Abstract
Brassica napus (oilseed rape) is one of the most important oil crops worldwide, but its growth is seriously threatened by Sclerotinia sclerotiorum. 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 S. sclerotiorum infection was involved in plant immunity. BnaA03.MKK5 overexpression lines exhibited decreased disease symptoms compared to wild-type plants, accompanied by the increased expression of camalexin-biosynthesis-related genes, including BnPAD3 and BnCYP71A13. In addition, two copies of BnMPK3 (BnA06.MPK3 and BnC03.MPK3) were induced by Sclerotinia 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 BnaA06.MPK3 and BnaC03.MPK3 overexpression lines and BnMPK3 gene editing lines mediated by CRISPR/Cas9 were generated; the results of the genetic transformation of BnaA06.MPK3/BnaC03.MPK3 indicate that BnMPK3 also has a positive role in Sclerotinia resistance. This study provides information about the potential mechanism of B. napus defense against S. Sclerotiorum mediated by a detailed BnaA03.MKK5-BnaA06.MPK3/BnaC03.MPK3 module.
Highlights
Brassica napus is an allotetraploid species derived from the natural hybridization of diploid ancestors Brassica rapa and Brassica oleracea about 7500 years ago; it contains 19 pairs of chromosomes (A01~A10 and C01~C09) [1]
In order to explore the response mechanism of oilseed rape to S. sclerotiorum infection, we focused on the mitogen-activated protein kinase (MAPK) cascade, including AtMKK5, the role of which in response to biotic and abiotic stress, especially in defense against B. cinerea in A. thaliana, has been well documented [15]
AtM3KoKf 414and AtMKK5 are similar in sequence and functionally redundant [11]
Summary
Brassica napus (genome AACC) is an allotetraploid species derived from the natural hybridization of diploid ancestors Brassica rapa (genome AA) and Brassica oleracea (genome CC) about 7500 years ago; it contains 19 pairs of chromosomes (A01~A10 and C01~C09) [1]. Oxalic acid secreted by S. sclerotiorum is considered to be one of the main causes of host disease [20]; Liu et al found that the heterologous overexpression of barley oxalate oxidase coding the BOXO gene in oilseed rape can partially enhance Sclerotinia resistance by reducing the oxalate level and increasing the hydrogen peroxide level in transgenic lines [21]. S. sclerotiorum causes great damage to the growth of B. napus, but our knowledge about the immune mechanism of oilseed rape against this pathogen is limited; in particular, the role of the MAPK cascade involved in is still obscure. We provide direct evidence that BnaA03.MKK5 and BnaA06.MPK3/BnaC03.MPK3 act as a module and positively contribute to Sclerotinia resistance
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