TM and ICD, marked as ZmWIK^ECD,TM (1-269 aa) and ZmWIK^ICD (270-594 aa), ZmWAKL^Y/ECD,TM (1-324 aa) and ZmWAKL^Y/ICD (325-665 aa), ZmWAKL^Q/ECD,TM (1-363 aa) and ZmWAKL^Q/ICD (364-704 aa). The N-terminal domain was marked as N-ZmRBOH4 (1-377 aa). The pairs of constructs were co-infiltrated into N. benthamiana leaves using the previously reported method⁵⁵. Two days after inoculation, 1 mM luciferin (Promega, E1601) was sprayed onto the inoculated leaves, and the luminescence signal was measured using the Chemiluminescent Imaging System (Tanon).

The luminescence signals were analyzed using ImageJ Launcher software (National Institutes of Health) to determine their intensities, so-called the mean gray value. This assay was immediately followed by protein extraction and immunoblotting with α-LUC antibody (Abcam, ab181640), and the immunoblot bands were measured using ImageJ Launcher as well. Total protein of the infiltrated leaf tissues was extracted and detected as described above.

Co-IP assay

To generate ZmWAKL^Y-Myc, ZmWAKL^Y-GFP, ZmWAKL^Q-Myc, ZmWAKL^Q-GFP, ZmWIK-Myc, ZmWIK^ECD/TM-GFP, ZmWIK^ICD-GFP and ZmBLK1-Myc, the CDS or gene segments for ZmWAKL, ZmWIK and ZmBLK1 were amplified and then cloned into pSuper1300 (Myc-tag or GFP-tag). The resulting plasmids were transformed into N. benthamiana leaves and expressed for about 48 h. Total protein from the infiltrated leaf tissues was extracted as described above, and the supernatant was incubated with the α-GFP magnetic beads (MBL, D153-11) at 4 °C for 2 h. The products were analyzed and detected by immunoblotting with α-Myc antibody (ABclonal, AE010) and α-LUC antibody (Abcam, ab181640). GFP and GFP-fusion proteins were detected by immunoblotting with α-GFP antibody (ABclonal, AE012).

IP–MS

The immunocomplexes were analyzed by mass spectrometry (MS) at the China Agricultural University Mass Spectrum Laboratory. Briefly, ~2 g leaf of Ubipro:ZmWAKL^Y-GFP transgenic plants were collected and ground in a mortar using liquid nitrogen. The GFP-tagged fusion protein complex was extracted as described above and separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS–PAGE). The gel lanes were cut, sliced and subjected to in-gel digestion with trypsin. After destaining and tryptic digestion, peptides were extracted and redissolved in 25 μl (0.1%) trifluoroacetic acid. In total, 6 μl of extracted peptides were analyzed by LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific). The Mascot search engine Mascot Server 2.3 (Matrix Science) was used for protein identification by searching against the UniProt protein database (https://www.uniprot.org/). The false discovery rate (FDR) was also set to 0.01 for protein identifications. The significance threshold was set at P < 0.05, and a minimum number of significant unique sequences was set to 1.

In vitro pull-down assay

In vitro pull-down assay was carried out as previously described⁵⁶. In brief, 0.5 µg of His-ZmWAKL^Y/ICD or His-ZmWAKL^Q/ICD or His-TF-ZmWAKL^Y/ICD or His-TF-ZmWAKL^Q/ICD or His-ZmWIK^ICD or His-ZmBLK1 protein was incubated with 5 µg of ZmWIK^ICD-MBP or N-ZmRBOH4-GST or N-ZmRBOH4-MBP and immunoprecipitated by MBP or GST agarose resin at 4 °C for 2 h. The mixture was gathered by centrifugation at 500g for 5 min, followed by washing with phosphate-buffered saline (PBS) buffer five times. Proteins were separated by 10% (wt/vol) SDS–PAGE and detected with α-MBP antibody (ABclonal, AE016), α-GST antibody (Yeasen, 30903ES10) and α-His antibody (Yeasen, 30404ES60).

