Functional Analysis Of CsMLO1 And CsMLO2 In The Regulation Of Cucumis Sativus Resistance To Corynespora Cassiicola

Cucumber(Cucumis sativus)is one of the main vegetable crops of facility cultivation in China.Corynespora cassiicola is an important vegetable infectious pathogen,which can cause cucumber target leaf spot(TLS)and seriously endangers the production of cucumber.Proteomics and transcriptome analysis in our lab showed that protein and gene levels of two MLO(Mildew Resistance Locus O)genes,CsMLO1(Cucsa.207280)and CsMLO2(Cucsa.308270),were significantly upregulated in the early stage under C.cassiicola infection.In this study,we investigated the expression patterns of CsMLO1 and CsMLO2 genes and the relationship between these two genes and C.cassiicola infection;CsMLO1/CsMLO2-overexpressing and CsMLO1/CsMLO2-silencing in cucumber cotyledons were successfully constructed for research the specific functions of the two genes in response to C.cassiicola infection;At the same time,yeast two-hybrid,firefly luciferase(LCI)complementation and bimolecular fluorescence complementation(Bi FC)were used to screen the proteins interactions,and to explore their mechanism of CsMLO1 and CsMLO2 in regulating cucumber resistance to C.cassiicola.This study will provide a basis for revealing the molecular mechanism of the interaction between cucumber and C.cassiicola,and will facilitate a more in-depth and accurate interpretation of the role of CsMLO1 and CsMLO2 proteins in signal transduction pathway against pathogen infection.The main results are as follows:(1)The DNA sequences and promoter sequences of CsMLO1 and CsMLO2 genes were cloned in cucumber.Bioinformatics analysis revealed that both CsMLO1 and CsMLO2 promoters contained the cis-acting element involvement in the abscisic acid(ABA)response and defense stress response.Conserved domains and motif analysis in the c DNA regions of the CsMLO1 and CsMLO2 genes revealed that they all contained seven transmembrane conserved domains and calmodulin binding site.The fusion proteins of CsMLO1-GFP and CsMLO2-GFP were transformed into tobacco leaves by Agrobacterium-mediated transient transformation,respectively.The subcellular results suggested that CsMLO1 and CsMLO2 were both localized in the plasma membrane.In conclusion,CsMLO1 and CsMLO2 are typical members of the MLO family and may be involved in regulating cucumber defense responses against C.cassiicola.(2)The expression patterns of CsMLO1 and CsMLO2 were determined in different resistant cultivars,different tissues and different stresses(C.cassiicola,H2O2,Ca Cl2,SA,Me JA and ABA)by RT-q PCR.The results suggested that CsMLO1 and CsMLO2 genes exhibited tissue-specific expression in cucumber.Cucumber CsMLO1 and CsMLO2 genes are closely related to Ca2+,reactive oxygen species(ROS)and ABA signaling pathways after abiotic stress.Under C.cassiicola infection,the expression levels of CsMLO1 and CsMLO2 genes in the susceptible cultivars were earlier and higher than those in the resistant cultivars.(3)The overexpressing vector and the virus-induced gene silencing vector of CsMLO1/CsMLO2 were constructed by In-fusion homologous recombination method,cucumber cotyledons with transient overexpressing and transient silencing of CsMLO1/CsMLO2 were successfully obtained by Agrobacterium transient transformation technique.The expression of disease resistance genes,disease index and lesion area analysis suggested that CsMLO1/CsMLO2 acted as negative modulators in cucumber resistance in response to C.cassiicola.(4)Combining ROS metabolism-related genes with enzyme activities,ABA signaling-related genes,endogenous ABA content and Ca M-related gene expression,it is speculated that CsMLO1 may regulate the expression of ROS,ABA and Ca2+ signaling pathway-related genes to regulate resistance of cucumber to C.cassiicola.The interaction between CsMLO1/CsMLO2 and Cs Ca M1/Cs Ca M2/Cs Ca M3 was investigatedby yeast two-hybrid,LCI and Bi FC.The results showed that CsMLO1 protein and Cs Ca M3 protein are stable interaction,the CsMLO2 protein does not interact with the Cs Ca M1/Cs Ca M2/Cs Ca M3 protein.Co-transformed with CsMLO1 and Cs Ca M3 in cucumber cotyledons indicated that CsMLO1 inhibited HR response of plants by blocking the gene expression of Cs Ca M3,resulting in weakened resistance of cucumber cotyledons to C.cassiicola.