Omics Dissection Of The Interactions Between Oilseed Rape And Sclerotinia Sclerotiorum

Oilseed rape(Brassica napus)is one of the most important oil crops in China.The white mould disease caused by Sclerotinia sclerotiorum,a necrotrophic fungal pathogen,is a major problem in rape production.Nevertheless,the resistance resources to against S.sclerotiorum are rather limited and the mechanisms underlying B.napus defense against this pathogen are poorly understood.Thus,identification of regulators of this resistance is urgently needed.In this study,taking advantage of the completion of the B.napus genome sequencing,multiple omics methods(miRNAomics,transcriptomics,proteomics and phosphoproteomics)were employed to reveal the mechanisms of interaction between rape and S.sclrotiorum.The main results are as follows.(1)The miRNAomics analysis for the interaction between rape and S.sclrotiorum was conducted and thereby the important role of miRNA and AGO-mediated RNA silencing in regulating rape-S.sclerotiorum interactions was demonstrated.227 conserved and 53 novel candidate miRNAs were identified by high-throughput deep sequencing in B.napus.Among them,target genes of 237 miRNAs were further obtained by sequencing of degradomes.MicroRNA microarray analysis revealed that 68 miRNAs were differentially expressed in response to S.sclerotiorum.A set of these miRNAs target genes involved in plant defense,such as NBS-LRR R genes and nitric oxygen and reactive oxygen species related genes.Additionally,three miRNAs targeted AGO1 and AGO2,key components of RNA silencing.Expression of several viral RNA silencing suppressors reduced the plant resistance to S.sclerotiorum.Arabidopsis mutants of AGO1 and AGO2 exhibited reduced resistance while transgenic lines over-expressing AGO1 displayed increased resistance to S.sclerotiorum.Moreover,over-expression of miRNAs targeting AGO1 and AGO2 decreased resistance to S.sclerotiorum in B.napus.(2)Gene family members of three key components of RNA silencing,Dicer-like(DCL),Argonaute(AGO)and RNA-dependent RNA polymerase(RDR)were identified in B.napus at genome-wide level.We first revealed that RNA silencing was regulated by CAMTA3,an important transcription factor in Ca2+ signaling pathway.Genome of B.napus possessed 8 DCL,27 AGO and 16 RDR genes and RDR5 group appears to have undergone further expansion through duplication during evolution.Twenty-one out of 51 DCL,AGO and RDR genes were predicted to contain CAMTA3-binding site(CGCG box).Moreover,we showed that CG-1 domain of BnCAMTA3 could directly bind to the promoter region of 15 CGCG box-containing genes by performing DNA electrophoretic mobility-shift assays(EMSA).S.sclerotiorum inoculation strongly induced the expression of BnCAMTA3 genes while significantly suppressed that of some of these genes.Furthermore,mutant analyses demonstrated that dcll-9,dcl4-2,ago9-1,rdrl-1,rdr6-11 and rdr6-15 mutants exhibited altered resistance to S.sclerotiorum.Our results further confirmed the importance of RNA silencing in plant resistance to S.sclerotiorum and revealed a C AMT A3-mediated novel mechanism of RNA silencing regulation.(3)Integrated analysis of miRNA-seq and mRNA-seq in Arabidopsis ago2 and WT plants in response to S.sclerotiorum was used to elucidate the mechanism of AG02-mediated S.sclerotiorum resistance.Through analyzing the miRNA-seq and mRNA-seq data of ago2 and WT plants both before and after S.sclerotiorum infection,we identified 32 differentially expressed miRNAs and 884 differentially expressed genes in comparison group ago2-Ss/WT-Ss.Among them,there were 553 negative correlation miRNA-mRNA pairs,with 16 differentially expressed miRNAs targeting disease resistance-related genes.The Arabidopsis mutant plants of the target genes GSTU2?GSTU5 and RBOHF were more susceptible to S.sclerotiorum.These results suggested that AG02 may regulate the plant resistance to S.sclerotiorum by both miRNA-dependent and-independent ways.(4)Comparative quantitative proteomics analyses were performed and thereby a number of proteins involved in regulating S.sclerotiorum resistance were identified,providing new insights into the molecular mechanisms of interactions between B.napus and S.sclerotiorum.The proteomes of B.napus leaves inoculated with S.sclerotiorum wild-type strain 1980 and nonpathogenic mutant strain Ep-1PB as well as empty agar plug as the control were analyzed using TMT label-based quantitative analysis technique by LTQ Orbitrap Elite.A total of 79,299 and 173 proteins consistently differentially expressed between Ep-1PB-and mock-inoculated leaves,1980-and mock-inoculated leaves,as well as 1980-and Ep-1PB-inoculated leaves,respectively,were identified.GO,KEGG and protein-protein interaction prediction analyses revealed that redox homeostasis,lipid signaling,calcium signaling,histone and DNA methylation-mediated transcription regulation and defense-related proteins such as defensin and defensin-like proteins and cyanate lyase,contributed to defense against S.sclerotiorum.(5)Quantitative proteomics analyses were performed in rape in response to S.sclerotiorum and thereby a number of phosphoproteins involved in regulation of S.sclerotiorum resistance were identified.The phosphoproteomes of B.napus leaves inoculated with S.sclerotiorum 1980 and Ep-1PB as well as the mock inoculation control were analyzed using TiO2-metal oxide affinity chromatography,coupled with label-free quantification techniques.A total of 2,11 and 12 phosphoproteins differentially expressed between Ep-1PB-and mock-inoculated leaves,1980-and mock-inoculated leaves,as well as 1980-and Ep-1PB-inoculated leaves,respectively,were identified.The phosphorylation regulatory mechanism and their possible involvement in the interaction between B.napus and S.sclerotiorum of MED26c,TPR1 and bZIP63 were indicated.Collectively,we carried out comprehensive omics dissection for the interactions between B.napus and S.sclerotiorum,unveiled the important role and underlying regulatory mechanism of RNA silencing in B.napus resistance to S.sclrotiorum.Moreover,a set of novel regulatory genes/proteins were identified.These results implied some new insights into the mechanisms of B.napus defense response to S.sclrotiorum.