Exploring the Relationship Between Pattern-Triggered Immunity and Quantitative Resistance to Xanthomonas vasicola pv. vasculorum in Maize.

Bacterial leaf streak (BLS) of maize is an emerging foliar disease of maize in the Americas. It is caused by the gram-negative nonvascular bacterium Xanthomonas vasicola pv. vasculorum. There are no chemical controls available for BLS, and thus, host resistance is crucial for managing X. vasicola pv. vasculorum. The objective of this study was to examine the genetic determinants of resistance to X. vasicola pv. vasculorum in maize, as well as the relationship between other defense-related traits and BLS resistance. Specifically, we examined the correlations among BLS severity, severity for three fungal diseases, flg-22 response, hypersensitive response, and auricle color. We conducted quantitative trait locus (QTL) mapping for X. vasicola pv. vasculorum resistance using the maize recombinant inbred line population Z003 (B73 × CML228). We detected three QTLs for BLS resistance. In addition to the disease resistance QTL, we detected a single QTL for auricle color. We observed significant, yet weak, correlations among BLS severity, levels of pattern-triggered immunity response and leaf flecking. These results will be useful for understanding resistance to X. vasicola pv. vasculorum and mitigating the impact of BLS on maize yields.

Two pathogen loci determine Blumeria graminis f. sp. tritici virulence to wheat resistance gene Pm1a.

Blumeria graminis f. sp. tritici (Bgt) is a globally important fungal pathogen of wheat that can rapidly evolve to defeat wheat powdery mildew (Pm) resistance genes. Despite periodic regional deployment of the Pm1a resistance gene in US wheat production, Bgt strains that overcome Pm1a have been notably nonpersistent in the United States, while on other continents, they are more widely established. A genome-wide association study (GWAS) was conducted to map sequence variants associated with Pm1a virulence in 216 Bgt isolates from six countries, including the United States. A virulence variant apparently unique to Bgt isolates from the United States was detected in the previously mapped gene AvrPm1a (BgtE-5612) on Bgt chromosome 6; an in vitro growth assay suggested no fitness reduction associated with this variant. A gene on Bgt chromosome 8, Bgt-51526, was shown to function as a second determinant of Pm1a virulence, and despite < 30% amino acid identity, BGT-51526 and BGTE-5612 were predicted to share > 85% of their secondary structure. A co-expression study in Nicotiana benthamiana showed that BGTE-5612 and BGT-51526 each produce a PM1A-dependent hypersensitive response. More than one member of a B. graminis effector family can be recognized by a single wheat immune receptor, and a two-gene model is necessary to explain virulence to Pm1a.

Multi-Omics Analyses Reveal the Regulatory Network and the Function of ZmUGTs in Maize Defense Response.

Maize is one of the major crops in the world; however, diseases caused by various pathogens seriously affect its yield and quality. The maize Rp1-D21 mutant (mt) caused by the intragenic recombination between two nucleotide-binding, leucine-rich repeat (NLR) proteins, exhibits autoactive hypersensitive response (HR). In this study, we integrated transcriptomic and metabolomic analyses to identify differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) in Rp1-D21 mt compared to the wild type (WT). Genes involved in pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) were enriched among the DEGs. The salicylic acid (SA) pathway and the phenylpropanoid biosynthesis pathway were induced at both the transcriptional and metabolic levels. The DAMs identified included lipids, flavones, and phenolic acids, including 2,5-DHBA O-hexoside, the production of which is catalyzed by uridinediphosphate (UDP)-dependent glycosyltransferase (UGT). Four maize UGTs (ZmUGTs) homologous genes were among the DEGs. Functional analysis by transient co-expression in Nicotiana benthamiana showed that ZmUGT9250 and ZmUGT5174, but not ZmUGT9256 and ZmUGT8707, partially suppressed the HR triggered by Rp1-D21 or its N-terminal coiled-coil signaling domain (CCD21). None of the four ZmUGTs interacted physically with CCD21 in yeast two-hybrid or co-immunoprecipitation assays. We discuss the possibility that ZmUGTs might be involved in defense response by regulating SA homeostasis.

The maize ZmMIEL1 E3 ligase and ZmMYB83 transcription factor proteins interact and regulate the hypersensitive defence response.

