Molecular Mechanism Of GhMKK9 Mediated MAPK Cascade Signaling Pathway In Regulating Resistance To Cotton Fusarium Wilt

Cotton is one of the most important fiber economic crops in our country,and it is also an important oil and high protein crop in the national economy.However,cotton yields and quality are frequently limited by many different diseases.Among them,cotton Fusarium wilt has caused huge losses to cotton production.Cotton Fusarium wilt is a soil-and seed-borne vascular fungal disease in cotton caused by Fusarium oxysporum f.sp.vasinfectum.Nevertheless,the mechanisms of the pathogenesis and the induction of wilt symptoms by the fungus remain largely unknown.Research on cloning and function of cotton resistant gene to Fusarium wilt is slow.To defend against potential pathogens,plants have evolved complicated signalling networks to translate extracellular stimuli into intracellular responses.Mitogen-activated protein kinase?MAPK?cascades play an important role in plant immunity in plants.At present,the establishment of the MAPK cascades signal pathway has become one of the hotspots in the study of plant disease-resistance.In plants,the typical MAPK pathway includes Mitogen-activated protein kinase kinase kinase?MAPKKK,MEKK?,Mitogen-activated protein kinase kinase?MAPKK,MKK,and MEK?and MAPK.MAPKKK receives signals from the receptor,and successively phosphorylates in the manner of MAPKKK-MAPKK-MAPK to amplify the signal and transmit signals to the downstream target molecule.As the intermediate component of the MAPK cascades,MKK integrate a variety of signals received by different MAPKKK,and play a signal hub in plant body signal transduction pathway.The study of MKK is the key to the study of MAPK cascade signal pathways in regulating plant growth,biotic and abiotic stresses and other biological processes.So far,reports on the biological functions and the molecular mechanism of MKK cascades in resistance to cotton Fusarium wilt disease are still very limited.Therefore,the cotton MKK function and action mechanism in cotton Fusarium wilt not only enrich the theoretical system of MAPK cascade signaling pathway,but also provide a basis for improving cotton resistance traits.In this study,a group D MKK gene?GhMKK9?was isolated from upland cotton,and its sequence characteristics,promoter activity,expression pattern,subcellular localization,and pathogen resistance of gene silencing cotton and overexpression of transgenic tobacco was preliminarily studied.For the first time,we found that GhMKK9 played an important role in the resistance to F.oxysporum,and we also studied the molecular mechanism of MAPK cascade signaling pathway in regulating resistance to cotton Fusarium wilt.The main results are as follows:?1?GhMKK9 was identificatied from upland cotton,and it encoded a protein including323 amino acid residues.Amino acid sequence analysis showed that the GhMKK9 protein exhibited 11 conserved subdomains,a plant MAPK cascade conserved S/TXXXXXS/T motif,conservative motif?GXGXXC motif?,an activation loop and a docking site.Phylogenetic analysis showed that Gh MKK9 belongs to the family of typical group D MKKs.Subcellular localization analysis showed that GhMKK9 localized in both the cytoplasm and the nucleus.?2?The PlantCARE database predicted various plant cis-acting regulatory elements including pathogen/elicitor-related elements?Box-W1?and plant hormone-responsive elements?ABRE,TCA-element,ERE?in GhMKK9 promoter region.By GUS histochemical staining,the activity of GhMKK9 promoter was analysed,the results showed that the promoter activity was enhanced after treatment with F.oxysporum,SA,MeJA and H₂O₂.qRT-PCR and western blot analysis showed that GhMKK9 transcript level was up-regulated by exposure to F.oxysporum,H₂O₂,SA,and MeJA.This work suggests that GhMKK9 may regulate F.oxysporum by modulating cellular ROS and SA/JA molecules.?3?GhMKK9-silenced cotton plants were obtained using the VIGS technique.After F.oxysporum inoculation,the TRV leaves appeared severe disease symptoms with bigger necrotic areas and the presence of brown lesions than