In vitro phosphorylation assay

ZmWAKL^Y/ICD-MBP, ZmWAKL^Q/ICD-MBP, His-ZmWIK^ICD, ZmBLK1-GST and their corresponding kinase-inactive variants, His-N-ZmRBOH4 fusion protein and MBP^a (EMB Millipore, 13-104) were used for in vitro phosphorylation assay in the presence of (³²P)-γ-ATP.

For the in vitro phosphorylation assay, the recombinant proteins were incubated in a kinase buffer (20 mM Tris–HCl (pH 7.5), 10 mM MgCl₂, 1 mM CaCl₂ and 1 mM DTT) in the presence of 1 mM unlabeled ATP and 1 µCi of (³²P)-γ-ATP for 30 min at 30 °C. The reactions were stopped by adding 5× SDS loading buffer (GenStar, E153-10). Proteins were separated by SDS–PAGE, followed by staining with CBB overnight with decolorization. The phosphorylation status of the fusion proteins was analyzed by autoradiography using a Typhoon 9410 Variable Mode Imager (GE HealthCare).

In vivo phosphorylation assay

ZmBLK1 protein phosphorylated in vivo was detected using α-phospho-(Ser/Thr) antibody (Abcam, ab117253). Briefly, proteins were extracted from the protoplasts of Ubipro:ZmWAKL^Y-GFP and Ubipro:ZmWAKL^Q-GFP transgenic plants co-expressing cLUC-ZmWIK and ZmBLK1-Myc. Proteins were also extracted from the N. benthamiana leaves co-expressing cLUC-ZmWIK, ZmBLK1-Myc and ZmWAKL-GFP or GFP. The ZmWAKL protein phosphorylation level was identified by western blotting with α-phospho-(Ser/Thr) antibody (Abcam, ab117253). The proteins were extracted from the samples collected at different times after C. zeina infection. Protein extraction and detection were consistent with the methods described above.

Sample preparation for phosphoproteomic and proteomic

The sample was first grinded with liquid nitrogen, then the powder was added to four volumes of lysis buffer (including 10 mM dithiothreitol, and 1% protease inhibitor cocktail, 50 μM PR-619, 3 μM tryptic soy agar (TSA), 50 mM N-acetylmuramic acid (NAM) and 1% phosphatase inhibitor), ultrasonic cracking. An equal volume of Tris-saturated phenol (pH 8.0) was added and centrifugated at 5,500g at 4 °C for 10 min. Then the supernatant was collected and five times the volume of 0.1 M ammonium acetate/methanol was added to precipitate overnight. The precipitates were washed with methanol and acetone, respectively. Finally, the precipitate was redissolved in 8 M urea, and the protein concentration was determined with bicinchoninic acid (BCA) kit according to the manufacturer’s instructions. All samples were labeled with TMT 6-plex reagent (Thermo Fisher Scientific, 90068) according to the manufacturer’s instructions. The labeled samples were combined and dried for further analysis.

Identification of peptides and phosphopeptides

The mixed TMT 6-labeled samples were analyzed by high pH reverse phase high-performance liquid chromatography (HPLC) with Agilent 300Extend C18 as the chromatographic column. The peptide segments were separated using a gradient ranging from 8% to 32% acetonitrile (pH 9.6) within 60 min. The separated peptide segments were combined into eight components. The combined components were subjected to vacuum freeze-drying, and subsequent operations were carried out. Peptide mixtures were first incubated with IMAC microspheres suspension with vibration in loading buffer (50% acetonitrile/0.5% acetic acid). To remove the nonspecifically adsorbed peptides, the immobilised metal affinity chromatography (IMAC) microspheres were washed with 50% acetonitrile/0.5% acetic acid and 30% acetonitrile/0.1% trifluoroacetic acid, sequentially. To elute the enriched phosphopeptides, an elution buffer containing 10% NH₄OH was added and the enriched phosphopeptides were eluted with vibration. The supernatant containing phosphopeptides was collected and lyophilized for liquid chromatography with tandem mass spectrometry (LC–MS/MS) analysis. All raw data were retrieved using Proteome Discoverer (v2.4.1.15). Database is Zea_ mays_ 4577_ PR_ 20221123 (6,3235 sequences). A reverse library was added to the database to calculate the false positive rate (FPR) caused by random matching, and common contaminated libraries were added to the database to eliminate the impact of contaminated proteins in identification results. The quantitative method is set to TMT 6-plex, and the FPR for protein, peptide and peptide-spectrum match (PSM) identification is set to 1%.

