CaWRKY30 Zoo Tswj Cov Tshuaj Tiv Thaiv Kab Mob Los Ntawm Lub Hom Phiaj CaWRKY40 Tawm Tsam Ralstonia Solanacearum Inoculation Los Ntawm Modulating Defense-Related Genes Part 1

Abstract:

WRKY transcription factors (TFs) network yog tsim los ntawm WRKY TFs 'subset, uas ua lub luag haujlwm tseem ceeb hauv kev tiv thaiv kev tiv thaiv ntawm cov nroj tsuag. Txawm li cas los xij, kev ua haujlwm ntawm WRKY TFs 'network tseem tsis meej, tshwj xeeb tshaj yog nyob rau hauv cov nroj tsuag uas tsis yog qauv xws li kua txob (Capsicum annum L.). Txoj kev tshawb no ua haujlwm zoo li CaWRKY30-tus tswv cuab ntawm pawg III Pepper WRKY protein-rau kev tiv thaiv ntawm kua txob tiv thaiv kab mob Ralstonia solanacearum. Lub CaWRKY30 tau kuaj pom nyob rau hauv lub nucleus, thiab nws cov qib transcriptional qhia tau nce siab los ntawm R. solanacearum inoculation (RSI), foliar application ethylene (ET), abscisic acid (ABA), thiab salicylic acid (SA). Virus-induced gene silencing (VIGS) ntawm CaWRKY30 amplified lub kua txob qhov tsis zoo rau RSI.

Tsis tas li ntawd, qhov ntsiag to ntawm CaWRKY30 los ntawm VIGS cuam tshuam HR-zoo li cell tuag tshwm sim los ntawm RSI thiab txo qis kev tiv thaiv cov cim cim npe, xws li CaPR1, CaNPR1, CaDEF1, CaABR1, CaHIR1, thiab CaWRKY40. Conversely, ib ntus tshaj tawm ntawm CaWRKY30 nyob rau hauv kua txob tawm instigated HR-zoo li cell tuag thiab upregulated tiv thaiv kab mob ntsig txog cov noob caj noob ces. Tsis tas li ntawd, ib ntus tshaj tawm ntawm CaWRKY30 upregulated transcriptional theem ntawm CaWRKY6, CaWRKY22, CaWRKY27, thiab CaWRKY40.

Ntawm qhov tod tes, kev nthuav qhia dhau los ntawm CaWRKY6, CaWRKY22, CaWRKY27, thiab CaWRKY40 upregulated transcriptional expression theem ntawm CaWRKY30. Cov txiaj ntsig tau pom zoo tias tus yam ntxwv tshiab CaWRKY30 zoo tswj cov kua txob tiv thaiv kab mob Ralstonia nres, uas yog tswj hwm los ntawm kev sib koom ua ke ntawm cov teeb liab los ntawm phytohormones xws li ET, ABA, thiab SA, thiab transcriptionally assimilating rau hauv WRKY TFs tes hauj lwm, muaj CaWRWRKY6, CaWRKY2, 40 . Ua ke, peb cov ntaub ntawv yuav pab txhawb kom nthuav tawm cov txheej txheem hauv qab ntawm kev sib tham ntawm cov kua txob kev tiv thaiv thiab cov lus teb rau RSI.

Kev hloov pauv hloov pauv ua lub luag haujlwm tseem ceeb hauv lub cev tiv thaiv kab mob, tswj kev tswj hwm ntawm cov noob qhia thiab tsim cov hom cell tshwj xeeb. Cov hauv qab no yog kev sib raug zoo ntawm kev hloov pauv thiab kev tiv thaiv kab mob:

1. Kev hloov pauv hloov pauv - NF-κB: NF-κB yog qhov tseem ceeb ntawm kev hloov pauv hauv kev tswj hwm kev tiv thaiv kab mob tiv thaiv kab mob ntawm T hlwb thiab B hlwb. NF-κB kev ua kom muaj zog tuaj yeem txhawb cov lus teb inflammatory, tiv thaiv kab mob, thiab cell apoptosis.

