The MI-1-mediated pest resistance requires Hsp90 and Sgt1

The tomato (Solanum lycopersicum) Mi-1 gene encodes a protein with putative coiled-coil nucleotide-binding site and leucine-rich repeat motifs. Mi-1 confers resistance to root-knot nematodes (Meloidogyne spp.), potato aphids (Macrosiphum euphorbiae), and sweet potato whitefly (Bemisia tabaci). To identify genes required in the Mi-1-mediated resistance to nematodes and aphids, we used tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) to repress candidate genes and assay for nematode and aphid resistance. We targeted Sgt1 (suppressor of G-two allele of Skp1), Rar1 (required for Mla12 resistance), and Hsp90 (heat shock protein 90), which are known to participate early in resistance gene signaling pathways. Two Arabidopsis (Arabidopsis thaliana) Sgt1 genes exist and one has been implicated in disease resistance. Thus far the sequence of only one Sgt1 ortholog is known in tomato. To design gene-specific VIGS constructs, we cloned a second tomato Sgt1 gene, Sgt1-2. The gene-specific VIGS construct TRV-SlSgt1-1 resulted in lethality, while silencing Sgt1-2 using TRV-SlSgt1-2 did not result in lethal phenotype. Aphid and root-knot nematode assays of Sgt1-2-silenced plants indicated no role for Sgt1-2 in Mi-1-mediated resistance. A Nicotiana benthamiana Sgt1 VIGS construct silencing both Sgt1-1 and Sgt1-2 yielded live plants and identified a role for Sgt1 in Mi-1-mediated aphid resistance. Silencing of Rar1 did not affect Mi-1-mediated nematode and aphid resistance and demonstrated that Rar1 is not required for Mi-1 resistance. Silencing Hsp90-1 resulted in attenuation of Mi-1-mediated aphid and nematode resistance and indicated a role for Hsp90-1. The requirement for Sgt1 and Hsp90-1 in Mi-1-mediated resistance provides further evidence for common components in early resistance gene defense signaling against diverse pathogens and pests.     

A single-amino acid substitution in the sixth leucine-rich repeat of barley MLA6 and MLA13 alleviates dependence on RAR1 for disease resistance signaling

Interactions between barley and the powdery mildew pathogen, Blumeria graminis f. sp. hordei, (Bgh) are determined by unique combinations of host resistance genes, designated Mildew-resistance locus (Ml), and cognate pathogen avirulence genes. These interactions occur both dependent and independent of Rar1 (required for Mla12 resistance) and Sgt1 (Suppressor of G-two allele of skp1), which are differentially required for diverse plant disease-resistance pathways. We have isolated two new functional Mla alleles, Rar1-independent Mla7 and Rar1-dependent Mla10, as well as the Mla paralogs, Mla6-2 and Mla13-2. Utilizing the inherent diversity amongst Mla-encoded proteins, we identified the only two amino acids exclusively conserved in RAR1-dependent MLA6, MLA10, MLA12, and MLA13 that differ at the corresponding position in RAR1-independent MLA1 and MLA7. Two- and three-dimensional modeling places these residues on a predicted surface of the sixth leucine-rich repeat (LRR) domain at positions distinct from those within the beta-sheets hypothesized to determine resistance specificity. Site-directed mutagenesis of these residues indicates that RAR1 independence requires the presence of an aspartate at position 721, as mutation of this residue to a structurally similar, but uncharged, asparagine did not alter RAR1 dependence. These results demonstrate that a single-amino acid substitution in the sixth MLA LRR can alter host signaling but not resistance specificity to B. graminis.     

Barley Rom1 reveals a potential link between race-specific and nonhost resistance responses to powdery mildew fungi

The Rar1 gene, identified in the context of race-specific powdery mildew resistance mediated by the Hordeum vulgare (barley) resistance (R) gene Mla12, is required for the function of many R-mediated defense responses in mono- and dicotyledonous plant species. Mla resistance is associated with an oxidative burst and a subsequent cell death reaction of attacked cells. Rar1 mutants are impaired in these responses and, to identify genetic elements which negatively regulate the Mla12-triggered response, we have screened mutagenized Mla12 rar1 mutant populations for restoration of the resistance response. Here we describe the restoration of Mla12-specified resistance (rom1) mutant that restores features of disease resistance to a Blumeria graminis f. sp. hordei isolate expressing the avirulence gene AvrMla12 and retains susceptibility to an isolate lacking AvrMla12. Histochemical analyses show that, in rom1 mutant plants, a whole-cell oxidative burst and cell death response in attacked epidermal cells is restored in the incompatible interaction. Defense responses against tested inappropriate powdery mildews, B. graminis f. sp. tritici and Golovinomyces orontii, were diminished in rar1 mutant plants and enhanced in rom1 mutant plants relative to the wild type. These findings indicate antagonistic activities of Rar1 and Rom1 and reveal their contribution to nonhost and race-specific resistance responses.     