Oxidative burst assay

To determine pathogen-induced ROS accumulation, the GLS pathogen C. zeina was used to activate the innate immune system. C. zeina was cultured on the corn leaf powder medium for 1–2 weeks. Then, everything on the surface of the medium, including spores and hyphae, was eluted with sterile water, and the eluted solution’s OD₆₀₀ was adjusted to 2. The oxidative burst assay was carried out as previously described with slight modifications²¹. The fourth fully expanded leaves were sliced into 3 mm strips and incubated in 200 µl 1% DMSO overnight. In total, 5 µl C. zeina was added and treated for 2 h. Then 1% DMSO was replaced by 100 µl 2× L-012 (Wako, 120-04891) in 0.05% Silwet L-77, and the strip samples were infiltrated for 1 h. Then 100 µl buffer containing 40 µg ml⁻¹ horseradish peroxidase (Sigma-Aldrich, V900503) was added into the reactions. The luminescence was recorded every 60 s for 1.25 h using the GLOMAX96 Luminometer (Promega).

Inclusion and ethics

This study’s data exclusively comes from corn, without involving any animal experiments. The transgenic planting and artificial inoculation processes are subject to strict regulation. All experimental data are included in the Data availability section.

Statistics analysis

P values and sample sizes (n) are indicated in individual figures and figure legends. Statistical analysis was performed by IBM SPSS Statistics SV26. Statistical differences between the two groups were analyzed by two-sided Student’s t test or paired t test. Statistical significance between more than two groups was analyzed based on one-way analysis of variance (ANOVA) with Tukey’s test or Fisher’s least significant difference (LSD) test. Different lowercase letters indicate a significant difference (P < 0.05).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The authors declare that the data supporting the findings of this study are available within the paper and its Supplementary Information files. The reported WAKs/WAKLs and RLCKs’ protein sequences are downloaded from the NCBI (http://www.ncbi.nlm.nih.gov/). The protein sequences of AtRBOHs are downloaded from the Arabidopsis Information Resource database (TAIR, https://www.arabidopsis.org/) and the protein sequences of ZmRBOHs are obtained from the Gramene database (https://www.gramene.org/). The expression data of ZmRBOHs and RLKs are obtained from the Plant Public RNA-seq database (http://ipf.sustech.edu.cn/pub/plantrna/). The B73 genomic sequences in the mapped qRgls1 region are collected from the MaizeGDB (https://www.maizegdb.org/). The sequences of two BAC clones (17-37-1-53 and 57-9-1-93) are available at GenBank accessions OQ435908 and OQ435909, respectively. The genomic sequences of ZmWAKL used for haplotype analysis are available at GenBank under accessions OQ425304–OQ425401. The coding sequences of ZmWAKL^Y and ZmWAKL^Q are available at GenBank accessions OQ421108 and OQ421109, respectively. The genomic and coding sequences of ZmPR5^Y and ZmPR5^Q are available at GenBank accessions OQ421106–OQ421107 and OQ421110–OQ421111, respectively. The coding sequence of ZmWIK is available at GenBank accession OQ421112. The coding sequences of ZmBLK1 and ZmBLK1-1 are available at GenBank accessions OQ421113 and OQ421114, respectively. The N-terminal of ZmRBOH4 is available at GenBank accession OQ421115. Source data are provided with this paper.