2. Cov ntsiab lus hloov pauv—STATs: STATs yog cov kev hloov pauv uas cuam tshuam cov neeg nqa khoom thiab cov lus teb ntawm tes. STATs tuaj yeem qhib tau, ua lub luag haujlwm tseem ceeb hauv kev ua haujlwm T hlwb thiab B hlwb, thiab koom nrog cov txheej txheem xws li kev tiv thaiv kab mob hauv lub cev, kev sib txawv, thiab ua kom muaj zog.

3. Kev hloov pauv hloov pauv - AP-1: AP-1 yog tsev neeg ntawm cov ntaub ntawv hloov pauv uas cuam tshuam nrog kev tswj hwm ntawm ntau lub xov tooj ntawm tes thiab lub cev tiv thaiv kab mob. AP-1 kev ua kom muaj peev xwm txhawb nqa cov txheej txheem xws li kev loj hlob ntawm tes, kev sib txawv, thiab kev hloov pauv, nrog rau kev tuav cov tshuaj tiv thaiv kab mob thiab tiv thaiv kab mob.

4. Cov ntsiab lus hloov pauv-CEBPs: CEBPs yog ib tsev neeg loj ntawm cov ntaub ntawv hloov pauv, suav nrog CEBP , , , , thiab lwm yam hauv ntau lub hlwb. Hauv lub cev tiv thaiv kab mob, CEBP koom nrog hauv kev ua kom cov macrophages thiab tso tawm cytokines, nrog rau kev sib txawv thiab ua kom cov B hlwb thiab T hlwb.

Sib koom ua ke, cov kev hloov pauv hloov pauv ua lub luag haujlwm tseem ceeb hauv kev tiv thaiv kab mob, koom nrog kev tswj hwm thiab kev saib xyuas kev tiv thaiv kab mob hauv ntau hom cell thiab kev ua haujlwm. Yog li ntawd, peb kuj yuav tsum tau xyuam xim rau kev txhim kho kev tiv thaiv kab mob hauv peb lub neej txhua hnub. Cistanche tuaj yeem txhim kho kev tiv thaiv kab mob, thiab cov polysaccharides hauv cov nqaij tuaj yeem tswj lub cev tiv thaiv kab mob ntawm tib neeg lub cev, txhim kho kev ntxhov siab ntawm lub cev tiv thaiv kab mob, thiab txhim kho kev ua kom tsis muaj menyuam ntawm lub cev tiv thaiv kab mob.

Nyem qhov txiaj ntsig kev noj qab haus huv ntawm cistanche

Ntsiab lus:

kua txob (Capsicum annuum); Ralstonia solanacearum; CAWRKY 30; WRKY TFs.

1. Taw qhia

Cov nroj tsuag nquag raug rau ntau yam kab mob biotic thiab abiotic kev nyuaj siab thaum lawv lub neej ncua vim lawv qhov xwm txheej tsis muaj zog [1,2]. Cov nroj tsuag tau tsim ntau lub tswv yim tiv thaiv hauv kev teb rau kev xaiv rov qab los ntawm kev txwv loj ecological thiab ib puag ncig. Kev sib txawv ntawm qhov sib txawv (TFs) sib cuam tshuam los tsim cov kev sib txuas lus zoo sib xws rau kev tswj hwm cov txheej txheem tiv thaiv classic ntawm qib transcriptional [3,4]. Kev tiv thaiv kev tiv thaiv rau ntau yam kev ntxhov siab yog ua kom zoo thiab tswj tau raws li lawv yog qhov tseem ceeb rau cov nroj tsuag hauv daim ntawv ntawm kev siv hluav taws xob thiab kev loj hlob [5]. Cov txheej txheem tiv thaiv tej zaum yuav txawv ntawm cov nroj tsuag sib txawv vim muaj qhov sib txawv ntawm ib puag ncig cuam tshuam rau lawv qhov kev ua tau zoo [6-8].

Yog li, kev tiv thaiv kev ua haujlwm los ntawm cov qauv nroj tsuag tsis tuaj yeem cuam tshuam tag nrho rau cov nroj tsuag uas tsis yog qauv. Yog li ntawd, kev tiv thaiv kab mob ntawm cov nroj tsuag tau ua qhov tseem ceeb hauv lub xyoo dhau los; Txawm li cas los xij, cov kev tshawb fawb no feem ntau tsom rau cov qauv nroj tsuag, piv txwv li, Arabidopsis thaliana thiab mov (Oryza sativa L.). Txawm li cas los xij, kev ua haujlwm sib luag ntawm cov kev hloov pauv no los tswj cov nroj tsuag cov lus teb rau ntau yam kev ntxhov siab tau raug soj ntsuam tsis zoo, feem ntau hauv cov nroj tsuag tsis yog qauv.