GhMKK9-silenced cotton plants.Using DAB staining,less H₂O₂ accumulated in TRV-GhMKK9 cotton leaves than in TRV leaves.Trypan blue staining and electrolytic leakage showed the TRV leaves appeared more serious cell membrane damage.The above results show that GhMKK9-silenced cotton plants enhanced resistance to F.oxysporum.In addition,GhMKK9 overexpression transgenic N.benthamiana lines were gained using agrobacterium mediated leaf disc method.Pathogeny resistance analysis indicates that the overexpression of GhMKK9 enhances the susceptibility to F.oxysporum infection in the transgenic plants.After treatment with F.oxysporum,the transcript levels of pathogenesis-related?PR?genes?NPR1,PR1?,SA signal transduction related genes?ICS1,EDS1,PAD4,and NOA1?,JA signaling pathway related genes?JAZ1,AOC4,and AOS?,and HR-associated genes?HIN1,HSR203J?were affected in GhMKK9-silenced cotton plants and GhMKK9 overexpression transgenic N.benthamiana lines.We speculate that GhMKK9 regulat resistance against F.oxysporum in cotton may be related to the expression level of resistance related genes.?4?Further investigation shows that the expression levels of ROS-related genes?SOD,POD and CAT?were significantly higher in the TRV-GhMKK9 plants than in the TRV plants after F.oxysporum infection,and the activities of these antioxidant enzymes?CAT,POD and SOD?were apparently higher in the TRV-GhMKK9 plants than in the TRV plants.The results show that the silencing of GhMKK9 activates the antioxidant system.However,after treatment with F.oxysporum,the expression levels of CAT,POD,SOD,RbohA,and RbohB were lower in GhMKK9 overexpression plants than in WT plants,and the activities of CAT,POD,and SOD in OE plants were significantly lower than WT plants.The results showed that the overexpression of GhMKK9 inhibited the antioxidant capacity of transgenic plants.?5?By the yeast two hybrid screening test,we obtained a sequence of HT1-like protein,which belongs to the Raf subfamily of the MAPKKK family,named GhRaf19.GhRaf19contains 1125bp open reading frame?ORF?,which encoded a 374-amino acid protein.Sequence analysis showed that GhRaf19 contains a protein kinase active site?VIHRDLKSNNLLL?in its C-terminal kinase domain(L⁷⁷-Y³³⁴)and a conserved GTXX?W/Y?MAPE?L/V?motif.GhRaf19 and GhMKK9 subcellular localization is consistent,they both localized in the cytoplasm and the nucleus.Yeast two-hybrid assay and bimolecular fluorescence complementation?BiFC?assay demonstrated that GhRaf19 may interact with GhMKK9.It indicates that GhRaf19 may have similar functions with GhMKK9.qRT-PCR and western blot analysis showed that the expression characteristics of GhRaf19 and GhMKK9 are similar.The GhRaf19 transcript level was also enhanced by F.oxysporum,H₂O₂,MeJA and SA in varying degrees.?6?Using the same method,we obtained the cotton GhRaf19 silencing plants and GhRaf19 overexpression transgenic N.benthamiana lines.After F.oxysporum inoculation,the results of DAB staining,trypan blue staining and electron leakage test show that GhRaf19-silenced cotton plants enhanced resistance to F.oxysporum and overexpression of GhRaf19 enhanced the susceptibility to F.oxysporum in transgenic plants.After F.oxysporum inoculation,the transcript levels of PR genes,SA signal transduction related genes,JA signaling pathway related genes,and HR-associated genes were affected in GhRaf19-silenced cotton plants and GhRaf19 overexpression transgenic N.benthamiana lines.?7?After F.oxysporum inoculation,the expression levels of ROS-related genes were higher in the GhRaf19-silenced plants than in the TRV plants and the activities of these antioxidant enzymes were higher in the GhRaf19-silenced plants than in the TRV plants.The results showed that the silencing of GhRaf19 activates the antioxidant system.However,the expression levels of POD,SOD,CAT,RbohA,and RbohB and the activities of antioxidant enzymes were both lower in GhRaf19 overexpression plants than in WT plants.The results indicated that the overexpression of GhRaf19 inhibited the antioxidant capacity of transgenic plants.These results are similar to GhMKK9.