| Summary: | <i>Brassica napus</i> (oilseed rape) is one of the most important oil crops worldwide, but its growth is seriously threatened by <i>Sclerotinia sclerotiorum</i>. The mechanism of oilseed rape response to this pathogen has rarely been studied. Here, it was identified that BnaA03.MKK5 whose expression was induced by <i>S. sclerotiorum</i> infection was involved in plant immunity. <i>BnaA03.MKK5</i> overexpression lines exhibited decreased disease symptoms compared to wild-type plants, accompanied by the increased expression of camalexin-biosynthesis-related genes, including <i>BnPAD3</i> and <i>BnCYP71A13</i>. In addition, two copies of <i>BnMPK3</i> (<i>BnA06.MPK3</i> and <i>BnC03.MPK3</i>) were induced by <i>Sclerotinia</i> incubation, and BnaA03.MKK5 interacted with BnaA06.MPK3/BnaC03.MPK3 in yeast. These interactions were confirmed using in vivo co-immunoprecipitation assays. In vitro phosphorylation assays showed that BnaA06.MPK3 and BnaC03.MPK3 were the direct phosphorylation substrates of BnaA03.MKK5. The transgenic oilseed rape plants including <i>BnaA06.MPK3</i> and <i>BnaC03.MPK3</i> overexpression lines and <i>BnMPK3</i> gene editing lines mediated by CRISPR/Cas9 were generated; the results of the genetic transformation of <i>BnaA06.MPK3/BnaC03.MPK3</i> indicate that BnMPK3 also has a positive role in <i>Sclerotinia</i> resistance. This study provides information about the potential mechanism of <i>B. napus</i> defense against <i>S. Sclerotiorum</i> mediated by a detailed BnaA03.MKK5-BnaA06.MPK3/BnaC03.MPK3 module.
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