Function And Molecular Mechanism Of GhCaM7 In Regulating Cotton Resistance To Verticillium Wilt

Object:Cotton（Gossypium hirsutum L.）is an important cash crop in the world and the main source of natural fiber in textile industry.Verticillium wilt is a major disease in cotton production.It occurs widely in cotton producing countries all over the world and seriously affects the yield and quality of cotton.Verticillium dahliae is the pathogenic bacterium causing Cotton Verticillium Wilt.It is a soil borne and semi living filamentous fungus.Due to its dormant form,micro sclerotia can survive in the soil for decades,It is still difficult to prevent and control Cotton Verticillium Wilt.Breeding and planting disease resistant varieties is the most economical and effective way to control Cotton Verticillium Wilt.However,the traditional breeding cycle is long and the pathogen variation is frequent,which makes the selected disease resistant varieties lose resistance quickly.Therefore,mining the key genes of Cotton Verticillium Wilt resistance and analyzing its disease resistance mechanism is the premise of using biological breeding technology to understand the harm of Cotton Verticillium Wilt.In the early stage of this study,using the real-time acetylation proteomic analysis of the infection of resistant variety Zhongzhimian 2 and susceptible variety Ji 11,it was found that there were significant differences in calmodulin GhCaM7（Gh＿A09G122000）between resistant and susceptible strains,suggesting that this protein may be involved in regulating the resistance of cotton to Verticillium wilt.This study intends to study the role of calmodulin GhCaM7 in cotton disease resistance and analyze its molecular mechanism of regulating Verticillium wilt resistance,in order to provide effective target genes and theoretical support for improving Cotton Verticillium wilt resistance through genetic engineering.Methods:（1）Virus induced gene silencing（VIGS）,real-time fluorescence quantitative（qRT-PCR）and physiological and biochemical methods were used to reveal the role of GhCaM7 in cotton resistance to Verticillium wilt.（2）GhCaM7 overexpression and gene editing plants were obtained by genetic transformation with upland cotton"HM-1"as receptor material.At the same time,GhCaM7 and its acetylation modification site mutant GhCaM7^K95R transgenic Arabidopsis were obtained by flower dipping method,and the disease resistance differences between the transgenic lines and the control were identified.（3）Yeast two hybrid（Y2H）,bimolecular fluorescence complementation（BiFC）and luciferase complementation（LCI）techniques verified the protein interacting with GhCaM7 and analyzed its protein regulatory network in cotton resistance to Verticillium wilt.（4）The subcellular localization vector of GhCaM7,GhCaM7 interaction protein and its acetylation modification site mutant GhCaM7^K95R was constructed,and its position in tobacco cells was observed by laser confocal microscope.（5）Through transcriptome sequencing（RNA-seq）technology,the differentially expressed genes of GhCaM7overexpression（CAM7）and gene editing（cam7）cotton inoculated with Verticillium dahliae at different time points were analyzed by GO,KEGG and WGCNA,and the regulatory network of cotton resistance to Verticillium wilt mediated by GhCaM7 gene was analyzed.Results:（1）qRT-PCR analysis showed that GhCaM7 was up-regulated after being induced by Verticillium dahliae,JA and H₂O₂.After GhCaM7 gene silencing and knockout,the resistance of cotton to Verticillium dahliae was weakened,and the contents of JA and ROS decreased significantly compared with the control.Overexpression of GhCaM7 could enhance the resistance of cotton to Verticillium dahliae,and the contents of JA and ROS were higher than those of the control.The above results suggest that GhCaM7 may regulate the disease resistance of cotton to Verticillium dahliae through JA and ROS signaling pathways.Heterologous overexpression of GhCaM7 in Arabidopsis also enhanced the disease resistance of plants to Verticillium dahliae.After the mutation of GhCaM7 acetylation modification site,the disease resistance of GhCaM7^K95R transgenic Arabidopsis was weaker than that of GhCaM7.In conclusion,the function of GhCaM7 in cotton and Arabidopsis is conservative in resistance to Verticillium wilt,and the acetylation modification site is very important for GhCaM7 to play a disease resistance function.（2）In order to further analyze the regulatory mechanism of GhCaM7 involved in cotton Verticillium wilt resistance,GhCaM7 was used as bait to screen the GhCaM7 interaction protein osmotic protein GhOSM34 from the cotton root c DNA library induced by Verticillium dahliae.The interaction between GhCaM7 and GhOSM34 was further verified by Y2H,BiFC and LCI techniques.BiFC analysis showed that and GhOSM34 interacted on the cell membrane.The results of Y2H and LCI analysis showed that the interaction between GhCaM7^K95R and GhOSM34 was weaker than that between GhCaM7 and GhOSM34.After silencing GhOSM34 by VIGS technology,the sensitivity of cotton to Verticillium dahliae was enhanced.Subcellular localization results showed that GhCaM7 was located in the nucleus and cell membrane,and GhCaM7 acetylation modification site mutants GhCaM7^K95R and GhOSM34 were located in the cell membrane.These results suggest that acetylation modification site play an important role in regulating the interaction between GhCaM7 and GhOSM34 and the subcellular localization of GhCaM7.（3）Go,KEGG and WGCNA analysis of the transcriptome differentially expressed genes of GhCaM7overexpression（CAM7）and gene editing mutant（cam7）plants at different time points showed that GhCaM7 was involved in cotton resistance to Verticillium wilt through JA,MAPK,Ca²⁺and ROS signaling pathways.Conclusion:（1）Calmodulin-encoding gene,GhCaM7 is a positive regulator of cotton and Arabidopsis in response to the infection of Verticillium dahliae.The mutation of GhCaM7 acetylation modification site reduced its disease resistance function in Arabidopsis（2）GhCaM7 can interact with the osmotic protein GhOSM34.The interaction between GhCaM7 and GhOSM34 was weakened by the mutation of the acetylation modification site of GhCaM7.（3）After sensing the pathogen infection,GhCaM7 binds to Ca²⁺as a secondary messenger to enhance the defense response of cotton to Verticillium wilt by activating JA,ROS and MAPK pathways and changing cell osmotic potential.