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Gray leaf spot, northern leaf blight and southern leaf blight are three of the most destructive foliar diseases affecting maize (Zea mays L.). Here we identified a gene, ZmCPK39, that encodes a calcium-dependent protein kinase and negatively regulates quantitative resistance to these three diseases. The ZmCPK39 allele in the resistant line displayed significantly lower pathogen-induced gene expression than that in the susceptible line. A marked decrease in ZmCPK39 abundance mitigated the phosphorylation and degradation of the transcription factor ZmDi19. This led to elevated expression of ZmPR10, a gene known to encode an antimicrobial protein, thereby enhancing maize resistance to foliar diseases. Moreover, the F 1 hybrid with reduced ZmCPK39 expression favored disease resistance, thereby increasing yield. Hence, the discovery of the ZmCPK39-ZmDi19-ZmPR10 immune module provides insight into the mechanisms underlying broad-spectrum quantitative disease resistance and also offers a new avenue for the genetic control of maize foliar diseases., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The common rust disease of maize is caused by the obligate biotrophic fungus Puccinia sorghi. The maize Rp1-D allele imparts resistance against the P. sorghi IN2 isolate by initiating a defense response that includes a rapid localized programmed cell death process, the hypersensitive response (HR). In this study, to identify AvrRp1-D from P. sorghi IN2, we employed the isolation of haustoria, facilitated by a biotin-streptavidin interaction, as a powerful approach. This method proves particularly advantageous in cases where the genome information for the fungal pathogen is unavailable, enhancing our ability to explore and understand the molecular interactions between maize and P. sorghi. The haustorial transcriptome generated through this technique, in combination with bioinformatic analyses such as SignalP and TMHMM, enabled the identification of 251 candidate effectors. We ultimately identified two closely related genes, AvrRp1-D.1 and AvrRp1-D.2, which triggered an Rp1-D-dependent defense response in Nicotiana benthamiana. AvrRp1-D-induced Rp1-D-dependent HR was further confirmed in maize protoplasts. We demonstrated that AvrRp1-D.1 interacts directly and specifically with the leucine-rich repeat (LRR) domain of Rp1-D through yeast two-hybrid assay. We also provide evidence that, in the absence of Rp1-D, AvrRp1-D.1 plays a role in suppressing the plant immune response. Our research provides valuable insights into the molecular interactions driving resistance against common rust in maize., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Plants usually employ resistance (R) genes to defend against the infection of pathogens, and most R genes encode intracellular nucleotide-binding, leucine-rich repeat (NLR) proteins. The recognition between R proteins and their cognate pathogens often triggers a rapid localized cell death at the pathogen infection sites, termed the hypersensitive response (HR). Metacaspases (MCs) belong to a cysteine protease family, structurally related to metazoan caspases. MCs play crucial roles in plant immunity. However, the underlying molecular mechanism and the link between MCs and NLR-mediated HR are not clear. In this study, we systematically investigated the MC gene family in maize and identified 11 ZmMCs belonging to two types. Further functional analysis showed that the type I ZmMC1 and ZmMC2, but not the type II ZmMC9, suppress the HR-inducing activity of the autoactive NLR protein Rp1-D21 and of its N-terminal coiled-coil (CC D21 ) signaling domain when transiently expressed in Nicotiana benthamiana. ZmMC1 and ZmMC2 physically associate with CC D21 in vivo. We further showed that ZmMC1 and ZmMC2, but not ZmMC9, are predominantly localized in a punctate distribution in both N. benthamiana and maize (Zea mays) protoplasts. Furthermore, the co-expression of ZmMC1 and ZmMC2 with Rp1-D21 and CC D21 causes their re-distribution from being uniformly distributed in the nucleocytoplasm to a punctate distribution co-localizing with ZmMC1 and ZmMC2. We reveal a novel role of plant MCs in modulating the NLR-mediated defense response and derive a model to explain it., (© 2020 Society for Experimental Biology and John Wiley & Sons Ltd.)

