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.     

Recognition specificity and RAR1/SGT1 dependence in barley Mla disease resistance genes to the powdery mildew fungus

A large number of resistance specificities to the powdery mildew fungus Blumeria graminis f. sp. hordei map to the barley Mla locus. This complex locus harbors multiple members of three distantly related gene families that encode proteins that contain an N-terminal coiled-coil (CC) structure, a central nucleotide binding (NB) site, a Leu-rich repeat (LRR) region, and a C-terminal non-LRR (CT) region. We identified Mla12, which encodes a CC-NB-LRR-CT protein that shares 89 and 92% identical residues with the known proteins MLA1 and MLA6. Slow Mla12-triggered resistance was altered dramatically to a rapid response by overexpression of Mla12. A series of reciprocal domains swaps between MLA1 and MLA6 identified in each protein recognition domain for cognate powdery mildew fungus avirulence genes (AvrMla1 and AvrMla6). These domains were within different but overlapping LRR regions and the CT part. Unexpectedly, MLA chimeras that confer AvrMla6 recognition exhibited markedly different dependence on Rar1, a gene required for the function of some but not all Mla resistance specificities. Furthermore, uncoupling of MLA6-specific function from RAR1 also uncoupled the response from SGT1, a protein known to associate physically with RAR1. Our findings suggest that differences in the degree of RAR1 dependence of different MLA immunity responses are determined by intrinsic properties of MLA variants and place RAR1/SGT1 activity downstream of and/or coincident with the action of resistance protein-containing recognition complexes.     

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.     

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.     

小麦类Mla基因的分离与特征分析

根据大麦MLa基因的保守区域设计了4对家族性引物.通过用家族性引物对小麦(Triticum aestivum L.)抗白粉病品系TAM104R在接种和未接种两种条件下的基因差异表达进行RT-PCR分析,获得了一个在接种条件下特异表达的基因片段RJ-3-3L,并用RACE方法获得了其cDNA全长,命名为TaMla1.序列比对显示:TaMlal与大麦MLa位点的基因家族成员具有高度同源性,TaMla1编码的氨基酸功能基序扫描表明其为一个CC-NBS-LRR型抗病蛋白.用一套中国春缺-四体材料将TaMla1定位到了小麦的1A染色体上,这正是大麦MLa基因位点在小麦中的同源区段所在的染色体.这些结果表明,TaMla1为一个类MLa抗白粉病基因.同时我们还获得了一个在不接种条件下特异表达的基因片段RW-2-3L,序列分析表明它与MLa基因也高度同源,推测其可能是一个小麦白粉病的敏感基因或抗性负调控因子.     

小麦类Mla基因的分离与特征分析

根据大麦MLa基因的保守区域设计了4对家族性引物。通过用家族性引物对小麦(Triticum aestivum L.)抗白粉病品系TAM104R在接种和未接种两种条件下的基因差异表达进行RT-PCR分析,获得了一个在接种条件下特异表达的基因片段RJ-3-3L, 并用RACE方法获得了其cDNA全长,命名为TaMla1。序列比对显示: TaMla1与大麦MLa位点的基因家族成员具有高度同源性,TaMla1编码的氨基酸功能基序扫描表明其为一个CC-NBS-LRR型抗病蛋白。用一套中国春缺-四体材料将TaMla1定位到了小麦的1A染色体上,这正是大麦MLa基因位点在小麦中的同源区段所在的染色体。这些结果表明,TaMla1为一个类MLa抗白粉病基因。同时我们还获得了一个在不接种条件下特异表达的基因片段RW-2-3L,序列分析表明它与MLa 基因也高度同源,推测其可能是一个小麦白粉病的敏感基因或抗性负调控因子。     

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.     

