Genome-Wide Analysis Of The Dirigent Gene Family And Functional Characterization Of CaDIR7 And CaDIR23 In Pepper (Capsicum Annuum L.) Defense Against Phytophthora Capsici And Abiotic Stresses

Pepper（Capsicum annuum L.）is an important solanaceous vegetable worldwide,that is liked by most of the people due to its major cuisines ingredient,essential vitamins and other healthy nutrients.There are many constraints in the cultivation and production of pepper,including both biotic（diseases and insects）and abiotic,but the most serious and dangerous threat is phytophthora blight caused by Phytophthora capsici.Many chemicals are used for the control of this blight,but it might cause pesticides residual in the production of pepper,as well as the pathogen is getting resistant to the chemicals.Thus,to develop the resistance of pepper plants to P.capsici through molecular techniques and genetic engineering is a crucial and realistic approach.Dirigent（DIR）is an important disease resistance responsive gene（DRRG）that plays a potential role in enhancing stress resistance in different crop plants and mediate the free radical coupling of monolignol plant phenols in plants to yield lignans and lignins,therefore believed to be involved in plant disease-resistance response.However,no study has been conducted on the function of DIR gene family in pepper until now.Therefore,the DIR family genes members were mined from pepper transcriptome database through bioinformatics.As a result,the discovered DIR family members（CaDIRs）were studied in the subsequent study,their expression in response to virulent and avirulent strains of P.capsici.The CaDIRs were further divided into 8 different groups on basis of their expression to P.capsici,and a representative CaDIR from each group was subjected to abiotic（Na Cl and mannitol）and hormonal（Salicylic acid and Methyl jasmonate）stresses.The CaDIR7and CaDIR23showed the differential expression in response to P.capsici strains as well abiotic and hormonal stresses,were further studied and analyzed through virus induced gene silencing（VIGS）.Main findings of the current study are given below:1.The DIR gene family was analyzed through bioinformatics.A total of 24 CaDIRs from the pepper genome were identified through a bioinformatics analysis and PCR testing and assigned names based on their chromosomal position and order.Five well-conserved characterized motifs were found in the amino acid sequences of 24 CaDIRs.The phylogenetic analysis showed that 24 CaDIRs were clearly separated into three distinct groups,DIR-a,DIR-b/d and DIR-e.In exon-intron analysis,only the members of DIR-e subfamily contain one intron each.The CaDIRs were distributed across all 12 chromosomes of pepper,and the paralogous analysis displayed that there were 10 pairs of segmental duplication and two pairs of tandem duplication in the pepper genome.Furthermore,a number of defense-related,stress stimulative and plant hormones responsive elements were found in the promoter regions of the CaDIRs.2.The tissue expressions of CaDIRs were analyzed,and the results showed that the expression patterns of CaDIRs in different tissues were different.The analysis showed that out of 24 CaDIRs,two genes（CaDIR2 and CaDIR5）were not expressed in all of the tested organs.Among all CaDIRs,CaDIR23 was expressed at the highest level in the stem（71.55）and green fruit（66.48）.Collectively,CaDIRs showed the highest expression in flowers,followed by the stem,leaf,green fruit and red fruit,whereas they were expressed at the lowest levels in roots.3.The expression analysis of the CaDIRs were also investigated under P.capsici,Na Cl,Mannitol,SA and Me JA treatments.The results revealed that some CaDIRs were up-regulated by virulent（CaDIR2,CaDIR3,CaDIR6,CaDIR7,CaDIR11,CaDIR14,CaDIR16,CaDIR22 and CaDIR23）and avirulent（CaDIR3,CaDIR5,CaDIR7,CaDIR16,CaDIR20,CaDIR22,CaDIR23 and CaDIR24）strains of P.capsici.The 8 candidate CaDIRs showed differential expression to Na Cl,Mannitol,SA and Me JA.Among the eight CaDIRs,CaDIR7,CaDIR10,CaDIR12 and CaDIR23 showed up-regulation after salt and mannitol stress,while the CaDIR7 and CaDIR23 were gradually up-regulated and reached to peak at 48 hours post treatment（hpt）.In response to hormonal treatments,CaDIR4,CaDIR10 and CaDIR14 showed no significant response,CaDIR7 was only up-regulated by Me JA,while CaDIR13 and CaDIR22 were up-regulated by SA.CaDIR23 exhibited a progressive increment in the expression levels and reached to maximum（4.56 and 3.94）at 48 hpt.In response to avirulent strain inoculation,the acid soluble lignin content was increased by 14.46%,36.09%,25.67%,90.40%and 43.93%,while in case of virulent strain inoculation,the increase was 21.68%,50.57%,33.07%,116.02%and 68.10%as compared to control plants at 6,12,24,48 and 72hours post inoculation（hpi）,respectively.4.The CaDIR7 showed the differential expression in response to P.capsici inoculation,was further investigated through VIGS.After silencing no obvious difference was observed in the phenotypes of the control（TRV2:00）and CaDIR7-silenced（TRV2:CaDIR7）plants.Silencing efficiency measurement revealed that the transcription of CaDIR7 was much lower（>60%）in the silence plants as compared to control.Silencing of CaDIR7 weakened plant defense by reducing（～50%）root activity and made plants more susceptible（35.7%）to P.capsici.This signifies that CaDIR7 is involved in defense response of pepper against P.capsici.Silencing of CaDIR7 also weakened pepper plants defense against salt stress.After exposure to salt（300 m M）stress,the expression of defense related genes（Ca DEF1 and Ca PO1）and root activity of the silenced plants was significantly lower as compared to control.Relative electrolyte leakage（REL）of the silenced plants was also higher than the control.Besides,leaf discs of the silenced and control plants exposed to Na Cl and mannitol（0,100 and 300 m M each）exhibited loss of chlorophyll,but this loss of chlorophyll was significantly higher in the silenced plant leaf discs.This indicates that CaDIR7 may positively regulate the defense response of pepper against salt and drought stress.5.The expression profile of CaDIR23 was investigated in response to signaling molecules and abiotic stresses.The results revealed that CaDIR23 was strongly induced by ABA and H₂O₂,reached to maximum at 48 hpt（4.15 folds）and 12 hpt（3.79）respectively.In response to heat stress,it was induced at 6 hpt,and peaked at 12 hpt（4.39 fold）post stress,followed by down-regulation.While cold stress resulted initially down-regulation,then abrupt up-regulation at 6 hpt（4.40 fold）,and again down-regulated.Silencing of CaDIR23 weakened pepper plants defense response against P.capsici by reducing the expression of defense related genes（Ca PR1 and Ca SAR82c）,root activity,lignin content,activities of enzymes related to lignin biosynthesis and more lesions on the detached leaves of the silenced plants as compared to control.Silencing of CaDIR23 attenuated the pepper defense response against mannitol（300m M）stress by reducing the expression of defense related genes（Ca PR1 and Ca SAR82c）,root activity,lignin content,and CaDIR23-silenced plants deprived of water for14 days showed no recovery.While responding Na Cl stress,the lower expression of defense related genes（Ca PR1 and Ca SAR82c）,root activity and more REL were observed in the silenced plants.Leaf discs of the CaDIR23-silenced plants exposed to Na Cl and mannitol（100 m M and 300 m M each）,exhibited a significant decrease in the chlorophyll content.These results suggested that CaDIR23 is involved in lignin biosynthesis and pepper defense response against pathogen and abiotic stresses.