The plant hypersensitive response (HR), a rapid cell death at the point of pathogenesis, is mediated by nucleotide-binding site, leucine-rich repeat (NLR) resistance proteins (R-proteins) that recognize the presence of specific pathogen-derived proteins. Rp1-D21 is an autoactive maize NLR R-protein that triggers HR spontaneously. We previously mapped loci associated with variation in the strength of HR induced by Rp1-D21. Here we identify the E3 ligase ZmMIEL1 as the causal gene at a chromosome 10 modifier locus. Transient ZmMIEL1 expression in Nicotiana benthamiana reduced HR induced by Rp1-D21, while suppression of ZmMIEL1 expression in maize carrying Rp1-D21 increased HR. ZmMIEL1 also suppressed HR induced by another autoactive NLR, the Arabidopsis R-protein RPM1D505V, in N. benthamiana. We demonstrated that ZmMIEL1 is a functional E3 ligase and that the effect of ZmMIEL1 was dependent on the proteasome but also that levels of Rp1-D21 and RPM1D505V were not reduced when coexpressed with ZmMIEL1 in the N. benthamiana system. By comparison to a similar system in Arabidopsis, we identify ZmMYB83 as a potential target of ZmMIEL1. Suppression of ZmMYB83 expression in maize lines carrying Rp1-D21 suppressed HR. Suppression of ZmMIEL1 expression caused an increase in ZmMYB83 transcript and protein levels in N. benthamiana and maize. Using coimmunoprecipitation and bimolecular fluorescence complementation assays, we demonstrated that ZmMIEL1 and ZmMYB83 physically interacted. Additionally, ZmMYB83 and ZmMIEL1 regulated the expression of a set of maize very long chain fatty acid (VLCFA) biosynthetic genes that may be involved in regulating HR.

Maize metacaspases modulate the defense response mediated by the NLR protein Rp1-D21 likely by affecting its subcellular localization.

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 (CCD21 ) signaling domain when transiently expressed in Nicotiana benthamiana. ZmMC1 and ZmMC2 physically associate with CCD21 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 CCD21 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.

Use of virus-induced gene silencing to characterize genes involved in modulating hypersensitive cell death in maize.

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.

A maize polygalacturonase functions as a suppressor of programmed cell death in plants.

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.

Maize Homologs of CCoAOMT and HCT, Two Key Enzymes in Lignin Biosynthesis, Form Complexes with the NLR Rp1 Protein to Modulate the Defense Response.

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.

Maize Homologs of Hydroxycinnamoyltransferase, a Key Enzyme in Lignin Biosynthesis, Bind the Nucleotide Binding Leucine-Rich Repeat Rp1 Proteins to Modulate the Defense Response.

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.

Cytoplasmic and Nuclear Localizations Are Important for the Hypersensitive Response Conferred by Maize Autoactive Rp1-D21 Protein.

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.

Targeted discovery of quantitative trait loci for resistance to northern leaf blight and other diseases of maize.

To capture diverse alleles at a set of loci associated with disease resistance in maize, heterogeneous inbred family (HIF) analysis was applied for targeted QTL mapping and near-isogenic line (NIL) development. Tropical maize lines CML52 and DK888 were chosen as donors of alleles based on their known resistance to multiple diseases. Chromosomal regions ("bins"; n = 39) associated with multiple disease resistance (MDR) were targeted based on a consensus map of disease QTLs in maize. We generated HIFs segregating for the targeted loci but isogenic at ~97% of the genome. To test the hypothesis that CML52 and DK888 alleles at MDR hotspots condition broad-spectrum resistance, HIFs and derived NILs were tested for resistance to northern leaf blight (NLB), southern leaf blight (SLB), gray leaf spot (GLS), anthracnose leaf blight (ALB), anthracnose stalk rot (ASR), common rust, common smut, and Stewart's wilt. Four NLB QTLs, two ASR QTLs, and one Stewart's wilt QTL were identified. In parallel, a population of 196 recombinant inbred lines (RILs) derived from B73 × CML52 was evaluated for resistance to NLB, GLS, SLB, and ASR. The QTLs mapped (four for NLB, five for SLB, two for GLS, and two for ASR) mostly corresponded to those found using the NILs. Combining HIF- and RIL-based analyses, we discovered two disease QTLs at which CML52 alleles were favorable for more than one disease. A QTL in bin 1.06-1.07 conferred resistance to NLB and Stewart's wilt, and a QTL in 6.05 conferred resistance to NLB and ASR.