Functional And RNAi Studies Of Sugar Metabolism Related Genes VdST And VdPT1 In Verticillium Dahliae From Cotton

【Object】Cotton（Gossypium hirsutum L.）is the most important cash crop in the world and is an important strategic material for national livelihoods.Cotton is often subjected to various biotic stresses in the production process,among which Verticillium wilt seriously affects the yield and quality of cotton and is known as the"cancer"of cotton.Verticillium dahliae is a soil-borne vascular fungal disease that is difficult to control due to its stable microsclerotia structure and variable physiological race.RNA interference techniques are increasingly being applied to crop protection against pathogenic fungi.In recent years,it has been found that sugar metabolism plays an important role in the growth and pathogenicity of pathogenic fungi,but relevant researches in V.dahliae are still few.In this study,we identified sugar metabolism related genes Vd ST and Vd PT1 in V.dahliae and investigated their functions to provide a theoretical basis for elucidation of the molecular mechanism of V.dahliae pathogenicity and to provide new targets for breeding new cotton germplasm with disease resistance by RNAi technology.【Methods】Sugar transporter family genes were identified from the whole genome of V.dahliae and analyzed by bioinformatics.The asi RNAs targeting Vd ST3（asi R201 and asi R1238）and Vd ST12（asi R815and asi R1436）were designed and co-cultured with V.dahliae respectively.The co-cultured V.dahliae were used to investigate various physiological indicators and pathogenicity.The knockout and complementary vectors of neutral trehalase gene Vd PT1 were constructed using homologous recombination and transformed into V.dahliae protoplasts using PEG-Ca Cl₂-mediated genetic transformation to obtain deletion mutant strains and complementary mutant strain,which were used to investigate various physiological indicators and pathogenicity.The TRV-mediated HIGS was used to silence of the Vd ST3,Vd ST12 and Vd PT1 genes in cotton,and the HIGS treated cotton plants inoculated with V.dahliae were used to investigate disease symptoms and disease indexes and to detect fungal biomass and gene expression levels using q RT-PCR.The pathogenicity related genes Vd ST3,Vd ST12 and Vd PT1 were used as new targets to create new cotton germplasm with disease resistance by RNAi technology.【Results and conclusions】（1）A total of 65 sugar transporter genes（Vd ST1-Vd ST65）were identified from the V.dahliae genome database and clustered into 8 evolutionary Clades,each corresponding to a specific sugar substrate.Nine Vd STs genes were found to be induced only by the root exudates from the susceptible cotton variety based on previous transcriptome data,including Vd ST3 and Vd ST12 that were selected for further functional study.After co-culture of Vd991 with asi RNAs targeting Vd ST3 and Vd ST12,the expression of Vd ST3 and Vd ST12 genes in Vd991 were found to be significantly reduced via q RT-PCR detection,and colony and mycelial growth as well as melanin formation were significantly inhibited,with a more significant effect observed for the asi RNAs targeting Vd ST12.Silencing of Vd ST12 by feeding asi R815+asi R1436significantly decreased the pathogenicity of Vd991.The results suggested that the sugar transporter genes Vd ST3 and Vd ST12 play important roles in the growth,development and pathogenesis of V.dahliae,and that asi RNAs can be used as a powerful tool for rapid screening of pathogenicity-related genes.（2）The Vd PT1 gene belongs to the GH37 subfamily of glycoside hydrolase and contains a typical structural domain of a neutral trehalase.Compared to wild-type Vd991,the Vd PT1 deletion mutant strains showed reduced trehalase activity,increased trehalose content and reduced glucose content,indicating that Vd PT1 was involved in the metabolism of endogenous trehalose.Compared with wild-type Vd991,the Vd PT1 deletion mutants exhibited reduced spore production,spore germination rate,colony growth,mycelial growth and penetration,increased melanin and decreased pathogenicity,indicating that the gene plays an important role in the growth,development and pathogenicity of V.dahliae.（3）RNA-Seq results revealed that a total of 3130 differentially expressed genes between wild-type Vd991 and Vd PT1-1 deletion mutant strains,including 1480 up-regulated genes and 1650 down-regulated genes.GO and KEGG analysis revealed that Vd PT1 gene deletion caused a significant decrease in the expression level of hydrolase activity,hydrolysis of O-glycosyl compounds related genes,most of which were cell wall degrading enzymes related genes,which was one of the reasons for the reduced mycelial penetration and pathogenicity of the deletion mutant strains.Vd PT1 deletion lead to a decrease in the expression level of genes related to glycolysis,pentose phosphate,glyoxylate cycle,nucleic acid and amino acid pathways,which were responsible for the reduced spore production,spore germination rate,mycelial and colony growth of deletion mutant strains.Vd PT1 deletion caused a increase in the expression level of genes related to glycerophospholipid and steroid metabolism pathways,which may be responsible for the changes in cell membrane components,resulting in increased stress resistance of deletion mutant strains.These changes in gene expression provide molecular evidence to explain the phenotypic differences between wild-type Vd991 and Vd PT1 deletion mutant strains.（4）Silencing of Vd ST3,Vd ST12 or Vd PT1 in the host plants by TRV-based HIGS effectively reduced fungal biomass,damage caused by pathogenic fungus and enhanced cotton disease resistance,indicating that all three genes could be used as targets for creating new cotton germplasm with disease resistance using RNAi.The RNAi vectors for Vd ST3,Vd ST12 and Vd PT1 genes were successfully constructed and transformed into cotton hypocotyl via Agrobacterium-mediated method.T0 generation transgenic plants have been obtained,providing new cotton germplasm for disease resistance breeding.