AtRDR-mediated Resistance To Sclerotinia Sclerotiorum In Arabidopsis Thaliana

Canola(Brassica napus)is a very important oil crop as a major source of edible oil in the world and plays an important role in China national economy.Sclerotinia stem rot is one of key factors influencing the canola production and thus one of main breeding objectives.However,it was found that the Sclerotinia disease severity is negatively correlated with early maturation required for crop rotation and this correlation has not yet been solved.In the previous canola flowering time QTLs study in our lab,a candidate gene was selected for functional study and its Arabidopsis ortholog AtRDR(At5g24290);producing two splicing variants of AtRDRl and AtRDR2)was genetically proved controlling earlyflowering in Arabidopsis.This gene contains rich stress-related cis elements in the promoter sequences,and thus was predicted for functioning in plant disease resistance.Therefore,objectives of this study were to investigate the function of AtRDR in Sclerotinia disease resistance,including identification of resistance intransgenic plants,effect of AtRDR on defence-related gene expression,the study of its interacting proteins,bioinformatics analysis of gene families of interacting proteins LTP1 and PDF2.2 and development of related transgenic plants.The results were obtained as follows:1.Resistance conferred by AtRDR.Transgenic analyses revealed that the lines of over-expression of AtRDR1 and AtRDR2 were more sensitive to the Sclerotiniadisease,but the knockout mutant rdr059 was more resistant 48h after inoculation and afterward.By analyzing the expression level of PR1,PDF1.2 and AtrbohB,marker genes for SA,JA and ROS signaling pathways,respectively,we found thatAtRDR1and AtRDR2over-expressing plants were significantly higher in AtPR1 expression than the knock-out mutant and WT;AtRbohB exhibited similar trend;Contrastly,AtPDF1.2 expression was significantly higher than that in over-expression plants and WT.These suggested that AtRDR negatively controls resistance through negatively regulating the JA pathway and positively regulating the SA pathway.2.Studies of interacting proteins with AtRDR.Yeast two-hybrid and BiFC revealed AtRDR direct interacted with AtLTP1 and AtPDF2.2,and further q-PCR experiments with transgenic plants showed that AtRDR inhibited(negatively regulated)expression of AtLTP1 andAtPDF2.2.3.Bioinformatic analysis of AtLTP1 and AtPDF2.2 and its family members.Based on genome sequences,we carried out the evolutionary analysis of LTP and PDF gene family members in A.thaliana,B.rapa,B.oleraceaand B.napus.Totally,we found 83 LTP family members which were divided into five classes and 56 PDF family members which were divided into three classes.The promoter regions of LTP and PDF family members are rich in TC-rich repeats,LTRE,ABRE,MYB,Box-W1 and other stress response cis-acting elements.These analyzes could be used for functional study of LTP gene and PDF gene in Brassica.4.Construction of transgenic materials.We conducted transgenic plant development torigorously prove function of both AtLTPl and AtPDF2.2,and the molecular mechanism of the resistance mediated by AtRDR.Transgenic arabidopsis of AtLTP1andAtPDF2.2,and AtRDRwere obtained,for both over-expression and RNAi,as well as transgenic canola with AtLTP1 and AtPDF2.2.Also AtRDR-transgenic plants were crossed with those of LTP1 and PDF2.2 and F1 plants were obtained.In addition,two homologous genes in B.napusof AtRDR were identified,named BnMEBl,BnEMB2 and the BnEMB2 overexpression vector was constructed and transformed into Arabidopsis thaliana.Because of time limit,I have not conducted resistance identification of these transgenic plants.This study found that Arabidopsis early flowering gene AtRDR negative regulation on Sclerotinia sclerotiorum,provides an important evidence for resistance to Sclerotinia related to flowering time,and provide reference for gene function identification of BnEMB2.