Screening And Functional Verification Of Disease-resistant Genes In Foxtail Millet And Grape

Millet and grape are one of the main cereal crops and economic crops in Shanxi province.However,plant fungal disease has become a severe threat to the millet and grape production.To breed new disease resistant cultivars,in this study we revealed the key resistance genes in millet and grape during interaction with fungal pathogens.Our study would lay the foundation for genetic breeding of millet and grape varieties resistant to fungal diseases.Foxtail millet is a characteristic coarse cereal in Shanxi province.However,the millet smut disease caused by basidiomycete fungus Ustilago crameri is a severe detriment to the millet production.Although there are some disease-resistant cultivars,their disease resistance descends year after year due to repeated cultivation.To breed new smut resistant millet cultivars using genetic engineering,a comprehensive understanding of gene expression during interaction between foxtail millet and smut fungus is needed.In this study,we utilized RNA-Seq to investigate transcriptional modulation in the resistant variety Jigu20 and the susceptible variety Changnong35.In total,1712 and1166 differentially expressed genes（DEGs）were characterized in Jigu20 and Changnong35,respectively.Among these DEGs,Jigu20 and Changnong 35 shared 288 common ones.The KEGG analysis showed that they were enriched in three pathways:the plant-pathogen interaction pathway,the MAPK signaling pathway,the plant hormone signaling pathway.Furthermore,through physiological and molecular biological analysis,we selected SiBGL,SiCHI,SiRPM1,SiSGT1,SiHSP,SiCDPK,SiRbohF and SiPAL as potential disease resistance genes.Additionally,we found that the expression of SiLEA14,a gene known to regulate abiotic stress in foxtail millet,was significantly upregulated in smut inoculated group of both Jigu20 and Changnong35.To assess the role of SiLEA14 in smut disease resistance,we overexpressed SiLEA14 in Arabidopsis,and the T1 generation plants had been obtained.Grape is widely grown worldwide.The European grape（Vitis vinifera L.）is the main grape variety cultivated in our province.Although it has rich nutritional value and good taste,it has the disadvantage of poor disease resistance,especially to the fungus pathogen Botrytis cinerea.It has been reported sulphur dioxide（SO₂）treatment could enhance the disease resistance of grapes,however,the underlying mechanisms are not clear.To identify the key disease resistance genes in grape,we performed RNA-Seq analysis using the grape berries with and without SO₂ treatment.We found several stress tolerance related transcription factors WRKY,bHLH、AP2/ERF and MYB were differentially expressed.In this study,we studied VvWRKY70 in detail.The coding sequence of VvWRKY70 was 969 bp in length.Furthermore,by fusion with GFP we found it localized at nucleus.In addition,we over-expressed VvWRKY70 in Arabidopsis.The phenotypic analysis showed that the homozygous T3 transgenic lines exhibited enhanced disease resistance to Botrytis cinerea infection.Above all,our results indicate that VvWRKY70may act as a positive regulator in the grape defense response against Botrytis cinerea.