Functional Analyses of the Diels-Alderase Gene sol5 of Ascochyta rabiei and Alternaria solani Indicate that the Solanapyrone Phytotoxins Are Not Required for Pathogenicity.

Ascochyta rabiei and Alternaria solani, the causal agents of Ascochyta blight of chickpea (Cicer arietinum) and early blight of potato (Solanum tuberosum), respectively, produce a set of phytotoxic compounds including solanapyrones A, B, and C. Although both the phytotoxicity of solanapyrones and their universal production among field isolates have been documented, the role of solanapyrones in pathogenicity is not well understood. Here, we report the functional characterization of the sol5 gene, which encodes a Diels-Alderase that catalyzes the final step of solanapyrone biosynthesis. Deletion of sol5 in both Ascochyta rabiei and Alternaria solani completely prevented production of solanapyrones and led to accumulation of the immediate precursor compound, prosolanapyrone II-diol, which is not toxic to plants. Deletion of sol5 did not negatively affect growth rate or spore production in vitro, and led to overexpression of the other solanapyrone biosynthesis genes, suggesting a possible feedback regulation mechanism. Phytotoxicity tests showed that solanapyrone A is highly toxic to several legume species and Arabidopsis thaliana. Despite the apparent phytotoxicity of solanapyrone A, pathogenicity tests showed that solanapyrone-minus mutants of Ascochyta rabiei and Alternaria solani were equally virulent as their corresponding wild-type progenitors, suggesting that solanapyrones are not required for pathogenicity.

Functional Analyses of the Diels-Alderase Gene sol5 of Ascochyta rabiei and Alternaria solani Indicate that the Solanapyrone Phytotoxins Are Not Required for Pathogenicity.

Ascochyta rabiei and Alternaria solani, the causal agents of Ascochyta blight of chickpea (Cicer arietinum) and early blight of potato (Solanum tuberosum), respectively, produce a set of phytotoxic compounds including solanapyrones A, B, and C. Although both the phytotoxicity of solanapyrones and their universal production among field isolates have been documented, the role of solanapyrones in pathogenicity is not well understood. Here, we report the functional characterization of the sol5 gene, which encodes a Diels-Alderase that catalyzes the final step of solanapyrone biosynthesis. Deletion of sol5 in both Ascochyta rabiei and Alternaria solani completely prevented production of solanapyrones and led to accumulation of the immediate precursor compound, prosolanapyrone II-diol, which is not toxic to plants. Deletion of sol5 did not negatively affect growth rate or spore production in vitro, and led to overexpression of the other solanapyrone biosynthesis genes, suggesting a possible feedback regulation mechanism. Phytotoxicity tests showed that solanapyrone A is highly toxic to several legume species and Arabidopsis thaliana. Despite the apparent phytotoxicity of solanapyrone A, pathogenicity tests showed that solanapyrone-minus mutants of Ascochyta rabiei and Alternaria solani were equally virulent as their corresponding wild-type progenitors, suggesting that solanapyrones are not required for pathogenicity.

Karyotype polymorphism and chromosomal rearrangement in populations of the phytopathogenic fungus, Ascochyta rabiei.

The fungus Ascochyta rabiei is the causal agent of Ascochyta blight of chickpea and the most serious threat to chickpea production. Little is currently known about the genome size or organization of A. rabiei. Given recent genome sequencing efforts, characterization of the genome at a population scale will provide a framework for genome interpretation and direction of future resequencing efforts. Electrophoretic karyotype profiles of 112 isolates from 21 countries revealed 12-16 chromosomes between 0.9 Mb and 4.6 Mb with an estimated genome size of 23 Mb-34 Mb. Three general karyotype profiles A, B, and C were defined by the arrangement of the largest chromosomes. Approximately one-third of isolates (group A) possessed a chromosome larger than 4.0 Mb that was absent from group B and C isolates. The ribosomal RNA gene (rDNA) cluster was assigned to the largest chromosome in all except four isolates (group C) whose rDNA cluster was located on the second largest chromosome (3.2 Mb). Analysis of progeny from an in vitro sexual cross between two group B isolates revealed one of 16 progeny with an rDNA-encoding chromosome larger than 4.0 Mb similar to group A isolates, even though a chromosome of this size was not present in either parent. No expansion of the rDNA cluster was detected in the progeny, indicating the increase in chromosome size was not due to an expansion in number of rDNA repeats. The karyotype of A. rabiei is relatively conserved when compared with published examples of asexual ascomycetes, but labile with the potential for large scale chromosomal rearrangements during meiosis. The results of this study will allow for the targeted sequencing of specific isolates to determine the molecular mechanisms of karyotype variation within this species.

Identification and function of a polyketide synthase gene responsible for 1,8-dihydroxynaphthalene-melanin pigment biosynthesis in Ascochyta rabiei.

Ascochyta rabiei produces and accumulates one of the well-known fungal polyketides, 1,8-dihydroxynaphthalene-melanin pigment (DHN-melanin), in asexual and sexual fruiting bodies. Degenerate PCR primers were used to isolate an ArPKS1 of A. rabiei encoding a polypeptide with high similarity to polyketide synthase (PKS) involved in biosynthesis of DHN-melanin in other ascomycetous fungi. Site-directed mutagenesis of ArPKS1 in A. rabiei generated melanin-deficient pycnidial mutants but caused no significant reduction of pathogenicity to chickpea. Pycnidiospores in ArPKS1-mutant pycnidia showed higher sensitivity to UV light exposure compared to pycnidiospores in melanized pycnidia of the wild-type progenitor isolate. Integration of an orthologous PKS1 gene from Bipolaris oryzae into the genome of the mutants complemented the dysfunctional ArPKS1 gene. This study demonstrated that A. rabiei uses a DHN-melanin pathway for pigmentation of pycnidia and this molecule may protect pycnidiospores from UV irradiation.

Development of codominant simple sequence repeat, single nucleotide polymorphism and sequence characterized amplified region markers for the pea root rot pathogen, Aphanomyces euteiches.

Three kinds of genetic markers including simple sequence repeats (SSRs), single nucleotide polymorphisms (SNPs) and sequence characterized amplified regions (SCARs) were developed from Aphanomyces euteiches. Of 69 loci tested, seven SSR, two SNP and two SCAR markers were codominantly polymorphic. These codominant markers and dominant markers described herein will facilitate population genetic and evolutionary studies of this important plant pathogen.