Analyzing LysM gene expression in entomopathogenic fungus

Twelve LysM domain-containing protein genes are encoded by B. bassiana [30 (link)]. To examine the expression of these genes, total RNA was extracted from the mycelia or blastospores harvested from SDB and the conidial spores or hyphae from the PDA plates. To determine gene expression during fungal in vivo infection, the last instar larvae of wax moth (G. mellonella) were individually injected with 10 μl of spore suspension (10⁷ spores/ml) for 60 hrs. Insect hemolymph was collected on ice, and fungal hyphal bodies were harvested by gradient centrifugation using Centricoll (Sigma-Aldrich) [30 (link)]. Each RNA sample was converted to cDNA using an AffinityScript multiple-temperature cDNA synthesis kit (Toyobo). qRT-PCR analysis was performed using a SYBR Premix Ex Taq kit (Takara) containing the primer pairs for different genes (S3 Table) on an ABI Prism 7000 system (Applied Biosystems). The β-tubulin gene (BBA_07018) of B. bassiana was amplified as an internal control. To determine insect antifungal gallerimycin gene expression, the last instar wax moth larvae were individually injected with the spore suspensions of WT and mutants for 36 hrs. Insect fat bodies were then dissected on ice and collected for RNA extraction to quantify the expression of the antifungal gene [9 (link)].