Code availability

All software used in this study is publicly available on the Internet as described in the Methods and Reporting summary.

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Acknowledgements

We thank the members of Xu Laboratory for their helpful discussions. We thank the Center for Crop Functional Genomics and Molecular Breeding of China Agricultural University for providing transgenic technology support. We also thank W. Zhu (China Agricultural University) and G. Bi (China Agricultural University) for their insightful discussions during manuscript preparation. This research was supported by the National Key Research and Development Program (grant 2022YFD1201800) and the National Natural Science Foundation of China (grant 31471500).

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Authors and Affiliations

State Key Laboratory of Plant Environmental Resilience/College of Agronomy and Biotechnology/National Maize Improvement Center/Center for Crop Functional Genomics and Molecular Breeding, China Agricultural University, Beijing, P.R. China
Tao Zhong, Mang Zhu, Qianqian Zhang, Suining Deng, Chenyu Guo & Mingliang Xu
Institute of Agricultural Biotechnology, Jilin Academy of Agricultural Sciences, Changchun, P.R. China
Yan Zhang
State Key Laboratory of Plant Environmental Resilience/College of Biological Sciences, China Agricultural University, Beijing, P.R. China
Ling Xu
Baoshan Institute of Agricultural Science, Baoshan, P.R. China
Tingting Liu & Yancong Li
Institute of Food Crops, Yunnan Academy of Agricultural Sciences, Kunming, P.R. China
Yaqi Bi & Xingming Fan
USDA-ARS Plant Science Research Unit, Raleigh NC and Department of Entomology and Plant Pathology, North Carolina State University, Raleigh, NC, USA
Peter Balint-Kurti

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Tao Zhong
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Mang Zhu
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Qianqian Zhang
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Yan Zhang
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Suining Deng
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Chenyu Guo
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Ling Xu
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Tingting Liu
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Yancong Li
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Yaqi Bi
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Xingming Fan
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Peter Balint-Kurti
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Mingliang Xu
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Contributions

M.L.X. and T.Z. conceived the project and designed the experiments. M.L.X. supervised this project. T.Z. performed most of the experiments. M.Z. was involved in most of the fieldwork. Y.Z. and L.X. were involved in the fine-mapping process and helped revise the manuscript. Q.Q.Z. was involved in the phosphorylation assay and S.N.D. was involved in confocal microscopy experiments and protein expression. C.Y.G. identified the interaction between ZmBLK1 and ZmRBOH4. T.T.L. and Y.C.L. contributed to the materials management. X.M.F. and Y.Q.B. provided the materials and supported the phenotypic identification. P.B.-K. participated in project discussions and manuscript revision. M.L.X. and T.Z. wrote the manuscript with input from all authors.

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Correspondence to Mingliang Xu.

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Extended data

Extended Data Fig. 1 Sequential fine-mapping of qRgls1.

(a) Previous mapping efforts located qRgls1 to a 1.4-Mb region on the short arm of maize chromosome 8. (b–d) Sequential fine-mapping of qRgls1 (n = 3290). The schematic diagrams to the right represent the genomic architecture of each recombinant type. The markers used in the fine-mapping are indicated, with red markers showing the limits of the qRgls1 region as defined at each fine-mapping step. The tables to the right summarize the DSI from homozygous and heterozygous backcross progeny of recombinants. Significant difference (P < 0.05) in DSI between heterozygous and homozygous offspring indicates the presence of the resistance allele at qRgls1 in the heterozygous region of their parental recombinant; otherwise, there is no qRgls1 (P > 0.05). Data are shown as means ± s.d. in (b)–(d) and the P value was determined based on two-sided Student’s t-test.

Source data

Extended Data Fig. 2 The genetic effect and phenotype of qRgls1.

