title: Identification and functional analysis of mitogen-activated protein kinase kinase kinase (MAPKKK) genes in canola (Brassica napus L.).
abstract: Mitogen-activated protein kinase (MAPK) signalling cascades, consisting of three types of reversibly phosphorylated kinases (MAPKKK, MAPKK, and MAPK), are involved in important processes including plant immunity and hormone responses. The MAPKKKs comprise the largest family in the MAPK cascades, yet only a few of these genes have been associated with physiological functions, even in the model plant Arabidopsis thaliana. Canola (Brassica napus L.) is one of the most important oilseed crops in China and worldwide. To explore MAPKKK functions in biotic and abiotic stress responses in canola, 66 MAPKKK genes were identified and 28 of them were cloned. Phylogenetic analysis of these canola MAPKKKs with homologous genes from representative species classified them into three groups (A-C), comprising four MAPKKKs, seven ZIKs, and 17 Raf genes. A further 15 interaction pairs between these MAPKKKs and the downstream BnaMKKs were identified through a yeast two-hybrid assay. The interactions were further validated through bimolecular fluorescence complementation (BiFC) analysis. In addition, by quantitative real-time reverse transcription-PCR, it was further observed that some of these BnaMAPKKK genes were regulated by different hormone stimuli, abiotic stresses, or fungal pathogen treatments. Interestingly, two novel BnaMAPKKK genes, BnaMAPKKK18 and BnaMAPKKK19, which could elicit hypersensitive response (HR)-like cell death when transiently expressed in Nicotiana benthamiana leaves, were successfully identified. Moreover, it was found that BnaMAPKKK19 probably mediated cell death through BnaMKK9. Overall, the present work has laid the foundation for further characterization of this important MAPKKK gene family in canola.
authors.affiliation: State Key Laboratory of Crop Stress Biology for Arid Areas and College of Life Sciences, Northwest A & F University, Yangling, Shaanxi 712100, China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Sun
authors.forename: Chen
authors.initials: C
authors.lastname: Wang
authors.forename: Bo
authors.initials: B
authors.lastname: Yang
authors.forename: Feifei
authors.initials: F
authors.lastname: Wu
authors.forename: Xueyu
authors.initials: X
authors.lastname: Hao
authors.forename: Wanwan
authors.initials: W
authors.lastname: Liang
authors.forename: Fangfang
authors.initials: F
authors.lastname: Niu
authors.forename: Jingli
authors.initials: J
authors.lastname: Yan
authors.forename: Hanfeng
authors.initials: H
authors.lastname: Zhang
authors.forename: Boya
authors.initials: B
authors.lastname: Wang
authors.forename: Michael K
authors.initials: MK
authors.lastname: Deyholos
authors.forename: Yuan-Qing
authors.initials: YQ
authors.lastname: Jiang
mesh_terms: D000595:Amino Acid Sequence; D029688:Brassica napus / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D020224:Expressed Sequence Tags / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D020930:MAP Kinase Kinase Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010802:Phylogeny; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013312:Stress, Physiological; D020798:Two-Hybrid System Techniques
chemical_list: D018076:DNA, Complementary; D010940:Plant Proteins; D020930:MAP Kinase Kinase Kinases
keywords: Abiotic stress; Brassica napus; MAPKKK; MKK; Sclerotinia sclerotiorum.; cell death