Functional Analysis Of Cotton Chitin Associated Genes And Verticillium Dahliae VdRGS1

Cotton?Gossypium spp.?,the most important source of natural textile fibers around the world,was severely damaged by Verticillium dahliae.Once the cotton plant was infected with the Verticillium wilt,resulted in severe yield and quality losses.In china,the average economic losses of the cotton output has reached several billion yuan.More seriously,Verticillium wilt has got worse and spread widely because of the transgenic pollution and environmental problems.So,how to prevent and control of cotton Verticillium wilt became more urgent.However,to date,the resistance mechanism to V dahilae in cotton is still poorly understood.Hence,excavated the key resistance genes and studied the mechanism in the cotton,and explored the functional characteristics of pathogenic genes in V dahliae,which better provided the theoretical basis for the study of the interaction mechanism between cotton and V dahliae,but applied important candidate genes for cotton germplasm innovation in resistant varieties.In this study,the resistant and susceptible cotton cultivars,Hai 7124 and Junmian 1,and the defoliating type strain Vd8 and V991,were mainly used to the functional analyse.We firstly excavated the resistant-related genes in the cotton and V dahliae,and further analyzed the functional mechanism and applied in the cotton production.The main results were as follows:1.Plant innate immunity relies on the pattern recognition receptors?PRRs?at the plant cell surface to successfully detect the microbe-associated molecular patterns?MAMPs?of invading microbes.Lysin motif?LysM?-containing proteins are unique to plants and serve important functions in MAMPs perception to participate in defense against pathogens attack.Verticillium dahliae is a destructive and soil-borne fungal pathogen in cotton.However,to date,the cotton LysM genes have not been systematically analyzed and effectively utilized in V dahliae resistance.Here,we identified 29,30,60,and 56 LysM genes in the sequenced cotton species,diploid Gossypium raimondii?D5?,G arboreum?A2?,tetraploid G.hirsutum acc.TM-1?AD1?,and G barbadense acc.3-79?AD2?,respectively.Based on the characteristics of the conserved domains and the subcellular localization predictions,the LysM gene family was divided into four categories,including the Lyks,Lyps,LysMes and LysMns.As the G.raimondii chromosomes had been integrated with the D=subgenome in G.hirsutum,and hence,on the basis of the reordered G.raimondii chromosomes,the LysM genes in G raimondii were named preferentially from GrLypl to GrLyp4,GrLykl to GrLyk8,GrLysMel to GrLysMe9,and GrLysMnl to GrLysMn8 following their chromosome orders.The expression patterns of LysM genes suggested their various expressions in different organs and tissues,chitin and chitin fragment N-acetylchitohexaose treatments,and in the V dahliae response.Intriguingly,the Lypl,Lyk7,and LysMe3,which had higher expression levels and were significantly induced after chitin,chitin fragment N-acetylchitohexaose,and V dahliae treatments.We further characterized the Lypl,Lyk7,and LysMe3,which were plasma membrane localization,suggesting these genes might play important roles in the chitin recognition and V dahliae defense.Silencing of Lyp1,Lyk7,and LysMe3 expression drastically impaired the salicylic acid,jasmonic acid,and reactive oxygen species generation,and defense gene activation,led to compromised resistance against cotton plants to V.dahliae.Collectively,our results indicated that Lyp1,Lyk7,and LysMe3 were important PRRs to recognize chitin signal and activate downstream defense processes to induce cotton defense against V dahliae.2.Chitinases,the typically resistance genes,which are found in a variety of organisms,catalyze the hydrolysis of the ?-1-4-linkage in the N-acetyl-D-glucosamine polymer of chitin a major component of fungal cell walls and arthropod exoskeletons.They play a major role in defense by directly attacking invading fungal pathogens.Here,we identified 47,49,92,and 116 Chis from four sequenced cotton species,diploid Gossypium raimondii?D5?,G arboreum?A2?,tetraploid G hirsutum acc.TM-1?AD1?,and G barbadense acc.3-79?AD2?,respectively.The orthologous genes were not one-to-one correspondence in the diploid and tetraploid cotton species,implying changes in the number of Chis during the evolution of Gossypium.The nomenclature of Chis in Gossypium followed their chromosome orders.As the G.raimondii chromosomes had been integrated with the D-subgenome in G.hirsutum,the Chis in G.raimondii were named preferentially from GrChil to GrChi47,based on the reordered G raimondii chromosomes.Phylogenetic classification indicated that these Chis could be classified into six groups,named as A to F,with distinguishable structural characteristics.The transcriptome data from G hirsutum acc.TM-1 vegetative tissues?root,stem,and leaf?,floral tissues?petal and anther?,ovule tissues?