Functional Characterization Of Cpubi4 Gene And The Ubiquitylome Study In Cryphonectria Parasitica

Ubiquitination is an important post-translation modification(PTM),which plays key roles in protein degradation,cell cycle,endocytosis,and stress adaption.The infection of hypo virus CHV1-EP713 in Cryphonectria parasitica leads to the secretion of ubiquitin.Sequence analysis of the whole genome revealed there are three ubiquitin coding genes,from which,two of the genes encode a hybrid protein with ubiquitin and ribosome,another one gene encodes a protein consists of three direct repeats of ubiquitin with a final amino acid after the last repeat.The transcript accumulation of cpubi4 was unregulated by the infection of hypovirus,while the other two genes showed no o'bvious change in transcription measured by realtime RT-PCR.The null mutants of cpubi4 gene were obtained by homologous recombination.Deletion of cpubi4 resulted in abnormal morphology,loss of aerial hyphae,reduced sporulation,and loss of virulence,while the accumulation of hypovirus was not affected.The global protein ubiquitination of Acpubi4 was significant reduced comparing to the original strain DK80.The concentration of protein of Acpubi4 was remarkably higher than DK80 at the same weight of hypha.We supposed the deletion of polyubiquitin gene may lead to a shortage of free ubiquitin in the strain,which will reduce the ubiquitination of global proteins,leading to the accumulation of proteins and disorder of ubiquitination regulation.To understand the ubiquitylome of C.parasitica,the specific antibody of K-s-GG were used to enrich the ubiquitinated peptides.The peptides were fractionated by HPLC followed by tandem mass spectrometry(MS/MS).A total of 2663 ubiquitinated peptides from 1181 proteins were identified,in which between 1 and 28 putative ubiquitination sites were identified,with about 55%containing only one ubiquitination site.Nine conserved ubiquitinated motifs were identified from the 2663 peptides.Multiple proteins relate to virulence were found to be ubiquitinated.To further confirm the affection of deletion of cpubi4 gene in protein ubiquitination,the difference of ubiquitination peptides was analyzed between Ocpubi4 and DK80.A total of 51 sites in 45 proteins were quantified as up-regulated targets and 378 sites in 236 proteins were quantified as down-regulated targets at a threshold of 2(P<0.05).These proteins were enriched in pathways involving in protein synthesis and metabolism,signaling transduction,metabolism of second metabolite and so on.These results indicated that the disruption of cpubi4 directly reduced the ubiquitination of many proteins,but did not change the relative expression of protein when the total protein amount was equal.Our research provides evidences of polyubiquitin gene are relative to the growth,virulence,ubiquitination in filamentous fungus.Furthermore,our research lay a foundation for understanding the function of ubiquitination in fungus.