Robert S. Brueggeman

The rpg4-Mediated Resistance to Wheat Stem Rust (Puccinia graminis) in Barley (Hordeum vulgare) Requires Rpg5, a Second NBS-LRR Gene, and an Actin Depolymerization Factor

The rpg4 gene confers recessive resistance to several races of wheat stem rust (Puccinia graminis f. sp. tritici) and Rpg5 provides dominant resistance against isolates of the rye stem rust (P. graminis f. sp. secalis) in barley. The rpg4 and Rpg5 genes are tightly linked on chromosome 5H, and positional cloning using high-resolution populations clearly separated the genes, unambiguously identifying Rpg5; however, the identity of rpg4 remained unclear. High-resolution genotyping of critical recombinants at the rpg4/Rpg5 locus, designated here as rpg4-mediated resistance locus (RMRL) delimited two distinct yet tightly linked loci required for resistance, designated as RMRL1 and RMRL2. Utilizing virus-induced gene silencing, each gene at RMRL1, i.e., HvRga1 (a nucleotide-binding site leucine-rich repeat [NBS-LRR] domain gene), Rpg5 (an NBS-LRR-protein kinase domain gene), and HvAdf3 (an actin depolymerizing factor-like gene), was individually silenced followed by inoculation with P. graminis f. sp. tritici race QCCJ. Silencing each gene changed the reaction type from incompatible to compatible, indicating that all three genes are required for rpg4-mediated resistance. This stem rust resistance mechanism in barley follows the emerging theme of unrelated pairs of genetically linked NBS-LRR genes required for specific pathogen recognition and resistance. It also appears that actin cytoskeleton dynamics may play an important role in determining resistance against several races of stem rust in barley.

Necrotrophic effector-triggered susceptibility (NETS) underlies the barley-Pyrenophora teres f. teres interaction specific to chromosome 6H.

Barley net form net blotch (NFNB), caused by the necrotrophic fungus Pyrenophora teres f. teres, is a destructive foliar disease in barley-growing regions worldwide. Little is known about the genetic and molecular basis of this pathosystem. Here, we identified a small secreted proteinaceous necrotrophic effector (NE), designated PttNE1, from intercellular wash fluids of the susceptible barley line Hector after inoculation with P. teres f. teres isolate 0-1. Using a barley recombinant inbred line (RIL) population developed from a cross between the sensitive/susceptible line Hector and the insensitive/resistant line NDB 112 (HN population), sensitivity to PttNE1, which we have named SPN1, mapped to a common resistance/susceptibility region on barley chromosome 6H. PttNE1-SPN1 interaction accounted for 31% of the disease variation when the HN population was inoculated with the 0-1 isolate. Strong accumulation of hydrogen peroxide and increased levels of electrolyte leakage were associated with the susceptible reaction, but not the resistant reaction. In addition, the HN RIL population was evaluated for its reactions to 10 geographically diverse P. teres f. teres isolates. Quantitative trait locus (QTL) mapping led to the identification of at least 10 genomic regions associated with disease, with chromosomes 3H and 6H harbouring major QTLs for resistance/susceptibility. SPN1 was associated with all the 6H QTLs, except one. Collectively, this information indicates that the barley-P. teres f. teres pathosystem follows, at least partially, an NE-triggered susceptibility (NETS) model that has been described in other necrotrophic fungal disease systems, especially in the Dothideomycete class of fungi.

The hijacking of a receptor kinase-driven pathway by a wheat fungal pathogen leads to disease

Activation of a wheat gene product by a fungal protein leads to cell death in the plant, allowing the pathogen to cause disease. Necrotrophic pathogens live and feed on dying tissue, but their interactions with plants are not well understood compared to biotrophic pathogens. The wheat Snn1 gene confers susceptibility to strains of the necrotrophic pathogen Parastagonospora nodorum that produce the SnTox1 protein. We report the positional cloning of Snn1, a member of the wall-associated kinase class of receptors, which are known to drive pathways for biotrophic pathogen resistance. Recognition of SnTox1 by Snn1 activates programmed cell death, which allows this necrotroph to gain nutrients and sporulate. These results demonstrate that necrotrophic pathogens such as P. nodorum hijack host molecular pathways that are typically involved in resistance to biotrophic pathogens, revealing the complex nature of susceptibility and resistance in necrotrophic and biotrophic pathogen interactions with plants.

