Dataset Information

A Single Nucleotide Mutation in Adenylate Cyclase Affects Vegetative Growth, Sclerotial Formation and Virulence of Botrytis cinerea.

ABSTRACT: Botrytis cinerea is a pathogenic fungus that causes gray mold disease in a broad range of crops. The high intraspecific variability of B. cinerea makes control of this fungus very difficult. Here, we isolated a variant B05.10M strain from wild-type B05.10. The B05.10M strain showed serious defects in mycelial growth, spore and sclerotia production, and virulence. Using whole-genome resequencing and site-directed mutagenesis, a single nucleotide mutation in the adenylate cyclase (BAC) gene that results in an amino acid residue (from serine to proline, S1407P) was shown to be the cause of various defects in the B05.10M strain. When we further investigated the effect of S1407 on BAC function, the S1407P mutation in bac showed decreased accumulation of intracellular cyclic AMP (cAMP), and the growth defect could be partially restored by exogenous cAMP, indicating that the S1407P mutation reduced the enzyme activity of BAC. Moreover, the S1407P mutation exhibited decreased spore germination rate and infection cushion formation, and increased sensitivity to cell wall stress, which closely related to fungal development and virulence. Taken together, our study indicates that the S1407 site of bac plays an important role in vegetative growth, sclerotial formation, conidiation and virulence in B. cinerea.

SUBMITTER: Chen X

PROVIDER: S-EPMC7215688 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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A Single Nucleotide Mutation in Adenylate Cyclase Affects Vegetative Growth, Sclerotial Formation and Virulence of Botrytis cinerea.

Chen Xue X Zhang Xiaohong X Zhu Pinkuan P Wang Yiwen Y Na Yantao Y Guo Han H Cai Yunfei Y Nie Haozhen H Jiang Yina Y Xu Ling L

International journal of molecular sciences 20200421 8

Botrytis cinerea is a pathogenic fungus that causes gray mold disease in a broad range of crops. The high intraspecific variability of B. cinerea makes control of this fungus very difficult. Here, we isolated a variant B05.10M strain from wild-type B05.10. The B05.10M strain showed serious defects in mycelial growth, spore and sclerotia production, and virulence. Using whole-genome resequencing and site-directed mutagenesis, a single nucleotide mutation in the ...[more]

PMID: 32326350

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Project description:Botrytis cinerea is an important plant pathogenic fungus with a wide range of host. It usually produces black-colored sclerotia (BS) due to deposition of 1,8-dihydroxynaphthalene melanin in sclerotial melanogenesis. Our previous study (Zhou et al., 2018) reported six B. cinerea isolates producing orange-colored sclerotia (OS) with deficiency in sclerotial melanogenesis. Comparison of ecological fitness (conidia, mycelia, sclerotia), natural distribution, and melanogenesis of selected BS and OS isolates suggests that sclerotia play an important role in the disease cycle caused by B. cinerea. However, the molecular mechanism for formation of the OS B. cinerea remains unknown. This study was done to unravel the molecular mechanism for the sclerotial melanogenesis deficiency in the OS isolates. We found that all the five sclerotial melanogenesis genes (bcpks12, bcygh1, bcbrn1/2, bcscd1) were down-regulated in OS isolates, compared to the genes in the BS isolates. However, the sclerotial melanogenesis-regulatory gene bcsmr1 had similar expression in both types of sclerotia, suggesting the sclerotial melanogenesis deficiency is due to loss-of-function of bcsmr1, rather than lack of expression of bcsmr1. Therefore, we cloned bcsmr1 from OS (bcsmr1OS ) and BS (bcsmr1BS ) isolates, and found a single-nucleotide deletion in bcsmr1OS . The single-nucleotide deletion caused formation of a premature stop codon in the open reading frame of bcsmr1OS , resulting in production of a 465-aa truncated protein. The transcription activity of the truncated protein was greatly reduced, compared to that of the 935-aa full-length protein encoded by bcsmr1BS in the BS isolates. The function of bcsmr1OS was partially complemented by bcsmr1BS . This study not only elucidated the molecular mechanism for formation of orange-colored sclerotia by the spontaneous mutant XN-1 of B. cinerea, but also confirmed the regulatory function of bcsmr1 in sclerotial melanogenesis of B. cinerea.

Project description:Autophagy is a conserved degradation process that maintains intracellular homeostasis to ensure normal cell differentiation and development in eukaryotes. ATG8 is one of the key molecular components of the autophagy pathway. In this study, we identified and characterized BcATG8, a homologue of Saccharomyces cerevisiae (yeast) ATG8 in the necrotrophic plant pathogen Botrytis cinerea Yeast complementation experiments demonstrated that BcATG8 can functionally complement the defects of the yeast ATG8 null mutant. Direct physical interaction between BcAtg8 and BcAtg4 was detected in the yeast two-hybrid system. Subcellular localization assays showed that green fluorescent protein-tagged BcAtg8 (GFP-BcAtg8) localized in the cytoplasm as preautophagosomal structures (PAS) under general conditions but mainly accumulated in the lumen of vacuoles in the case of autophagy induction. Deletion of BcATG8 (ΔBcAtg8 mutant) blocked autophagy and significantly impaired mycelial growth, conidiation, sclerotial formation, and virulence. In addition, the conidia of the ΔBcAtg8 mutant contained fewer lipid droplets (LDs), and quantitative real-time PCR (qRT-PCR) assays revealed that the basal expression levels of the LD metabolism-related genes in the mutant were significantly different from those in the wild-type (WT) strain. All of these phenotypic defects were restored by gene complementation. These results indicate that BcATG8 is essential for autophagy to regulate fungal development, pathogenesis, and lipid metabolism in B. cinereaIMPORTANCE The gray mold fungus Botrytis cinerea is an economically important plant pathogen with a broad host range. Although there are fungicides for its control, many classes of fungicides have failed due to its genetic plasticity. Exploring the fundamental biology of B. cinerea can provide the theoretical basis for sustainable and long-term disease management. Autophagy is an intracellular process for degradation and recycling of cytosolic materials in eukaryotes and is now known to be vital for fungal life. Here, we report studies of the biological role of the autophagy gene BcATG8 in B. cinerea The results suggest that autophagy plays a crucial role in vegetative differentiation and virulence of B. cinerea.

Dataset Information

A Single Nucleotide Mutation in Adenylate Cyclase Affects Vegetative Growth, Sclerotial Formation and Virulence of Botrytis cinerea.

Publications

A Single Nucleotide Mutation in Adenylate Cyclase Affects Vegetative Growth, Sclerotial Formation and Virulence of <i>Botrytis cinerea</i>.

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