Heat stress (HS) influences the growth and development of organisms. Thus, a comprehensive understanding of how organisms sense HS and respond to it is required. Ganoderma lucidum , a higher basidiomycete with bioactive secondary metabolites, has become a potential model system due to the complete sequencing of its genome, transgenic systems, and reliable reverse genetic tools. In this study, we found that HS inhibited mycelium growth, reduced hyphal branching, and induced the accumulation of ganoderic acid biosynthesis and heat shock proteins (HSPs) in G. lucidum . Our data showed that HS induced a significant increase in cytosolic Ca ²⁺ concentration. Further evidence showed that Ca ²⁺ might be a factor in the HS-mediated regulation of hyphal branching, ganoderic acid (GA) biosynthesis, and the accumulation of HSPs. Our results further showed that the calcium-permeable channel gene ( cch )-silenced and phosphoinositide-specific phospholipase gene ( plc )-silenced strains reduced the HS-induced increase in HSP expression compared with that observed for the wild type (WT). This study demonstrates that cytosolic Ca ²⁺ participates in heat shock signal transduction and regulates downstream events in filamentous fungi. IMPORTANCE Ganoderma lucidum , a higher basidiomycete with bioactive secondary metabolites, has become a potential model system for evaluating how environmental factors regulate the development and secondary metabolism of basidiomycetes. Heat stress (HS) is an important environmental challenge. In this study, we found that HS inhibited mycelium growth, reduced hyphal branching, and induced HSP expression and ganoderic acid biosynthesis in G. lucidum . Further evidence showed that Ca ²⁺ might be a factor in the HS-mediated regulation of hyphal branching, GA biosynthesis, and the accumulation of HSPs. This study demonstrates that cytosolic Ca ²⁺ participates in heat shock signal transduction and regulates downstream events in filamentous fungi. Our research offers a new way to understand the mechanism underlying the physiological and metabolic responses to other environmental factors in G. lucidum . This research may also provide the basis for heat shock signal transduction studies of other fungi.

Ganoderma lucidum is one of the most important medicinal mushrooms; however, molecular genetics research on this species has been limited due to a lack of reliable reverse genetic tools. In this study, the endogenous orotidine 5′-monophosphate decarboxylase gene (URA3) was cloned as a silencing reporter, and four gene-silencing methods using hairpin, sense, antisense, and dual promoter constructs, were introduced into G. lucidum through a simple electroporation procedure. A comparison and evaluation of silencing efficiency demonstrated that all of the four methods differentially suppressed the expression of URA3. Our data unequivocally indicate that the dual promoter silencing vector yields the highest rate of URA3 silencing compared with other vectors (up to 81.9%). To highlight the advantages of the dual promoter system, we constructed a co-silencing system based on the dual promoter method and succeeded in co-silencing URA3 and laccase in G. lucidum. The reduction of the mRNA levels of the two genes were correlated. Thus, the screening efficiency for RNAi knockdown of multiple genes may be improved by the co-silencing of an endogenous reporter gene. The molecular tools developed in this study should facilitate the isolation of genes and the characterization of the functions of multiple genes in this pharmaceutically important species, and these tools should be highly useful for the study of other basidiomycetes.