Association mapping of seedling resistance to spot form net blotch in a worldwide collection of barley.

Spot form net blotch (SFNB), caused by the necrotrophic fungal pathogen Pyrenophora teres f. maculata, is an important foliar disease of barley in major production regions around the world. Deployment of adequate host resistance is challenging because the virulence of P. teres f. maculata is highly variable and characterized minor-effect resistances are typically ineffective against the diverse pathogen populations. A world barley core collection consisting of 2,062 barley accessions of diverse origin and genotype were phenotyped at the seedling stage with four P. teres f. maculata isolates collected from the United States (FGO), New Zealand (NZKF2), Australia (SG1), and Denmark (DEN 2.6). Of the 2,062 barley accessions phenotyped, 1,480 were genotyped with the Illumina barley iSelect chip and passed the quality controls with 5,954 polymorphic markers used for further association mapping analysis. Genome-wide association mapping was utilized to identify and map resistance loci from the seedling disease response data and the single nucleotide polymorphism (SNP) marker data. The best among six different regression models was identified for each isolate and association analysis was performed separately for each. A total of 138 significant (-log10P value>3.0) marker-trait associations (MTA) were detected. Using a 5 cM cutoff, a total of 10, 8, 13, and 10 quantitative trait loci (QTL) associated with SFNB resistance were identified for the FGO, SG1, NZKF2, and DEN 2.6 isolates, respectively. Loci containing from 1 to 34 MTA were identified on all seven barley chromosomes with one locus at 66 to 69 cM on chromosome 2H common to all four isolates. Six distinct loci were identified by the association mapping (AM) analysis that corresponded to previously characterized SFNB resistance QTL identified by biparental population analysis (QRpt4, QRpt6, Rpt4, Rpt6, Rpt7, and a QTL on 4H that was not given a provisional gene or QTL nomenclature). The 21 putative novel loci identified may represent a broad spectrum of resistance and or susceptibility loci. This is the first comprehensive AM study to characterize SFNB resistance loci underlying broad populations of the barley host and P. teres f. maculata pathogen.

Evaluation of a Pyrenophora teres f. teres mapping population reveals multiple independent interactions with a region of barley chromosome 6H.

The necrotrophic fungal pathogen Pyrenophora teres f. teres causes the foliar disease net form net blotch (NFNB) on barley. To investigate the genetics of virulence in the barley- P. teres f. teres pathosystem, we evaluated 118 progeny derived from a cross between the California isolates 15A and 6A on the barley lines Rika and Kombar, chosen based on their differential reactions to isolates 15A and 6A for NFNB disease. Genetic maps generated with SNP, SSR, and AFLP markers were scanned for quantitative trait loci (QTL) associated with virulence in P. teres f. teres. Loci underlying two major QTL, VR1 and VR2, were associated with virulence on Rika barley, accounting for 35% and 20% of the disease reaction type variation, respectively. Two different loci, VK1 and VK2, were shown to underlie two major QTL associated with virulence on Kombar barley accounting for 26% and 19% of the disease reaction type variation, respectively. Progeny isolates harboring VK1, VK2, or VR2 alone were inoculated onto a Rika×Kombar recombinant inbred line mapping population and the susceptibility induced by each pathogen genotype corresponded to the same region on barley chromosome 6H as that identified for the parental isolates 15A and 6A. The data presented here indicate that the P. teres f. teres - barley interaction can at least partially be explained by pathogen-produced necrotrophic effectors (NEs) that interact with dominant barley susceptibility genes resulting in NE triggered susceptibility (NETS).

Evaluation of a barley core collection for spot form net blotch reaction reveals distinct genotype-specific pathogen virulence and host susceptibility.

