RESUMEN
Botrytis cinerea is a typical necrotrophic plant pathogenic fungus which can deliberately acidify host tissues and trigger oxidative bursts therein to facilitate its virulence. The white collar complex (WCC), consisting of BcWCL1 and BcWCL2, is recognized as the primary light receptor in B. cinerea. Nevertheless, the specific mechanisms through which the WCC components, particularly BcWCL2 as a GATA transcription factor, control virulence are not yet fully understood. This study demonstrates that deletion of BcWCL2 results in the loss of light-sensitive phenotypic characteristics. Additionally, the Δbcwcl2 strain exhibits reduced secretion of citrate, delayed infection cushion development, weaker hyphal penetration, and decreased virulence. The application of exogenous citric acid was found to restore infection cushion formation, hyphal penetration, and virulence of the Δbcwcl2 strain. Transcriptome analysis at 48 h post-inoculation revealed that two citrate synthases, putative citrate transporters, hydrolytic enzymes, and reactive oxygen species scavenging-related genes were down-regulated in Δbcwcl2, whereas exogenous citric acid application restored the expression of the above genes involved in the early infection process of Δbcwcl2. Moreover, the expression of Bcvel1, a known regulator of citrate secretion, tissue acidification, and secondary metabolism, was down-regulated in Δbcwcl2 but not in Δbcwcl1. ChIP-qPCR and electrophoretic mobility shift assays revealed that BcWCL2 can bind to the promoter sequences of Bcvel1. Overexpressing Bcvel1 in Δbcwcl2 was found to rescue the mutant defects. Collectively, our findings indicate that BcWCL2 regulates the expression of the global regulator Bcvel1 to influence citrate secretion, tissue acidification, redox homeostasis, and virulence of B. cinerea.IMPORTANCEThis study illustrated the significance of the fungal blue light receptor component BcWCL2 protein in regulating citrate secretion in Botrytis cinerea. Unlike BcWCL1, BcWCL2 may contribute to redox homeostasis maintenance during infection cushion formation, ultimately proving to be essential for full virulence. It is also demonstrated that BcWCL2 can regulate the expression of Bcvel1 to influence host tissue acidification, citrate secretion, infection cushion development, and virulence. While the role of organic acids secreted by plant pathogenic fungi in fungus-host interactions has been recognized, this paper revealed the importance, regulatory mechanisms, and key transcription factors that control organic acid secretion. These understanding of the pathogenetic mechanism of plant pathogens can provide valuable insights for developing effective prevention and treatment strategies against fungal diseases.
Asunto(s)
Botrytis , Ácido Cítrico , Proteínas Fúngicas , Factores de Transcripción GATA , Regulación Fúngica de la Expresión Génica , Homeostasis , Oxidación-Reducción , Botrytis/genética , Botrytis/patogenicidad , Botrytis/metabolismo , Virulencia , Ácido Cítrico/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Factores de Transcripción GATA/metabolismo , Factores de Transcripción GATA/genética , Enfermedades de las Plantas/microbiología , Eliminación de Gen , Hifa/crecimiento & desarrollo , Hifa/genética , Hifa/metabolismo , Perfilación de la Expresión GénicaRESUMEN
Botrytis cinerea is a devastating pathogen causing gray mold in fruits and vegetables if not properly managed. Although the mechanisms remain unclear, we previously revealed that the safe food additive calcium propionate (CP) could suppress gray mold development on grapes. The present study reports that sub-lethal dose of CP (0.2 % w/v) could allow growth with substantial reprograming the genome-wide transcripts of B. cinerea. Upon CP treatment, the genes related to fungal methylcitrate cycle (responsible for catabolizing propionate) were upregulated. Meanwhile, CP treatment broadly downregulated the transcript levels of the virulence factors. Further comparative analysis of multiple transcriptomes confirmed that the CP treatment largely suppressed the expression of genes related to development and function of infection cushion. Collectively, these findings indicate that CP can not only reduce fungal growth, but also abrogate fungal virulence factors. Thus, CP has significant potential for the control of gray mold in fruit crops.