Cell-autonomous expression of barley Mla1 confers race-specific resistance to the powdery mildew fungus via a Rar1-independent signaling pathway

The barley Mla locus encodes 28 characterized resistance specificities to the biotrophic fungal pathogen barley powdery mildew. We describe a single-cell transient expression assay using entire cosmid DNAs to pinpoint Mla1 within the complex 240-kb Mla locus. The MLA1 cDNA encodes a 108-kD protein containing an N-terminal coiled-coil structure, a central nucleotide binding domain, and a C-terminal leucine-rich repeat region; it also contains a second short open reading frame at the 5' end that has a possible regulatory function. Although most Mla-encoded resistance specificities require Rar1 for their function, we used the single-cell expression system to demonstrate that Mla1 triggers full resistance in the presence of the severely defective rar1-2 mutant allele. Wheat contains an ortholog of barley Mla, designated TaMla, that is tightly linked to (0.7 centimorgan) but distinct from a tested resistance specificity at the complex Pm3 locus to wheat powdery mildew. Thus, the most polymorphic powdery mildew resistance loci in barley and wheat may have evolved in parallel at two closely linked homeoloci. Barley Mla1 expressed in wheat using the single-cell transformation system failed to trigger a response to any of the wheat powdery mildew Avr genes tested, indicating that AvrMla1 is not genetically fixed in wheat mildew strains.     

A high throughput barley stripe mosaic virus vector for virus induced gene silencing in monocots and dicots

Barley stripe mosaic virus (BSMV) is a single-stranded RNA virus with three genome components designated alpha, beta, and gamma. BSMV vectors have previously been shown to be efficient virus induced gene silencing (VIGS) vehicles in barley and wheat and have provided important information about host genes functioning during pathogenesis as well as various aspects of genes functioning in development. To permit more effective use of BSMV VIGS for functional genomics experiments, we have developed an Agrobacterium delivery system for BSMV and have coupled this with a ligation independent cloning (LIC) strategy to mediate efficient cloning of host genes. Infiltrated Nicotiana benthamiana leaves provided excellent sources of virus for secondary BSMV infections and VIGS in cereals. The Agro/LIC BSMV VIGS vectors were able to function in high efficiency down regulation of phytoene desaturase (PDS), magnesium chelatase subunit H (ChlH), and plastid transketolase (TK) gene silencing in N. benthamiana and in the monocots, wheat, barley, and the model grass, Brachypodium distachyon. Suppression of an Arabidopsis orthologue cloned from wheat (TaPMR5) also interfered with wheat powdery mildew (Blumeria graminis f. sp. tritici) infections in a manner similar to that of the A. thaliana PMR5 loss-of-function allele. These results imply that the PMR5 gene has maintained similar functions across monocot and dicot families. Our BSMV VIGS system provides substantial advantages in expense, cloning efficiency, ease of manipulation and ability to apply VIGS for high throughput genomics studies.     

The Barley stripe mosaic virus system used for virus-induced gene silencing in cereals differentially affects susceptibility to fungal pathogens in wheat

Barley stripe mosaic virus (BSMV) has emerged as a vector for virus-induced gene silencing (VIGS) in cereals, having been used to study a number of genes involved in resistance in both wheat and barley. However, the effects of the BSMV vector on plant physiology and disease resistance in plants remains unexplored. The BSMV inoculation control vector, BSMV:GFP was shown to cause severe viral symptoms in wheat, displaying chlorosis, leaf curling and growth inhibition typical of the symptoms seen in BSMV-infected barley. These viral symptoms were accompanied by induction of genes implicated in defense against pathogens, namely PR1, PR4, PR5, PR10 and PAL. Subsequent inoculation of BSMV:GFP-infected wheat with a wheat pathotype of Magnaporthe oryzae, the blast pathogen, resulted in decreased susceptibility. Penetration of epidermal cells and subsequent multiple cell colonization by M. oryzae was significantly reduced. This increased restriction of pathogen growth observed for BSMV:GFP infections with and without the viral coat protein gene. However, prior infection with BSMV:GFP had no effect on the development of a compatible isolate of Blumeria graminis f. sp. tritici, the causal agent of powdery mildew.     

Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required for N-mediated resistance to tobacco mosaic virus

The tobacco N gene confers resistance to tobacco mosaic virus (TMV) and encodes a Toll-interleukin-1 receptor/nucleotide binding site/leucine-rich repeat (TIR-NBS-LRR) class protein. We have developed and used a tobacco rattle virus (TRV) based virus induced gene silencing (VIGS) system to investigate the role of tobacco candidate genes in the N-mediated signalling pathway. To accomplish this we generated transgenic Nicotiana benthamiana containing the tobacco N gene. The transgenic lines exhibit hypersensitive response (HR) to TMV and restrict virus spread to the inoculated site. This demonstrates that the tobacco N gene can confer resistance to TMV in heterologous N. benthamiana. We have used this line to study the role of tobacco Rar1-, EDS1-, and NPR1/NIM1- like genes in N-mediated resistance to TMV using a TRV based VIGS approach. Our VIGS analysis suggests that these genes are required for N function. EDS1-like gene requirement for the N function suggests that EDS1 could be a common component of bacterial, fungal and viral resistance signalling mediated by the TIR-NBS-LRR class of resistance proteins. Requirement of Rar1- like gene for N-mediated resistance to TMV and some powdery mildew resistance genes in barley provide the first example of converging points in the disease resistance signalling pathways mediated by TIR-NBS-LRR and CC-NBS-LRR proteins. The TRV based VIGS approach as described here to study N-mediated resistance signalling will be useful for the analysis of not only disease resistance signalling pathways but also of other signalling pathways in genetically intractable plant systems.     

Disruption of barley immunity to powdery mildew by an in-frame Lys-Leu deletion in the essential protein SGT1

Barley (Hordeum vulgare L.) Mla (Mildew resistance locus a) and its nucleotide-binding, leucine-rich-repeat receptor (NLR) orthologs protect many cereal crops from diseases caused by fungal pathogens. However, large segments of the Mla pathway and its mechanisms remain unknown. To further characterize the molecular interactions required for NLR-based immunity, we used fast-neutron mutagenesis to screen for plants compromised in MLA-mediated response to the powdery mildew fungus, Blumeria graminis f. sp. hordei. One variant, m11526, contained a novel mutation, designated rar3 (required for Mla6 resistance3), that abolishes race-specific resistance conditioned by the Mla6, Mla7, and Mla12 alleles, but does not compromise immunity mediated by Mla1, Mla9, Mla10, and Mla13. This is analogous to, but unique from, the differential requirement of Mla alleles for the co-chaperone Rar1 (required for Mla12 resistance1). We used bulked-segregant-exome capture and fine mapping to delineate the causal mutation to an in-frame Lys-Leu deletion within the SGS domain of SGT1 (Suppressor of G-two allele of Skp1, Sgt1_{ΔKL308–309}), the structural region that interacts with MLA proteins. In nature, mutations to Sgt1 usually cause lethal phenotypes, but here we pinpoint a unique modification that delineates its requirement for some disease resistances, while unaffecting others as well as normal cell processes. Moreover, the data indicate that the requirement of SGT1 for resistance signaling by NLRs can be delimited to single sites on the protein. Further study could distinguish the regions by which pathogen effectors and host proteins interact with SGT1, facilitating precise editing of effector incompatible variants.     

The MLA6 coiled-coil, NBS-LRR protein confers AvrMla6-dependent resistance specificity to Blumeria graminis f. sp. hordei in barley and wheat

The barley Mla locus confers multiple resistance specificities to the obligate fungal biotroph, Blumeria (= Erysiphe) graminis f. sp. hordei. Interspersed within the 240 kb Mla complex are three families of resistance gene homologs (RGHs). Probes from the Mla-RGH1 family were used to identify three classes of cDNAs. The first class is predicted to encode a full-length CC-NBS-LRR protein and the other two classes contain alternatively spliced, truncated variants. Utilizing a cosmid that contains a gene corresponding to the full-length candidate cDNA, two single-cell expression assays were used to demonstrate complementation of AvrMla6-dependent, resistance specificity to B. graminis in barley and wheat. The first of these assays was also used to substantiate previous genetic data that the Mla6 allele requires the signaling pathway component, Rar1, for function. Computational analysis of MLA6 and the Rar1-independent, MLA1 protein reveals 91.2% identity and shows that the LRR domain is subject to diversifying selection. Our findings demonstrate that highly related CC-NBS-LRR proteins encoded by alleles of the Mla locus can dictate similar powdery mildew resistance phenotypes yet still require distinct downstream signaling components.     