Spot form net blotch (SFNB) caused by Pyrenophora teres f. maculata is a major foliar disease of barley (Hordeum vulgare) worldwide. SFNB epidemics have recently been observed in major barley producing countries, suggesting that the local barley cultivars are not resistant and that virulence of the local pathogen populations may have changed. Here we attempt to identify sources of resistance effective against four diverse isolates of P. teres f. maculata collected from around the world. A total of 2,062 world barley core collection accessions were phenotyped using isolates of the pathogen collected in the United States (FGO), Australia (SG1), New Zealand (NZKF2), and Denmark (DEN 2.6). Isolate-specific susceptibility was identified in several of the barley accessions tested, indicating variability in both pathogen virulence and host resistance/susceptibility. Collectively, only 15 barley accessions were resistant across all isolates tested. These resistant accessions will be used to generate mapping populations and for germplasm development. Future research will involve the characterization of host resistance, pathogen virulence, and the host-pathogen interaction associated with SFNB of barley.

Validation of Genome-Wide Association Studies as a Tool to Identify Virulence Factors in Parastagonospora nodorum

Parastagonospora nodorum is a necrotrophic fungal pathogen causing Septoria nodorum blotch on wheat. We have identified nine necrotrophic effector-host dominant sensitivity gene interactions, and we have cloned three of the necrotrophic effector genes, including SnToxA, SnTox1, and SnTox3. Because sexual populations of P. nodorum are difficult to develop under lab conditions, genome-wide association study (GWAS) is the best population genomic approach to identify genomic regions associated with traits using natural populations. In this article, we used a global collection of 191 P. nodorum isolates from which we identified 2,983 single-nucleotide polymorphism (SNP) markers and gene markers for SnToxA and SnTox3 to evaluate the power of GWAS on two popular wheat breeding lines that were sensitive to SnToxA and SnTox3. Strong marker trait associations (MTA) with P. nodorum virulence that mapped to SnTox3 and SnToxA were first identified using the marker set described above. A novel locus in the P. nodorum genome associated with virulence was also identified as a result of this analysis. To evaluate whether a sufficient level of marker saturation was available, we designed a set of primers every 1 kb in the genomic regions containing SnToxA and SnTox3. Polymerase chain reaction amplification was performed across the 191 isolates and the presence/absence polymorphism was scored and used as the genotype. The marker proximity necessary to identify MTA flanking SnToxA and SnTox3 ranged from 4 to 5 and 1 to 7 kb, respectively. Similar analysis was performed on the novel locus. Using a 45% missing data threshold, two more SNP were identified spanning a 4.6-kb genomic region at the novel locus. These results showed that the rate of linkage disequilibrium (LD) decay in P. nodorum and, likely, other fungi is high compared with plants and animals. The fast LD decay in P. nodorum is an advantage only if sufficient marker density is attained. Based on our results with the SnToxA and SnTox3 regions, markers are needed every 9 or 8 kb, respectively, or in every gene, to guarantee that genes associated with a quantitative trait such as virulence are not missed.

Comparative Transcriptome Analysis of Resistant and Susceptible Common Bean Genotypes in Response to Soybean Cyst Nematode Infection

Soybean cyst nematode (SCN; Heterodera glycines Ichinohe) reproduces on the roots of common bean (Phaseolus vulgaris L.) and can cause reductions in plant growth and seed yield. The molecular changes in common bean roots caused by SCN infection are unknown. Identification of genetic factors associated with SCN resistance could help in development of improved bean varieties with high SCN resistance. Gene expression profiling was conducted on common bean roots infected by SCN HG type 0 using next generation RNA sequencing technology. Two pinto bean genotypes, PI533561 and GTS-900, resistant and susceptible to SCN infection, respectively, were used as RNA sources eight days post inoculation. Total reads generated ranged between ~ 3.2 and 5.7 million per library and were mapped to the common bean reference genome. Approximately 70–90% of filtered RNA-seq reads uniquely mapped to the reference genome. In the inoculated roots of resistant genotype PI533561, a total of 353 genes were differentially expressed with 154 up-regulated genes and 199 down-regulated genes when compared to the transcriptome of non- inoculated roots. On the other hand, 990 genes were differentially expressed in SCN-inoculated roots of susceptible genotype GTS-900 with 406 up-regulated and 584 down-regulated genes when compared to non-inoculated roots. Genes encoding nucleotide-binding site leucine-rich repeat resistance (NLR) proteins, WRKY transcription factors, pathogenesis-related (PR) proteins and heat shock proteins involved in diverse biological processes were differentially expressed in both resistant and susceptible genotypes. Overall, suppression of the photosystem was observed in both the responses. Furthermore, RNA-seq results were validated through quantitative real time PCR. This is the first report describing genes/transcripts involved in SCN-common bean interaction and the results will have important implications for further characterization of SCN resistance genes in common bean.

