The Transcription Factor StuA Regulates Central Carbon Metabolism, Mycotoxin Production, and Effector Gene Expression in the Wheat Pathogen Stagonospora nodorum

The Stagonospora nodorum StuA transcription factor gene SnStuA was identified by homology searching in the genome of the wheat pathogen Stagonospora nodorum. Gene expression analysis revealed that SnStuA transcript abundance increased throughout infection and in vitro growth to peak during sporulation. To investigate its role, the gene was deleted by homologous recombination. The growth of the resulting mutants was retarded on glucose compared to the wild-type growth, and the mutants also failed to sporulate. Glutamateas a sole carbon source restored the growth rate defect observed on glucose, although sporulation remained impaired. The SnstuA strains were essentially nonpathogenic, with only minor growth observed around the point of inoculation. The role of SnstuA was investigated using metabolomics, which revealed that this gene's product played a key role in regulating central carbon metabolism, with glycolysis, the TCA cycle, and amino acid synthesis all affected in the mutants. SnStuA was also found to positively regulate the synthesis of the mycotoxin alternariol. Gene expression studies on the recently identified effectors in Stagonospora nodorum found that SnStuA was a positive regulator of SnTox3 but was not required for the expression of ToxA. This study has uncovered a multitude of novel regulatory targets of SnStuA and has highlighted the critical role of this gene product in the pathogenicity of Stagonospora nodorum

Comprehensive annotation of the Parastagonospora nodorum reference genome using next-generation genomics, transcriptomics and proteogenomics

Parastagonospora nodorum, the causal agent of Septoria nodorum blotch (SNB), is an economically important pathogen of wheat (Triticum spp.), and a model for the study of necrotrophic pathology and genome evolution. The reference P. nodorum strain SN15 was the first Dothideomycete with a published genome sequence, and has been used as the basis for comparison within and between species. Here we present an updated reference genome assembly with corrections of SNP and indel errors in the underlying genome assembly from deep resequencing data as well as extensive manual annotation of gene models using transcriptomic and proteomic sources of evidence (https://github.com/robsyme/Parastagonospora_nodorum_SN15). The updated assembly and annotation includes 8,366 genes with modified protein sequence and 866 new genes. This study shows the benefits of using a wide variety of experimental methods allied to expert curation to generate a reliable set of gene models

Regulation of proteinaceous effector expression in phytopathogenic fungi

Effectors are molecules used by microbial pathogens to facilitate infection via effector-triggered susceptibility or tissue necrosis in their host. Much research has been focussed on the identification and elucidating the function of fungal effectors during plant pathogenesis. By comparison, knowledge of how phytopathogenic fungi regulate the expression of effector genes has been lagging. Several recent studies have illustrated the role of various transcription factors, chromosome-based control, effector epistasis, and mobilisation of endosomes within the fungal hyphae in regulating effector expression and virulence on the host plant. Improved knowledge of effector regulation is likely to assist in improving novel crop protection strategies

A quantitative PCR approach to determine gene copy number

Here, we report on the use of quantitative PCR (qPCR) to determine gene copy number in filamentous fungi. Using the sequenced dothideomycete Stagonospora nodorum, qPCR was used to unequivocally confirm the presence of single, two and three copy regions as predicted by in silico PCR. Further validation of the technique was demonstrated by verifying the copy numbers of introduced gene cassettes in previously characterised transformants of S. nodorum. Apart from increased sensitivity, this technique offers a high-throughput alternative to Southern blots for determining gene copy number, a significant factor when screening fungal mutants and transformants

A specific fungal transcription factor controls effector gene expression and orchestrates the establishment of the necrotrophic pathogen lifestyle on wheat

The fungus Parastagonospora nodorum infects wheat through the use of necrotrophic effector (NE) proteins that cause host-specific tissue necrosis. The Zn Cys transcription factor PnPf2 positively regulates NE gene expression and is required for virulence on wheat. Little is known about other downstream targets of PnPf2. We compared the transcriptomes of the P. nodorum wildtype and a strain deleted in PnPf2 (pf2-69) during in vitro growth and host infection to further elucidate targets of PnPf2 signalling. Gene ontology enrichment analysis of the differentially expressed (DE) genes revealed that genes associated with plant cell wall degradation and proteolysis were enriched in down-regulated DE gene sets in pf2-69 compared to SN15. In contrast, genes associated with redox control, nutrient and ion transport were up-regulated in the mutant. Further analysis of the DE gene set revealed that PnPf2 positively regulates twelve genes that encode effector-like proteins. Two of these genes encode proteins with homology to previously characterised effectors in other fungal phytopathogens. In addition to modulating effector gene expression, PnPf2 may play a broader role in the establishment of a necrotrophic lifestyle by orchestrating the expression of genes associated with plant cell wall degradation and nutrient assimilation. 2