Shunwen Lu

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.

The cysteine rich necrotrophic effector SnTox1 produced by Stagonospora nodorum triggers susceptibility of wheat lines harboring Snn1.

The wheat pathogen Stagonospora nodorum produces multiple necrotrophic effectors (also called host-selective toxins) that promote disease by interacting with corresponding host sensitivity gene products. SnTox1 was the first necrotrophic effector identified in S. nodorum, and was shown to induce necrosis on wheat lines carrying Snn1. Here, we report the molecular cloning and validation of SnTox1 as well as the preliminary characterization of the mechanism underlying the SnTox1-Snn1 interaction which leads to susceptibility. SnTox1 was identified using bioinformatics tools and verified by heterologous expression in Pichia pastoris. SnTox1 encodes a 117 amino acid protein with the first 17 amino acids predicted as a signal peptide, and strikingly, the mature protein contains 16 cysteine residues, a common feature for some avirulence effectors. The transformation of SnTox1 into an avirulent S. nodorum isolate was sufficient to make the strain pathogenic. Additionally, the deletion of SnTox1 in virulent isolates rendered the SnTox1 mutated strains avirulent on the Snn1 differential wheat line. SnTox1 was present in 85% of a global collection of S. nodorum isolates. We identified a total of 11 protein isoforms and found evidence for strong diversifying selection operating on SnTox1. The SnTox1-Snn1 interaction results in an oxidative burst, DNA laddering, and pathogenesis related (PR) gene expression, all hallmarks of a defense response. In the absence of light, the development of SnTox1-induced necrosis and disease symptoms were completely blocked. By comparing the infection processes of a GFP-tagged avirulent isolate and the same isolate transformed with SnTox1, we conclude that SnTox1 may play a critical role during fungal penetration. This research further demonstrates that necrotrophic fungal pathogens utilize small effector proteins to exploit plant resistance pathways for their colonization, which provides important insights into the molecular basis of the wheat-S. nodorum interaction, an emerging model for necrotrophic pathosystems.

Molecular characterization and genomic mapping of the pathogenesis-related protein 1 (PR-1) gene family in hexaploid wheat (Triticum aestivum L.)

The group 1 pathogenesis-related (PR-1) proteins, known as hallmarks of defense pathways, are encoded by multigene families in plants as evidenced by the presence of 22 and 32 PR-1 genes in the finished Arabidopsis and rice genomes, respectively. Here, we report the initial characterization and mapping of 23 PR-1-like (TaPr-1) genes in hexaploid wheat (Triticum aestivum L.), which possesses one of the largest (>16,000 megabases) genomes among monocot crop plants. Sequence analysis revealed that the 23 TaPr-1 genes all contain intron-free open reading frames that encode a signal peptide at the N-terminus and a conserved PR-1-like domain. Phylogenetic analysis indicated that TaPr-1 genes form three major monophyletic groups along with their counterparts in other monocots; each group consists of genes encoding basic, basic with a C-terminal extension, and acidic PR-1 proteins, respectively, suggesting diversity and conservation of PR-1 gene functions in monocot plants. Mapping analysis assisted by untranslated region-specified discrimination (USD) markers and various cytogenetic stocks located the 23 TaPr-1 genes to seven different chromosomes, with the majority mapping to chromosomes of homoeologous groups 5 and 7. Reverse transcriptase (RT)-PCR analysis revealed that 12 TaPr-1 genes were induced or up-regulated upon pathogen challenge. Together, this study provides insights to the origin, evolution, homoeologous relationships, and expression patterns of the TaPr-1 genes. The data presented provide critical information for further genome-wide characterization of the wheat PR-1 gene family and the USD markers developed will facilitate genetic and functional analysis of PR-1 genes associated with plant defense and/or other important traits.

Mating type locus-specific polymerase chain reaction markers for differentiation of Pyrenophora teres f. teres and P. teres f. maculata, the causal agents of barley net blotch.

Fourteen single nucleotide polymorphisms (SNPs) were identified at the mating type (MAT) loci of Pyrenophora teres f. teres (Ptt), which causes net form (NF) net blotch, and P. teres f. maculata (Ptm), which causes spot form (SF) net blotch of barley. MAT-specific SNP primers were developed for polymerase chain reaction (PCR) and the two forms were differentiated by distinct PCR products: PttMAT1-1 (1,143 bp) and PttMAT1-2 (1,421 bp) for NF MAT1-1 and MAT1-2 isolates; PtmMAT1-1 (194 bp) and PtmMAT1-2 (939 bp) for SF MAT1-1 and MAT1-2 isolates, respectively. Specificity was validated using 37 NF and 17 SF isolates collected from different geographic regions. Both MAT1-1 and MAT1-2 SNP primers retained respective specificity when used in duplex PCR. No cross-reactions were observed with DNA from P. graminea, P. tritici-repentis, or other ascomycetes, or barley. Single or mixed infections of the two different forms were also differentiated. This study provides the first evidence that the limited SNPs at the MAT locus are sufficient for distinguishing closely related heterothallic ascomycetes at subspecies levels, thus allowing pathogenicity and mating type characteristics of the fungus to be determined simultaneously. Methods presented will facilitate pathogen detection, disease management, and epidemiological studies.