Association mapping utilizing diverse barley lines reveals net form net blotch seedling resistance/susceptibility loci

Key messageA diverse collection of barley lines was phenotyped with three North American Pyrenophora teres f. teres isolates and association analyses detected 78 significant marker-trait associations at 16 genomic loci.AbstractPyrenophora teres f. teres is a necrotrophic fungal pathogen and the causal agent of the economically important foliar disease net form net blotch (NFNB) of barley. The deployment of effective and durable resistance against P. teres f. teres has been hindered by the complexity of quantitative resistance and susceptibility. Several bi-parental mapping populations have been used to identify QTL associated with NFNB disease on all seven barley chromosomes. Here, we report the first genome-wide association study (GWAS) to detect marker-trait associations for resistance or susceptibility to P. teres f. teres. Geographically diverse barley genotypes from a world barley core collection (957) were genotyped with the Illumina barley iSelect chip and phenotyped with three P. teres f. teres isolates collected in two geographical regions of the USA (15A, 6A and LDNH04Ptt19). The best of nine regression models tested were identified for each isolate and used for association analysis resulting in the identification of 78 significant marker-trait associations (MTA; −log10p value >3.0). The MTA identified corresponded to 16 unique genomic loci as determined by analysis of local linkage disequilibrium between markers that did not meet a correlation threshold of R2 ≥ 0.1, indicating that the markers represented distinct loci. Five loci identified represent novel QTL and were designated QRptts-3HL, QRptts-4HS, QRptts-5HL.1, QRptts-5HL.2, and QRptts-7HL.1. In addition, 55 of the barley lines examined exhibited a high level of resistance to all three isolates and the SNP markers identified will provide useful genetic resources for barley breeding programs.

Fine Mapping of the Barley Chromosome 6H Net Form Net Blotch Susceptibility Locus

Net form net blotch, caused by the necrotrophic fungal pathogen Pyrenophora teres f. teres, is a destructive foliar disease of barley with the potential to cause significant yield loss in major production regions throughout the world. The complexity of the host–parasite genetic interactions in this pathosystem hinders the deployment of effective resistance in barley cultivars, warranting a deeper understanding of the interactions. Here, we report on the high-resolution mapping of the dominant susceptibility locus near the centromere of chromosome 6H in the barley cultivars Rika and Kombar, which are putatively targeted by necrotrophic effectors from P. teres f. teres isolates 6A and 15A, respectively. Utilization of progeny isolates derived from a cross of P. teres f. teres isolates 6A × 15A harboring single major virulence loci (VK1, VK2, and VR2) allowed for the Mendelization of single inverse gene-for-gene interactions in a high-resolution population consisting of 2976 Rika × Kombar recombinant gametes. Brachypodium distachyon synteny was exploited to develop and saturate the susceptibility region with markers, delimiting it to ∼0.24 cM and a partial physical map was constructed. This genetic and physical characterization further resolved the dominant susceptibility locus, designated Spt1 (susceptibility to P. teres f. teres). The high-resolution mapping and cosegregation of the Spt1.R and Spt1.K gene/s indicates tightly linked genes in repulsion or alleles possibly targeted by different necrotrophic effectors. Newly developed barley genomic resources greatly enhance the efficiency of positional cloning efforts in barley, as demonstrated by the Spt1 fine mapping and physical contig identification reported here.