| Black Sigatoka, caused by the fungus Mycosphaerella fijiensis, is considered the most economically damaging banana disease. Despite its importance, little is known about the molecular basis of pathogenicity. Some of its close relatives produce polyketide toxins that kill host tissue and enable fungal nutrient acquisition. The goal of this research was to identify putative pathogenicity genes in this fungus, with focus on possible polyketide toxins. Bioinformatics analyses predicted seven polyketide synthases (PKS) and one hybrid PKS/non-ribosomal peptide synthase (NRPS) in the M. fijiensis genome. Putative biosynthetic gene clusters were also identified. The phylogenetics analyses and putative biosynthetic cluster comparisons predict that the M. fijiensis PKS10-1 cluster encodes proteins involved in DHN melanin synthesis, and also show that the PKS2-1, PKS8-2, and PKS10-2 gene clusters are similar to clusters in other fungi that produce alternapyrone, fumonisin, and solanapyrone, respectively. RNA-Seq analysis of M. fijiensis-infected leaf tissue in the necrotrophic phase showed that PKS7-1, PKS8-2, Hybrid8-3, PKS8-4, and PKS10-2 are more highly expressed in infected leaf tissue than in culture medium.;Two polyketide synthases were selected for further analysis. Promoter:GFP fusion experiments suggested that PKS8-4 is expressed in developing pseudothecia in the sexual reproductive cycle. Phylogenetic analysis indicated that it is homologous to PKS enzymes in other fungi that are required for female fertility. A pks8-4 disruption mutant was generated but was not altered in pathogenicity. GC-MS profiling identified significant changes in nonpolar metabolites between the wild type and pks8-4 mutant. Phylogenetic analysis of the PKS8-1 cluster showed similarity to monodictyphenone-producing clusters in other fungi. PKS8-1 was expressed in cultures producing a red-fluorescent compound with properties similar to perylenequinone toxins in other fungi, and expression of the MFS transporter gene in the PKS8-1 cluster correlated with production of the compound. From extracts of the redfluorescing M. fijiensis hyphae, fourteen compounds that had not previously been reported from M. fijiensis were identified, including an idebenone-like metabolite, 1,2-dihydroxy-3,4- epoxy-1,2,3,4-tetrahydronaphthalene, and pulverochromenol, which are similar to molecules produced by polyketide biosynthetic pathways. Overexpression of a transcription factor gene from the PKS8-1 gene cluster resulted in increased expression of many, but not all, genes in this cluster.;Transcriptome analysis enabled the identification of additional putative pathogenicity genes beyond those encoding polyketides that may be involved in the necrotrophic stage of disease. Genes with higher expression in infected leaf tissue include: genes encoding salicylate hydroxylase-like proteins, CFEM domain-containing proteins, hydrophobic surface binding proteins, proteins with characteristics common in effectors, proteins with Domain of Unknown Function 3328, and enzymes commonly associated with secondary metabolism. Analysis of differentially expressed gene clusters identified clusters for a NRPS and a novel fusicoccane. Transcriptome analysis also confirmed previous reports that 14 scaffolds in the M. fijiensis genome have characteristics common in dispensable chromosomes, as 14 putative dispensable scaffolds were identified with a distinctly different expression pattern than 'core' scaffolds. These included two putative dispensable scaffolds with a high percentage of genes with higher expression in infected leaf tissue, suggesting that those scaffolds may be involved in pathogenicity. No transcripts were mapped to two other scaffolds, and PCR analysis suggested that these scaffolds are largely absent in the M. fijiensis isolate used for RNA-Seq. Overall, this research provides important insight into the pathogenicity mechanisms for an under-studied pathogen, and provides many exciting new avenues for future research. |
