DOI: 10.5061/dryad.2bvq83bzx
title: RNAseq, virulence, and phylogenetics studies of the gene easR of Metarhizium brunneum

description: 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 &lt;
 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.
description: <strong>RNAseq analysis</strong>
 Larvae of <em>Galleria mellonella</em> were infected
 as described previously (Panaccione and Arnold, 2017; Steen et al., 2021),
 with four treatment groups of five wild-type <em>Metarhizium
 brunneum</em>, <em>M. brunneum easR</em> knockout strain
 1, <em>M. brunneum easR</em> 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 <em>M.
 brunneum</em> ARSEF 3297 reference genome, assembly MBR_1.0 (GenBank
 accession GCA_000814965.1).  The presence of zeros in the mRNA count data
 of the <em>easR</em> 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).
 <strong>Phylogenetics</strong> The
 sequence of AurF of <em>Calcarisporium arbuscula</em> and
 amino acid<strong> </strong>sequences encoded by 15 genes
 homologous to <em>easR </em>of <em>M. brunneum
 </em>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. <strong>Virulence tests
 in insects</strong> Spore suspensions were
 prepared at a concentration of 5 conidia/µL in modified PBS solution
 containing 9 mM Na<sub>2</sub>PO<sub>4</sub>, 1.6
 mM KH<sub>2</sub>PO<sub>4</sub>, 123 mM NaCl,
 0.01% wt/vol Tween 20, and 10 µg/ml rifampin.  <em>Galleria
 mellonella </em>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<em> </em>larvae infected with wild-type <em>M.
 brunneum </em>and an <em>M. brunneum easR </em>knockout
 strain.  All statistical analyses were performed in JMP Pro 14 (SAS; Cary,
 NC). <strong>References</strong>
 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 <em>Metarhizium
 brunneum</em> with insects. Appl Environ Microbiol
 87:e00748–21.
description: # 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 of the indicated ergot alkaloid synthesis genes.  Values are the ratio (times 1000) for total counts of each of the indicated ergot alkaloid synthesis gene divided by the total counts of tub2 from that same sample.  The data in the lower table are same data as above but with rows and columns transposed to prepare data for entry into JMP table.  Data in the “survival” tab of MS Excel file are arranged in four columns.  The first column lists the hour (after inoculation of the insect) at which survival was observed.  The second list the number of new dead larvae observed at that time, with the exception in some cases of the last time observation at which all remaining live larvae are counted and then ‘censored’ by inclusion of the character ‘1’ in column 3. The fourth column simply lists the treatment.  The data are in a form that is prepared for entry into JMP table.  Amino acid sequence data used for generation of the phylogenetic tree in the article are represented in the text file.  The first line of entry is the taxon name and accession number (ncbi.nlm.nih.gov) from which the data are derived, and the remainder of each entry are the amino acid sequences in IUPAC code.  Data are ready to be copied and pasted for alignment and phylogenetic analyses.