Functional Characterization Of The MOUBP14 Deubiquitinating Enzyme In Rice Blast Fungus

The rice blast disease caused by ascomycete fungus Magnaporthe oryzae is one of the most destructive diseases posing great threat to rice production,which renders huge rice production lose up to 30% of the total yield wordwide annually.Currently,the most effective and sustainable way to control the disease is the deployment of resistant cultivars.However,for the rapid evolution and high field selective pressure,the new virulent population will emerge in several years,which in turn overcome the resistance.Therefore,It is indispensable for the researchers to study the mechanisms how the rice blast fungus infects the host,in order to develop new targets in the fungus for the green agrochemicals treatment.With the genomic sequences of M.oryzae published,It has accelerated the gene functional study in M.oryzae,and numerous mechanisms underlying the important pathogenic processes have been unveiled.The ubiquitination is a conserved and vital protein posttranslational modification in the eukaryotic cells,and participate in a wide array of cellular processes,including proteasome mediated protein degradation,DNA repair,endocytosis,signal transduction,etc.The ubiquitination modification can be reversibly removed by the related deubiquitinating enzymes,which results in fine-tuning control for the ubiquitination homeostasis.It was reported that the ubiquitination mechineries play imperative roles in the growth,development,pathogenicity of the model plant fungal pathogen M.oryzae,but the functional characterization of deubiquitinating enzyme in rice blast fungus has not yet been reported to date.In this study,A gene homologous to Saccharomyces cerevisiae UBP14 was identified,and named as MoUBP14 in M.oryzae.MoUBP14 was knockout by homologous recombination strategy.Biological phenotypes and molecular function studies have been analysed.Results show that MoUBP14 gene knockout render severe pathogenicity defect,reduced growth rate and conidial production,retarded appressorium development,and abnormal appressorium turgor pressure,more sensitive to diverse ambient stresses.In the global proteome level,ΔMoubp14 mutant contains more free ubiquitin chains in contrast to the wide type strain,namely disorder in the ubiquitin pool in the deletion mutant.Two key rate-limiting enzymes involved in the gluconeogenesis pathway,Pck1 and Fbp1,were identified as the MoUbp14-binding proteins.Further experimental evidence revealed that Pck1 and Fbp1 showed higher ubiquitination level in mutant strain,which suggested they are potential client proteins of MoUbp14 deubiquitinase.In glucose induced gluconeogenesis enzymes proteolysis assay,Pck1 and Fbp1 displayed higher aboundance in ΔMoubp14 mutant,which demonstrated MoUbp14 is involved in UPS(Ubiquitin proteosome system)mediated Pck1 and Fbp1 degradation.This is the first functional characterization of deubiquitinase MoUbp14 in plant pathogenic fungus.MoUbp14 plays an pleiotropic role in rice blast fungus growth,conidiation,appressorium development,anti-stresses,and carbon assimilation,etc.This study provide experimental basis and theory support for further deubiquitinase functional characterization.