-3,0,and 3 DPA?,and fiber tissues?10,20,and 25 DPA?at different developmental stages were used to investigate the gene expression patterns.With FPKM>1.0,a total of 59 GhChis were found to be expressed,and showed diverse developmental and spatial regulation in the various tissues.The chitinase genes share diverse but overlapping expression patterns in various tissues and organs,suggesting that chitinase genes have conserved structures but diverse functions.To confirm the induction of chitinase genes after V.dahliae inoculation,the expression patterns of 23 chitinase genes with detectable expression levels in root tissues following transcriptome analysis were investigated.Of them,eight genes including Chi2,Chil4,Chi17,Chi23,Chi25,Chi28,Chi32 and Chi47 showed high expression levels at various treatment time points,showing that they were significantly induced by V.dahliae,and quickly reached a peak at different time points.Therefore,the eight genes might be responsible for V.dahliae resistance in cotton.To further investigate the function of Chis in V.dahliae resistance,we selected three candidate genes,Chi23,Chi32,and Chi47,with different expression levels that were significantly induced after V.dahliae inoculation,for functional identification through VIGS analysis.In a result,silencing of Chi23,Chi32,or Chi47 in cotton significantly impaired the resistance to V dahliae,suggesting these three genes might act as positive regulators in disease resistance to V dahliae.Of them,about 95%of the Chi32-and Chi47-silenced plants showed leaves wilting or exfliation that were similar to the Junmian 1 susceptible control plants,of which 100%were diseased 35 days after inoculation,suggesting Chi32 and Chi47 are important genes in resistance to V dahliae infection in cotton,and have potential uses in cotton disease-resistant breeding.3.Heterotrimeric guanine-nucleotide binding protein?G-protein?signaling,one of the most important signaling network,by which eukaryotic cells sense extracellular signals and integrate them into intrinsic signal transduction pathways.In fungi,G-proteins are mainly involved in the regulation of a variety of cellular functions in pathogenic development and vegetative growth,such as infection structure differentiation,pathogenicity,and conidiation.RGS proteins,the regulator of G protein signaling,which primarily acted as GTPase accelerators that promoted GTP hydrolysis by the Ga subunits,thereby inactivating the G protein and rapidly switching off G protein-coupled signaling pathways.Here,the 8 RGS genes were confirmed and named as VdRGSl?VDAG₀0683?,VdRGS2?VDAG₁0098?,VdRGSS?VDAG₀0979?,VdRGS4?VDAG₀2225?,VdRGS5?VDAG₀9977?,VdRGS6?VDAG₀2523?,VdRGS7?VDAG₀8194?,and VdRGS8?VDAG₀7045?.To further ascertain the possible functions of the RGS genes in V dahliae,we analyzed the expression patterns of the 8 RGS genes in the cotton seedlings induction in the different stages.All eight RGS genes showed increased expression levels except the VdRGS4 and VdRGSS;interestingly,only the increase in VdRGS1 was significant?14-fold at 12 hours?h?,20-fold at 24h?,in comparison to the VdRGS2?3-fold at 12h?,VdRGS3?5-fold at 12h?,VdRGS6?2.6-fold at 12h?,VdRGS7?2.7-fold at 24h?,and VdRGS8?2.4-fold at 24h?.These results indicate that VdRGS1 functions as virulence of V dahliae.To better ascertain the function of VdRGSl in V dahliae,a targeted gene replacement strategy basing on homologous recombination was used to generate VdRGS1 deletion mutants.We replaced the ORF?Open Reading Frame?of this gene with a selective maker gene?the bacterial phosphotransferase gene,HPH?using transformation of spores of the wild-type V.dahliae strain Vd8 via A.tumefaciens mediated transformation?ATMT?.In a result,compared the phenotypes of the wild-type V dahliae strain Vd8,VdRGS1 deletion mutants?? VdRGS1?,and VdRGSl complemented mutants?VdRGSl-com?,VdRGSl showed that clearly affected the microsclerotia and mycelia development,and spore production.Also,VdRGS1 deletion mutants lost the virulence,strongly suggesting that VdRGS1 was essential for the pathogenicity of V dahliae.These results indicated that,the VdRGS1 played crucial roles in the spore production,microsclerotia development and virulence of V dahliae.TRV-based virus-induced gene silencing?VIGS?in the G.hirsutum cv.Junmian 1 compromised V dahliae VdRGS1 expression,and effectively impaired the Verticillium wilt susceptibility.