EDS1-kev sib cuam tshuam J Protein 1 yog qhov tseem ceeb tsis zoo ntawm cov nroj tsuag Innate Immunity hauv Arabidopsis
Nov 10, 2023
Abstract
Cov nroj tsuag tau hloov kho cov txheej txheem meej los txhim kho lub cev tiv thaiv kab mob tiv thaiv kab mob. ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) plays lub luag haujlwm tseem ceeb hauv cov nroj tsuag hauv lub cev tiv thaiv kab mob los ntawm kev tswj hwm kev tiv thaiv basal thiab effector-triggered immunity. Nucleocytoplasmic kev lag luam ntawm EDS1 yog qhov yuav tsum tau ua kom muaj zog tiv thaiv, tab sis cov txheej txheem molecular tseem tsis pom. Ntawm no, peb qhia tau hais tias EDS1-INTERACTING J PROTEIN1 (EIJ1), uas ua raws li DnaJ protein-zoo li chaperone nyob rau hauv teb rau cov kab mob, ua hauj lwm raws li ib tug tseem ceeb tsis zoo regulator ntawm cog kev tiv thaiv los ntawm kev cuam tshuam nrog EDS1. Kev poob-ntawm-kev hloov pauv ntawm EIJ1 tsis cuam tshuam rau kev loj hlob ntawm cov nroj tsuag tab sis txhim kho cov kab mob tsis zoo. Thaum tus kab mob kis tau tus kab mob, EIJ1 tau hloov chaw los ntawm chloroplast mus rau cytoplasm, qhov chaw uas nws cuam tshuam nrog EDS1, yog li txwv tsis pub cov kab mob tshwm sim ntawm EDS1 mus rau lub nucleus thiab cuam tshuam kev tiv thaiv thaum ntxov kis kab mob. Thaum lub sij hawm kev loj hlob kab mob, EIJ1 tau maj mam degraded, tso cai rau lub nuclear tsub zuj zuj ntawm EDS1 rau transcriptional resistance reinforcement. Tus kab mob avirulent Pst DC3000 (AvrRps4) tshem tawm qhov kev tawm tsam ntawm EIJ1 los ntawm kev ua kom nws qhov kev puas tsuaj sai sai hauv cov lus teb cuam tshuam rau kev tiv thaiv kab mob. Yog li, peb qhov kev tshawb pom pom tau tias EIJ1 yog ib qho tseem ceeb EDS1- nyob ntawm qhov tsis zoo ntawm cov nroj tsuag tiv thaiv kab mob thiab muab kev nkag siab txog kev ua haujlwm ntawm nuclear piv rau cytoplasmic tis ntawm EDS1 raug tswj thaum lub sij hawm tiv thaiv kab mob.
cistanche cov txiaj ntsig-ua kom muaj zog tiv thaiv kab mob
Taw qhia
Cov nroj tsuag tau hloov kho lub network nyuaj los hloov kho lawv lub cev tiv thaiv kab mob. Txhawm rau tiv thaiv lawv tus kheej tawm tsam cov kab mob, cov nroj tsuag siv ob lub tswv yim tswj hwm lub ntsiab lus: qauv tiv thaiv kab mob (PTI), thiab kev tiv thaiv kab mob cuam tshuam (ETI). PTI yog tswj hwm los ntawm tus qauv lees paub receptors (PRRs) uas paub txog kab mob- lossis microbe-txuas nrog cov qauv molecular (Zipfel, 2008). Txhawm rau pab txhawb kev kis tus kab mob, cov kab mob virulent suppress PTI los ntawm kev zais cov kab mob cuam tshuam, thiab lawv tswj hwm cov hlwb kom pab txhawb kev tau txais cov khoom noj thiab thaum kawg kev yug me nyuam (Tsuda and Katagiri, 2010). Qib qis uas cov nroj tsuag qhia rau cov kab mob phem no yog hu ua basal resistance (Jones and Dangl, 2006; Klessig et al., 2018). Ntawm qhov tod tes, ETI yog kho los ntawm cov nroj tsuag-encoded kab mob tiv thaiv (R) cov proteins, uas tuaj yeem ncaj qha lossis ncaj qha paub txog qhov muaj cov nyhuv zais cia los ntawm cov kab mob (Tsuda thiab Katagiri, 2010). Feem ntau R cov proteins muaj cov nucleotide-binding site thiab leucine-nplua nuj rov sau npe (NB-LRR). Cov NB-LRR proteins uas muaj N-terminal Tus Xov Tooj / Interleukin-1 receptor (TIR) protein-protein kev sib koom ua ke npe hu ua TIR-NB-LRR (TNL) cov proteins, thiab lawv suav nrog pawg loj tshaj plaws ntawm NB- LRR proteins (Caplan et al., 2008). Piv nrog rau basal kuj, ETI induces ib tug muaj zog thiab ceev tiv thaiv teb tiv thaiv kab mob thiab yog feem ntau nrog los ntawm lub zos cell tuag, ib tug yam ntxwv feature ntawm hypersensitive teb (HR; Dodds thiab Rathjen, 2010). ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), lub hauv paus tswj hwm ntawm cov nroj tsuag tiv thaiv kab mob, pab tswj kev tiv thaiv basal los ntawm kev txwv kev cuam tshuam ntawm cov kab mob biotrophic thiab hemibiotrophic (Wiermer li al., 2005). EDS1 tseem ua lub luag haujlwm tseem ceeb hauv ETI, uas feem ntau yog kho los ntawm TNL chav kawm ntawm R proteins (Heidrich li al., 2012). EDS1 ua haujlwm zoo nrog nws cov coregulators PHYTOALEXIN DEFICIENT 4 thiab SENESCENCE-ASSOCIATED GENE 101 nyob rau hauv cytoplasm thiab / los yog nucleus los tswj intracellular reactive oxygen hom (ROS) ntau lawm, salicylic acid (SA) tsub zuj zuj, thiab lwm yam txheej txheem, yog li txhawb kev tiv thaiv kab mob. 'rucci et al., 2001; Rietz et al., 2011; Wagner et al., 2013). Raws li, Arabidopsis eds1 mutant yog qhov raug rau Pseudomonas syringae pv. Txiv lws suav (Pst) strain DC3000 thiab qhia tau hais tias muaj kev cuam tshuam TNL-mediated ETI, uas ISOCHORISMATE SYNTHASE1 (ICS1; SA biosynthetic gene) qhia thiab SA biosynthesis yog inhibited (Parker li al., 1996; Feys et 20et al. ib., 2006). Tsis tas li ntawd, EDS1 nthuav tawm cov khoom lag luam protein ntawm cov cytoplasm thiab nucleus mediated los ntawm nuclear thauj receptors (Garcı'a li al., 2010). Txawm hais tias lub zog nuclear ntawm EDS1 koom nrog basal tsis kam thiab ROS ntau lawm, programmed cell tuag thiab tiv thaiv kev tiv thaiv yuav tsum muaj kev sib koom tes ntawm nuclear tiv thaiv cytoplasmic localization ntawm EDS1 (Heidrich li al., 2011). Qhov sib npaug ntawm cov nyiaj EDS1 hauv cytosol thiab cov nyiaj hauv lub nucleus yog qhov tseem ceeb rau kev ua haujlwm zoo basal thiab TNL-triggered immunity (Garcı'a et al., 2010). Txawm li cas los xij, yuav ua li cas EDS1 tswj cov qauv nucleocytoplasmic faib tawm thaum lub sij hawm cog qoob loo hauv lub cev tsis paub meej. DnaJ proteins (los yog J proteins) yog chaperone proteins (Pulido thiab Leister, 2018) uas yog nyob rau hauv lub cysteine (Cys) - nplua nuj domain superfamily thiab koom nyob rau hauv lub folding thiab sib sau ua ke ntawm nascent proteins, thauj cov proteins nyob rau hauv daim nyias nyias nrog tom qab refolding, thiab Kev pov tseg ntawm cov proteins uas tsis tau nthuav tawm lossis tsis zoo (Bukau li al., 2006). Cov txheej txheem no yuav tsum muaj rau kev ua haujlwm ntawm cov cellular feem ntau ntawm cov theem sib txawv (Pulido thiab Leister, 2018). Kev tshawb fawb nyob rau hauv ntau yam kab mob qhia tau hais tias DnaJ proteins kuj muaj ntau lub luag hauj lwm nyob rau hauv biotic thiab abiotic stress teb, tshwj xeeb tshaj yog nyob rau hauv lub cev tiv thaiv kab mob. Hauv Nicotiana benthamiana, DnaJ protein Nb-MIP1 ua haujlwm ua tus cochaperone hauv cov lus teb rau cov kab mob kis (Du li al., 2013). Soybean (Glycine max L.) Gm-heat shock protein 40 (HSP40.1), a nucleus-localized DnaJ protein, yog tus tswj hwm zoo ntawm kev tiv thaiv kab mob soybean mosaic (Liu thiab Whitham, 