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

[https://doi.org/10.5061/dryad.2bvq83bzx](https://doi.org/10.5061/dryad.2bvq83bzx)

Data supporting Fabian SJ, Steen CR, Damron FH, DeRiggi CA, and Panaccione DG. A gene regulating ergot alkaloid biosynthesis in *Metarhizium brunneum*, submitted to *Applied and Environmental Microbiology* submitted for review May 2024

## Description of the data and file structure

 Data on “total counts” tab of MS Excel file are arranged as follows.  Columns A through P contain total counts for each gene listed in rows 3 through 15 of that tab.  Columns are labeled ‘wild type’ for Metarhizium brunneum strain ARSEF 9354 and easR ko 1 or easR ko2 for eaR knockouts 1 or 2 made in that M. brunneum ARSEF 9354 background. 

 Data in the upper table of the tab labeled “eas relative to tub2” of the MS Excel file are presented with columns named for the fungal strains (as described in the paragraph above) and rows for each ...