Jack B. Rasmussen

Emergence of a new disease as a result of interspecific virulence gene transfer

New diseases of humans, animals and plants emerge regularly. Enhanced virulence on a new host can be facilitated by the acquisition of novel virulence factors. Interspecific gene transfer is known to be a source of such virulence factors in bacterial pathogens (often manifested as pathogenicity islands in the recipient organism) and it has been speculated that interspecific transfer of virulence factors may occur in fungal pathogens. Until now, no direct support has been available for this hypothesis. Here we present evidence that a gene encoding a critical virulence factor was transferred from one species of fungal pathogen to another. This gene transfer probably occurred just before 1941, creating a pathogen population with significantly enhanced virulence and leading to the emergence of a new damaging disease of wheat.

A unique wheat disease resistance-like gene governs effector-triggered susceptibility to necrotrophic pathogens

Plant disease resistance is often conferred by genes with nucleotide binding site (NBS) and leucine-rich repeat (LRR) or serine/threonine protein kinase (S/TPK) domains. Much less is known about mechanisms of susceptibility, particularly to necrotrophic fungal pathogens. The pathogens that cause the diseases tan spot and Stagonospora nodorum blotch on wheat produce effectors (host-selective toxins) that induce susceptibility in wheat lines harboring corresponding toxin sensitivity genes. The effector ToxA is produced by both pathogens, and sensitivity to ToxA is governed by the Tsn1 gene on wheat chromosome arm 5BL. Here, we report the cloning of Tsn1, which was found to have disease resistance gene-like features, including S/TPK and NBS-LRR domains. Mutagenesis revealed that all three domains are required for ToxA sensitivity, and hence disease susceptibility. Tsn1 is unique to ToxA-sensitive genotypes, and insensitive genotypes are null. Sequencing and phylogenetic analysis indicated that Tsn1 arose in the B-genome diploid progenitor of polyploid wheat through a gene-fusion event that gave rise to its unique structure. Although Tsn1 is necessary to mediate ToxA recognition, yeast two-hybrid experiments suggested that the Tsn1 protein does not interact directly with ToxA. Tsn1 transcription is tightly regulated by the circadian clock and light, providing further evidence that Tsn1-ToxA interactions are associated with photosynthesis pathways. This work suggests that these necrotrophic pathogens may thrive by subverting the resistance mechanisms acquired by plants to combat other pathogens.

SnTox3 Acts in Effector Triggered Susceptibility to Induce Disease on Wheat Carrying the Snn3 Gene

The necrotrophic fungus Stagonospora nodorum produces multiple proteinaceous host-selective toxins (HSTs) which act in effector triggered susceptibility. Here, we report the molecular cloning and functional characterization of the SnTox3-encoding gene, designated SnTox3, as well as the initial characterization of the SnTox3 protein. SnTox3 is a 693 bp intron-free gene with little obvious homology to other known genes. The predicted immature SnTox3 protein is 25.8 kDa in size. A 20 amino acid signal sequence as well as a possible pro sequence are predicted. Six cysteine residues are predicted to form disulfide bonds and are shown to be important for SnTox3 activity. Using heterologous expression in Pichia pastoris and transformation into an avirulent S. nodorum isolate, we show that SnTox3 encodes the SnTox3 protein and that SnTox3 interacts with the wheat susceptibility gene Snn3. In addition, the avirulent S. nodorum isolate transformed with SnTox3 was virulent on host lines expressing the Snn3 gene. SnTox3-disrupted mutants were deficient in the production of SnTox3 and avirulent on the Snn3 differential wheat line BG220. An analysis of genetic diversity revealed that SnTox3 is present in 60.1% of a worldwide collection of 923 isolates and occurs as eleven nucleotide haplotypes resulting in four amino acid haplotypes. The cloning of SnTox3 provides a fundamental tool for the investigation of the S. nodorum–wheat interaction, as well as vital information for the general characterization of necrotroph–plant interactions.

Duplication and partitioning in evolution and function of homoeologous Q loci governing domestication characters in polyploid wheat

