Centricoll

RNA samples of M. robertsii were extracted using the RNeasy plant minikit (Qiagen, Germantown, MD) from spores harvested from 2-week-old PDA cultures, mycelia harvested from SDB, and appressorium cells induced on soldier fly wings. Wax moth larvae were individually injected with a spore suspension (10 μL containing 1 × 10⁷ conidia/mL in 0.05% Tween 20) for 3 days for collection of insect hemolymph samples on ice. Fungal cells (hyphal bodies) were then separated for RNA extraction by centrifugation in Centricoll (Sigma-Aldrich) at 4°C (46 (link)). To examine the induction of MrHelA expression by bacteria, we germinated the WT strain spores in LB broth for 24 h, and the samples were divided into aliquots of 20 mL each. Fresh cells of S. aureus were then added at a final OD₆₀₀ of 0.4 in each sample for incubation in a rotary shaker for different times at 25°C and 210 rpm prior to collection for extracting fungal RNAs. First-strand cDNA of each sample was converted using 1 μg of total RNA with the RT master mix kit (TransGen Biotech, China), and quantitative PCR analysis of MrHelA expression was conducted using a SYBR mix (Toyobo, Japan). The β-tubulin gene of M. robertsii was amplified and used as a reference (47 (link)).

RNA samples of M. robertsii were extracted using the RNeasy plant minikit (Qiagen, Germantown, MD) from spores harvested from 2-week-old PDA cultures, mycelia harvested from SDB, and appressorium cells induced on soldier fly wings. Wax moth larvae were individually injected with a spore suspension (10 μL containing 1 × 10⁷ conidia/mL in 0.05% Tween 20) for 3 days for collection of insect hemolymph samples on ice. Fungal cells (hyphal bodies) were then separated for RNA extraction by centrifugation in Centricoll (Sigma-Aldrich) at 4°C (46 (link)). To examine the induction of MrHelA expression by bacteria, we germinated the WT strain spores in LB broth for 24 h, and the samples were divided into aliquots of 20 mL each. Fresh cells of S. aureus were then added at a final OD₆₀₀ of 0.4 in each sample for incubation in a rotary shaker for different times at 25°C and 210 rpm prior to collection for extracting fungal RNAs. First-strand cDNA of each sample was converted using 1 μg of total RNA with the RT master mix kit (TransGen Biotech, China), and quantitative PCR analysis of MrHelA expression was conducted using a SYBR mix (Toyobo, Japan). The β-tubulin gene of M. robertsii was amplified and used as a reference (47 (link)).

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)].