Isolation And Functional Identification Of The Genes Associated With Mating And Pathogenicity Of Sporisorium Scitamineum

Sugarcane smut caused by Sporisorium scitamineum occurs worldwide,causing serious loss in both yield and quality.Isolation and functional identification of the genes involved in mating and pathogenicity in S.scitamineum are essential for understanding the pathogenicity mechanism of this fungus and the interaction of pathogen-host at molecular level.These are helpful for the design of novel,safe,and environment-friendly fungicides and for the development of new strategies to control sugarcane smut.In this study,25056 transformants of Sporisorium scitamineum with hygromycin resistance marker were obtained by Agrobacterium tumefaciens-mediated transformation(ATMT),in which 9183 transformants were derived from JG35 and 15873 from JG36,two compatible mating type strains.PCR analysis of hph and GFP confirmed that T-DNA were inserted into the S.scitamineum genome in the mutants.Southern blotting analysis of 18randomly selected independent transformants showed that the T-DNA was integrated randomly into the nuclear genome with a single copy.A total of 18 mutants with mating defect were obtained by screening the transformants using mating tests.Among them,mutants 91F9、91F10、205G8、205D10 and 208E6 showed reduced pathogenicity and mutants 131D11 and 243D1 that were derived from JG35 and 248E3 that was derived from JG36 were completely nonpathogenic when inoculated to the sugarcane seedlings in combinations with their compatible mating type strains.By high-efficiency TAIL-PCR(hi TAIL-PCR),T-DNA insertion sites in the mutants131D11,243D1 and 248E3 were determined,and full-length c DNA of the corresponding genes were obtained by RT-PCR and 3’RACE and the gemomic sequences of the genes were obtained by referring to the genome sequence of S.scitamineum strains JG35 and JG36.Then,function of the genes in mating and pathogenicity were confirmed by gene complementation and inoculation to the sugarcane seedlings.The detailed results were presented below:In mutant 131D11,T-DNA was found to insert into the promoter region of a putative gene SSCI＿3902.The full-length of SSCI＿3902 is 2184bp,with no intron.The gene encodes a 727 amino-acid(aa)protein and was tentatively named Ss DLIC,since it contains a conserved domain of dynein light intermediate chain(DLIC)and its function may be related with DLIC.Ss DLIC was found in both JG35 and JG36 with a single copy in the genome.Mating and pathogenicity of the mutant could be restored to the level of the wild type by complementation with copy of the wild-type Ss DLIC.In mutant 243D1,T-DNA was found to insert into the promoter region of a putative gene SSCI＿4698.The full-length of SSCI＿4698 is 1101bp,composed of two exons and one intron.The gene encodes a 325aa protein and was tentatively named Ss SURF4,since it contains a conserved domain of SURF4.Ss SURF4 was found to present in both JG35 and JG36 with a single copy in the genome.Mating and pathogenicity of the mutant could be restored to the level of the wild type by complementation with copy of the wild-type Ss SURF4.In mutant 248E3,T-DNA was found to insert inside the ORF region of a putative gene SSCI＿2082.The full-length of SSCI＿2082 is 2310bp in length encoding a 722aa protein,with no intron.This gene was tentatively named Ss Prf1,since it contains a highly conserved domain of MATA＿HMG box and is homolous to the pheromone response factor(Prf1).Ss Prf1 was found to present in both JG35 and JG36 with a single copy in the genome.Mating and pathogenicity of the mutant could be restored to the level of the wild type by complementation with copy of the wild-type Ss Prf1.To dissect the function of Ss Prf1 in pheromone signaling and pathogenicity,Ss Prf1null mutants were constructed by homologous recombination.Similar to the T-DNA insertion mutant 248E3,the Ss Prf1 null mutants,JG35Δprf1 and JG36Δprf1,failed to form Fuz~+colonies when co-spotted with a compatible partner strain and were nonpathogenic.q PCR analyses showed that Ss Prf1 was not only essential for the basal transcription but also essential for pheromone stimulated transcription of three putative mating type genes,namely mfa1.2,mfa1.3 and pra1 in a1 locus.In addition,Ss Prf1 regulates the transcription of cutinase,which is an important factor for pathogenic development in fungus.Taken to gether,results presented in this work showed that Ss DLIC,Ss SURF4 and Ss Prf1 were involved in mating and pathogenicity in S.scitamineum.The results also indicated that Ss Prf1 would place a key role in pheromone signalling and filamentous growth.