Cpkk2, a MEK from Cryphonectria parasitica is necessary for maintenance of CHV1 virus infection

We have recently obtained and characterized the knock out strains of the three MEKs present in the Cryphonectria parasitica genome, Cpkk1, Cpkk2 and Cpkk3, homologues of yeast Mkk1p/Mkk2p, Ste7p and Pbs2p, respectively. We tried to infect each of the knock out strain with Cryphonectria hypovirus 1 (CHV1), a mycovirus causing hypovirulence: ?cpkk1 and ?cpkk3 were easily infected by CHV1 through anastomosis, but we failed to infect ?cpkk2. We then showed that hyphal fusion was prevented in such knock out strain: for this reason we attempted at infecting the ?cpkk2 strain with two alternative protocols that overcome the hyphal phusion impairment: stable transformation of protoplasts with a cDNA infectious clone and transfection of protoplasts with viral RNA transcripts obtained in vitro from a cDNA infectious clone. We originated infected strains with both protocols using wild-type C. parasitica protoplasts, whereas no stable infected strain was obtained starting from ?cpkk2 protoplasts, which, on the contrary, could be transformed with the empty vector carrying only the resistance gene for selection. Given the uniqueness of such result, we are now trying to show what is the specific molecular impairment that prevents CHV1 maintenance in ?cpkk2 strain. A proteomic approach was undertaken using 2-DE MALDI-TOF MS/MS and shotgun coupled to LC-MS/MS to compare the WT and ?cpkk2 strains. A number of metabolic pathways are heavily impacted in the mutant. Of interest, proteins involved in folding, transport and trafficking, are up-regulated suggesting an altered protein turnover. Defence machinery is also up-regulated, indicating that the fungus perceives a stress situation. Moreover, a strong down-regulation of proteins involved in energy production and conversion was detected, indicating a possible reduction of the energetic metabolism. Among them are some GAPDH isoforms. Given the recent discovery of the role of GAPDH in viral replication complexes of RNA viruses, we obtained anti-GAPDH antibodies in order to study its possible role in CHV1 viral replication.