Functional Analysis Of Two NAC Transcription Factor Genes In Regulation Of Defense Responses In Arabidopsis Thaliana

Activation of defense responses against pathogen infection is usually regulatedthrough modulation of a large set of defense genes at transcriptional level by specifictranscription factors.A large body of evidence has implicated that several groups oftranscription factors have functions directly or indirectly in regulating plant defenseresponses to pathogen attack.Recently,transcription factors of NAC family have beenreported to be involved in various biological processes of plant growth anddevelopment and in responses to hormones and abiotic stresses.In this study,Iidentified two defense-related NAC genes in Arabidopsis thaliana.One is a newlyidentified NAC gene DRN1(defense-related NAC1) and another one is the previouslyreported stress-responsive ATAF1.I studied in detail by functional analysis of these twoNAC genes for their biological roles in plant defense responses against necrotrophicand biotrophic pathogens.Analyses of public microarray data revealed that expression of DRN1 gene wasup-regulated after infection with Botrytis incerea,Pseudomonas syringae pv.tomato(Pst) DC3000 and by treatment with salicylic acid(SA),flg22,suggesting that DRN1may be involved in regulation of defense response in Arabidopsis.The DRN1 geneencodes a 275 amino acid protein containing a highly conserved NAC domain.RT-PCRanalysis showed that expression of DRN1 was induced in Arabidopsis plants afterinoculation with Pst DC3000 or B.cinerea and that the expression level wassignificantly up-regulated during 24～48 hr after inoculation.Likely,the expression ofDRN1 was also induced in Arabidopsis plants after treatment with some well-knowndefense-related chemical inducers such as salicylic acid(SA),benzothaidiazole(BTH),jasmonic acid(JA) and 1-aminocyclopropane-1-carboxylic acid(ACC,a precursor ofethylene biosynthesis).These results provide preliminary evidence that DRN1 is involved in Arabidopsis defense response.Subcellular localization analysis using GFP fusion protein showed that the DRN1protein is localized in the nucleus of cells.Transactivation analysis demonstrated thatDRN1 can activate.reporter gene expression in yeast,indicating that DRN1 is atranscriptional activator.Furthermore,deletion analysis of transactivation revealed thattransactivation activity of the DRN1 protein is localized in a region of 168-275 aminoacids at its C-terminal domain,and that the N-terminal region containing the conservedNAC domain does not have transactivation activity.Therefore,DRN1 is atranscriptional activator and its C-terminal region contributes to the transactivationactivity.To study the function of DRN1 in disease resistance response,a T-DNA insertion linedrn1-1 was identified from genotyping screening.RT-PCR analysis indicated thattranscript of the DRN1 gene was undetectable in drn1-1 plants,suggesting that drn1-1is a knockout mutant of the DRN1 gene.However,the phenotypes of the drn1-1 plantsafter inoculation with Pst DC3000 or B.cinerea showed slight but not significanthigher levels of diseases than those in wild-type plants.Considering redundancy ofgene function in NAC family,I constructed a chimeric repressor vector35S::DRN1-SRDX and an overexpression vector 35S::DRN1,and introduced them intowild-type plants using floral dip transformation protocol.Seven 35S::DRN1 and six35S::DRN1-SRDX transgenic lines with single copy of the transgene were obtained.Homozygous lines were identified from the 3rd generation through screening and threehomozygous lines for 35S::DRN1(DRN1-OE#2,#6 and #11) and 35S::DRN1-SRDX(DRN1-SRDX#1,#3 and #8) were chosen for further work.Disease phenotypes of thewild-type,drn1-1,DRN1-OE and DRN1-SRDX plants were compared afterinoculation with B.cinerea,Pst DC3000 or Altenaria brassicicola.As compared withthe wild-type plants,the drn1-1 plants