Barley stripe mosaic virus-induced gene silencing in a monocot plant

RNA silencing of endogenous plant genes can be achieved by virus-mediated, transient expression of homologous gene fragments. This powerful, reverse genetic approach, known as virus-induced gene silencing (VIGS), has been demonstrated only in dicot plant species, where it has become an important tool for functional genomics. Barley stripe mosaic virus (BSMV) is a tripartite, positive-sense RNA virus that infects many agriculturally important monocot species including barley, oats, wheat and maize. To demonstrate VIGS in a monocot host, we modified BSMV to express untranslatable foreign inserts downstream of the gammab gene, in either sense or antisense orientations. Phytoene desaturase (PDS) is required for synthesizing carotenoids, compounds that protect chlorophyll from photo-bleaching. A partial PDS cDNA amplified from barley was 90, 88 and 74% identical to PDS cDNAs from rice, maize and Nicotiana benthamiana, respectively. Barley infected with BSMV expressing barley, rice or maize PDS fragments became photo-bleached and accumulated phytoene (the substrate for PDS) in a manner similar to plants treated with the chemical inhibitor of PDS, norflurazon. In contrast, barley infected with wild-type BSMV, or BSMV expressing either N. benthamiana PDS or antisense green fluorescent protein (GFP), did not photo-bleach or accumulate phytoene. Thus BSMV silencing of the endogenous PDS was homology-dependent. Deletion of the coat protein enhanced the ability of BSMV to silence PDS. This is the first demonstration of VIGS in a monocot, and suggests that BSMV can be used for functional genomics and studies of RNA-silencing mechanisms in monocot plant species.     

The Hsp90 Co-Chaperone Sgt1 Governs Candida albicans Morphogenesis and Drug Resistance

The molecular chaperone Hsp90 orchestrates regulatory circuitry governing fungal morphogenesis, biofilm development, drug resistance, and virulence. Hsp90 functions in concert with co-chaperones to regulate stability and activation of client proteins, many of which are signal transducers. Here, we characterize the first Hsp90 co-chaperone in the leading human fungal pathogen, Candida albicans. We demonstrate that Sgt1 physically interacts with Hsp90, and that it governs C. albicans morphogenesis and drug resistance. Genetic depletion of Sgt1 phenocopies depletion of Hsp90, inducing yeast to filament morphogenesis and invasive growth. Sgt1 governs these traits by bridging two morphogenetic regulators: Hsp90 and the adenylyl cyclase of the cAMP-PKA signaling cascade, Cyr1. Sgt1 physically interacts with Cyr1, and depletion of either Sgt1 or Hsp90 activates cAMP-PKA signaling, revealing the elusive link between Hsp90 and the PKA signaling cascade. Sgt1 also mediates tolerance and resistance to the two most widely deployed classes of antifungal drugs, azoles and echinocandins. Depletion of Sgt1 abrogates basal tolerance and acquired resistance to azoles, which target the cell membrane. Depletion of Sgt1 also abrogates tolerance and resistance to echinocandins, which target the cell wall, and renders echinocandins fungicidal. Though Sgt1 and Hsp90 have a conserved impact on drug resistance, the underlying mechanisms are distinct. Depletion of Hsp90 destabilizes the client protein calcineurin, thereby blocking crucial responses to drug-induced stress; in contrast, depletion of Sgt1 does not destabilize calcineurin, but blocks calcineurin activation in response to drug-induced stress. Sgt1 influences not only morphogenesis and drug resistance, but also virulence, as genetic depletion of C. albicans Sgt1 leads to reduced kidney fungal burden in a murine model of systemic infection. Thus, our characterization of the first Hsp90 co-chaperone in a fungal pathogen establishes C. albicans Sgt1 as a global regulator of morphogenesis and drug resistance, providing a new target for treatment of life-threatening fungal infections.     

Chromosome landing at the barley Rar1 locus

The barley Rar1 gene is an essential component of the race-specific, Mla-12-specified powdery mildew resistance reaction. As part of a map-based cloning strategy designed to isolate Rar1, five barley yeast artificial chromosomes (YACs) have been identified, ranging in size from 300 to 1100?kb. PCR-based YAC end-specific markers have been established and were employed to construct a local YAC contig. Four out of five YAC clones were found to be non-colinear with the source DNA. High-resolution genetic mapping of the YAC ends demonstrated that the set of five overlapping YAC clones encompasses the barley Rar1 gene.