Plant disease resistance proteins (R-proteins) detect specific pathogen-derived molecules, triggering a defence response often including a rapid localized cell death at the point of pathogen penetration called the hypersensitive response (HR). The maize Rp1-D21 gene encodes a protein that triggers a spontaneous HR causing spots on leaves in the absence of any pathogen. Previously, we used fine mapping and functional analysis in a Nicotiana benthamiana transient expression system to identify and characterize a number of genes associated with variation in Rp1-D21-induced HR. Here we describe a system for characterizing genes mediating HR, using virus-induced gene silencing (VIGS) in a maize line carrying Rp1-D21. We assess the roles of 12 candidate genes. Three of these genes, SGT1, RAR1, and HSP90, are required for HR induced by a number of R-proteins across several plant-pathogen systems. We confirmed that maize HSP90 was required for full Rp1-D21-induced HR. However, suppression of SGT1 expression unexpectedly increased the severity of Rp1-D21-induced HR while suppression of RAR1 expression had no measurable effect. We confirmed the effects on HR of two genes we had previously validated in the N. benthamiana system, hydroxycinnamoyltransferase and caffeoyl CoA O-methyltransferase. We further showed the suppression the expression of two previously uncharacterized, candidate genes, IQ calmodulin binding protein (IQM3) and vacuolar protein sorting protein 37, suppressed Rp1-D21-induced HR. This approach is an efficient way to characterize the roles of genes modulating the hypersensitive defence response and other dominant lesion phenotypes in maize., (© 2020 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Background: The hypersensitive defense response (HR) in plants is a fast, localized necrotic response around the point of pathogen ingress. HR is usually triggered by a pathogen recognition event mediated by a nucleotide-binding site, leucine-rich repeat (NLR) protein. The autoactive maize NLR gene Rp1-D21 confers a spontaneous HR response in the absence of pathogen recognition. Previous work identified a set of loci associated with variation in the strength of Rp1-D21-induced HR. A polygalacturonase gene homolog, here termed ZmPGH1, was identified as a possible causal gene at one of these loci on chromosome 7., Results: Expression of ZmPGH1 inhibited the HR-inducing activity of both Rp1-D21 and that of another autoactive NLR, RPM1(D505V), in a Nicotiana benthamiana transient expression assay system. Overexpression of ZmPGH1 in a transposon insertion line of maize was associated with suppression of chemically-induced programmed cell death and with suppression of HR induced by Rp1-D21 in maize plants grown in the field., Conclusions: ZmPGH1 functions as a suppressor of programmed cell death induced by at least two autoactive NLR proteins and by two chemical inducers. These findings deepen our understanding of the control of the HR in plants.

Main Conclusion: The gene GRMZM2G318346 which encodes a cytochrome b-c1 complex subunit 7 is associated with variation in strength of the hypersensitive response in maize. We previously identified a QTL at 3,545,354 bp (B73 reference genome V2) on maize chromosome 5 associated with variation in the hypersensitive response (HR) conferred by the autoactive R-gene Rp1-D21 (Olukolu et al. in PLoS Genet 10:e1004562 2014). In this study, we show that a gene at this locus, GRMZM2G318346 which encodes a cytochrome b-c1 complex subunit seven (ZmQCR7), an important part of the mitochondrial electron transport chain, can suppress HR mediated by Rp1-D21 in a transient expression assay. ZmQCR7 alleles from two maize lines, W22 and B73 differ for the encoded proteins at just two sites, amino acid 27 (threonine and alanine in B73 and W22, respectively) and amino acid 109 (asparagine and serine), however, the B73 allele is much more effective at suppressing HR. We show that variation at amino acid 27 controlled this variation in HR-suppressing effects. We furthermore demonstrate that the B73 allele of ZmQCR7 can suppress HR induced by RPM1(D505 V), another autoactive R-gene, and that Arabidopsis homologs of ZmQCR7 can also suppress NLR-induced HR. The implications of these findings are discussed.

Disease resistance (R) genes encode nucleotide binding Leu-rich-repeat (NLR) proteins that confer resistance to specific pathogens. Upon pathogen recognition they trigger a defense response that usually includes a so-called hypersensitive response (HR), a rapid localized cell death at the site of pathogen infection. Intragenic recombination between two maize (Zea mays) NLRs, Rp1-D and Rp1-dp2, resulted in the formation of a hybrid NLR, Rp1-D21, which confers an autoactive HR in the absence of pathogen infection. From a previous quantitative trait loci and genome-wide association study, we identified genes encoding two key enzymes in lignin biosynthesis, hydroxycinnamoyltransferase (HCT) and caffeoyl CoA O-methyltransferase (CCoAOMT), adjacent to the nucleotide polymorphisms that were highly associated with variation in the severity of Rp1-D21-induced HR We have previously shown that the two maize HCT homologs suppress the HR conferred by Rp1-D21 in a heterologous system, very likely through physical interaction. Here, we show, similarly, that CCoAOMT2 suppresses the HR induced by either the full-length or by the N-terminal coiled-coil domain of Rp1-D21 also likely via physical interaction and that the metabolic activity of CCoAOMT2 is unlikely to be necessary for its role in suppressing HR. We also demonstrate that CCoAOMT2, HCTs, and Rp1 proteins can form in the same complexes. A model is derived to explain the roles of CCoAOMT and HCT in Rp1-mediated defense resistance., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

