RESUMO
Mango is an important tropic fruit, but its production is highly restricted by anthracnose diseases. Mango anthracnose development is related to the fruit-ripening hormone ethylene, but how the pathogen senses ethylene and affects the infection remains largely unknown. In this study, mango pathogen Colletotrichum asianum strain TYC-2 was shown to sense ethylene to enhance spore germination, appressorium formation and virulence. Upon further analysis of ethylene sensing signaling, three histidine kinase genes (CaHKs) and a G-protein gene (CaGα1) were functionally characterized. Ethylene upregulated the expression of the three CaHKs but had no influence on CaGα1 expression. No function in ethylene sensing was identified for the three CaHKs. Ethylene enhanced spore germination and multiple appressorium formation of the wild-type TYC-2 but not CaGα1 mutants. TYC-2 has extremely low germination in water, where self-inhibition may play a role in ethylene sensing via CaGα1 signaling. Self-inhibitors extracted from TYC-2 inhibited spore germination of TYC-2 and CaGα1 mutants, but ethylene could not rescue the inhibition, indicating that the self-inhibition was not mediated by CaGα1 and had no interactions with ethylene. Interestingly, spore germination of CaGα1 mutants was significantly enhanced in water on hydrophobic but not hydrophilic surfaces, suggesting that CaGα1 is involved in surface sensing. In the pathogenicity assay, CaGα1 mutants showed less virulence with delayed germination and little appressorium formation at early infection on mango leaves and fruit. Transcriptome and qRT-PCR analyses identified several pathogenicity-related genes regulated by ethylene, indicating that ethylene may regulate TYC-2 virulence partially by regulating the expression of these genes.
RESUMO
Alternaria alternata relies on the ability to produce a host-selective toxin and to detoxify reactive oxygen species to successfully colonize the host. An A. alternata major facilitator superfamily transporter designated AaMFS54 was functionally characterized by analysis of loss- and gain-of-function mutations to better understand the factors required for fungal pathogenesis. AaMFS54 was originally identified from a wild-type expression library after being subtracted with that of a mutant impaired for the oxidative stress-responsive transcription regulator Yap1. AaMFS54 contains 14 transmembrane helixes. Fungal mutant lacking AaMFS54 produced fewer conidia and increased sensitivity to many potent oxidants (potassium superoxide and singlet-oxygen generating compounds), xenobiotics (2,3,5-triiodobenzoic acid and 2-chloro-5-hydroxypyridine), and fungicides (clotrimazole, fludioxonil, vinclozolin, and iprodione). AaMFS54 mutant induced necrotic lesion sizes similar to those induced by wild-type on leaves of susceptible citrus cultivars after point inoculation with spore suspensions. However, the mutant produced smaller colonies and less fluffy hyphae on the affected leaves. Virulence assays on citrus leaves inoculated by spraying with spores revealed that AaMFS54 mutant induced less severe lesions than wild-type, indicating the requirement of AaMFS54 in pathogenesis. All defective phenotypes were restored in a strain re-acquiring a functional copy of AaMFS54. Northern blotting analysis revealed that the expression of AaMFS54 was suppressed by xenobiotics. The current studies indicate that the Yap1-mediated transporter plays a role in resistance to toxic oxidants and fungicides in A. alternata. The relationships of MFS transporters with other regulatory components conferring stress resistance and A. alternata pathogenesis are also discussed.