WRKY proteins suav nrog tsev neeg TFs loj tshaj plaws hauv cov nroj tsuag. WRKY TFs muaj ib lossis ob lub WRKY domains thiab feem ntau WRKYGQK ib ntu ntawm N-terminus ua raws li C2HC lossis C2H2 zinc ntiv tes motif [4,9,10]. WRKY proteins yog phylogenetically muab faib ua peb pawg loj (piv txwv li, pawg I-III) raws li ob peb WRKY domains thiab cov qauv ntawm zinc-ntiv tes motif.

Pawg II ntawm WRKY TFs tau muab faib ua tsib pawg (IIa, IIb, IIc, IId, thiab IIe) [11–13]. Cov tswv cuab ntawm WRKY feem ntau khi nrog W-boxes [TTGAC(C/T)] pom nyob rau hauv cov cheeb tsam txhawb nqa ntawm ntau lub hom phiaj cov noob los ntawm kev ntxig ib qho tshwj xeeb vertically rau hauv DNA qhov loj zawj [14] los ntawm WRKY GQK motif ntawm qhov thib ob b strand, uas txhawb nqa. transcriptional modulation ntawm kev qhia ntawm lub hom phiaj noob [14–16]. WRKYs tau koom nrog ntau lub tshuab lom neeg hauv cov nroj tsuag, suav nrog cov noob dormancy, noob germination, senescence, thiab kev loj hlob ntawm cov noob. Ib yam li ntawd, cov no tseem koom nrog hauv cov lus teb rau biotic thiab abiotic stresses thiab hloov pauv ntawm lawv cov hom phiaj hom phiaj [17–20].

Nws tau tshaj tawm tias ib pawg ntawm WRKY TFs lossis ib qho WRKY TF transcriptionally induced los ntawm tsuas yog ib qho kev nyuaj siab modulates ntau qhov kev ntxhov siab. W-boxes yog enriched hauv cov cheeb tsam txhawb nqa [21–24]. Cov kev tshawb fawb no piav qhia txog qhov muaj WRKY tes hauj lwm cuam tshuam los ntawm kev teb rau ib qho kev ntxhov siab, lossis kev sib txuas ntawm ob lossis ntau dua. Lub luag haujlwm ntawm WRKY cov proteins thiab cov txheej txheem hauv qab hauv cov lus teb rau biotic thiab abiotic stresses tau tshawb xyuas ntau zaus hauv kaum xyoo dhau los; Txawm li cas los xij, qhov tseem ceeb ntawm cov kev tshawb fawb no yog feem ntau ntawm ib lub noob hauv kev teb rau ib qho kev ntxhov siab hauv cov qauv nroj tsuag, xws li A. thaliana thiab O. sativa. Tsis ntev los no, ib qho tseem ceeb ua haujlwm sib txawv ntawm cov homologs ze ntawm WRKY TFs los ntawm ntau hom nroj tsuag tau pom zoo [24,25]. Kev ua haujlwm ntawm WRKYs thiab WRKY lub vev xaib hauv cov nroj tsuag tsis yog qauv xws li Capsicum annuum L., hauv kev teb rau ib leeg lossis ua ke kev ntxhov siab, tau tshawb nrhiav tsis zoo txog tam sim no.

