Functional Analysis Of Five Key Genes In Two-component Histidine Kinase Signaling Pathway In Botrytis Cinerea

Two-component histidine kinase (TCHK) signaling pathway is involved in a variety of life activities of fungi, but to date, little is known about the TCHK in Botrytis cinerea, which causes grey mold in more than 200 economically important crops. Previous studies have showed that the dicarboximides fungicides (DCFs) act as antifungal compounds, in part, through activation of the TCHK in B. cinerea. However, the target of DCFs remains unclear. In this study, thus, we investigated functions of the five important TCHK components:BOS1, BRRG-1, BOS4, BOS5 and BOS2 in Botrytis cinerea using a gene knockout and complementary strategy. Results of this study showed that 1) BRRG-1, BOS4, BOSS and BOS2 knockout mu-tants could not produce aerial hyphae and conidia. The BOS5 deletion mutant formed seriously distorted hyphal and swelling tips with more branches.2) The disruption mutant of BOS1 showed increased resistance to DCFs, while mutants of other four elements exhibited more sensitive to these fungicides compared to the wild type strain. Additionally, mutations in BOS1 resulted to resistance to DCFs.3) Sensitivity to osmotic stress showed that all five gene deletion mutants revealed increased sensitiv-ity to ionic osmotic stresses produced by NaCl and KC1, but not by the neutral os-motic stress mediated by sorbitol.4) Sensivity to oxidative stress indicated that the mutants of BRRG-1, BOS4, BOS5 and BOS2 became more sensitive to oxidative stress generated by H2O2 except that of BOS1as compared to the wild-type strain.5) Inoculation on cucumber and rapeseed leaves showed that the three mitogen-activated protein (MAP) kinases BOS4, BOS5 and BOS2 in TCHK pathway were essential for the pathogenicity. These gene disruption mutants could penetrate the host surface but couldn't form lesions.6) Western blotting analysis showed that the phosphorylation of BOS2 protein was not detected in BRRG-1, BOS4 and BOS5 deletion mutants un-der normal cultural condition, suggesting that these elements were located at the BOS2 upstream of TCHK signaling pathway.Results of this study indicate that 1) the TCHK signaling pathway is involved in regulating vegetative differentiation, pathogenicity, and the sensitivity to osmotic and oxidative stresses in B. cinerea; 2) BOS1 could be the target of DCFs, meanwhile be-cause BOS4, BOS5 and BOS2 are important virulence determinants in B. cinerea, they could become potential targets for the development of new antifungal com-pounds.