2013). Hauv cov txiv lws suav (Solanum lycopersicum), chloroplast-localized DnaJ protein, Le-CDJ2, tau tshaj tawm los txhim kho kev tiv thaiv kab mob, Pseudomonas solanacearum (Wang et al., 2014). Tsis tas li ntawd, Tsi1- interacting protein1 (Tsip1), ib tug haus luam yeeb (Nicotiana tabacum) DnaJ protein, tau tshaj tawm los tswj Tsi1- mediated transcriptional activation nyob rau hauv teb rau kev ntxhov siab (Ham et al., 2006). Cov txiaj ntsig no qhia meej tias DnaJ cov proteins ua lub luag haujlwm tseem ceeb hauv kev tiv thaiv cov nroj tsuag, txawm hais tias cov txheej txheem hauv qab yuav tsum tau tshawb xyuas ntxiv. Hauv txoj kev tshawb no, peb cais Arabidopsis thaliana EDS1 INTERACTING J PROTEIN 1 (EIJ1), HSP40-zoo li DnaJ protein, thiab pom tias EIJ1 cuam tshuam nrog EDS1 hauv planta. Kev poob-ntawm-kev ua haujlwm eij1 cov nroj tsuag mutant pom qhov kev loj hlob ib txwm muaj, muaj zog tiv thaiv kab mob, thiab kev qhia ntau dua ntawm cov tshuaj tiv thaiv kab mob thaum kis kab mob los ntawm cov kab mob piv nrog cov nroj tsuag qus. Txawm li cas los xij, qhov poob ntawm kev ua haujlwm hloov pauv ntawm EIJ1 tsis tau cawm qhov cuam tshuam phenotype ntawm eds1 mutant raws li kev sib tw nrog Pst DC3000. Tsis tas li ntawd, EIJ1 protein, uas ib txwm nyob rau hauv chloroplast, tau tso tawm los ntawm chloroplast mus rau cytoplasm raws li cov kab mob inoculation, qhov uas nws cuam tshuam nrog EDS1, yog li tiv thaiv nws cov kab mob ua rau kev lag luam mus rau lub nucleus thiab thiaj li cuam tshuam cov lus teb thaum ntxov. theem kab mob. Cov kev tshawb pom no qhia meej cov ntsiab lus tseem ceeb ntawm cov txheej txheem tswj hwm hauv qab ntawm nucleocytoplasmic faib qauv ntawm EDS1 thiab muab lub hom phiaj muaj txiaj ntsig zoo rau kev tiv thaiv kab mob hauv cov qoob loo.
cistanche cov txiaj ntsig-ua kom muaj zog tiv thaiv kab mob
Cov txiaj ntsig
EIJ1 cuam tshuam nrog EDS1 hauv vitro thiab hauv vivo
Raws li ib qho tseem ceeb ntawm cov lus teb tiv thaiv cov nroj tsuag, EDS1 yog nyob rau ntawm ib qho tseem ceeb ntawm cov nroj tsuag qhov kev tiv thaiv kab mob hauv lub cev. Txhawm rau txheeb xyuas cov neeg muaj peev xwm sib cuam tshuam cov neeg koom tes ntawm EDS1, peb tau ua qhov kev tshuaj ntsuam poov xab ob-hybrid. EDS1 tau pom muaj kev sib cuam tshuam nrog Arabidopsis HSP40- zoo li chaperone protein (AT2G24860) nrog qhov tsis paub ua haujlwm (Daim duab 1, A thiab B), koom nrog DnaJ superfamily (Cov duab ntxiv 1A); Cov protein no yog tom qab ntawd hu ua EIJ1. Kev soj ntsuam ib ntus tau pom tias EIJ1 muaj 144 amino acid residues, suav nrog plaub Cys-nplua nuj Zn ntiv tes motifs (CXXCXGXG; Ntxiv daim duab 1A). Txawm hais tias lwm hom kab mob angiosperm muaj homologs ntawm EIJ1, qhov ze tshaj plaws homologs tshwm sim tsuas yog rau Brassicaceae (Ntxiv daim duab 1B thiab File S1). Qhov tseem ceeb, EIJ1 tau pom qhov sib luag zoo sib xws nrog N. tabacum Tsip1 protein, uas koom nrog cov kab mob tsis zoo (Cov duab ntxiv 1B; Ham et al., 2006). Txhawm rau txheeb xyuas cov haujlwm tseem ceeb ntawm lub luag haujlwm rau kev sib cuam tshuam ntawm EDS1 thiab EIJ1, ob qhov kev hloov pauv ntawm cov proteins no tau siv rau hauv cov poov xab ob-hybrid assays (Daim duab 1A; Ntxiv daim duab 1A). N terminus ntawm EIJ1 tau pom muaj kev sib cuam tshuam nrog tag