The Role Of CsRSF1 And CsRSF2 In The Regulation Of Cucumis Sativus Resistance To Sphaerotheca Fuliginea

Cucumber(Cucumis sativus)is one of the largest protected vegetable cultivation areas in China.Cucumber powdery mildew caused by Sphaerotheca fuliginea is an important disease in cucumber,which seriously affects the yield and quality of cucumber.The research shows two NBS-LRR proteins in cucumber(Cucsa.102240 and Cucsa123410)are predicted and have similar domain to Arabidopsis powdery mildew resistance protein RPW8.We named these two proteins Cs RSF1 and Cs RSF2 respectively.Nucleotide-binding leucine-rich repeat(NBS-LRR)-type proteins can directly or indirectly recognize pathogen effectors from different sources.They play a key role in plant defense mechanisms.However,the role of Cs RSF1 and Cs RSF2 genes in regulating powdery mildew is still unclear.In this study,the CDS sequences of Cs RSF1 and Cs RSF2 were cloned and the sequence characteristics and expression patterns of Cs RSF1 and Cs RSF2 were clarified.Cs RSF1 and Cs RSF2 transient silencing and overexpressing cucumbers were used to initially reveal the functions of the two genes in response to S.fuliginea infection of cucumber.The main results are as follows:(1)The CDS sequences of Cs RSF1 and Cs RSF2 were cloned from cucumber two sister lines B21-a-2-1-2 and B21-a-2-2-2 respectively.We compared the sequences of the two sister cucumber lines and found no differences between them.Bioinformatics analysis showed that Cs RSF1 and Cs RSF2 were typical NBS-LRR proteins and both contained NBS,LRR,and RPW8 domains;the cis-acting elements of the promoter both contained defense and stress response elements and various plant hormone response elements.The subcellular localization results showed that Cs RSF1 and Cs RSF2 were both located in membrane and cytoplasm,and Cs RSF1 was also found in the nucleus.(2)The q RT-PCR technology was used to analyze the gene expression patterns of Cs RSF1 and Cs RSF2 in different tissues of the two sister cucumber lines,under different treatments(ABA,Me JA,SA,ETH,GA,and H2O2)and S.fuliginea.The results showed that Cs RSF1 and Cs RSF2 had tissue expression specificity;the expression of Cs RSF1 was highest in roots of disease-resistant varieties,lowest in leaves,highest in leaves of susceptible varieties,and lower in roots and cotyledons.The expression of Cs RSF2 was highest in cotyledons of diseaseresistant and susceptible varieties,lowest in stems of disease-resistant varieties and lowest in roots of susceptible varieties.Cs RSF1 and Cs RSF2 are closely related to ABA and GA signals;The cucumber Cs RSF1 and Cs RSF2 genes are involved in the response of S.fuliginea infection;In the early stage of S.fuliginea infection,the expression levels of Cs RSF1 and Cs RSF2 in disease-resistant varieties are higher than in susceptible varieties.(3)The transient silencing vector and transient overexpression vector of Cs RSF1 and Cs RSF2 genes were constructed by In-fusion homologous recombination method.Cs RSF1 and Cs RSF2 transient overexpressing and silencing cotyledons were successfully obtained through the transient transformation technology medieted by Agrobacterium.After inoculation with S.fuliginea,the cotyledon phenotype,disease index,hypha number and the expression patterns of defense-related genes were analyzed.Studies have shown that transient silencing of CsRSF1 and Cs RSF2 can reduce the resistance of cucumber to S.fuliginea,while transient overexpression of Cs RSF1 and Cs RSF2 can increase the resistance of cucumber to S.fuliginea.Thus,Cs RSF1 and Cs RSF2 have been initially identified to play a positive regulatory role in response to S.fuliginea in cucumber.