Kua txob yog thoob ntiaj teb paub txog nws qhov tseem ceeb ntawm kev lag luam. Nws yog cog rau hauv toj siab thaum lub caij ntuj sov [26,27]. Kua txob raug rau ntau yam kab mob hauv av xws li kua txob blight thiab kab mob wilt tshwm sim los ntawm cov kab mob pathogens, piv txwv li, Phytophthora capsicum thiab Ralstonia solanacearum, feem [28,29]. Qhov xwm txheej ntawm cov kab mob no ua rau muaj kev cuam tshuam loj heev rau kev tsim kua txob nyob rau hauv qhov kub thiab txias (HTHH). HTHH cuam tshuam cov kua txob kev tiv thaiv kab mob los ntawm R protein thiab ua kom cov kab mob loj hlob thiab kev loj hlob. Tsis tas li ntawd, cov kab mob tawm tsam thiab HTHH ib txhij ua rau lub siab xaiv cov kua txob, uas tau cuam tshuam cov kua txob cov kev hloov pauv yav dhau los [30–32]. Hloov pauv, qhov tshwm sim ib txhij ntawm cov kab mob thiab HTHH yuav tsis cuam tshuam rau kev hloov pauv ntawm cov qauv nroj tsuag, xws li A. thaliana thiab O. sativa. Yog li, kua txob zoo li tsim nyog rau kev tshawb fawb txog kev tiv thaiv kab mob biotic lossis abiotic stresses, piv txwv li, RSI lossis HTHH.

Ralstonia solanacearum ua rau cov kab mob wilt nyob rau hauv ntau cov qoob loo, piv txwv li, kua txob, txiv lws suav, qos yaj ywm, thiab lwm yam. Nws yog ib tug avborne gram-negative -proteobacterium [33]. Nws tsim cov kab mob sai sai hauv cov ntaub so ntswg xylem, uas ua rau cov kab mob wilt kab mob hauv cov nroj tsuag kab mob [34].

Cov lus teb ntawm cov nroj tsuag rau biotic thiab abiotic kev nyuaj siab yog paub tias yuav tswj los ntawm phytohormones [35]. Phytohormones yog me me molecules koom nrog hauv kev tswj kev loj hlob ntawm cov nroj tsuag, cog qoob loo, thiab ciaj sia. Salicylic acid (SA) thiab jasmonic acid (JA) yog qhov tseem ceeb rau kev tiv thaiv kab mob ntawm cov nroj tsuag. Ob qho no suav hais tias yog lub hauv paus tseem ceeb ntawm kev tiv thaiv kab mob ntawm cov nroj tsuag tiv thaiv kab mob. Biosynthesis thiab signaling ntawm SA yog qhov tseem ceeb rau kev tiv thaiv cov kab mob biotrophic, thaum JA muab kev tiv thaiv tawm tsam necrotrophic pathogens [36,37]. Ethylene (ET) pab tiv thaiv cov lus teb tiv thaiv ntau yam nroj tsuag-pathogen cuam tshuam [37].

Ntau yam kua txob hauv paus hauv paus cultivars ntawm cov cheeb tsam subtropical ua kom pom cov kab mob nrawm, txawm tias nyob hauv HTHH [38]. Heat-related cis-element HSE feem ntau koom nrog phytohormones, suav nrog SA, JA, ET, ABA, lossis cov kab mob ntsig txog cis-cov ntsiab lus hauv cov cheeb tsam txhawb nqa ntawm feem ntau MAPKs thiab CDPKs koom nrog hauv kev tiv thaiv cov nroj tsuag [39–41]. Nws pom zoo kom muaj crosstalk ntawm kev tiv thaiv kab mob thiab HTHH. Pepper genome yog 27 thiab 7.5 npaug loj dua piv rau A. thaliana thiab O. sativa, feem. Txawm li cas los xij, nws tsuas muaj 73 WRKY cov noob, thaum cov genomes me ntawm A. thaliana thiab O. sativa muaj 72 thiab 122 WRKY genes, feem [9,42,43]. Peb yav dhau los kuaj pom tias CaWRKY6 [44], CaWRKY22 [9], CaWRKY40 [45], thiab CaWRKY58 [46] tau koom nrog cov kua txob cov lus teb tawm tsam RSI.

Ntawm cov no, CaWRKY6, -22, -27, thiab -40 ua raws li kev tswj hwm zoo ntawm cov nroj tsuag tiv thaiv kab mob, whereas CaWRKY58 yog qhov tsis zoo regulator. Cov noob no muaj cov txheej txheem ntawm HSE cov ntsiab lus thiab W-boxes hauv lawv cheeb tsam txhawb nqa thiab cov neeg txhawb nqa ntawm lwm cov WRKY TFs, inferring lub luag hauj lwm ntawm WRKY tes hauj lwm nyob rau hauv cov kev cai ntawm kua txob cov lus teb rau RSI. Tsis tas li ntawd, CaWRKY40 tau ncaj qha tswj hwm los ntawm CaWRKY6 [44] thiab CabZIP63 [16], thaum CaWRKY40 tau tswj tsis ncaj los ntawm CaCDPK15 [47]. Hloov pauv, feem ntau ntawm cov kua txob WRKY TFs tseem tsis tau paub txog lawv cov lus teb rau cov kab mob tawm tsam.