The Q gene encodes an AP2-like transcription factor that played an important role in domestication of polyploid wheat. The chromosome 5A Q alleles (5AQ and 5Aq) have been well studied, but much less is known about the q alleles on wheat homoeologous chromosomes 5B (5Bq) and 5D (5Dq). We investigated the organization, evolution, and function of the Q/q homoeoalleles in hexaploid wheat (Triticum aestivum L.). Q/q gene sequences are highly conserved within and among the A, B, and D genomes of hexaploid wheat, the A and B genomes of tetraploid wheat, and the A, S, and D genomes of the diploid progenitors, but the intergenic regions of the Q/q locus are highly divergent among homoeologous genomes. Duplication of the q gene 5.8 Mya was likely followed by selective loss of one of the copies from the A genome progenitor and the other copy from the B, D, and S genomes. A recent V329-to-I mutation in the A lineage is correlated with the Q phenotype. The 5Bq homoeoalleles became a pseudogene after allotetraploidization. Expression analysis indicated that the homoeoalleles are coregulated in a complex manner. Combined phenotypic and expression analysis indicated that, whereas 5AQ plays a major role in conferring domestication-related traits, 5Dq contributes directly and 5Bq indirectly to suppression of the speltoid phenotype. The evolution of the Q/q loci in polyploid wheat resulted in the hyperfunctionalization of 5AQ, pseudogenization of 5Bq, and subfunctionalization of 5Dq, all contributing to the domestication traits.

Role of the Arginyl-Glycyl-Aspartic Motif in the Action of Ptr ToxA Produced by Pyrenophora tritici-repentis

A fundamental problem of plant science is to understand the biochemical basis of plant/pathogen interactions. The foliar disease tan spot of wheat (Triticum aestivum), caused byPyrenophora tritici-repentis, involves Ptr ToxA, a proteinaceous host-selective toxin that causes host cell death. The fungal gene ToxA encodes a 17.2-kD pre-pro-protein that is processed to produce the mature 13.2-kD toxin. Amino acids 140 to 142 of the pre-pro-protein form an arginyl-glycyl-aspartic (RGD) sequence, a motif involved in the binding of some animal proteins and pathogens to transmembrane receptor proteins called integrins. Integrin-like proteins have been identified in plants recently, but their role in plant biology is unclear. Our model for Ptr ToxA action predicts that toxin interacts with a putative host receptor through the RGD motif. Mutant clones of a ToxA cDNA, created by polymerase chain reaction such that the RGD in the pro-toxin was changed to arginyl-alanyl-aspartic or to arginyl-glycyl-glutamic, were expressed in Escherichia coli. Extracts containing mutated forms of toxin failed to cause host cell death, but extracts from E. coliexpressing both a wild-type pro-protein cDNA and a control mutation away from RGD were active in cell death development. In competition experiments, 2 mm RGD tripeptide reduced the level of electrolyte leakage from wheat leaves by 63% when co-infiltrated with purified Ptr ToxA (15 μg mL−1) obtained from the fungus, but the control peptide arginyl-glycyl-glutamyl-serine provided no protection. These experiments indicate that the RGD motif of Ptr ToxA is involved with toxin action, possibly by interacting with a putative integrin-like receptor in the host.

Two putatively homoeologous wheat genes mediate recognition of SnTox3 to confer effector-triggered susceptibility to Stagonospora nodorum

The pathogen Stagonospora nodorum produces multiple effectors, also known as host-selective toxins (HSTs), that interact with corresponding host sensitivity genes in an inverse gene-for-gene manner to cause the disease Stagonospora nodorum blotch (SNB) in wheat. In this study, a sensitivity gene was identified in Aegilops tauschii, the diploid D-genome donor of common wheat. The gene was mapped to the short arm of chromosome 5D and mediated recognition of the effector SnTox3, which was previously shown to be recognized by the wheat gene Snn3 on chromosome arm 5BS. Comparative mapping suggested that Snn3 and the gene on 5DS are probably homoeologous and derived from a common ancestor. Therefore, we propose to designate these genes as Snn3-B1 and Snn3-D1, respectively. Compatible Snn3-D1-SnTox3 interactions resulted in more severe necrosis in both effector infiltration and spore inoculation experiments than compatible Snn3-B1-SnTox3 interactions, indicating that Snn3-B1 and Snn3-D1 may have different affinities in SnTox3 recognition or signal transduction. Wheat bin-mapped expressed sequence tags and good levels of collinearity among the wheat Snn3 regions, rice (Oryza sativa), and Brachypodium distachyon were exploited for saturation and fine mapping of the Snn3-D1 locus. Markers delineating the Snn3-D1 locus to a 1.4 cM interval will be useful for initiating positional cloning. Further characterization of how these homoeologous genes mediate recognition of the same pathogen effector should enhance understanding of host manipulation by necrotrophic pathogens in causing disease.

