Genome-wide Identification Of Papain-like Cysteine Proteases In Gossypium Hirsutum And Functional Characterization Of GhRD21-7 In Response To Verticillium Dahliae

Verticillium wilt(VW)is the main disease affecting the quality and yield of cotton.Therefore,it is very necessary to study the key resistance genes against cotton VW and to elucidate its molecular mechanism.Papain-like cysteine proteases(PLCPs),which belong to the family C1 A of clan CA,play crucial roles in plant defense against pathogens.In this study,the biology information of PLCPs was analyzed by using the TM-1 genomic data of upland cotton.The overexpression and RNAi vector of GhRD21-7 were constructed,and used for generating transgenic cotton lines.The transgenic lines were used for the subsequent functional analysis.The main findings obtained are as follows:1.Using the TM-1(The allotetraploid cotton G.hirsutum L.acc.Texas Marker-1)genomic data,we identified 78 PLCPs in upland.The phylogenetic tree was constructed using upland cotton and Arabidopsis PLCPs.According to the clustering results,all members were divided into 9 subfamilies,which were named subfamily RD21,CEP,XCP,XBCP3,THI,SAG12,RD19,ALP and CTB,respectively.Genes in each subfamily exhibit a similar structure and motif composition,indicating the evolutionary similarity of members of the same family.2.Tissue expression profiles showed that PLCP genes were predominantly expressed in petal and anther,followed by 10 days fiber,cotyledons germinating after 24 hours,stems,seeds after 10 days of flowering,relatively low expressed in stigma,ovary and root.The expression patterns were investigated in response to heat,cold,salt and polyethylene glycol(PEG).Thirty-five PLCP genes were induced by multiple stresses.There were 21,24,10 and 8 genes significantly changed after treatment with heat,cold,salt and PEG respectively.The expression of twenty-nine PLCP genes were significantly changed after V.dahliae infection.Further analysis showed that sixteen PLCP genes were up-regulated in response to V.dahliae.Among those,GhRD21-7 was down-regulated at 6 h and up-regulated from 12 h to 24 h.GhRD21-7 also showed a higher transcription level than most other PLCP genes.3.We cloned GhRD21-7,which encodes a protein of 457 amino acids.GhRD21-7 was abundantly expressed in anther,subsequently in leaf,root,stem,and petal.In order to study the function of GhRD21-7 in cotton,we constructed the overexpression and interference vector of GhRD21-7,and obtained the overexpressing lines(OE154,OE173)and the RNAi lines(Ri294,Ri381).Infection assay showed that over-expression of GhRD21-7 led to enhanced resistance and RNAi lines were more susceptible to V.dahliae in cotton.4.Totally,1522 differentially expressed genes were identified in overexpressing line OE154 compared with the wild type,and 804 DEGs showed up-regulated expression patterns and 718 genes showed down-regulated.After inoculation with V.dahliae,more differentially expressed genes were identified in overexpressing line OE154 than in wild type.There were 2172 DEGs were identified,and 1387 DEGs showed up-regulated expression patterns and 785 genes were down-regulated.We found that the phenylpropanoid metabolic pathway was enriched in differentially expressed genes.4-coumarate:CoA ligase(4CL),caffeoyl-CoAO-methyltransferase(CCoAOMT),cinnamyl alcohol dehydrogenase(CAD),and cinnamate 4-hydroxylase(C4H),which were key enzymes in the lignin synthesis pathway,and 4CL and C4 H are key enzymes in the core of the phenylpropanoid metabolic pathway,CCoAOMT and CAD are key enzymes in the lignin G-type and S-type monomer synthesis pathways.Before and after induced by V.dahliae,the expression levels of 4CL and C4 H were up-regulated in overexpression line,indicating that GhRD21-7 medicated cotton resistance to V.dahliae was associated with activation of lignin synthesis pathway.The study provided a wealth of information on members of the cotton Papain-like cysteine proteases gene family and provided a reference for subsequent research in this family member.At the same time,the over-expression and inhibition of GhRD21-7 expression lines were used to identify the resistance of cotton to V.dahliae and over-expression of GhRD21-7 led to enhanced resistance and RNAi lines were more susceptible to V.dahliae in cotton.The transcriptome data indicating that GhRD21-7 mediated cotton resistance to V.dahliae was associated with activation of lignin synthesis pathway.