(a) Genetic effect of qRgls1 on maize resistance to gray leaf spot in field tests. (b,c) GLS symptoms (b) and GLS scale (c) (n = 98) of NIL-Q11 and NIL-Y32 plants at 44 dpi in the field. Scale bar, 15 cm. (d) C. zeina hyphae on the leaf surface of NIL-Q11 and NIL-Y32 plants at 44 dpi. Scale bars, 500 μm (overall view); 100 μm (detailed view). At least three separate leaves were observed with similar results. (e,f) Pairwise comparison of flowering-related traits of two NILs in Beijing (long days) (n = 5) and Sanya (short days) (n = 3). Data are shown as means ± s.d. in (a), means ± s.e. in (f). In (c), data are shown as violin plots with individual data points. In (a), (c) and (e) the P value was determined based on two-sided Student’s t-test. Data in (e) are displayed as box and whisker plots with individual data points. The error bars represent maximum and minimum values. Center line, median; box limits, 25^th and 75^th percentiles.

Source data

Extended Data Fig. 3 The schematic diagrams of ZmWAKL and ZmPR5L loci.

Schematic diagrams of ZmWAKL (a) and ZmPR5L (b) loci, as determined by aligning the full-size complementary DNA (cDNA) with the corresponding genomic sequence. Rectangles, exons; lines, introns. The colors indicate different domains: yellow, SP (signal peptide); light orange, GUB (galacturonan-binding domain of wall-associated receptor kinase); pink, TM (transmembrane domain); blue, Ser/Thr kinase. Gray shading between the Y32 and Q11 alleles represents the syntenic region; red lines denote SNPs; black spaces or lines represent InDels.

Extended Data Fig. 4 Transgenic functional validation of the susceptible ZmWAKL^Q allele and the chimeric ZmWAKL^C gene in GLS resistance.

(a) Schematic diagram of the Ubipro:ZmWAKL^Q-GFP construct. (b) Relative expression of ZmWAKL in ZmWAKL^Q overexpressing plants (n = 3). (c,d) GLS resistance of the ZmWAKL^Q overexpression transgenic plants in backcross populations (n = 397, T₁BC₁F₁; n = 455, T₁BC₃F₁). (e) Schematic diagram of the Ubipro:ZmWAKL^C construct. The chimeric ZmWAKL^C gene was constructed by combining the ZmWAKL^Y ECD/TM domains (1-324 aa) in-frame with the ZmWAKL^Q intracellular domain (364-704 aa). (f) Relative expression of ZmWAKL in ZmWAKL^C overexpressing plants (n = 3). (g) GLS resistance of the ZmWAKL^C overexpression transgenic plants in backcross populations (n = 918). (h) DSI values resulting from the overexpression of ZmWAKL^Y (n = 28) or ZmWAKL^C (n = 38). Data are shown as means ± s.e. in (c), (d), (g) and (h); means ± s.d. in (b) and (f). Statistical significance was determined by a two-sided Student’s t-test in (b) and (f) or a paired t-test in (c), (d), (g) and (h).

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Extended Data Fig. 5 Transgenic functional validation of ZmPR5L in GLS resistance.

(a) Relative expression of ZmPR5L in plants overexpressing ZmPR5L (n = 3). (b,c) GLS symptoms (b) and DSI (c) in segregating T₂ transgenic plants (n = 365). N, non-transgenic plant; T, transgenic plant. Scale bars, 15 cm. (d) Schematic diagram of ZmPR5L and CRISPR/Cas9 editing target (red box). (e,f) GLS symptoms (e) and GLS scale (n = 148) (f) of the wild-type B73 and homozygous Zmpr5l knockout plants. Scale bar, 15 cm. Data are shown as means ± s.d. in (a); means in (c). Statistical significance was determined by a two-sided Student’s t-test in (a) and (c). Data in (f) are displayed as box and whisker plots, and different lowercase letters indicate a significant difference (P < 0.05) based on one-way ANOVA with Fisher’s least significant difference (LSD) test. The asterisk denotes the mean, and the box limits indicate the interquartile range.

Source data

Extended Data Fig. 6 Molecular characterization of ZmWAKL.