Nyob rau hauv txoj kev tshawb no tam sim no, tag nrho-ntev cDNA rau WRKY TFs tsev neeg ntawm kua txob raug cais thiab hu ua CaWRKY30. Peb tus cwj pwm nws nthuav tawm thiab ua haujlwm tau zoo thiab pom tias CaWRKY30 raug ntxias los ntawm RSI thiab foliar daim ntawv thov ntawm SA, JA, ET, thiab ABA. Kev nthuav qhia dhau ntawm CaWRKY30 hauv cov kua txob nplooj ua rau HR-zoo li cell tuag thiab H2O2 ntau lawm. Silencing ntawm CaWRKY30 hauv kua txob cuam tshuam kev tiv thaiv ntawm kua txob rau RSI. Cov txiaj ntsig no qhia tias CaWRKY30 zoo tswj cov kua txob tiv thaiv kab mob RSI.

2. Cov txiaj ntsig

2.1. Cloning thiab Sequence Analysis ntawm CaWRKY30

Ib qho tshiab WRKY gene CaWRKY30 (CA01g34480) tau txheeb xyuas los ntawm genome-wide tsom xam. Lub CaWRKY30 noob tau raug xaiv rau kev ua haujlwm zoo txij li thaum muaj kev tiv thaiv kab mob ntsig txog cis-cov ntsiab lus xws li TGA (TGACG motif-binding factor), TGACG-motif, TATC-box, TATAbox, thiab W-box nyob rau hauv cheeb tsam txhawb nqa nws ua tau. lub luag haujlwm hauv kua txob tiv thaiv kab mob (Daim duab S1). Ib qho cDNA fragment ntawm CaWRKY30 (CA01g34480) ntawm 924 bp qhib-nyeem-thav duab (ORF) tau cloned los ntawm kev siv cov noob tshwj xeeb primers (Table S1). Qhov ntev ntawm qhov txiav tawm amino acid ib ntus ntawm CaWRKY30 yog 307 amino acid residues, muaj kev txuag WRKY domain, thiab nws tau muab faib ua pawg III (Daim duab 1). Cov protein tau kwv yees muaj qhov loj me thiab theoretical pI ntawm 34.55 kDa thiab 7.32, feem. Lub CaWRKY30 qhia 88, 60, 68, thiab 57 feem pua ​​​​ amino acid tus kheej nrog NsWRKY53, CcWRKY53, NaWRKY53, thiab StWRKY53, raws li (Daim duab S2).

2.2. Transcriptional Expression of CaWRKY30 nyob rau hauv RSI thiab Kev Kho Mob nrog Ntau Yam Phytohormones

Lub xub ntiag ntawm kev tiv thaiv kab mob cuam tshuam nrog cis-cov ntsiab lus hauv cheeb tsam txhawb nqa ntawm CaWRKY30 qhia nws lub luag haujlwm muaj peev xwm hauv kua txob kev tiv thaiv RSI. Txhawm rau txheeb xyuas qhov kev xav, kev tshuaj xyuas qRT-PCR tau ua los ntsuas cov qib kev hloov pauv ntawm CaWRY30 raws li RSI. Cov qib transcriptional ntawm CaWRKY30 tau nce nyob rau hauv Ralstonia-kho nplooj piv rau mock-kho nplooj (Daim duab 2A). Qhov kev txhim kho CaWRKY30 cov lus qhia kev hloov pauv tau zoo ib yam ntawm 0 h txog 48 h tom qab kho (HPT). Qhov siab tshaj plaws transcriptional qhia theem tau sau tseg ntawm 48 hpt (Daim duab 2A).