Dimerization and protease resistance: New insight into the function of PR-1

The group 1 pathogenesis-related (PR-1) proteins have long been considered hallmarks of hypersensitive response/defense pathways in plants, but their biochemical functions are still obscure despite resolution of the NMR/X-ray structures of several PR-1-like proteins, including P14a (the prototype PR-1). We report here the characterization of two basic PR-1 proteins (PR-1-1 and PR-1-5) recently identified from hexaploid wheat (Triticum aestivum). Both proteins were expressed in Pichia pastoris as a single major species of ∼15 kDa. Sequence identity of the expressed PR-1 proteins was verified by MALDI-TOF/TOF analysis. Accumulation of the native PR-1-5 protein in pathogen-challenged wheat was confirmed by protein gel blot analysis. Low-temperature SDS-PAGE and yeast two-hybrid assays revealed that PR-1-1 exists primarily as a monomer whereas PR-1-5 forms homodimers. Both PR-1 proteins are resistant to proteases compared to bovine serum albumin, but PR-1-1 shows resistance mainly to subtilisin and protease K (serine proteases) whereas PR-1-5 shows resistance to subtilisin, protease K and papain (a cysteine protease). Site-specific mutations at the five putative active sites in the PR-1 domain all affected dimerization, with the mutations at Glu-72 and Glu-102 (in the PR-1-5 numeration) also diminishing protease resistance. Sequence analysis revealed that the Glu-72 and Glu-102 residues are located in motif-like sequences that are conserved in both PR-1 and the human apoptosis-related caspase proteins. These findings prompt us to examine the function of PR-1 for a role in protease-mediated programmed cell death pathways in plants.

Genome-Wide Analysis of Small Secreted Cysteine-Rich Proteins Identifies Candidate Effector Proteins Potentially Involved in Fusarium graminearum-Wheat Interactions.

Pathogen-derived, small secreted cysteine-rich proteins (SSCPs) are known to be a common source of fungal effectors that trigger resistance or susceptibility in specific host plants. This group of proteins has not been well studied in Fusarium graminearum, the primary cause of Fusarium head blight (FHB), a devastating disease of wheat. We report here a comprehensive analysis of SSCPs encoded in the genome of this fungus and selection of candidate effector proteins through proteomics and sequence/transcriptional analyses. A total of 190 SSCPs were identified in the genome of F. graminearum (isolate PH-1) based on the presence of N-terminal signal peptide sequences, size (≤200 amino acids), and cysteine content (≥2%) of the mature proteins. Twenty-five (approximately 13%) SSCPs were confirmed to be true extracellular proteins by nanoscale liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) analysis of a minimal medium-based in vitro secretome. Sequence analysis suggested that 17 SSCPs harbor conserved functional domains, including two homologous to Ecp2, a known effector produced by the tomato pathogen Cladosporium fulvum. Transcriptional analysis revealed that at least 34 SSCPs (including 23 detected in the in vitro secretome) are expressed in infected wheat heads; about half are up-regulated with expression patterns correlating with the development of FHB. This work provides a solid candidate list for SSCP-derived effectors that may play roles in mediating F. graminearum-wheat interactions. The in vitro secretome-based method presented here also may be applicable for identifying candidate effectors in other ascomycete pathogens of crop plants.

Molecular Cloning and Comparative Analysis of a PR-1-RK Hybrid Gene from Triticum urartu , the A-Genome Progenitor of Hexaploid Wheat

Wheat genomes encode pathogenesis-related protein 1 (PR-1)/receptor-like kinase (RK) hybrid proteins as first reported for hexaploid wheat. To date, no PR-1-RK-like proteins have been identified in the diploid wild wheat Triticum urartu, the A-genome progenitor of hexaploid wheat. Here, we report the cloning and characterization of a PR-1-RK hybrid gene (TuPr-1-rk) and a related pseudogene (TuPr-1-rkP) from T. urartu and comparative analysis of the wheat Pr-1-rk genes. TuPr-1-rk and TuPr-1-rkP were found to distribute independently among the natural populations of T. urartu, and the accession G1812 (the source of the published genome) was found to contain TuPr-1-rkP only. Sequence analysis revealed that TuPr-1-rkP originated from TuPr-1-rk through repetitive DNA-associated recombination. Transcriptional analysis confirmed that TuPr-1-rk is expressed in response to salinity stress and pathogen attack and is subject to alternative splicing (AS) as are the Pr-1-rk genes in hexaploid wheat, whereas TuPr-1-rkP is completely silenced. Thirteen AS-derived TuPr-1-rk mRNA isoforms were identified, and a comparable abundance was found between one encoding the full-length protein and those encoding C-terminally truncated proteins. Comparative analysis revealed that the wheat PR-1-RK proteins are highly conserved despite the substantial genomic variations of the coding genes. The identification of the TuPr-1-rk gene adds an important ancestral member to the wheat PR-1-RK gene family, and the observed commonality in expression patterns and the conservation of the PR-1-RK proteins support the notion that the Pr-1-rk gene may play important roles in stress response-related pathways in wheat.