Tsn1-Mediated Host Responses to ToxA from Pyrenophora tritici-repentis

The toxin sensitivity gene Tsn1 interacts with Ptr ToxA (ToxA), a host-selective toxin produced by the necrotrophic fungus Pyrenophora tritici-repentis. The molecular mechanisms associated with cell death in sensitive wheat cultivars following ToxA application are not well understood. To address this question, we used the Affymetrix GeneChip Wheat Genome Array to compare gene expression in a sensitive wheat cultivar possessing the Tsn1 gene with the insensitive wheat cv. Nec103, which lacks the Tsn1 gene. This analysis was performed at early timepoints after infiltration with ToxA (e.g., 0.5 to 12 h postinfiltration [hpi]); at this time, ToxA is known to internalize into mesophyll cells without visible cell death symptoms. Gene expression also was monitored at later timepoints (24 to 48 hpi), when ToxA causes extensive damage in cellular compartments and visible cell death. At both early and late timepoints, numerous defense-related genes were induced (2- to 197-fold increases) and included genes involved in the phenylpropanoid pathway, lignification, and the production of reactive oxygen species (ROS). Furthermore, a subset of host genes functioning in signal transduction, metabolism, and as transcription factors was induced as a consequence of the Tsn1-ToxA interaction. Nine genes known to be involved in the host defense response and signaling pathways were selected for analysis by quantitative real-time polymerase chain reaction, and the expression profiles of these genes confirmed the results obtained in microarray experiments. Histochemical analyses of a sensitive wheat cultivar showed that H(2)O(2) was present in leaves undergoing cell death, indicating that ROS signaling is a major event involved in ToxA-mediated cell death. The results suggest that recognition of ToxA via Tsn1 triggers transcriptional reprogramming events similar to those reported for avirulence-resistance gene interactions, and that host-derived genes play an important role in the modulation of susceptibility to P. tritici-repentis.

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.

Variable Expression of the Stagonospora nodorum Effector SnToxA Among Isolates Is Correlated with Levels of Disease in Wheat

Most research on host?pathogen interactions is focused on mechanisms of resistance, but less is known regarding mechanisms of susceptibility. The wheat?Stagonospora nodorum pathosystem involves pathogen-produced effectors, also known as host-selective toxins, that interact with corresponding dominant host genes to cause disease. Recognition of the S. nodorum effectors SnToxA and SnTox2 is mediated by the wheat genes Tsn1 and Snn2, respectively. Here, we inoculated a population of wheat recombinant inbred lines that segregates for Tsn1 and Snn2 with conidia from two S. nodorum isolates, Sn4 and Sn5, which both produce SnToxA and SnTox2 to compare the effects of compatible Tsn1?SnToxA and Snn2?SnTox2 interactions between the two isolates. Genetic analysis revealed that the two interactions contribute equally to disease caused by isolate Sn4 but the Tsn1?SnToxA interaction contributed substantially more to disease conferred by Sn5 than did the Snn2?SnTox2 interaction. Sequence analysis of the SnToxA locus from Sn4 and Sn5 indicated that they were 99.5% identical, with no polymorphisms in the coding region or the predicted promoters. Analysis of transcription levels showed that expression levels of SnToxA peaked at 26 h postinoculation for both isolates but SnToxA expression in Sn5 was more than twice that of Sn4. This work demonstrates that necrotrophic effectors of different isolates can be expressed at different levels in planta, and that higher levels of expression lead to increased levels of disease in the wheat?S. nodorum pathosystem.

Requirement of Host Signaling Mechanisms for the Action of Ptr ToxA in Wheat

Ptr ToxA, the host-selective toxin produced by Pyrenophora tritici-repentis, is genetically associated with the development of tan spot disease of wheat. The toxin was shown previously to cause a programmed cell death in the host that requires de novo mRNA and protein synthesis. In the present study, inhibitors of plant signaling mechanisms protected wheat leaves from toxin action, as determined by electrolyte leakage bioassays, when applied to leaves with toxin. Okadaic acid, calyculin A and phenylarsine oxide, all inhibitors of protein phosphatase activity, reduced toxin-induced electrolyte leakage by more than 90%. Inorganic calcium channel blockers (LaCl3 and CoCl2 reduced toxin-induced electrolyte leakage by 78–95%, depending on inhibitor and time of measurement. By comparison, about 50% protection was achieved by the application of the protein kinase inhibitors staurosporine and K-252A. Nonetheless, the reduction in toxin-induced electrolyte leakage by protein kinase inhibitors was reproduced in multiple trials and was statistically significant. The data indicate that host signaling mechanisms, including calcium fluxes and a protein phosphorylation cascade, are required for the Ptr ToxA-induced cell death in wheat. Our current model holds that the signaling events occur between toxin perception by the cell and the toxin-directed gene expression in the host associated with cell death. As an alternative, the toxin-induced mRNA synthesis required for cell death may be for protein phosphatase and/or protein kinase genes. Additional work is required to resolve these possibilities.