(a) Relative ZmWAKL expression in tissues collected from NIL-Q11 and NIL-Y32 plants (n = 3). (b,c) Relative expression levels of ZmWAKL in NIL-Q11 and NIL-Y32 after inoculation with C. zeina in the greenhouse (b) or field (c). (d,e) Subcellular localization of ZmWAKL in onion epidermal cells (d) and ZmWAKL-GFP overexpression transgenic plants (e). Plasmolysis was achieved by incubating cells in 0.3 g/mL sucrose for 5 min. Scale bars, 50 μm. DIC, differential interference contrast. The membrane was stained with the membrane-impermeable dye FM4-64. This experiment was repeated two times with the same results. In (a–c), values are means ± s.d., statistical significance was determined by a two-sided Student’s t-test.

Source data

Extended Data Fig. 7 ZmWIK interacts with ZmWAKL^ICD and regulates GLS resistance.

(a,c) Interactions of plasma membrane-tethered kinases with two ZmWAKL isoforms in SLC assays. (b,d) Analysis of relative luminescence units (RLUs) representing the interaction strengths between two ZmWAKL isoforms and plasma membrane-tethered kinases in pairwise combinations (n = 8). (e) Relative expression of ZmPK2 and ZmWIK in leaves of NILs (n = 3). (f,g) Plasma-membrane localization of ZmWIK in onion epidermal cells (f) and N. benthamiana leaves (g). Plasmolysis was achieved by incubating onion epidermal cells in 0.3 g/mL sucrose for 5 min. In N. benthamiana leaves, AtPIP2-mCherry (Plasma membrane intrinsic protein 2) was used as the membrane localization marker. DIC, differential interference contrast. Scale bars, 50 μm (onion epidermal cells); 100 μm (N. benthamiana leaves). This experiment was repeated two times with the same results. (h) Relative expression of ZmWIK in B73 and two homozygous overexpression transgenic lines (n = 3). (i,j) GLS symptoms (i) and scales (n = 265) (j) of B73 and transgenic lines. Scale bar, 15 cm. Data are shown as means ± s.d. in (b), (d), (e) and (h), statistical significance was determined by a two-sided Student’s t-test. Data in (j) are displayed as box and whisker plots with individual data points, the asterisk denotes the mean and the box limits indicate the interquartile range. In (b), (d) and (h), different lowercase letters indicate a significant difference (P < 0.05) based on one-way ANOVA with Tukey’s test. In (j), different lowercase letters indicate a significant difference (P < 0.05) based on one-way ANOVA with LSD test. The SLC assays were repeated at least four times.

Source data

Extended Data Fig. 8 ZmBLK1 interacts with the ZmWAKL/ZmWIK complex for phosphorylation signaling.

(a) Determination of the interaction between ZmBLK1/ZmBLK1-1 and ZmWAKL or ZmWIK. (b) Phosphorylation of ZmBLK1 by ZmWAKL^Y/ZmWIK or ZmWAKL^Q/ZmWIK in N. benthamiana leaves. ZmBLK1 was immunoprecipitated with α-Myc magnetic beads (IP, α-Myc). The immunoprecipitated and input samples were immunoblotted with the indicated antibodies. α-pS/T, anti-phospho-(Ser/Thr) antibody. (c) Phosphorylation of ZmBLK1 by the mixed ZmWAKL^Y/ZmWIK and ZmWAKL^Q/ZmWIK in vivo. ZmBLK1 was immunoprecipitated with α-Myc magnetic beads (IP, α-Myc). The immunoprecipitated and input samples were immunoblotted with the indicated antibodies. α-pS/T, anti-Phospho-(Ser/Thr) antibody. (d) Determination of ZmBLK1 kinase activity. The immunoprecipitated ZmBLK1 in (c) can phosphorylate the substrate MBP^a. Green ‘Y’ and blue ‘Q’ denote ZmBLK1 immunoprecipitated from the protoplasts of Ubipro:ZmWAKL^Y-GFP and Ubipro:ZmWAKL^Q-GFP, respectively. Phosphorylation of MBP^a was detected by autoradiography (upper). Equal protein loading is shown by CBB staining (lower). The protein amounts were quantified by densitometry of band intensities, with the value to the right set to 1. The SLC assays were repeated at least two times and the in vivo kinase assays were repeated two times.

