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The basal transcription factor II H (TFIIH) is a multicomponent complex. In the present study, we characterized a TFIIH subunit Tfb5 by analysing loss- and gain-of-function mutants to gain a better understanding of the molecular mechanisms underlying stress resistance and pathogenicity in the citrus fungal pathogen Alternaria alternata. Tfb5 deficiency mutants (ΔAatfb5) decreased sporulation and pigmentation, and were impaired in the maintenance of colony surface hydrophobicity and cell wall integrity. ΔAatfb5 increased sensitivity to ultraviolet light, DNA-damaging agents, and oxidants. The expression of Aatfb5 was up-regulated in the wild type upon infection in citrus leaves, implicating the requirement of Aatfb5 in fungal pathogenesis. Biochemical and virulence assays revealed that ΔAatfb5 was defective in toxin production and cellwall-degrading enzymes, and failed to induce necrotic lesions on detached citrus leaves. Aatfb5 fused with green fluorescent protein (GFP) was localized in the cytoplasm and nucleus and physically interacted with another subunit, Tfb2, based on yeast two-hybrid and co-immunoprecipitation analyses. Transcriptome and Antibiotics & Secondary Metabolite Analysis Shell (antiSMASH) analyses revealed the positive and negative roles of Aatfb5 in the production of various secondary metabolites and in the regulation of many metabolic and biosynthetic processes in A. alternata. Aatfb5 may play a negative role in oxidative phosphorylation and a positive role in peroxisome biosynthesis. Two cutinase-coding genes (AaCut2 and AaCut15) required for full virulence were down-regulated in ΔAatfb5. Overall, this study expands our understanding of how A. alternata uses the basal transcription factor to deal with stress and achieve successful infection in the plant host., (© 2020 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

The tangerine pathotype of the ascomycete fungus Alternaria alternata is the causal agent of citrus brown spot, which can result in significant losses of both yield and marketability for tangerines worldwide. A conditionally dispensable chromosome (CDC), which harbours the host-selective ACT toxin gene cluster, is required for tangerine pathogenicity of A. alternata. To understand the genetic makeup and evolution of the tangerine pathotype CDC, we isolated and sequenced the CDCs of the A. alternata Z7 strain and analysed the function and evolution of their genes. The A. alternata Z7 strain has two CDCs (~1.1 and ~0.8 Mb, respectively), and the longer Z7 CDC contains all but one contig of the shorter one. Z7 CDCs contain 254 predicted protein-coding genes, which are enriched in functional categories associated with 'metabolic process' (55 genes, P = 0.037). Relatively few of the CDC genes can be classified as carbohydrate-active enzymes (CAZymes) (4) and transporters (19) and none as kinases. Evolutionary analysis of the 254 CDC proteins showed that their evolutionary conservation tends to be restricted within the genus Alternaria and that the CDC genes evolve faster than genes in the essential chromosomes, likely due to fewer selective constraints. Interestingly, phylogenetic analysis suggested that four of the 25 genes responsible for the ACT toxin production were likely transferred from Colletotrichum (Sordariomycetes). Functional experiments showed that two of them are essential for the virulence of the tangerine pathotype of A. alternata. These results provide new insights into the function and evolution of CDC genes in Alternaria., (© 2019 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)