showed a slight but not significant increasedsusceptibility to B.cinerea,Pst DC3000 or A.brassicicola.However,the transgenicDRN1-SRDX#1,#3 and #8 plants showed significant increased susceptibility to theabove-mentioned pathogens;while the DRN1-OE#2,#6 and #11 plants showed asignificant reduced susceptibility to the patogens.Furthermore,growth of B.cinerea and Pst DC3000 in leaves of the DRN1-SRDX#1,#3 and #8 plants was much greaterthan in the wild-type plants;whereas growth of B.cinerea and Pst DC3000 in leaves ofthe DRN1-OE#2,#6 and #11 plants was significantly reduced.After inoculation with B.cinerea,expression of PR-1 and PR-5 genes was markedly up-regulated in theDRN1-SRDX#1 and #8 plants,and expression of PDF1.2 and BIK1 genes wasup-regulated in the DRN 1-OE#2 and # 11 plants.By contrast,after inoculation with PstDC3000,the expression of PDF1.2 was up-regulated in the DRN1-SRDX#1 and #8plants,and the expression of PR-1,PR-5 and NPR1 genes was up-regulated in theDRN1-OE#2 and #11 plants.Moreover,DRN1-overexpressing transgenic plantsshowed an enhanced ABA sensitivity.These results demonstrate that DRN1 is apositive regulator of defense responses in Arabidopsis against both necrotrophic andbiotrophic pathogens through activation of defense gene expression.ATAF1 is one of the first identified NAC genes in Arabidopsis thaliana and waspreviously reported to be involved in abiotic stress response.Because ATAF2,a NACgene that is closely related to ATAF1,plays an important role in defense responseagainst vascular Fusarium wilt pathogen,I therefore studied the possible involvementof ATAF1 in Arabidopsis defense responses against necrotrophic and biotrophicpathogens.The ATAF1 gene encodes a 289 amino acid protein containing a highlyconserved NAC domain.RT-PCR analysis showed that expression of the ATAF1 genewas suppressed in Arabidopsis plants after inoculation with Pst DC3000 and B.cinerea,or treatment with several well-known defense-related chemical inducers including SA,BTH,ACC and JA.To study the function of ATAF1 in defense response,I constructeda chimeric repressor vector 35S::ATAF1-SRDX and an overexpression vector35S::ATAF1 and introduced them into Arabidopsis through floral dip transformationprotocol.Two independent transgenic lines with a single copy of the transgene wereidentified for 35S::ATAF1(ATAF1-OE#1 and #7) and 35S::ATAF1-SRDX(ATAF1-SRDX#3 and #10),respectively,and chosen for further studies.Diseasephenotypes of the wild-type,atafl(a T-DNA insertion mutant ofATAF1),ATAF1-OEand ATAF1-SRDX plants were compared after inoculation with B.cinerea,Pst DC3000and A.brassicicola.After inoculation,the atafl plants showed a slight but not significant reduced disease level than the wild-type plants.The disease levels in theATAF1-SRDX#3 and #10 plants were significantly reduced,whereas the disease levelsin the ATAF1-OE#1 and #7 plants were markedly increased.Furthermore,whencompared with those in the wild-type plants,growth of B.cinerea and Pst DC3000 inleaves of the ATAF1-SRDX#3 and #10 plants was significantly decreased,whilegrowth of the pathogens in leaves of the ATAF1-OE#1 and #7 plants was significantlyincreased.After inoculation with B.cinerea,expression of PR-1,PR-5 and NPR1 wasup-regulated in the ATAF1-SRDX#3 and #10 plants,and the expression of BIK1 wasup-regulated in the ATAF1-OE#1 and #7 plants.However,expression of PR-1,PR-5and NPR1 was up-regulated in the ATAF1-SRDX#3 and #10 plants,and the expressionof PDF1.2 was up-regulated in the ATAF1-OE#1 and #7 plants after inoculation withPst DC3000.Moreover,significant accumulation of reactive oxygen species(H₂O₂ andsuperoxide anion) in leaves of the ATAF1-OE#1 and #7 plants,but not in leaves ofATAF1-SRDX#3 and #10 plants after Botrytis infection.These results clearlydemonstrate that ATAF1 is a negative regulator of defense responses in Arabidopsisagainst necrotrophic and biotrophic pathogens.