Phytohormones, suav nrog ET, ABA, SA, thiab JA-mediated signaling pathways, yog cov tswj hwm tseem ceeb ntawm cov nroj tsuag cuam tshuam rau biotic thiab abiotic stresses. Cov kev cai ntawm CaWRKY30 los ntawm phytohormones-mediated signaling pathways tau kawm los ntawm foliar daim ntawv thov ntawm ET, ABA, SA, thiab JA siv qRT-PCR tsom xam. Cov txiaj ntsig tau qhia tias cov txheeb ze ntawm cov lus piav qhia theem ntawm CaWRKY30 tau txhim kho los ntawm 0 h mus rau 48 hpt nrog 100 µM ET piv rau qhov kev thuam. Cov qib kev hloov pauv no tau siab tshaj ntawm 48 hpt nrog ET (Daim duab 2B). Foliar daim ntawv thov ntawm 100 µM ABA tau nce qib ntawm kev hloov pauv ntawm CaWRKY30 piv rau kev thuam. Kev hloov pauv mus txog qib siab tshaj plaws ntawm 12 hpt nrog ABA (Daim duab 2C). Cov txiaj ntsig tau qhia tias kev sib sau ua ke ntawm CaWRKY30 tau nce ntxiv tom qab daim ntawv thov ntawm 1 mM SA, piv rau qhov kev thuam. Cov txheej txheem kev hloov pauv hloov pauv tau zoo tshaj plaws ntawm 24 hpt (Daim duab 2D).

Daim duab 2. Kev ntsuam xyuas qRT-PCR ntawm CaWRKY30 txheeb ze ntawm cov lus qhia txog qib hauv cov kua txob nplooj raug rau Ralstonia solanacearum inoculation (RSI), thiab foliar daim ntawv thov ntawm ntau yam phytohormones. Kev tshuaj xyuas qRT-PCR tau siv los tshuaj xyuas cov qib transcriptional ntawm CaWRKY30 hauv cov kua txob nplooj raug rau ntau yam kev kho mob suav nrog RSI (A), daim ntawv thov ntawm 100 µm ET (B), daim ntawv thov ntawm 100 µm ABA (C), thiab daim ntawv thov ntawm 1 mM SA (D) ntawm lub sijhawm sib txawv. Cov kev hloov pauv ntau ntxiv hauv RSI-kho nplooj tau muab piv nrog MgCl2 - kho cov nplooj (mock), uas nws tus txheeb ze qhia theem tau teeb tsa rau "1". (B-D) Cov theem kev hloov pauv hauv phyto-hormone-kho nplooj tau muab piv nrog ddH2O-kho nplooj (mock), uas nws qib qhia tau teem rau "1". Qhov siab ntawm lub bar qhia txhais tau tias thiab yuam kev tuav qhia tus qauv yuam kev ntawm txhais tau tias. Cov ntawv sib txawv saum toj no qhia qhov sib txawv ntawm qhov txhais tau tias raws li Fisher's tiv thaiv LSD xeem.

2.3. CaWRKY30 tau pom muaj nyob rau hauv lub Nucleus

Kev soj ntsuam ib ntus los ntawm kev siv WoLFPSORT tau piav qhia tias qhov kev kwv yees CaWRKY30 protein ib ntus muaj lub teeb liab tso rau hauv thaj chaw nuclear (Daim duab 1), qhia nws lub peev xwm lub hom phiaj hauv lub nucleus. Txhawm rau kom ntseeg tau qhov kev xav no, ib qho kev tsim kho aCaWRKY30 GFP fusion tau tsim tawm uas tau tsav los ntawm kev txhawb nqa ntawm CaMV35S. Tom qab ntawd, qhov teeb meem no tau hloov mus rau hauv Agrobacterium strainGV3101. Peb tau tshaj tawm dhau los ntawm CaWRKY30 GFP tsim nyob rau hauv nplooj ntawm Nicofiamabenthamiana los ntawm A. tumefacient txhaj, thiab GFP cov cim tau tshuaj xyuas los ntawm confocal fluores.science microscope. Cov txiaj ntsig tau piav qhia tias GFP cov cim ntawm CaWRKY30-GFP muaj nyob rau hauv lub nuclei, thaum GFP ntawm kev tswj tau kuaj pom nyob rau hauv ntau yam subcellular, xws li cytoplasm thiab nuclei (Daim duab 3).

