Functional Analysis Of CreC,CreB And CreD,carbon Catabolite Repression Genes In Magnaporthe Oryzae

Carbon catabolism repression(carbon catabolite repression,CCR)is a regulatory mechanism that involves utilization of carbon source(usually glucose)by microorganisms thereby inhibiting uptake and utilization of alternative carbon sources in the fermentation mixture.The carbon catabolite repression genes encoded by CreA,CreC,CreB and CreD are major components of glucose repression and depression pathways in Aspergillus nidulans.In the rice blast fungus,this process may also be regulated by their orthologs.Previously,we found that MoCreA is an important repressor in the carbon catabolite of this fungus.To clarify the molecular function of other three genes in Magnaporthe oryzae,we cloned and functionally characterized them using gene knockout strategies.MoCreC,MoCreB and MoCreD mutants were obtained and further tested for growth rate in minimal medium containing various carbon sources(glucose,sucrose,starch,xylose,fructose,and maltose)and our results reveal that ΔMoCreC mutant growth rate was drastically reduced compared to Guy 11 WT.Other developmental stages of disease life cycle including conidial germination,conidia morphology and appressorium formation in ΔMoCreC mutant strain were largely impaired in this mutation.When tested for pathogenicity,ΔMoCreC virulence was significantly reduced on susceptible rice cultivar CO39 and barley.Furthermore,majority of MoCreC mutant produced conidia with abnormal morphologies and reduced numbers compared to the wild type strain.ΔMoCreC and wild type strains,were cultured on minimal media supplemented with CMC and 20mg/ml 2-DOG,from our results ΔMoCreC mutant was resistant to 2-DOG.ΔMoCreC mutant was also sensitive to 100 mM Ally Alcohol compared with the wild type.We also tested for the effects of the cell wall degrading agents 200μg/ml Congo red and 0.01%SDS on the MoCreC mutant,and observed that ΔMocreC mutant was sensitive to these reagents.This indicates that MoCreC gene play an important role in maintaining cell wall integrity.QRT-PCR results revealed that expression of βglucosidase,feruloyl esterase and exoglucanase enzymes involved in cell wall degradation,were up regulated in MoCreC mutant by 1.6 fold,0.4 fold and 1.3 fold respectively.Deletion of MoCreB and MoCreD mutants did not result to significant difference in pathogenicity,conidia germination,and conidia morphology and appressorium formation compared to wild type strain.Moreover,we tested MoCreB and MoCreD mutants’ in the minimal media with different carbon source still utilized as a wild type.We generated MoCreB/MoCreD double knockout mutant,and analyzed pathogenic defects in this mutants.From our observation there is reduction in virulence compared to single mutants and wild type strains.In addition,MoCreC/MoCreD double knock out mutant displayed similar phenotypes to those of MoCreC single mutant.In order to investigate if these proteins(CreA,CreB,CreC and CreD)are capable of interacting we used yeast two hybrids tests and our results revealed that there is no interactions between these four proteins in M.oryzae suggesting they might be playing independent roles in carbon catabolite pathway.