Localization of Ptr ToxA Produced by Pyrenophora tritici-repentis Reveals Protein Import into Wheat Mesophyll Cells

Pyrenophora tritici-repentis, causal agent of tan spot of wheat, produces host-selective toxins that are determinants of pathogenicity or virulence. Ptr ToxA (ToxA), a proteina-ceous toxin produced by P. tritici-repentis, is a necrotizing toxin produced by the most common races isolated from infected wheat. Recent studies have shown that ToxA is internalized into the mesophyll cells and localizes to chloroplasts of sensitive wheat cultivars only. We employed a yeast two-hybrid screen in an effort to determine plant proteins that interact with ToxA and found that ToxA interacts with a chloroplast protein, designated ToxA binding protein 1 (ToxABP1). ToxABP1 contains a lysine-rich region within a coiled-coil domain that is similar to phosphotidyl-inositol binding sites present in animal proteins involved in endocytosis. In both ToxA-sensitive and -insensitive cultivars, ToxABP1 is expressed at similar levels and encodes an identical protein. ToxABP1 protein is present in both chloroplast membranes and chloroplast stroma. ToxA appears to interact primarily with a multimeric complex of ToxABP1 protein associated with the chloroplast membrane.

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The Arg-Gly-Asp–Containing, Solvent-Exposed Loop of Ptr ToxA Is Required for Internalization

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-21-3-0315 ◽

2008 ◽

Vol 21 (3) ◽

pp. 315-325 ◽

Cited By ~ 55

Author(s):

Viola A. Manning ◽

Sara M. Hamilton ◽

P. Andrew Karplus ◽

Lynda M. Ciuffetti

Keyword(s):

Phosphorylation Site ◽

Affinity Binding ◽

Mesophyll Cells ◽

High Affinity ◽

Treatment Zone ◽

Ptr Toxa ◽

High Affinity Binding Site ◽

Toxin Uptake

Internalization of the proteinaceous host-selective toxin, Ptr ToxA (ToxA), into sensitive wheat mesophyll cells is correlated with toxin activity. The solvent-exposed, Arg-Gly-Asp (RGD)-containing loop of ToxA is a candidate for interaction with the plasma membrane, which is a likely prerequisite to toxin internalization. Based on the percentage of cells affected by a given number of ToxA molecules in a treatment zone, the number of ToxA molecules bound to high-affinity sites was estimated at 3 × 106 per cell and the Kd for binding was estimated to be near 1 nM. An improved heterologous expression method of proteins that contain mutations in ToxA, coupled with a newly developed semiquantitative bioassay, revealed that some amino acids in the RGD-containing loop contribute more to toxin activity than others. Protease protection assays that detect internalized protein and inhibition of toxin uptake indicated that, for each ToxA variant tested, the extent of toxin activity correlates with the amount of internalized protein. RGD-containing peptide inhibition of both activity and internalization supported these findings. These data support the hypothesis that ToxA interacts with a high-affinity binding site on wheat mesophyll cells through the RGD-containing, solvent-exposed loop, resulting in toxin internalization and eventual cell death. The inability to detect phosphorylation of ToxA in vitro and in vivo suggests that a putative CKII phosphorylation site in the RGD-containing loop is required for internalization, not phosphorylation.

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A Novel Source of Resistance in Wheat to Pyrenophora tritici-repentis Race 1

Plant Disease ◽

10.1094/pdis-92-1-0091 ◽

2008 ◽

Vol 92 (1) ◽

pp. 91-95 ◽

Cited By ~ 22

Author(s):

Sukhwinder Singh ◽

William W. Bockus ◽

Indu Sharma ◽

Robert L. Bowden

Keyword(s):