Daim duab 3. Subcellular localization ntawm CaWRKY30. Lub CaWRKY30 muaj tag nrho nyob rau hauv lub nucleus ntawm nicotiana benthaminna nplooj. Cov xim ntsuab qhia GFP Xiav xim qhia DAPI staining ntawm lub nucleus. Cov xim cyan qhia txog kev sib xyaw ntawm ntsuab CFP thiab xiav DAPI-stained nuclei. CPP signaGreen) rau kev tswj, N. benthamiana nplooj raug kuaj pom thoob plaws ntawm tes. Cov duab raug ntes los ntawm confocal microscopy ntawm 48 h tom qab inoculation. Bars=25 um.

2.4. Silencing ntawm CaWRKY30 los ntawm VIGS Curtailed Pepper's Immunity rau RSI thiab Downregulated Immunity Associated Marker Genes.

Lub CaWRKY30 tau ntsiag to los ntawm tus kab mob-induced gene silencing (VIGS) los kawm txog nws lub luag haujlwm hauv kev tiv thaiv kua txob. Tau txais tag nrho 50 CaWRKY30-unsilenced (TRV:00) thiab 50 CaWRKY30- silenced (TRV: CaWRKY30) cov nroj tsuag tau txais. Rau CaWRKY30 silenced nroj tsuag raug randomly xaiv los xyuas lawv cov efficiency ntawm noob silencing los ntawm cov hauv paus hniav inoculation nrog cov hlwb ntawm tshaj virulent R. solanacearum strain. Peb qhov kev tshawb pom tau qhia tias qib kev hloov pauv ntawm CaWRKY30 tau txo qis los ntawm ~ 30 feem pua ​​​​hauv Ralstonia-kho CaWRKY30 silenced nroj tsuag piv rau cov nroj tsuag uas tsis muaj suab nrov, ua pov thawj qhov kev ntsiag to ntawm CaWRKY30 (Daim duab 4A).

Lub CaWRKY30 silenced nroj tsuag pom tau ntau dua susceptibility rau RSI piv rau unsilenced nroj tsuag. Kua txob qhov kev raug mob rau cov kab mob tau nce los ntawm kev qhia txog kev nce hauv Ralstonia cov pej xeem kev loj hlob, cim los ntawm ntau dua cfu qhov tseem ceeb hauv CaWRKY30 silenced kua txob cov nroj tsuag piv nrog cov nroj tsuag tsis muaj suab nrov ntawm 3 d thiab 5 hnub tom qab inoculation (dpi) (Daim duab 4B). Histochemical staining assay tau ua tiav los kuaj xyuas H2O2 ntau lawm thiab cell necrosis hauv Ralstonia inoculated CaWRKY30 silenced (TRV: CaWRKY30), thiab unsilenced (TRV:00) kua txob nplooj. Qhov tsaus nti DAB staining (ib qho cim ntawm H2O2 ntau lawm) thiab HR-zoo li cell tuag qhia los ntawm tsaus trypan xiav staining tau pom nyob rau hauv unsilenced kua txob nplooj ntawm 48 hpi (teev tom qab inoculation). Hloov pauv, qhov concentrations ntawm trypan xiav thiab DAB staining tau txo qis hauv CaWRKY30 silenced kua txob nplooj (Daim duab 4C). Hluav taws xob conductivity - qhov taw qhia ntawm ion to - raug xam los kawm plasma membrane puas thiab cell tuag tom qab RSI.

