Isolation Of GhVQ1 And GhVQ28 Genes From Cotton And Analysis Of Their Function Against Fusarium Oxysporum

Cotton is one of the most important crops in the world.But a variety of diseases have caused huge losses to cotton production.Fusarium wilt,as one of the most destructive diseases on cotton,has seriously restricted the yield and quality of cotton.However,the molecular mechanism of cotton against Fusarium oxysporum is still unclear.In order to explore the molecular mechanism of cotton against Fusarium oxysporum,RNA-seq analysis was used at the early stage,which indicated that the expression of 5 cotton VQ genes was significantly upregulated after Fusarium oxysporum treatment.Recent studies have shown that the function of VQ proteins was closely related to the MAPK cascade pathways which played an important role in plant immune response.To explore the function of VQ proteins in cotton against Fusarium oxysporum and its relationship with the MAPK cascade pathway,we identified two VQ proteins GhVQ1 and GhVQ28 related to the MAPK cascade pathway through yeast twohybrid screening.Their expression characteristics and biological functions were analyzed.The main results are as follows:(1)The results of RNA-seq analysis showed that the expression of many genes,including5 VQ genes,were induced by Fusarium oxysporum,which suggested that these 5 VQ genes may play an important role in resistance to Fusarium oxysporum.(2)Bioinformatics analysis showed that all of these 5 VQ proteins were highly conserved at VQ-motif,but poorly conserved in other regions,showing common structural characteristics of VQ proteins.5 VQ genes were respectively named as GhVQ1,GhVQ7,GhVQ10,GhVQ23 and GhVQ28 by referencing to Arabidopsis thaliana genome database.Phylogenetic tree analysis of above cotton VQ proteins and 34 Arabidopsis thaliana VQ proteins showed that GhVQ1 and GhVQ10 belonged to group VIII VQ proteins,GhVQ7 and GhVQ28 belonged to group IX VQ proteins,GhVQ23 belonged to group VI VQ proteins.(3)Yeast two-hybrid assays of 5 VQ proteins and 6 MAPK proteins related to disease resistance indicated that GhVQ1 interacted with GhMAPK6 a,and GhVQ28 interacted with GhMAPK20.The mutations of valine(V)and glutamine(Q)in VQ-motif did not affect the interactions of GhVQ1 with GhMAPK6 a and GhVQ28 with GhMAPK20,while the deletion of the N-terminus and C-terminus affected the interactions between them.The above results were further verified by Bi FC.(4)Subcellular localization analysis revealed that GhVQ1 and GhVQ28 were both localized in the nucleus.Expression pattern analysis showed that both GhVQ1 and GhVQ28 were expressed in cotton roots at a high level.Moreover,the expression of GhVQ1 and GhVQ28 was affected by Fusarium oxysporum,methyl jasmonate and salicylic acid to varying degrees.(5)GhVQ1-silenced and GhVQ28-silenced cotton plants were obtained through virusinduced gene silencing technique.After treatment with the Fusarium oxysporum,compared with the control,gene-silenced cotton leaves accumulated less hydrogen peroxide and caused less damage.Therefore,GhVQ1-silenced and GhVQ28-silenced cotton plants enhanced the resistance to Fusarium oxysporum.GhVQ1 or GhVQ28 transgenic Arabidopsis thaliana plants was obtained using floral-dip method.After treatment with Fusarium oxysporum,contrasted with wild-type,the area of diseased spots on the leaves of the transgenic plants was larger and the disease severity was more serious.Thus,overexpression of GhVQ1 or GhVQ28 enhanced the susceptibility to Fusarium oxysporum in the transgenic Arabidopsis thaliana plants.(6)In yeast two-hybrid experiments,it was found that in addition to GhMAPK20,GhVQ28 also interacted with GhMKK4,but not with GhWRKY40.VQ-motif mutation of GhVQ28 did not affect its interaction with GhMKK4,while deletion of N-terminus or Cterminus affected their interaction.Previous studies have shown that the GhMKK4-GhMAPK20-GhWRKY40 pathway was of great significance for the resistance of cotton to Fusarium oxysporum.Therefore,GhVQ28 may affect the GhMKK4-GhMAPK20-GhWRKY40 pathway as a scaffold protein.(7)The GhVQ1 interaction protein GhWRKY33 a was obtained through yeast two-hybrid library screening.The mutation of VQ-motif affected its interaction with GhWRKY33 a,while the deletion of N-terminus or C-terminus did not affect the interaction of them,which showed that VQ-motif was extremely important for their interaction.The above results indicated that there may be a GhMAPK6a-GhVQ1-GhWRKY33 a cascade pathway in cotton,which regulated the resistance of cotton to Fusarium oxysporum.In addition,the yeast two-hybrid library screening also indicated that GhVQ1 may function by forming a homodimer.