Great Plains ◽

Recombinant Inbred Lines ◽

The United States ◽

Interval Mapping ◽

Tan Spot ◽

Sources Of Resistance ◽

Pyrenophora Tritici Repentis ◽

Ptr Toxa ◽

Chromosome 5B ◽

Race 1

Tan spot, caused by the fungus Pyrenophora tritici-repentis, causes serious yield losses in wheat (Triticum aestivum) and many other grasses. Race 1 of the fungus, which produces the necrosis toxin Ptr ToxA and the chlorosis toxin Ptr ToxC, is the most prevalent race in the Great Plains of the United States. Wheat genotypes with useful levels of resistance to race 1 have been deployed, but this resistance reduces damage by only 50 to 75%. Therefore, new sources of resistance to P. tritici-repentis are needed. Recombinant inbred lines developed from a cross between the Indian spring wheat cvs. WH542 (resistant) and HD29 (moderately susceptible) were evaluated for reaction to race 1 of the fungus. Composite interval mapping revealed quantitative trait loci (QTL) on the short arm of chromosome 3A explaining 23% of the phenotypic variation, and the long arm of chromosome 5B explaining 27% of the variation. Both resistance alleles were contributed by the WH542 parent. The QTL on 5BL is probably tsn1, which was described previously. The 3AS QTL (QTs.ksu-3AS) on 3AS is a novel QTL for resistance to P. tritici-repentis race 1. The QTL region is located in the most distal bin of chromosome 3AS in a 2.2-centimorgan marker interval. Flanking markers Xbarc45 and Xbarc86 are suitable for marker-assisted selection for tan spot resistance.

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

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-22-9-1056 ◽

2009 ◽

Vol 22 (9) ◽

pp. 1056-1068 ◽

Cited By ~ 31

Author(s):

Tika B. Adhikari ◽

Jianfa Bai ◽

Steven W. Meinhardt ◽

Suraj Gurung ◽

Mary Myrfield ◽

...

Keyword(s):

Gene Expression ◽

Cell Death ◽

Molecular Mechanisms ◽

Wheat Cultivar ◽

Expression Profiles ◽

Phenylpropanoid Pathway ◽

Host Responses ◽

Mesophyll Cells ◽

Pyrenophora Tritici Repentis ◽

Host Defense Response

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 H2O2 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.

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Virulence of Pyrenophora tritici-repentis: a minireview

10.22616/rrd.27.2021.003 ◽

2021 ◽

Author(s):

Janis Kaneps ◽

◽

Biruta Bankina ◽

Inga Moročko-Bičevska ◽

Keyword(s):

Single Copy ◽

Host Susceptibility ◽

Gene Encoding ◽

Pyrenophora Tritici Repentis ◽

Ptr Toxa ◽

Triticum Spp ◽

Copy Gene ◽

Polar Low ◽

Available Information ◽

Necrotrophic Effectors

Pyrenophora tritici-repentis is a major wheat pathogen in all wheat (Triticum spp.) growing areas worldwide. Up to date, eight P. tritici-repentis races have been described based on chlorosis, necrosis, or both symptoms caused on race differential wheat genotypes: ‘Glenlea’, 6B662, 6B365, and ‘Salamouni’. Symptom development on differential genotypes depends on the interaction of the pathogen’s necrotrophic effectors named Ptr ToxA, Ptr ToxB, and Ptr ToxC with host susceptibility genes. Ptr ToxA is encoded by the single copy gene ToxA and induces necrosis on sensitive wheat cultivars. Ptr ToxB causes chlorosis and is encoded by the multicopy gene ToxB. The Ptr ToxC is the non-proteinaceous, polar, low molecular mass molecule that also induces chlorosis, but up to date, the gene encoding this toxin is unknown. Races producing Ptr ToxA are predominant in the global Ptr population. There are several reports about new putative races of P. tritici-repentis that do not conform with the current race system, so further research is required. This study aims to collect and systematise available information about the virulence and races of P. tritici-repentis.

