Molecular Mechanism Of BnaA03.WRKY28 And BnWRKY33 Involved In Sclerotinia Sclerotiorum Resistance In Brassica Napus

Rapeseed（Brassica napus）is one of the most important oil crops worldwide.However,Sclerotinia sclerotiorum is generally considered one of the most economically damaging diseases on rapeseed.The plant innate immune system may be the primary solution to defense against S.sclerotiorum for rapeseed.BnWRKY33 positinely contributes to Sclerotinia resistance of rapeseed.In the present study,two WRKY transcription factors,BnaA03.WRKY28 and BnWRKY33 could bind to the promoter of BnWRKY33,but they had the opposite effect on the Sclerotinia resistance in Brassica napus.By analyzing the induced expression patterns of BnaA03.WRKY28 and BnWRKY33 after inoculation with S.sclerotiorum,and the function action when the two WRKY transcription factors interacted with BnWRKY33 promoter,the mechanism of Sclerotinia resistance mediated by BnaA03.WRKY28 and BnWRKY33 was explained.And a relatively complete signal pathway related to Sclerotinia resistance,trade-off between growth and defense in rapeseed was proposed.The main results are as follows:1.Identification of the biological function of BnaA03.WRKY28.Five homologous copies of At WRKY28 were isolated from two rapeseed lines,namely,Jia9709and Westar,which were located on A01,C01,A03,A08 and C08 chromosomes,respectively.Among them,the A03 copy,BnaA03.WRKY28,was induced by infection of S.sclerotiorum,and the expression of BnaA03.WRKY28 reached the peak after 48 h inoculation.The overexpression vectors driven by Ca MV35S and gene editing vectors mediated by CRISPR/Cas9 of BnaA03.WRKY28 were constructed and transformed into Jia9709 and Westar,and homozygous transgenic lines were obtained.BnaA03.WRKY28transgenic lines and wild-type plants were inoculated with S.sclerotiorum.In vitro leaf inoculation and stem inoculation experiments showed that the lesion degree of BnaA03.WRKY28 overexpression lines was more serious than that of wild-type plants,while the lesion degree of BnaA03.WRKY28 gene editing lines was less than that of wild-type plants.These results indicate that BnaA03.WRKY28 is a negative factor of Sclerotinia resistance in Brassica napus.2.Identification of the downstream target genes of BnaA03.WRKY28.The results of RNA-seq and ChIP-seq analysis showed that BnWRKY33 may be the downstream target gene of BnaA03.WRKY28.The direct binding of BnaA03.WRKY28and BnWRKY33 promoter was proved by the in vivo and in vitro experiments such as ChIP-q PCR,Y1H,dual-luciferase transient transcriptional activity assays and EMSA.Further analysis showed that two W-boxes（W1 and W3）of BnWRKY33 promoter were binding sites of BnaA03.WRKY28.BnaA03.WRKY28 promoted the expression of BnWRKY33 by directly binding to BnWRKY33 promoter.BnWRKY33^DD was a phosphorylated and activated form of BnWRKY33.BnWRKY33^DD could also directly bind to the promoter of BnWRKY33 and promote the expression of BnWRKY33.However,the transcriptional activity of BnWRKY33^DD was significantly higher than that of BnaA03.WRKY28.3.Analysis of MAPK cascade which phosphorylated and activated BnWRKY33.BnaA03.MKK4,BnaA06.MAPK3 and Bna C03.MAPK3 were induced by Sclerotinia.It was found that BnaA03.MKK4 interacted with BnaA06.MAPK3 or Bna C03.MAPK3,and BnWRKY33 interacted with BnaA06.MAPK3 or Bna C03.MAPK3.Further,through in vivo dual-luciferase transient transcriptional activity assays and in vitro phosphorylation test,it was found that BnWRKY33 was the phosphorylation substrate of BnaA03.MKK4-BnaA06.MAPK3/Bna C03.MAPK3 module.The transcription activity of BnWRKY33 was significantly enhanced after phosphorylation.Genetic transformation provedthattheMAPKcascademediatedby