Study On The Role Of F-box Protein SsPof7 In The Sexual Mating Of Sporisorium Scitamineum

Sugarcane smut caused by Sporisorium scitamineum has become one of the most important diseases in sugar cane production.Understanding the growth and development and pathogenic mechanism of S.scitamineum can help effectively prevent and control the disease.Ubiquitination is an important way for eukaryotes to regulate biological functions of proteins.The classic ubiquitination is that ubiquitin activating enzyme E1activates ubiquitin and transfers ubiquitin to ubiquitin-binding enzyme E2.Ubiquitin ligase E3 connects E2-binding ubiquitin to the substrate protein.The substrate protein is recognized and degraded by the 26S proteasome.There are many E3 ubiquitin ligases,which are mainly divided into three families:HECT,RING and RBR,according to their characteristic domains and the mechanism of action of ubiquitin transfer to substrate proteins.The Skp1-Cullins-F-box（SCF）protein complex is an important member of the RING family and plays an important role in the cell life cycle,where the F-box protein determines the specificity of the substrate.In this study,an F-box protein SsPof7 was identified in S.scitamineum.Its function is related to sexual mating.The main results are as follows:1.Through bioinformatics analysis,an unknown function gene（LK056695.1）was identified in the genome of S.scitamineum,which encodes a protein containing an F-box（motif）（CDR88992.1）.According to the ortholog gene POF7 in the Ustilago maydis,the gene was named SsPOF7 in the S.scitamineum.2.The homologous knockout method was used to knock out the SsPOF7 gene to obtain knockout mutants sspof7Δ,namely sspof7Δ-17（wild-type WT17 background）and sspof7Δ-18（wild-type WT18 background）.In YEPS liquid medium,the mutant sspof7Δspores became longer,and multiple spore aggregation components were branched;In YEPS solid medium and MM solid medium,the mutants have slower sexual mating with the wild type;The results of live inoculation showed that the pathogenicity of the mutants was reduced.3.The SsPof7 protein is unstable.After the protein is extracted,it will be degraded at room temperature after 30 minutes.In the control experiment with 26S proteasome inhibitor MG132,the protein remained stable.Under low temperature（ice bath）,SsPof7 is stable,and the protein begins to degrade in 60 minutes,while adding MG132 can maintain SsPof7 for 120 minutes.The results suggest that the stability of F-box protein SsPof7 is related to the degradation pathway of 26S ubiquitin proteasome.4.SsPof7 was used as a bait protein to screen the c DNA library of S.scitamineum,and it was found that the protein can interact with Ss Rbx1 and Ss Skp1 in the yeast system,and then the Ss Skp1 and Ss Rbx1 interactions were proved by the one-to-one yeast co-transformation method.At the same time,Predicting 5 Cullin proteins in S.scitamineum,the results of one-to-one co-rotation experiments with Ss Skp1 were found that Ss Cullin-1interacts with Ss Skp1.The yeast two-hybrid system was preliminarily identified to form the SCF^SsPof7protein complex with Ss Skp1,Ss Rbx1 and Ss Cullin-1.5.Further experiments were performed on the components of the SCF^SsPof7protein complex Ss Skp1,Ss Rbx1 and Ss Cullin-1 in the context of wild-type WT17 to obtain knockout mutants ssskp1Δ,sscullin-1Δ,and ssrbx1Δ,respectively.Observation of growth and micromorphology of each knockout mutant revealed that each mutant had slower growth,ssskp1Δhaploid spores became longer and both ends were sharp and slender.sscullin-1Δhaploid spore division is abnormal,mostly budding,forming a tridentate branch,vesicles enlarged,and abnormal morphology.ssrbx1Δhaploid spores became longer and the shapes of the two ends were sharper.The knockout mutants ssskp1Δ,sscullin-1Δand ssrbx1Δwere analyzed for sexual mating with wild-type WT18,and the sexual mating of each mutant was slowed.6.Each mutant was inoculated with sugarcane.After 3 days,the biomass of the inoculated part was tested for biomass,and the GADPH gene of sugarcane was used as an internal reference to detect the relative expression of Ss ACTIN in S.scitamineum.The results showed that the sspof7Δ,ssrbx1Δand ssskp1Δbiomass were all lower than those of the wild type.At the same time,the bacterial cells at the inoculation site were separated and then cultured.The counting results showed that the survival amount of the mutant was lower than that of the wild type.It indicates that the mutants have weakened immune response against host after vaccination.The pathogenic vaccination results also showed that the pathogenicity of the mutants of the wild-type SCF^SsPof7protein complex components were all reduced.In this study,the F-box protein SsPof7 and its formed protein complex SCF^SsPof7were identified in Sporisorium scitamineum for the first time,participating in the regulation of sexual mating,growth and development,stress resistance and virus-inducing ability,etc.The development of this study will lay the foundation for the development of disease prevention and control of the S.scitamineum.