| The constitutive photomorphogenesis 9 signalosome(CSN)is a protein complex consisting of 1-8 subunits,that was first discovered and reported to function as a regulator of light-mediated development in Arabidopsis thaliana seedlings,but later it was found to be involved in other essential biological processes in a wide range of organisms.However,their roles in conidiation,appressorium and hyphae formation,which are the key structures required for successful dissemination and pathogenicity of most filamentous fungi have not been studied in Magnaporthe oryzae.In this study,we identified and characterized three CSN homologs namely mocsn2,mocsn5 and moscn8 from M.oryzae genome by generating their corresponding knockout mutants through targeted gene deletion approach.Our results showed that mocsn2,mocsn5 and mocsn8 are all required for normal growth rate in M.oryzae as they exhibited a slowed growth when compared to the wild type.The Δmocsn5 mutant cultured on complete media revealed a defective hyphal growth rate and morphology when compared to the wild type.Δmocsn2,Δmocsn5 mutants had a dramatic reduction in the number of conidia produced as the amount of conidia produced were significantly reduced to levels less than 5%inΔmocsn2 mutant and about 50%in Δmocsn8 mutants as compared to the wild type.However,Δmocsn5 mutant completely lost the ability to produce conidia.Moreover,Δmocsn2 and Δmocsn5 could not cause infection on barley leaves and susceptible rice cultivar(CO-39).Δmocsn8 mutant retained its virulence on both barley and rice leaves although to a reduced degree compared to the wild type.mocsn2-gfp,mocsn5-gfp revealed their predominant localization in the nucleus as well as in the cytoplasmic space as they co-localized with His-RFP while moscn8-gfp only localized in the nucleus of both the conidia and appressorium.Finally our analysis of the predicted domain architecture showed that mocsn2 and mocsn8 had a Proteosome-Cop9 signalosome Initiation factor 3 domains(PCI)domain in their structure that’s important for scaffolding during proteinprotein interaction.Mocsn5 had a JAB domain that consists of a JAMM motif that confers an enzymatic activity.Therefore these results not only provides useful information on the distinct roles of the individual subunits and that the disruption of an individual subunit confers a significant effect on M.oryzae development but also indicates that the subunits through their domains and binding motifs could also be functioning in a complex.However,the role of the entire complex and the pattern of the subunit-subunit interactions will only become more clear when all the 8 subunits have been studied in Magnaporthe oryzae. |
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