Functional Characterization Of Ccr4-NOT Complex Components In The Rice Blast Fungus

Ccr4-NOT(carbon catabolite repression-negative on TATA-less)complex is a global regulator,which regulates mRNA decay.The components and structure of this complex are conserved from yeast to human basically.In fungi,Ccr4-NOT complex has been characterized to be important for virulence in pathogenic species.However,the target of this kind of regulators has not yet been identified,especially the roles in plant pathogenic fungi.In this study,we screened the ATMT mutant library of the wild-type P131 in Magnaporthe oryzae,and obtained a mutant LF21 defecti ve in pathogenicity.The defective phenotype of the mutant was co-segregated with hygromycin-resistant marker by genetic analysis,meanwhile,the mutant had a single insertion of this hygromycin-resistant marker by Southern blot analysis.We identified a putative pathogenic gene,MNOT2(MGG₁2164),which encodes a protein shares 42%overall amino acid identity with Not2 in Saccharomyces cerevisiae,and 39%overall amino acid identity with Schizosaccharomyce spombe Not2.The core components of Ccr4-NOT in yeast include nine subunits,Not1,Not2,Not3,Not4,Not5,Ccr4,Caf1,Caf40 and Caf1 30.The results from the blast database indicated that there are seven components of the Ccr4-NOT complex in M.oryzae,which are MGG₀2476,MGG₁2164,MGG₈101,MGG₁1229,MGG₁3020,MGG₀1666 and MGG₀0632.These seven proteins are named MNot1,MNot2,MNot3,MNot4,MCcr4,MCaf1 and MCaf40,respectively,based on the similarity of their counterparts in S.cerevisiae.In M.oryzae,the function of MNot3 overlaps with MNot5,and there is no homolog of Caf130.In this study,we focused on three components of Ccr4-NOT complex(Not2,Not3,Caf40)and found out that they are important in vegetative growth,conidiation and plant infection.To determine the function of the Ccr4-NOT complex in Magnaporthe oryzae,the deletion mutants of MNOT2,MNOT3 and MCAF40 were generated by the homologous approach.We characterized the roles of MNOT2,MNOT3 and MCAF40 in vegetative growth and pathogenesis.Compared to the wild-type P131,deletion of MNOT2,MNOT3 and MCAF40 lead to defects in vegetative growth,conidiation and pathogenicity,especially the deletion of MNOT3 caused the defects much more severe.Besides,the deletion mutants of MNOT2,MNOT3 and MCAF40 were much more sensitive to Calcofluor White(CFW)than the wild-type P131,suggesting MNOT2,MNOT3 and MCAF40 are very important for cell wall integrity.The DAB experiment showed that MNOT2 and MNOT3 mutants can overcome reactive oxygen species by plant defence responses,suggesting that they have a funtion in infectious growth in host cells.We combined the MCAF40 with GFP,and transformed the vector to the MCAF40 null mutants.Twenty-four complementary transformants were isolated,and all of them,including CDW3,formed the same phenotype as the wild-type P131.The GFP signals were uniformly distributed to the cytoplasm of conidia,germ tubes,appressorium and infection hyphae,indicating a role in cytoplasm.It has been reported that the MAPK signalling pathway,cAMP signalling pathway and Ca2+signalling pathway involve in appressorium formation,and cutin monomers can also induce appressorium formation.We added IBMX,DAG and Diol to induce appressorium of the wild-type and the mutants in hydrophobic plastic surface,and found that only Diol can induce MNOT2 appressorium formation partially.These results indicated that MNOT2 and MCAF40 might not affect appressorium formation by the MAPK signalling pathway,cAMP signalling pathway and Ca2+signalling pathway,they might also have a function in the downstream of these signalling pathway.To uncover the functions of Ccr4-NOT complex,we use RNAseq to compare the cumulative amount of mRNA between the wild-type P131 and the mutant MNOT2 from transcriptomics level.Data shows that a total of 1601 and 1542 genes were up-and down-regulated for over two-fold in the mutant MNOT2.78%of these genes are hypothetical genes with unknown function.After characterizing the proteins of known function which changed over two-fold,we discovered that among the 261 up-regulated genes and 428 down-regulated genes could be classified into 17 subgroups.From all the genes which cumulative amount of mRNA have changed significantly,we focused on one subgroup involving in RNA degradation.Except for 2 up-regulated genes,17 genes have been down regulated significatly,including MGG₁2164(CCR4-NOT transcription complex subunit 2),three other components of the Ccr4-NOT complex,MGG 02476(CCR4-NOT transcription complex subunit 1),MGG₁1229(CCR4-NOT transcription complex subunit 4),MGG₁3020(CCR4-NOT transcription complex subunit Ccr4),Pan2(MGG₁7449)-Pan3(MGG₀2939)complex,MGG₀9505(polyadenylate-binding protein PRBP1),MGG 06410(mRNA-decapping enzyme 2),and MGG₀3388(ATP-dependent RNA helicase DHH1)etc.Furthermore,we used the 3×Flag vector to identify proteins interact with MCaf40 by pull-down and MS analysis.We identified 503 proteins identified in twice independent pull-down experiments.4 proteins were homologous to the components of the Ccr4-NOT complex.We used the yeast two-hybrid experiment to verify the physical interaction betweeen these proteins,and identified that MCaf40 directly interacted with MCcr4.Overall,this study may firstly indicate three components of the Ccr4-NOT complex have important roles in vegetative growth,conidiation,pathogenicity in M.oryzae.We also explicit the relationship between the complex and the known MAPK signalling pathway,cAMP signalling pathway and Ca2+ signalling pathway.We uncover different types of genes affected by the Ccr4-NOT complex preliminary.All these results provide a fundation for the further study of Ccr4-NOT in regulating the pathogenicity,growth and development.