Cov txiaj ntsig tau nthuav tawm tias cov nroj tsuag tsis muaj qhov tsis hnov ​​​​tsw kho nrog RSI nthuav tawm cov ions ntau dua li Ralstonia-kho CaWRKY30 silenced nroj tsuag ntawm 24 thiab 48 dpi (Daim duab 4D). Tus txheeb ze tus kab mob Performance index tau kwv yees txog li 10 dpi rau inferring theem ntawm tus kab mob nyob rau hauv CaWRKY30-silenced thiab un-silenced nroj tsuag tom qab RSI (Sab Ntxiv Table S2). Cov tsos mob tseem ceeb tau pom nyob rau hauv CaWRKY30 silenced nroj tsuag ntawm 10 dpi, thaum cov nroj tsuag unsilenced qhia cov tsos mob me me (Daim duab 4E). Rau CaWRKY30-silenced thiab un-silenced nroj tsuag tau random xaiv thiab infiltrated nrog R. solanacearum nyob rau hauv cov hauv paus hniav rau phenotype assay. Cov tsos mob ntawm tus kab mob wilting pom tseeb tau pom nyob rau hauv CaWRKY30 silenced kua txob ntawm 10 dpi, thaum cov nroj tsuag unsilenced qhia cov tsos mob tsis tshua muaj wilting (Daim duab 4F). Kev tshuaj xyuas qRT-PCR tau ua tiav los kawm txog kev sib sau ntawm kev tiv thaiv kab mob ntsig txog cov noob caj noob ces thiab cov txiaj ntsig tau qhia tias qib kev hloov pauv ntawm kev tiv thaiv kab mob cuam tshuam nrog cov cim npe xws li CaPR1, CaNPR1, CaDEF1, CaABR1, thiab CaHIR1 tau txo qis hauv CaWRKY{{ 25}}cov nroj tsuag uas ntsiag to piv rau cov nroj tsuag tsis ntsiag to ntawm 48 dpi (Daim duab 4G).

Daim duab 4. Silencing of CaWRKY30 los ntawm VIGS curtailed pepper's resistance to RSI and upregulated immunity-associated marker genes. Qhov kev ntsuam xyuas qRT-PCR ntawm CaWRKY30 qhia theem hauv R. solanacearum-inoculated, thuam (iniculated nrog MgCl2 tov) CaWRKY30-silenced pepper plant (TRV: CaWRKY30), thiab tswj cov nroj tsuag (TRV:00) (A). Qhov txawv ntawm kev loj hlob ntawm R. solanacearum ntawm CaWRKY30-silenced thiab un-silenced (tswj) kua txob nroj tsuag inoculated nrog R. solanacearum ntawm 3 thiab 5 dpi (B). Histochemical staining (DAB thiab trypan xiav staining) hauv R. solanacearum-inoculated CaWRKY30-silenced (TRV: CaWRKY30) thiab un-silenced (TRV:00) kua txob tawm ntawm 48 dpi. Scale bar=50 µm (C). Electrolyte to ntsuas ntsuas raws li ion conductivity los ntsuas cov cell-tuag teb nyob rau hauv cov nplooj ntawv discs ntawm CaWRKY30-silenced (TRV: CaWRKY22) thiab un-silenced (TRV:00) kua txob nroj tsuag ntawm 24 thiab 48 hpi nrog thiab tsis muaj R. solanacearum (D). CaWRKY30- ntsiag to los ntawm VIGS txhim kho cov kua txob cov nroj tsuag 'ua rau R. solanacearum kab mob.

Tus kab mob Performance index nce zuj zus nrog lub sijhawm. CaWRKY30-silenced (TRV: CaWRKY30) cov nroj tsuag qhia tau hais tias muaj kev cuam tshuam rau R. solanacearum kab mob piv rau un-silenced (TRV:00) cov nroj tsuag nrog sij hawm. Qhov nruab nrab yog raws li plaub qhov kev hloov pauv lom neeg nrog tsib tsob nroj ib qho kev rov ua dua (E). Cov nyhuv phenotypic ntawm R. solanacearum kev kho mob ntawm CaWRKY30-silenced (TRV: CaWRKY30) thiab un-silenced (TRV:00) kua txob nroj tsuag ntawm 10 dpi (F). qRT-PCR kev soj ntsuam ntawm cov qib transcriptional ntawm kev tiv thaiv-txuas nrog cov cim cim noob hauv CaWRKY30-silenced (TRV: CaWRKY30) thiab un-silenced (TRV:00) kua txob nroj tsuag ntawm 48 h tom qab inoculation nrog R. solanacearum (G). Tus txheeb ze transcriptional qhia theem ntawm mock-kho un-silenced nroj tsuag tau teem rau "1". Qhov siab ntawm lub bar qhia txhais tau tias thiab yuam kev tuav qhia tus qauv yuam kev ntawm txhais tau tias. Cov ntaub ntawv sawv cev txhais tau tias ± SE los ntawm plaub qhov kev hloov pauv lom neeg. Cov ntawv sib txawv saum toj ntawm cov tuav qhia qhov sib txawv ntawm cov txhais tau tias.

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