RNAseq, virulence, and phylogenetics studies of the gene easR of Metarhizium brunneum

Ergot alkaloid synthesis (eas) gene clusters found in several fungi encode biosynthesis of agriculturally and pharmaceutically important ergot alkaloids. Whereas the biosynthetic genes of the ergot alkaloid pathway have been well characterized, regulation of those genes is unknown. We characterized a gene with sequence similarity to a putative transcription factor and that was found adjacent to the eas cluster of Metarhizium brunneum, a plant symbiont and insect pathogen. The function of the novel gene, easR, was explored by CRISPR-Cas9-derived gene knockouts. To maximize potential for ergot alkaloid accumulation, strains of M. brunneum were injected into larvae of the insect Galleria mellonella.  Larvae infected with the wild type contained abundant ergot alkaloids, but those infected with easR knockouts lacked detectable ergot alkaloids.  The easR knockout strains had significantly reduced or no detectable mRNA from eas cluster genes in RNAseq and qualitative RT-PCR analyses, whereas the wild-type strain contained abundant mRNA from all eas genes. These data demonstrate that the product of easR is required for ergot alkaloid accumulation and provide evidence it has a role in expression of ergot alkaloid biosynthesis genes. Larvae infected with an easR knockout survived significantly longer than those infected with the wild type (P < 0.0001), indicating a role for EasR, and indirectly confirming a role for ergot alkaloids, in virulence of M. brunneum to insects. Homologs of easR were found associated with eas clusters of at least 15 other ergot alkaloid-producing fungi, indicating EasR homologs may contribute to regulation of ergot alkaloid synthesis in additional fungi.

Methods
RNAseq analysis

Larvae of Galleria mellonella were infected as described previously (Panaccione and Arnold, 2017; Steen et al., 2021), with four treatment groups of five wild-type Metarhizium brunneum, M. brunneum easR knockout strain 1, M. brunneum easR knockout strain 2, and control larvae injected with only the PBS solution.  Larvae were harvested six days post inoculation, and mRNA was extracted by utilizing the Qiagen RNeasy Plant kit with on-column DNase I treatment (Qiagen Sciences Inc.; Germantown, MD).  Libraries for RNAseq analysis were prepared from 500 ng of total RNA with the KAPA mRNA HyperPrep kit (Roche Molecular Systems; Pleasanton, CA) and Illumina compatible iTru adapters.  Samples of 100 bp each from paired ends were sequenced by Illumina HiSeq technology at the Marshall University Genomics Core (Huntington, WV).  Total counts from individual genes were derived by comparison to the M. brunneum ARSEF 3297 reference genome, assembly MBR_1.0 (GenBank accession GCA_000814965.1).  The presence of zeros in the mRNA count data of the easR knockout strains resulted in unequal variances among treatments.  For this reason, RNAseq data were analyzed nonparametrically with Wilcoxon tests, and when Wilcoxon tests indicated a significant treatment effect, Steel-Dwass multiple comparison tests.  Statistical analyses were conducted in JMP Pro 14 (SAS, Cary, NC).

Phylogenetics

The sequence of AurF of Calcarisporium arbuscula and amino acid sequences encoded by 15 genes homologous to easR of M. brunneum were collected from ncbi.nlm.nih.gov.  Accession numbers for each sequence are listed next to each taxon in the text document that contains the sequences.  Amino acid sequences were aligned by MUSCLE and trimmed by eye to yield 249 residues of well-aligning amino acids ending near the carboxy terminus of EasR.  Maximum likelihood analyses were performed in MEGA X (Kumar et al., 2018) with the model JTT + G and 1000 bootstrap replications.

Virulence tests in insects

Spore suspensions were prepared at a concentration of 5 conidia/µL in modified PBS solution containing 9 mM Na2PO4, 1.6 mM KH2PO4, 123 mM NaCl, 0.01% wt/vol Tween 20, and 10 µg/ml rifampin.  Galleria mellonella larvae were injected with 20 µL of the spore suspension via an insulin syringe into the hindmost left proleg (Panaccione and Arnold, 2017; Steen et al., 2021).  Control larvae were inoculated using the same protocol but with 20 µL of modified PBS solution lacking spores.  Survival was recorded every few hours.  Data were graphed on Kaplan-Meier survival curves, and differences in survival rates were assessed with log rank tests with a Bonferonni corrected α set at 0.016.  One-way ANOVA was used to compare the average time to 50% lethality for larvae infected with wild-type M. brunneum and an M. brunneum easR knockout strain.  All statistical analyses were performed in JMP Pro 14 (SAS; Cary, NC).

References

Kumar S, Stecher G, Li M, Knyaz C, Tamura K. 2018. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 35:1547–1549.
Panaccione DG, Arnold SL. 2017. Ergot alkaloids contribute to virulence in an insect model of invasive aspergillosis. Sci Rep 7:8930.
Steen CR, Sampson JK, Panaccione DG. 2021. A Baeyer–Villiger monooxygenase gene involved in the synthesis of lysergic acid amides affects the interaction of the fungus Metarhizium brunneum with insects. Appl Environ Microbiol 87:e00748–21.