Regulation Of The Membrane Anchor Protein Mr-OPY2 Controls The Switch Between Saprophytic And Pathogenic Lifestyles Of The Insect Pathogenic Fungus, Metarhizium Robertsii

Metarhizium robertsii is a common insect pathogen. Unlike bacteria and viruses that need to be ingested to cause disease, it infects insects by direct penetration of the cuticle. During infection processes the fungus produces toxins and proliferates in the hemolymph which will kill the insect host. Increasing attention has been paid to M. robertsii since it became an alternative option to traditional chemical pesticides. Therefore, the research on the pathogenic mechanism of entomopathogenic fungi contributes to invent efficient and safe fungal pesticides by using the genetic recombination technology.From a random genome-wide T-DNA insertion library of M. robertsii, we identified a mutant (M2880) that showed abolished virulence to insects but displayed no change with saprophytic growth. The disrupted gene (Mr-OPY2) in M2880 is a homolog of the membrane anchor protein Sc-OPY2 from Saccharomyces cerevisiae.For functional analysis, we disrupted Mr-OPY2 to get M.robertsii ΔMr-OPY2. No appressorium formation was observed in ΔMr-OPY2 on the insect cuticle and the Petri dish that result in the disability of penetrating the cuticle. The growth and germination rate were influencedunder the hyperosmotic stress, whereΔMr-OPY2 germlings were severely deformed. In addition, the conidia yield was affected due to the damaged conidiophores and some of the conidia were bigger than the WT’s.Only a long mRNA transcript of Mr-ΔPY2 named MrOPY2-L was obtained from mycelium grown in the saprophytic lifetsyle; while an extra shorter Mr-OPY2mRNA transcript named MrOPY2-S was cloned from the appressorium-forming germlings and mycelium grown in the hemolymph. The only difference in Mr-OPY2 transcription between the saprophytic lifetsyle and the pathogenic lifetsyle is that the latter lifetsyle had a 384bp-shorter mRNA isoform, which thus could be responsible for the increased amount of Mr-OPY2 protein. Two upstream-ORFs (uORFs) exist in the 5’UTR of MrOPY2-L, which did not existed in MrOPY2-S. We suspected that the uORFs in MrOPY2-L interfere the translation of Mr-OPY2 protein. Mr-OPY2 produced the two mRNA isoforms during pathogenic lifetsyle using alternative transcription start sites. The 5’UTR regions of two mRNA isoforms have different RNA secondary structures so that the translational efficiency are different. The Ste50 scaffold protein binding to the accumulated OPY2 membrane anchor mediates the MAPK pathways since the insect-parasitic lifestyle start.In summary, Mr-OPY2 was functionally studied by gene deletion and other studies. It was found that the mechanisms of transcriptional and translational regulation were precise and the importance of switching between parasitic and saprophytic lifestyle of M.robertsii. The results of this thesis facilitate the understanding of the mechanisms of fungal pathogenicity.