Functional Analysis Of Mating-related Gene SsGpa In Sporisorium Scitamineum

Sporisorium scitamineum(Syd.) M. Piepenber, M. Stoll & F. Oberw is an important fungal pathogen causing sugarcane smut disease which threatens the sugarcane production worldwide. However, little informations to develop the molecular mechanisms of the sexual mating and pathogenicity of S. scitamineum, Understanding the molecular basis of S. scitamineum mating is essential for developing new disease control methods. For the aims, the Ss GPA gene was identified by sequencing the flanking sequence of T-DNA insertion in mutant A9 with mating deficiency. Then the gene functions were analyzed by genetic deletion and complementation assays. The main results are as follows:1. Analyzing the flanking sequences of T-DNA insertion site in mutant A9.Bioinformatics analysis showed that the disrupted gene contains an open reading frame of1365-bp with one intron of 29 bp and encodes a peptide of 454 amino acids, with 66% and57% identity to the uncharacterized protein genes of Sporisorium reilianum, and Ustilago maydis, respectively. As it contains a G-Patch domain, we tentatively named it as Ss Gpa.2. Generating Ss GPA knockout mutant and complementary strain. Three pairs of locus-specific primers were designed according to Ss Gpa sequence and its upstream and downstream sequence. Then the vectors used in Ss Gpa knockout and complement were constructed by ligation with PCR amplifiedn fragments. The ?Ss Gpa mutant and complementary strain Ss Gpa-c were generated by ATMT. The gene disrupted by T-DNA in mutant A9 was confirmed as Ss Gpa by PCR, Southern blot and q RT-PCR.3. Gene Ss Gpa affects S. scitamineum mating but not vegetative growth. Analysis of the mutants and wild type showed that morphology and haploid growth rate of the ?Ss Gpa mutant were comparable with wild type. Mating assay showed that ?Ss Gpa was defective in mating, which could be restored in Ss Gpa-c strain, indicating that gene Ss Gpa isessential S. scitamineum mating.4. Transcriptome analysis shows that Ss GPA affects the expression of Pheromone response factor gene(prf1). a locus and b locus activate by Prf1 as a activating transcription factor, transcriptome analysis shows that the expression of pheromone precursor gene(mfa)and b East(b E) significant decrease. We analyzed the expression level of mfa and pra by real-time PCR. The results showed that mfa was barely detected in ?Ss Gpa, and the expression of pra is comparable with WT, which indicates that Ss Gpa affects expression of gene mfa that encodes mating pheromone, resulting in mating defect in ?Ss Gpa.5. Ss GPA is important for S. scitamineum pathogencity. Pathogencity tests showed pathogencity of ?Ss Gpa mixed with WT17 was significantly attenuated as disease incidence was nearly 3.33%. In contrast, pathogencity of Ss Gpa-c mixed with WT17 was similar with wild type(73.33%).In summary, we have proven that gene Ss GPA is essential for sexual mating of S.scitamineum. Deletion of Ss GPA results in significant reduction of mfa expression, causing defective mating and reduced virulence in S. scitamineum.