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Heterologous Expression of Functional Ptr ToxA

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2000.13.4.456 ◽

2000 ◽

Vol 13 (4) ◽

pp. 456-464 ◽

Cited By ~ 37

Author(s):

Robert P. Tuori ◽

Thomas J. Wolpert ◽

Lynda M. Ciuffetti

Keyword(s):

Fusion Protein ◽

Heterologous Expression ◽

Specific Activity ◽

Maximal Activity ◽

Cysteine Residue ◽

Binding Studies ◽

Reading Frame ◽

Pyrenophora Tritici Repentis ◽

Ptr Toxa

Ptr ToxA, a proteinaceous host-selective toxin (HST) produced by the fungus Pyrenophora tritici-repentis, was expressed in Escherichia coli and purified as a polyhistidine-tagged, fusion protein (NC-FP). NC-FP, consisting of both the N and C domains of the ToxA open reading frame (ORF), is produced as an insoluble protein in E. coli at approximately 10 to 16 mg per liter of culture. Following in vitro refolding, NC-FP elicits cultivar-specific necrosis in wheat, with a specific activity similar to that of native Ptr ToxA. A fusion protein consisting of only the C domain has approximately 10 to 20% of the activity of native Ptr ToxA. These data suggest that (i) the N domain is important for maximal activity of Ptr ToxA, (ii) the N domain does not function to eliminate activity of the protoxin, and (iii) post-translational modifications of Ptr ToxA are not essential for activity. A C domain construct with a cysteine residue mutated to glycine is inactive. This, plus the observation that toxin activity is sensitive to reducing agents, provides evidence that the two cysteine residues in Ptr ToxA are involved in a disulfide bond that is essential for activity. The heterologous expression of Ptr ToxA provides a valuable tool for addressing a number of issues such as receptor binding studies, structure/function studies, and screening wheat cultivars for disease resistance.

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Host-selective toxins, Ptr ToxA and Ptr ToxB, as necrotrophic effectors in the Pyrenophora tritici-repentis-wheat interaction

New Phytologist ◽

10.1111/j.1469-8137.2010.03362.x ◽

2010 ◽

Vol 187 (4) ◽

pp. 911-919 ◽

Cited By ~ 125

Author(s):

Lynda M. Ciuffetti ◽

Viola A. Manning ◽

Iovanna Pandelova ◽

Melania Figueroa Betts ◽

J. Patrick Martinez

Keyword(s):

Pyrenophora Tritici Repentis ◽

Ptr Toxa ◽

Necrotrophic Effectors

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Role of Host Sensitivity to Ptr ToxA in Development of Tan Spot of Wheat

Phytopathology ◽

10.1094/phyto.2003.93.4.397 ◽

2003 ◽

Vol 93 (4) ◽

pp. 397-401 ◽

Cited By ~ 51

Author(s):

T. L. Friesen ◽

S. Ali ◽

S. Kianian ◽

L. J. Francl ◽

J. B. Rasmussen

Keyword(s):

Genetic Variance ◽

Dominant Gene ◽

Tan Spot ◽

Wild Type ◽

Pyrenophora Tritici Repentis ◽

Ptr Toxa ◽

Recombinant Inbred Population ◽

Race 2 ◽

Wheat Lines

Pyrenophora tritici-repentis race 2 produces Ptr ToxA, a host-selective toxin previously described as a pathogenicity factor for tan spot on wheat. The objective of this research was to evaluate the role of host sensitivity to toxin, conditioned by a single dominant gene on chromosome 5BL, in the disease development by race 2. An F2-derived F6 recombinant inbred population of 108 wheat lines, produced from crosses of toxin-sensitive, disease-susceptible cv. Kulm with the toxin-insensitive, disease-resistant cv. Erik segregated 1:1 for toxin reaction. However, the population was skewed toward resistance to race 2 of the fungus. Toxin reaction accounted for 24.4% of the genetic variance for disease. Heritability estimates suggested the presence of four to five genes that influence disease reaction in the population. Toxin-insensitive mutants, previously derived Kulm, were susceptible to race 2, although disease developed more slowly on the mutants than it did on the wild-type Kulm. The data indicate that sensitivity to Ptr ToxA influences disease severity in some host genotypes without defining susceptibility.

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