In plants, most disease resistance genes encode nucleotide binding Leu-rich repeat (NLR) proteins that trigger a rapid localized cell death called a hypersensitive response (HR) upon pathogen recognition. The maize (Zea mays) NLR protein Rp1-D21 derives from an intragenic recombination between two NLRs, Rp1-D and Rp1-dp2, and confers an autoactive HR in the absence of pathogen infection. From a previous quantitative trait loci and genome-wide association study, we identified a single-nucleotide polymorphism locus highly associated with variation in the severity of Rp1-D21-induced HR. Two maize genes encoding hydroxycinnamoyltransferase (HCT; a key enzyme involved in lignin biosynthesis) homologs, termed HCT1806 and HCT4918, were adjacent to this single-nucleotide polymorphism. Here, we show that both HCT1806 and HCT4918 physically interact with and suppress the HR conferred by Rp1-D21 but not other autoactive NLRs when transiently coexpressed in Nicotiana benthamiana. Other maize HCT homologs are unable to confer the same level of suppression on Rp1-D21-induced HR. The metabolic activity of HCT1806 and HCT4918 is unlikely to be necessary for their role in suppressing HR. We show that the lignin pathway is activated by Rp1-D21 at both the transcriptional and metabolic levels. We derive a model to explain the roles of HCT1806 and HCT4918 in Rp1-mediated disease resistance., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

Disease resistance (R) genes have been isolated from many plant species. Most encode nucleotide binding leucine-rich repeat (NLR) proteins that trigger a rapid localized programmed cell death called the hypersensitive response (HR) upon pathogen recognition. Despite their structural similarities, different NLR are distributed in a range of subcellular locations, and analogous domains play diverse functional roles. The autoactive maize NLR gene Rp1-D21 derives from an intragenic recombination between two NLR genes, Rp1-D and Rp1-dp2, and confers a HR independent of the presence of a pathogen. Rp1-D21 and its N-terminal coiled coil (CC) domain (CCD21) confer autoactive HR when transiently expressed in Nicotiana benthamiana. Rp1-D21 was predominantly localized in cytoplasm with a small amount in the nucleus, while CCD21 was localized in both nucleus and cytoplasm. Targeting of Rp1-D21 or CCD21 predominantly to either the nucleus or the cytoplasm abolished HR-inducing activity. Coexpression of Rp1-D21 or CCD21 constructs confined, respectively, to the nucleus and cytoplasm did not rescue full activity, suggesting nucleocytoplasmic movement was important for HR induction. This work emphasizes the diverse structural and subcellular localization requirements for activity found among plant NLR R genes.

Plant disease resistance is often mediated by nucleotide binding-leucine rich repeat (NLR) proteins which remain auto-inhibited until recognition of specific pathogen-derived molecules causes their activation, triggering a rapid, localized cell death called a hypersensitive response (HR). Three domains are recognized in one of the major classes of NLR proteins: a coiled-coil (CC), a nucleotide binding (NB-ARC) and a leucine rich repeat (LRR) domains. The maize NLR gene Rp1-D21 derives from an intergenic recombination event between two NLR genes, Rp1-D and Rp1-dp2 and confers an autoactive HR. We report systematic structural and functional analyses of Rp1 proteins in maize and N. benthamiana to characterize the molecular mechanism of NLR activation/auto-inhibition. We derive a model comprising the following three main features: Rp1 proteins appear to self-associate to become competent for activity. The CC domain is signaling-competent and is sufficient to induce HR. This can be suppressed by the NB-ARC domain through direct interaction. In autoactive proteins, the interaction of the LRR domain with the NB-ARC domain causes de-repression and thus disrupts the inhibition of HR. Further, we identify specific amino acids and combinations thereof that are important for the auto-inhibition/activity of Rp1 proteins. We also provide evidence for the function of MHD2, a previously uncharacterized, though widely conserved NLR motif. This work reports several novel insights into the precise structural requirement for NLR function and informs efforts towards utilizing these proteins for engineering disease resistance.