BnaA03.MKK4-BnaA06.MAPK3/Bna C03.MAPK3 enhanced the Sclerotinia resistance of rapeseed,which was an important pathway for defense response against S.sclerotiorum in Brassica napus.4.BnaA09.VQ12 was a co-factor of BnaA03.WRKY28.VQ protein usually plays a role by forming protein complexes with WRKY transcription factors.BnaA09.VQ12and BnaA03.WRKY28 showed similar induced expression profile when inoculated with S.sclerotiorum.The interaction between BnaA09.VQ12 and BnaA03.WRKY28 was confirmed by Y2H,Bi FC,pull down and Co-IP.And BnaA09.VQ12 formed a protein complex with BnaA03.WRKY28 in the nucleus through the DNA domain of BnaA03.WRKY28.In addition,overexpression of BnaA09.VQ12 weakened Sclerotinia resistance of transgenic plants,while knockout of BnaA09.VQ12 enhanced resistance.In response to Sclerotinia infection,BnaA09.VQ12 and BnaA03.WRKY28 showed similar biological functions.5.The relationship between BnWRKY33 and BnaA03.WRKY28 in Sclerotinia resistance.BnWRKY33 was highly expressed at the early stage of infection by Sclerotinia,and BnaA03.WRKY28 was higher at the later stage of incubation.Additionally,the expression level of BnWRKY33 at the late stage of defense was dramatically lower than that at the early stage.Overexpression of BnaA03.WRKY28 and BnaA09.VQ12 inhibited the expression of BnWRKY33 at the early stage of defense,while the expression of BnWRKY33 remained at a high level in BnaA03.WRKY28 gene editing lines and BnaA09.VQ12 gene editing lines.In vitro EMSA showed that BnaA09.VQ12 enhanced the binding ability of BnaA03.WRKY28 to BnWRKY33 promoter by binding to the DNA domain of BnaA03.WRKY28.Further,In vivo analysis of dual-luciferase transient transcriptional activity assays showed that BnaA03.WRKY28 had stronger affinity to BnWRKY33 promoter than BnWRKY33 by forming protein complex with BnaA09.VQ12.6.BnaA03.WRKY28 promoted the formation of lateral branches of Brassica napus.BnaA03.WRKY28 overexpression lines produced more branches,especially two or more branches in one leaf axil,and produced more high-order branches.GUS staining showed that BnaA03.WRKY28 was expressed in the leaf axil.q PCR analysis showed that the expression of BnBRC1 and BnBRC2,which are closely related to branching,and the auxin mediated branching related gene BnAXR1 were down-regulated,while the auxin transporter gene BnPIN1 was up-regulated in BnaA03.WRKY28 overexpression lines after48 h inoculation with S.sclerotiorum.In addition,EMSA showed that BnaA03.WRKY28directly bound to the promoter of Bna C03.BRC1.Therefore,it is suggested that BnaA03.WRKY28 may be involved in the branching of rapeseed by directly regulating the expression of Bna C03.BRC1 and influencing auxin mediated branching.In conclusion,when threatened by S.sclerotiorum,the innate immune system of rapeseed plants was activated.Activated MAPK cascade directly acted on transcription factor BnWRKY33.The transcriptional activity of phosphorylated BnWRKY33 was significantly enhanced,and the expression level of BnWRKY33 was significantly increased by binding to its own promoter,thus promoting disease resistance.Over constant infection,BnaA03.WRKY28 and BnaA09.VQ12 were induced.The interaction between BnaA09.VQ12 and BnaA03.WRKY28 enhanced the binding ability of BnaA03.WRKY28 to BnWRKY33 promoter.BnaA03.WRKY28-BnaA09.VQ12preferentially bound to BnWRKY33 promoter compared with BnWRKY33,which weakening disease resistance.In addition,at the late stage of defense,BnaA03.WRKY28,expressed in the the leaf axil,promoted the formation of lateral branches by regulating the expression of branching related genes such as Bna C03.BRC1 and BnPIN1,so as to realize the trade-off between growth and defense in response to S.sclerotiorum in Brassica napus.