The Molecular Mechanism Of Functional Differentiation Of Cdc2A And Cdc2B Kinases In Sexual Development In Fusarium Graminearum

Fusarium head blight(FHB)caused by Fusarium graminearum is a worldwide disease of cereal crops,seriously threating the yield and quality of grains.In the recent years,due to climate warming and changes in farming practices,the incidence area of FHB in China has been increasing,and China has become one of the countries with the largest incidence area of the FHB.Besides causing yield loss significantly,the mycotoxin produced by F.graminearum during infection is harmful to human and animal health.Ascospores of F.graminearum are the primary inocula of FHB disease,playing an important role in the infection cycle.Elucidating the molecular mechanism of sexual reproduction in F.graminearum is crucial for developing effective disease prevention and control strategies.Cyclin-dependent protein kinase is the key regulator of cell cycle.There are many CDK in animals,but only one CDK in fungi(CDC28 in Saccharomyces cerevisiae and CDC2 in Schizosaccharomyces pombe).Previous studies by our laboratory found that there were two CDC2 orthologous genes(CDC2A and CDC2B)in F.graminearum.Δcdc2A and Δcdc2B knockout mutants have no obvious phenotype in vegetative growth stage,while only Δcdc2A have serious defects in sexual development.To elucidate the molecular mechanisms of functional differentiation between CDC2 A and CDC2 B during sexual development,we divided the sequences of Cdc2 A and Cdc2 B into five variation regions: I,II,III,IV and V,based on their sequence alignment,and replaced each region of CDC2 A with the corresponding CDC2 B sequence respectively.Phenotypic analysis showed that only the replacement mutant of the III region has serious defects in sexual reproduction,similar to cdc2 A knockout mutants.We further subdivided the III region into III1,III2,III3,III4 and III5 to perform similar analysis,and found that only replaced the III2 region affected sexual development.There are four different amino acid sites in III2 region and each of them was replaced with the amino acid of Cdc2 B.The results showed that the 126 th amino acid substitution affected sexual development.Alternatively,we mutated the 126 th amino acid of Cdc2 B to the amino acid of Cdc2 A and used this sequence to complement the Δcdc2A mutant.We found the complemented transformants had similar phenotypes with the wild type.These results suggest that the difference at the 126 th amino acid site in Cdc2 A and Cdc2 B is the main determinant for their function differentiation during sexual development.To identify the potential interaction of Cyclin specifically to Cdc2 A,we performed knockout and phenotypic analysis of each of the three Cdc2-related Cyclin genes CLN1,CLN2 and CLN3 encoded in F.graminearum genome.The results showed that the absence of CLN1leads to a slower growth and reduction of ascospore cells.The absence of CLN3 has no effect on the growth of hyphae,but resulted in a serious defect in sexual development.The ascus development is stunted at an early stage and no normal ascus is formed inΔcln3 mutant.These results revealed that the CLN1 mainly acts in the vegetative growth phase,while CLN3 mainly functions in the sexual reproduction.CLN2 may be an essential gene as no knockout mutant was obtained after multiple independent transformation.Protein subcellular localization analysis showed weaker fluorescence signals of Cln1 and Cln3 in both cytoplasm and nucleus.Interestingly,the fluorescence signals of Cln3 are obvious in the microtubule organizing center(MTOC).The fluorescence signal of Cln2 is located in the nucleus and obvious in the MTOC.Previous studies found that although fluorescence signal of Cdc2A is localized in both cytoplasm and nucleus,the fluorescence signal in the nucleus was significantly higher than that in cytoplasm,especially in the MTOC.Therefore,based on the phenotype and protein subcellular localization,Cln3 may have specific interaction with Cdc2A during sexual reproduction.The interaction between Cdc2A and three Cyclin was further studied by Co-IP,and the results showed that the interaction between Cdc2A and all three Cyclin proteins in the vegetative growth stage.Their interaction during sexual reproduction remains to be determined.Collectedly,this study demonstrated that the difference of amino acid at 126 th is the main determinant of functional differentiation between Cdc2 A and Cdc2 B during sexual reproduction,and imply that there may be a specific interaction between Cln3 and Cdc2 A during sexual reproduction.Our results helps to shed light on the molecular mechanism of functional difference between Cdc2 A and Cdc2 B.