Disruptions of the genes involved in lysine biosynthesis, iron acquisition, and secondary metabolisms affect virulence and fitness in Metarhizium robertsii

Authors: DONZELLI, BRUNO GIULIANO - Cornell University; TURGEON, B. GILLIAN - Cornell University; Gibson, Donna; Krasnoff, Stuart

Submitted to: Fungal Genetics and Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/18/2016
Publication Date: 11/19/2016
Citation: Donzelli, B.G., Turgeon, B., Gibson, D.M., Krasnoff, S. 2016. Disruptions of the genes involved in lysine biosynthesis, iron acquisition, and secondary metabolisms affect virulence and fitness in Metarhizium robertsii. Fungal Genetics and Biology. DOI: 10.1016/j.fgb.2016.11.005.

Interpretive Summary: Metarhizium fungi infect a wide range of agricultural pest insects. Thus, these fungi are important because of their potential as natural biocontrol agents of insects and several species of Metarhizium have been commercialized as environmentally friendly biopesticides. Metarhizium fungi produce a large number of unique small molecule metabolites, aka secondary metabolites. Some of these molecules may be critical to the performance of the fungus as a biocontrol agent. To evaluate the role played by these molecules in pathogenic processes, several master genes that control the production of the majority of secondary metabolites by the biocontrol species Metarhizium robertsii were identified by analysis of the M. robertsii genome and then mutant strains in which these critical genes were deactivated were produced by genetic engineering. The consequences of losing these genes were evaluated by comparing the pathogenic and fitness characteristics of the mutant strains to the naturally occurring strain. One of the mutant strains displayed a 19-fold loss of virulence based on assays against the Spotted-winged drosophila fruit fly. This established the critical role played by secondary metabolites in the performance of this fungus as a biocontrol agent and as a biopesticide.

Technical Abstract: To evaluate the total contribution of polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) pathways to M. robertsii fitness and virulence, mutants deleted for mrpptA, a gene required for their activation were generated. 'mrpptA strains failed to produce any of the nonribosomal peptides or polyketides known from M. robertsii and were also auxotrophic for lysine. Two other mutant strains were generated: 'mraarA, in which the a-aminoadipate reductase gene critical for lysine biosynthesis was disrupted, and 'mrsidA, in which the gene that is critical for siderophore biosynthesis was disrupted. The phenotypes of these mutants were compared to those of 'mrpptA to separate effects of the loss of lysine or siderophore production from the overall effect of losing all polyketide and non-ribosomal peptide production. Loss of lysine alone marginally increased resistance to H2O2 while it had little effect on the sensitivity to the cell wall disruptor sodium dodecyl sulfate (SDS) and no effect on sensitivity to iron deprivation. In contrast, loss of siderophores by inactivation of mrsidA resulted in hypersensitivity to H2O2, iron deprivation, and SDS, that were either identical or marginally higher in 'mrpptA strains. Differently from 'mrpptA, loss of mrsidA did not completely abolish siderophore activity, which suggested the presence of non-hydroxamate iron-chelating secondary metabolites in M. robertsii. Compared to WT, 'mrpptA strains displayed ~19-fold reduction in virulence against Drosophila suzukii. In contrast, lysine auxotrophy and loss of siderophores accounted for ~2 and ~6-fold decreases in virulence, respectively. Our results suggest that PKS and NRPS metabolism in total plays a significant role in M. robertsii virulence and contributes marginally to resistance to oxidative stress and iron homeostasis.