Asunto(s)
Propionatos , Factores de Virulencia , Botrytis , Enfermedades de las Plantas/microbiologíaRESUMEN
Adenylate cyclase (AC) regulates growth, reproduction, and pathogenicity in many fungi by synthesizing cyclic adenosine monophosphate (cAMP) and activating downstream protein kinase A (PKA). Botrytis cinerea is a typical necrotrophic plant-pathogenic fungus. It shows a typical photomorphogenic phenotype of conidiation under light and sclerotia formation under dark; both are important reproduction structures for the dispersal and stress resistance of the fungus. The report of B. cinerea adenylate cyclase (BAC) mutation showed it affects the production of conidia and sclerotia. However, the regulatory mechanisms of the cAMP signaling pathways in photomorphogenesis have not been clarified. In this study, the S1407 site was proven to be an important conserved residue in the PP2C domain which poses a remarkable impact on the phosphorylation levels and enzyme activity of the BAC and the overall phosphorylation status of total proteins. The point mutation bacS1407P , complementation bacP1407S , phosphomimetic mutation bacS1407D , and phosphodeficient mutation bacS1407A strains were used for comparison with the light receptor white-collar mutant Δbcwcl1 to elucidate the relationship between the cAMP signaling pathway and the light response. The comparison of photomorphogenesis and pathogenicity phenotype, evaluation of circadian clock components, and expression analysis of light response transcription factor genes Bcltf1, Bcltf2, and Bcltf3 showed that the cAMP signaling pathway could stabilize the circadian rhythm that is associated with pathogenicity, conidiation, and sclerotium production. Collectively, this reveals that the conserved S1407 residue of BAC is a vital phosphorylation site to regulate the cAMP signaling pathway and affects the photomorphogenesis, circadian rhythm, and pathogenicity of B. cinerea.
RESUMEN
Ethylene (ET) represents a signal that can be sensed by plant pathogenic fungi to accelerate their spore germination and subsequent infection. However, the molecular mechanisms of responses to ET in fungi remain largely unclear. In this study, Colletotrichum gloeosporioides was investigated via transcriptomic analysis to reveal the genes that account for the ET-regulated fungal development and virulence. The results showed that ET promoted genes encoding for fungal melanin biosynthesis enzymes, extracellular hydrolases, and appressorium-associated structure proteins at 4 h after treatment. When the germination lasted until 24 h, ET induced multiple appressoria from every single spore, but downregulated most of the genes. Loss of selected ET responsive genes encoding for scytalone dehydratase (CgSCD1) and cerato-platanin virulence protein (CgCP1) were unable to alter ET sensitivity of C. gloeosporioides in vitro but attenuated the influence of ET on pathogenicity. Knockout of the G-protein-coupled receptors CgGPCR3-1/2 and the MAPK signaling pathway components CgMK1 and CgSte11 resulted in reduced ET sensitivity. Taken together, this study in C. gloeosporioides reports that ET can cause transcription changes in a large set of genes, which are mainly responsible for appressorium development and virulence expression, and these processes are dependent on the GPCR and MAPK pathways.
RESUMEN
In filamentous fungi, 1,8-dihydroxynaphthalene (DHN) melanin is a major component of the extracellular matrix, endowing fungi with environmental tolerance and some pathogenic species with pathogenicity. However, the subcellular location of the melanin biosynthesis pathway components remains obscure. Using the gray mold pathogen Botrytis cinerea, the DHN melanin intermediate scytalone was characterized via phenotypic and chemical analysis of mutants, and the key enzymes participating in melanin synthesis were fused with fluorescent proteins to observe their subcellular localizations. The Δbcscd1 mutant accumulated scytalone in the culture filtrate rather than in mycelium. Excessive scytalone appears to be self-inhibitory to the fungus, leading to repressed sclerotial germination and sporulation in the Δbcscd1 mutant. The BcBRN1/2 enzymes responsible for synthesizing scytalone were localized in endosomes and found to be trafficked to the cell surface, accompanied by the accumulation of BcSCD1 proteins in the cell wall. In contrast, the early-stage melanin synthesis enzymes BcPKS12/13 and BcYGH1 were localized in peroxisomes. Taken together, the results of this study revealed the subcellular distribution of melanin biosynthetic enzymes in B. cinerea, indicating that the encapsulation and externalization of the melanin synthetic enzymes need to be delicately orchestrated to ensure enzymatic efficiency and protect itself from the adverse effect of the toxic intermediate metabolite.IMPORTANCE The devastating gray mold pathogen Botrytis cinerea propagates via melanized conidia and sclerotia. This study reveals that the sclerotial germination of B. cinerea is differentially affected by different enzymes in the melanin synthesis pathway. Using gene knockout mutants and chemical analysis, we found that excessive accumulation of the melanin intermediate scytalone is inhibitory to B. cinerea. Subcellular localization analysis of the melanin synthesis enzymes of B. cinerea suggested two-stage partitioning of the melanogenesis pathway: the intracellular stage involves the steps until the intermediate scytalone was translocated to the cell surface, whereas the extracellular stage comprises all the steps occurring in the wall from scytalone to final melanin formation. These strategies make the fungus avert self-poisoning during melanin production. This study opens avenues for better understanding the mechanisms of secondary metabolite production in filamentous fungi.