The Biological Function And Regulatory Mechanism Of GhMKK6 Mediated MAPK Cascade Signaling Pathway In Cotton

Cotton is one of the most important economic crops in China,but cotton production is constrained by various cotton disease.Fusarium wilt is considered to be one of the most serious threat to the yield and quality of cotton.MAPK cascade signaling pathway is the highly conservative signaling pathway in eukaryotes which is the major modules identifying and amplifying external signals into intracellular components.Many studies reported that activation of MAPK cascade signaling pathway in plants was one of the earliest events in plant immune response,and they controled the biosynthesis of phytoalexins and the expression of defense genes.Therefore,study on the molecular mechanism of MAPK cascade signaling pathway regulating plant immune responses has important theoretical significance for guiding crop disease resistance breeding.However,MAPK cascade signaling pathways in plants are mostly carried out in Arabidopsis.The research on the function of MAPK cascade signaling pathway in other plants,especially in cotton and other crops,is still relatively limited,and the regulatory mechanism of MAPK cascade signaling pathway is rarely reported.In this study,upland cotton(Gossypium hirsutum L.)was used as the experimental material.Through the integration of biochemical,molecular biology,bioinformatics experiments,we clarified the role of GhMKK6-mediated MAPK cascade signaling pathway in the regulation of Fusarium wilt resistance in cotton.The regulatory mechanism of GhMKK6-mediated MAPK cascade signaling pathway by miRNA and MAPK scaffold proteins was discussed.The major results are as follows:(1)The GhMKK6-mediated MAPK cascade signaling pathway played important roles in regulating the resistance of cotton to Fusarium oxysporum,but excessive activation of GhMKK6 may be detrimental to plants.GhMKK6 is a group A MEKs gene in cotton.Subcellular localization analysis showed that the gene was located in the nucleus.Expression pattern analysis revealed that F.oxysporum,SA,and MeJA could affect the expression level of GhMKK6,indicating that GhMKK6 was involved in regulating the immune response of cotton.Silencing GhMKK6 in cotton using VIGS revealed that the resistance of cotton to F.oxysporum significantly decreased,and the expression level of SA-mediated plant resistance signaling pathway related genes were significantly reduced.GhMKK6 overexpressing transgenic tobacco was obtained using Agrobacterium-mediated leaf disc transformation.In transgenic tobaccos,although the expression levels of SA/JA pathway related genes were significantly increased,the transgenic tobaccos still showed a sensitive phenotype to F.oxysporum.Using site-directed mutagenesis,constitutively activated GhMKK6 and inactived GhMKK6 were obtained.The transient expression of GhMKK6 with different activities in tobacco leaves revealed that although all of tobacco leaves showed chlorosis,the constitutively activation of GhMKK6 could increase the resistance to F.oxysporum by activating the expression of downstream resistance genes,ROS generation,and SA/JA-mediated plant resistance signaling pathways.While excessive activation of GhMKK6will lead to a large accumulation of ROS,resulting in lesions and affecting plant growth.(2)ghr-miR5272a and GhMKK6 constituted a feedback regulation system.ghr-miR5272a regulated the expression level of GhMKK6 through feedback regulation to preventing excessive GhMKK6 activation.Analysis of the deep sequencing of miRNA in cotton revealed that the 3′non-coding region of GhMKK6 had high homology with ghr-miR5272a.Further experiments demonstrated that GhMKK6 was the target gene of ghr-miR5272a,and ghr-miR5272a regulates the expression level of GhMKK6 by recognizing the 3′non-coding region of GhMKK6.Expression pattern analysis showed that SA could not induce the expression of ghr-miR5272a,while MeJA and F.oxysporum could induce the expression of ghr-miR5272a.The expression pattern of ghr-miR5272a was complementary to GhMKK6.This result indicated that ghr-miR5272a might regulate cotton resistance to F.oxysporum by affecting the expression level of GhMKK6.Silencing ghr-miR5272a in cotton significantly inhibited the response of GhMKK6 to F.oxysporum,and the expression pattern of SA/JA-mediated plant resistance signaling pathway related genes were similar to that in GhMKK6 overexpressing tobaccos.The transcriptional level and protein level of GhMKK6decreased significantly in miR5272 overexpressing cotton.The resistance of miR5272overexpressing cotton to F.oxysporum was significantly lower than in control,and the expression of SA/JA pathway related genes were similar to that in GhMKK6 silenced cotton.(3)The yeast two-hybrid was used to screen the GhMKK6 interaction protein.A cotton yeast two-hybrid library was constructed using total cotton RNA.The capacity of this library was 1.4×10~6 and the average length of insert fragments were approximately 1000bp.The bait vector was constructed using the full-length ORF of GhMKK6.The autoactivation and toxicity test showed that the bait vector had no autoactivation activity and toxicity.Using this bait vector,the interaction protein of GhMKK6 was screened by the yeast two-hybrid technology.After removing duplicate genes,untranslatable fragments,and sequencing failed fragments,ten significant gene fragments were obtained.Through sequence analysis,the screened interaction proteins of GhMKK6 were mainly related to gene transcription and translation,signal transduction,carbohydrate metabolism and redox process.(4)GhMORG1 was one of the GhMKK6 interaction proteins obtained from the yeast two-hybrid screen.GhMORG1,as a scaffold protein of MAPK cascade signaling pathway,affected cotton resistance to F.oxysporum by regulating GhMKK6-mediated MAPK cascade signaling pathway.The ORF of GhMORG1 is 900bp in length and encodes a polypeptide consisting of 299 amino acid residues.Sequence analysis and protein structure prediction revealed that this gene contained seven tandem WD40 domains,which can form a circularβ-helix structure.Therefore,it can be used as a MAPK scaffold protein to regulate the MAPK cascade signaling pathway.Subcellular localization analysis showed that GhMORG1 was located in the nucleus as well as GhMKK6.The yeast two-hybrid and BiFC results showed that GhMORG1 interacted with GhMKK6 in the nucleus.The expression of GhMORG1 was induced by F.oxysporum and MeJA,indicating that GhMORG1 was involved in regulating cotton immune response.GhMORG1 silenced cottons were obtained by using VIGS.The resistance of GhMORG1 silenced cotton to F.oxysporum was significantly decreased,and the expression level of SA/JA-mediated plant resistance signaling pathway related genes were lower than that in control cotton.Further experiments showed that the resistance of GhMORG1 overexpressing transgenic tobaccos to F.oxysporum was significantly increased,and the expression pattern of SA/JA-mediated plant resistance signaling pathway related genes were similar to that in GhMKK6 overexpressing transgenic tobaccos.