Source data

Extended Data Fig. 9 Investigation of the interactions between N-ZmRBOH4 and ZmWAKL (or ZmWIK).

(a) Phylogenetic analysis demonstrated that ZmRBOH4 is closely related to AtRBOHD. (b) Determination of the interaction between ZmBLK1/ZmBLK1-1 and ZmRBOH4. (c,d) N-ZmRBOH4 (1-377aa) does not interact with ZmWAKL^Y, ZmWAKL^Q or ZmWIK in a SLC assay (c) or GST pull-down assay in vitro (d). (e) Interactions of cLUC-ZmBLK1 with ZmRBOHs-nLUC in an SLC assay. (f) Relative expression levels of ZmRBOHs in NIL-Q11 and NIL-Y32 after inoculation with C. zeina (n = 3). hpi, hours post inoculation. The SLC and pull-down assays were repeated at least three times.

Source data

Extended Data Fig. 10 Knockout of ZmRBOH4 by CRISPR/Cas9 results in changes to plant morphology.

(a) Schematic diagram of ZmRBOH4 and the sgRNA target site in the first exon for CRISPR/Cas9-mediated editing. The sequences of two homozygous knockout lines with insertion (ko #1) and deletion (ko #2) are shown. The wild-type B73 sequence is shown on top. The target sites and PAM are highlighted in red and gray, respectively, with deletions indicated by dashes. (b,c) Plant morphology (b) and height (c) of B73 and two homozygous knockout lines (n = 183). The knockout lines exhibit significantly reduced height. Scale bar, 30 cm. Data in (c) are displayed as box and whisker plots with individual data points, the asterisk denotes the mean, and the box limits indicate the interquartile range. The center line indicates the median, and different lowercase letters indicate a significant difference (P < 0.05) based on one-way ANOVA with LSD test.

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Zhong, T., Zhu, M., Zhang, Q. et al. The ZmWAKL–ZmWIK–ZmBLK1–ZmRBOH4 module provides quantitative resistance to gray leaf spot in maize. Nat Genet 56, 315–326 (2024). https://doi.org/10.1038/s41588-023-01644-z

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Received: 31 January 2023
Accepted: 08 December 2023
Published: 18 January 2024
Issue Date: February 2024
DOI: https://doi.org/10.1038/s41588-023-01644-z

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Abstract

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Main

Fine-mapping of qRgls1 against GLS

ZmWAKL is the causal gene underlying qRgls1

Molecular characterization of ZmWAKL

The self-association and kinase activity of ZmWAKL

ZmWIK is a coreceptor of ZmWAKL

ZmWAKL/ZmWIK interacts with ZmBLK1 for immune signaling

ZmBLK1 is phosphorylated by ZmWAKL/ZmWIK

ZmBLK1 interacts with and phosphorylates ZmRBOH4

ZmWAKL-mediated immunity against GLS in vivo

Working model of maize WAKL–WIK–BLK1–RBOH4 immune module

Discussion

Methods

Plant materials

Field assays

Sequential fine-mapping of qRgls1

Genetic transformation

Plant protein extraction and immunoblot analysis

Subcellular localization

Phylogenetic analysis

Yeast two-hybrid assay

SLC assay

Co-IP assay

IP–MS

In vitro pull-down assay

In vitro phosphorylation assay

In vivo phosphorylation assay

Sample preparation for phosphoproteomic and proteomic

Identification of peptides and phosphopeptides

Oxidative burst assay

Inclusion and ethics

Statistics analysis

Reporting summary

Data availability

Code availability

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Acknowledgements

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