DNA polymorphism among barley NILs of cv. Pallas, carrying genes for resistance to powdery mildew (Blumeria graminis f. sp. hordei)

Barley powdery mildew, caused by the pathogen Blumeria graminis f. sp. hordei is an important disease of barley (Hordeum vulgare L.). The random amplified polymorphic DNA (RAPD) method was used to detect DNA polymorphism among 7 Pallas near-isogenic lines (NILs) carrying Mla3, Mla12, Mlk, Mlp, Mlat, Mlg and MlLa genes for resistance to B. graminis f. sp. hordei. From among 500 random 10-mer primers tested, 3 were specific for NIL P2 (Mla3), 1 for P10 (Mla12), 6 for P17 (Mlk), 46 for P19 (Mlp), 4 for P20 (Mlat), 6 for P21 (Mlg), and 4 for P23 (MlLa). The results of this study demonstrated that the RAPD technique is a useful tool for detecting DNA polymorphism among Pallas NILs.     

Recombination of alleles conferring specific resistance to powdery mildew at the Mla locus in barley.

In barley (Hordeum vulgare L.), the Mla locus conditions reaction to the powdery mildew fungus Erysiphe graminis f.sp. hordei. Enrichment for genetic recombinants in the Mla region is possible by screening for recombination events between the flanking endosperm storage proteins hordeins C and B. Reciprocal crosses were made between the Franger (C.I. 16151) and Rupee (C.I. 16155) lines carrying the (Mla6 + Mla14) and Mla13 alleles, respectively. Recombinants were identified from F2 segregants by analyzing the extracted hordein polypeptides by sodium dodecyl sulphate - polyacrylamide gel electrophoresis. Two hundred and seventy-six recombinant gametes were identified from the 1800 seeds that were screened. Recombination of Mla alleles was analyzed by inoculating F4 recombinant lines with three isolates of E. graminis (A27, 5874, and CR3), which recognize specific Mla alleles. The linkage order established is Hor1-Mla6-Mla13-Mla14-Hor2. The genetic distances between Hor1-Mla6, Mla6-Mla13, and Mla13-Hor2, obtained using Mapmaker 3.0b F3 intercross analysis, are 3.9, 0.2, and 5.2 cM, respectively.     

Resistance genes for rye stem rust (SrR) and barley powdery mildew (Mla) are located in syntenic regions on short arm of chromosome.

Genetic stocks were developed for the localization and eventual cloning of the stem rust resistance gene SrR that occurs in wheat lines carrying the 1RS translocation from Secale cereale 'Imperial' rye. We have used a mutation-based approach for molecular analysis of the SrR region in rye. Forty-one independent mutants resulting in loss of SrR resistance were isolated: many of these were deletions of various sizes that were used to locate SrR with respect to chromosome group 1S markers. The analysis of the mutants showed that markers about 1 Mb apart flanking the barley Mla locus also flank SrR. Additionally, three of the approximately 20 closely related sequences of Mla in rye are deleted in each of six interstitial deletion mutants of SrR. The results indicate that the SrR region in rye is syntenic to the Mla region in barley or that SrR is possibly orthologous to the Mla locus.     

Barley Rom1 antagonizes Rar1 function in Magnaporthe oryzae-infected leaves by enhancing epidermal and diminishing mesophyll defence

* Barley (Hordeum vulgare) is a host for Blumeria graminis f. sp. hordei (Bgh), which causes powdery mildew, and for the rice blast pathogen Magnaporthe oryzae. It has previously been shown that Rar1, initially identified in a mutational screen as being required for Mla12-specified Bgh-resistance, also controlled pathogenic growth of M. oryzae in barley. Here, we tested whether the rom1 mutation (restoration of Mla12-specified resistance), which restored resistance against Bgh in a susceptible rar1-2 genetic background, also influences the interaction between barley and M. oryzae. * Disease severity after infection with M. oryzae was analysed on rar1-2 mutants and rar1-2 rom1 double mutants. Microscopy and northern analysis were used to gain insight into cellular and molecular events. * On rar1-2 rom1 double mutant plants, the number of M. oryzae disease lesions was increased in comparison to the wild-type and the rar1-2 mutant which correlated with augmented epidermal penetration. However, a decrease in the lesion diameter, apparently conditioned in the mesophyll, was also observed. * These results highlight the impact of Rom1 in basal defence of barley against different pathogens. Importantly, a tissue-specific function for Rom1 with contrasting effects on epidermal and mesophyll defence was demonstrated.     

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.