BcHK71 and BcHK67, Two-Component Histidine Kinases, Regulate Conidial Morphogenesis, Glycerol Synthesis, and Virulence in Botrytis cinerea

Fungal two-component signaling systems comprise histidine kinases (HKs), phosphotransfer intermediates, and response regulators. HKs are classified into eleven groups based on domain architecture; however, Group XI members in <i>Botrytis cinerea</i> remain uncharacterized. In this study, we investigated the functions of two Group XI histidine kinase genes, <i>BcHK71</i> and <i>BcHK67</i>, in <i>B. cinerea</i> via gene replacement. Phenotypic analysis revealed that <i>BcHK71</i> and <i>BcHK67</i> regulate conidiation, infection structures formation, and glycerol synthesis. Notably, <i>BcHK71</i> maintained cell wall integrity. Both genes also modulated expression of high osmolarity glycerol mitogen-activated protein kinase (HOG-MARK) signaling pathway components (<i>BcYpd1</i>, <i>BcSkn7</i>, <i>BcBos4</i>), while <i>BcHK67</i> uniquely upregulated <i>BcBrrg1</i> and enhanced BcHog1 phosphorylation. Transcriptomics analysis further indicated that <i>BcHK71</i> and <i>BcHK67</i> participated in pathways related to carbohydrate and lipid transport, metabolism and secondary metabolite biosynthesis. Disruption of these processes reduced pathogenicity and altered fungicide sensitivity in <i>B. cinerea</i>, with the Δ<i>BcHK71</i> mutant exhibiting more severe pronounced defects. Collectively, our findings underscore the critical roles of <i>BcHK71</i> and <i>BcHK67</i> in fungal development and pathogenicity, highlighting their potential as novel targets for controlling fungal diseases and managing fungicide resistance.