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 B05.10 M strain from wild-type B05.10. The B05.10 M 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 B05.10 M 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.(MAPK) pathway, have been deeply studied in B. cinerea [8]. In B. cinerea, key components of the cAMP pathway include bac (the gene encoding adenylate cyclase in B. cinerea), bcpkaR and bcpka1, knockout mutants of which are defective in growth and pathogenicity [9]. Adenylate cyclase (AC) is activated by the G-alpha subunit BCG1. Activated AC converts ATP to cAMP, which is recognized by its primary receptor, cAMP-dependent protein kinase (PKA) [10]. However, the regulatory mechanisms that act downstream of PKA and PKA-independent signaling cascades remain to be explored. BCG1 can also activate the Ca 2+ signaling pathway through phospholipase C (BcPLC1), and knockdown of bcplc1 in B. cinerea leads to defects in spore germination, sclerotium formation and virulence [11]. In the Ras superfamily, knockout of bcras1 and bcras2 attenuates mycelial growth and pathogenicity [9,12]. The MAPK signaling pathway transmits signals through the sequential activation of three interacting protein kinases. In B. cinerea, it has been reported that three MAPKs, Bmp1, Bmp3, and Bcsak1, are involved in growth and pathogenicity [13][14][15][16]. Furthermore, the filamentous fungi have evolved light signaling pathways which regulate growth, development and metabolism and allow them to adapt to changes in the environment. In B. cinerea, exposure to white light regulates the development of reproductive structures; the production of asexual conidia is induced by white light, whereas sclerotia, which are dormant structures, are formed in darkness [8]. It has been reported that the blue light receptor White collar complex and the downstream light-responsive transcription factors BcLTF1 and BcLTF2 are involved in these responses [17,18]. Moreover, ...

Phosphorylation status of a conserved residue in the adenylate cyclase of Botrytis cinerea is involved in regulating photomorphogenesis, circadian rhythm, and pathogenicity

“…Supplementary Table S1 lists the wild-type and mutant strains of B. cinerea used in the present study. The wild type of B. cinerea strain B05.10 ( Büttner et al, 1994 ), the BAC single mutant strain bac S1407P , and the complemented strain bac P1407S ( Chen et al, 2020 ) were used in the present experiments. The bac S1407A and bac S1407D strains were generated in this work by introducing homologous recombination fragments into the B05.10 strain according to the previous method ( Schumacher et al, 2012 ).…”

“…The pathogenicity assay was performed as described previously ( Chen et al, 2020 ). The healthy mature fruits of grape ( Vitis vinifera ), apple ( Malus pumila Mill ), tomato fruit ( Solanum lycopersicum ), 4-week-old tomato leaves, and 2-week-old A. thaliana leaves were used in the inoculation assay.…”

“…In our early molecular genetics study with B. cinerea , a spontaneously variant mutant strain with defects in pathogenicity, conidiation, and sclerotium reproduction was characterized to be caused by a single nucleotide mutation at the S1407 site of adenylate cyclase (BAC) ( Chen et al, 2020 ), in the PP2C (protein phosphatase, family 2C) domain, which is a negative regulatory domain of protein kinase cascade through dephosphorylation. Besides, BAC contains a Gα binding domain that can bind with upstream G protein; a RAS interacting domain; an LRR domain that can participate in a variety of biological processes; and a catalytic domain of adenosine cyclase type III that catalyzes the transformation of ATP into cAMP ( Bassler et al, 2018 ).…”

“…However, the relationship between the B. cinerea adenylate cyclase (BAC)–mediated cAMP signaling pathway and the formation of conidia and sclerotia and the circadian clock still needs to be explored. We previously reported that the growth rate, conidia, sclerotia production, and pathogenicity are seriously defective in association with the BAC S1407 site mutation ( Chen et al, 2020 ). In this study, the analysis of the S1407 site of BAC demonstrated that the phosphorylation level of BAC could significantly affect some protein phosphorylation levels, B. cinerea pathogenicity at different times of the day, and photomorphogenesis.…”

cAMP Signalling Pathway in Biocontrol Fungi

“…AC is activated by Gα and then catalyses ATP conversion to cAMP. AC can regulate the growth, development, mating, morphological characteristics, conidiation, metabolite production, and resistance to environmental stress of fungi [ 84 , 85 , 86 , 87 ], as well as the pathogenicity of fungal pathogens, including the formation of appressoria and virulence to the host [ 88 , 89 , 90 ]. Moreover, in the medical area, ACs can regulate pathological cardiac fibrosis and monocyte inflammation [ 91 , 92 , 93 ].…”