Background: Rp1 is a complex locus of maize, which carries a set of genes controlling race-specific resistance to the common rust fungus, Puccinia sorghi. The resistance response includes the "Hypersensitive response" (HR), a rapid response triggered by a pathogen recognition event that includes localized cell death at the point of pathogen penetration and the induction of pathogenesis associated genes. The Rp1-D21gene is an autoactive allelic variant at the Rp1 locus, causing spontaneous activation of the HR response, in the absence of pathogenesis. Previously we have shown that the severity of the phenotype conferred by Rp1-D21 is highly dependent on genetic background., Results: In this study we show that the phenotype conferred by Rp1-D21 is highly dependent on temperature, with lower temperatures favoring the expression of the HR lesion phenotype. This temperature effect was observed in all the 14 genetic backgrounds tested. Significant interactions between the temperature effects and genetic background were observed. When plants were grown at temperatures above 30°C, the spontaneous HR phenotype conferred by Rp1-D21 was entirely suppressed. Furthermore, this phenotype could be restored or suppressed by alternately reducing and increasing the temperature appropriately. Light was also required for the expression of this phenotype. By examining the expression of genes associated with the defense response we showed that, at temperatures above 30°C, the Rp1-D21 phenotype was suppressed at both the phenotypic and molecular level., Conclusions: We have shown that the lesion phenotype conferred by maize autoactive resistance gene Rp1-D21 is temperature sensitive in a reversible manner, that the temperature-sensitivity phenotype interacts with genetic background and that the phenotype is light sensitive. This is the first detailed demonstration of this phenomenon in monocots and also the first demonstration of the interaction of this effect with genetic background. The use of temperature shifts to induce a massive and synchronous HR in plants carrying the Rp1-D21 genes will be valuable in identifying components of the defense response pathway.

The partially dominant, autoactive maize disease resistance gene Rp1-D21 causes hypersensitive response (HR) lesions to form spontaneously on leaves and stems in the absence of pathogen recognition. The maize nested association mapping (NAM) population consists of 25 200-line subpopulations each derived from a cross between the maize line B73 and one of 25 diverse inbred lines. By crossing a line carrying the Rp1-D21 gene with lines from three of these subpopulations and assessing the F(1) progeny, we were able to map several novel loci that modify the maize HR, using both single-population quantitative trait locus (QTL) and joint analysis of all three populations. Joint analysis detected QTL in greater number and with greater confidence and precision than did single population analysis. In particular, QTL were detected in bins 1.02, 4.04, 9.03, and 10.03. We have previously termed this technique, in which a mutant phenotype is used as a "reporter" for a trait of interest, Mutant-Assisted Gene Identification and Characterization (MAGIC).

Plant oxylipins, produced via the lipoxygenase (LOX) pathway, function as signals in defense and development. In fungi, oxylipins are potent regulators of mycotoxin biosynthesis and sporogenesis. Previous studies showed that plant 9-LOX-derived fatty acid hydroperoxides induce conidiation and mycotoxin production. Here, we tested the hypothesis that oxylipins produced by the maize 9-LOX pathway are required by pathogens to produce spores and mycotoxins and to successfully colonize the host. Maize mutants were generated in which the function of a 9-LOX gene, ZmLOX3, was abolished by an insertion of a Mutator transposon in its coding sequence, which resulted in reduced levels of several 9-LOX-derived hydroperoxides. Supporting our hypothesis, conidiation and production of the mycotoxin fumonisin B1 by Fusarium verticillioides were drastically reduced in kernels of the lox3 mutants compared with near-isogenic wild types. Similarly, conidia production and disease severity of anthracnose leaf blight caused by Colletotrichum graminicola were significantly reduced in the lox3 mutants. Moreover, lox3 mutants displayed increased resistance to southern leaf blight caused by Cochliobolus heterostrophus and stalk rots caused by both F. verticillioides and C. graminicola. These data strongly suggest that oxylipin metabolism mediated by a specific plant 9-LOX isoform is required for fungal pathogenesis, including disease development and production of spores and mycotoxins.

A correction to the article “Molecular and Functional Analyses of a Maize Autoactive NB-LRR Protein Identify Precise Structural Requirements for Activity" that was published in the journal is presented.