Functions And Molecular Mechanisms Of MiR1885 And MiR6030 In Regulating Brassica Napus Resistance To Sclerotinia Sclerotiorum

Oilseed rape（Brassica napus）is one of the most important oil crops in China,which has high economic value.Stem rot disease caused by Sclerotinia sclerotiorum is a devastating disease in oil crops.This disease causes huge economic losses every year due to the lack of highly resistant rape varieties.The in-depth analysis of the mechanism underlying rape resistance to S.sclerotiorum can provide new strategy for green management of stem rot disease.miRNAs（micro RNAs）are a class of non-coding RNAs with a length of about 22 nt.The regulatory functions and mechanisms of miRNAs in rape resistance to S.sclerotiorum are not yet clear.In this study,the regulatory functions and mechanisms of Brassica-specific miRNAs,miR1885 and miR6030 in the resistance to S.sclerotiorum were analyzed.The main results are as follows.（1）The function and mechanisms of two members of the miR1885 family,miR1885a and miR1885b,in rape resistance to S.sclerotiorum were revealed.Disease resistance analysis in rape plants in which miR1885a and miR1885b were transiently silenced or overexpressed and in their stably transgenic rape plants demonstrated that miR1885a and miR1885b negatively regulated rape resistance to S.sclerotiorum.The5’RNA ligation mediated-rapid amplification of c DNA ends（5’RLM-RACE）analysis identified a Toll/interleukin-1 receptor-like-nucleotide binding site-leucine-rich repeats（TIR-NBS-LRR）protein-encoding gene Bn RSS1（Brassica napus resistance to Sclerotinia sclerotiorum 1）as a target of both miR1885a and miR1885b.Virus induced gene silencing（VIGS）assay and disease resistance analysis in transgenic rape plants over-expressing Bn RSS1 showed that Bn RSS1 played a positive role in rape resistance to S.sclerotiorum.Together,these results revealed that the miR1885-Bn RSS1 module played an important role in rape resistance to S.sclerotiorum.Further analyses verified that this module also functioned in other Brassica crops such as B.pekinensis and B.rapa.Both single and dual Luciferase reporter assays and EMSA analysis confirmed that Bn CAMTA3 could directly bind to MIR1885 promoter and negatively regulated its transcription.Disease resistance analysis in transgenic plants with Bn CAMTA3 over-expression or RNAi（RNA interference）showed that Bn CAMTA3 positively regulated rape resistance to S.sclerotiorum.Collectively,the Bn CAMTA3-miR1885-Bn RSS1 module was involved in rape resistance to S.sclerotiorum.（2）The function and mechanisms of miR6030 in rape resistance to S.sclerotiorum were elucidated.Resistance evaluation in rape plants in which miR6030was transiently silenced or overexpressed and in its stably transgenic rape plants in which miR6030 was silenced made clear that miR6030 negatively regulated rape resistance to S.sclerotiorum.The GUS staining assay identified two Coiled-coil-nucleotide binding site-leucine-rich repeats（CC-NBS-LRR）protein-encoding genes,Bna Cnng64090D and Bna A09g10520D as target genes of miR6030.Transient overexpression analysis indicated that these two target genes played a positive role in rape resistance to S.sclerotiorum.Both single and dual Luciferase reporter assays and EMSA analysis verified that Bn CAMTA3 could directly bind to MIR6030 promoter and negatively regulated its transcription.Together,theseresultsdemonstratedthatthe Bn CAMTA3-miR6030-Bna Cnng64090D/Bna A09g10520D module could function in regulating rape resistance to S.sclerotiorum.Collectively,in this study,the functions and mechanisms of miR1885 and miR6030 in rape resistance to S.sclerotiorum were dissected.It was found that miR1885 and miR6030 regulated rape resistance to S.sclerotiorum via targeting different types of NBS-LRR（NLR）protein-encoding genes,and the transcription of both MIRNA genes was negatively regulated by Bn CAMTA3,an important transcription factor in Ca²⁺signaling pathway.This study unveiled the pivotal role of the Bn CAMTA3-miRNA-NLR module in regulating rape resistance to S.sclerotiorum and thereby connected the Ca²⁺signaling pathway-transcriptional regulation-post-transcrptional regulation-NLR and resistance to S.sclerotiorum.Together,this study provided some insights into the mechanisms underlying miRNAs,NLR and CAMTA3-mediated regulation of resistance and the mechanisms underlying rape resistance to S.sclerotiorum,thereby provided theoretical guidance and gene resources for molecular breeding of varieties highly resistant to stem rot disease.