nrho-ntev EDS1 nrog rau EDS1 N terminus tab sis tsis muaj zog cuam tshuam nrog EDS1 C terminus (Daim duab 1B). Qhov no qhia tau hais tias N terminus ntawm EDS1, uas muaj lipase-zoo li tus sau, thiab N terminus ntawm EIJ1 yog qhov tsim nyog thiab txaus rau lawv cov kev sib cuam tshuam. Txawm hais tias C terminus ntawm EIJ1 muaj qhov khaws cia Cys-nplua nuj Zn ntiv tes uas tuaj yeem pab txhawb nws txoj kev khi rau DNA lossis lwm cov proteins, nws tsis ua rau muaj kev cuam tshuam nrog EDS1. Pull-down kev soj ntsuam ntxiv txheeb xyuas qhov kev cuam tshuam ntawm EDS1 thiab EIJ1 hauv vitro (Daim duab 1C), thaum co-immunoprecipitation (Co-IP) kev soj ntsuam siv 35Spro: EIJ1- 6HA 35Spro: EDS1-3FLAG transgenic nroj tsuag lees paub lawv cov kev sib cuam tshuam hauv planta (Daim duab 1D). Ua ke, cov txiaj ntsig no qhia tau tias EDS1 cuam tshuam nrog EIJ1 ob qho tib si hauv vitro thiab hauv vivo. Tus cwj pwm ntawm EIJ1 Txhawm rau txiav txim siab subcellular localization ntawm EIJ1, peb fused EIJ1 qhib kev nyeem ntawv nrog mCherry reporter gene nyob rau hauv kev tswj hwm ntawm cov kab mob kab mob kab mob 35S los tsim 35Spro: EIJ1-mCherry tsim. Qhov kev tsim no tau hloov pauv mus rau N. benthamiana nplooj los ntawm agroinfiltration. Fluorescence microscopy tau pom tias EIJ1-mCherry tau nyob rau hauv ob qho tib si chloroplast thiab nucleus (Daim duab 2A). Cov qauv hauv cheeb tsam no tau lees paub los ntawm kev tshuaj ntsuam immunoblot ntawm transgenic 35Spro: EIJ1-6HA cov nroj tsuag (Cov duab ntxiv 2). Txhawm rau tshawb xyuas cov qauv qhia ntawm EIJ1, genomic sequence ntawm EIJ1 tau fused nrog ß-glucuronidase (GUS) reporter gene los tsim EIJ1pro: EIJ1-GUS transgenic Arabidopsis kab. Muaj zog GUS kev ua haujlwm tau kuaj pom hauv rosette nplooj thiab cov hauv paus hniav; Txawm li cas los xij, GUS kev ua haujlwm tsis muaj zog hauv siliques thiab tsis tuaj yeem kuaj pom hauv stems thiab paj (Daim duab 2B). Cov txiaj ntsig no tau ua raws li cov lus qhia rau EIJ1, uas tau tshuaj xyuas los ntawm ntau qhov rov qab hloov pauv-PCR (RT-PCR) hauv ntau cov ntaub so ntswg (Daim duab 2C). Muab hais tias EDS1 yog lub hauv paus tswj hwm ntawm cov nroj tsuag tiv thaiv kab mob (Saikat li al., 2011), peb tau tshawb xyuas seb EIJ1, ib tug putative cofactor ntawm EDS1, kuj koom nrog cov lus teb rau cov kab mob. Cov ntawv sau tseg ntawm EIJ1 tau raug ntxias los ntawm kev kho mob nrog cov kab mob hemibiotrophic Pst DC3000 lossis SA (Daim duab 2D). GUS staining qhia tias EIJ1 raug ntxias thiab ua kom pom tseeb nyob ib puag ncig cov trichomes hauv Pst DC3000-inoculated nplooj (Daim duab 2E–G), qhia txog qhov muaj feem cuam tshuam txog kev lom neeg ntawm EIJ1 hauv kev tiv thaiv teb (Xin thiab Nws, 2013). Tom ntej no, peb tshuaj xyuas qib EIJ1 hauv 35Spro: EIJ1-HA cov nroj tsuag kho nrog Pst DC3000 lossis SA. Interestingly, raws li Pst DC3000 los yog SA kev kho mob, EIJ1 tau maj mam degraded tom qab ib tug ceev induction (Daim duab 2, H thiab kuv). Cov txiaj ntsig no tsis zoo ib yam nrog cov qauv qhia ruaj khov ntawm EIJ1-HA tau pom nyob rau hauv 35Spro: EIJ1-HA cov nroj tsuag thaum kis kab mob (Cov duab ntxiv 3), qhia txog kev tswj hwm kev hloov pauv ntawm EIJ1. Ua ke, cov txiaj ntsig no qhia tias EIJ1 koom nrog hauv cov nroj tsuag tiv thaiv kab mob.
cistanche cov txiaj ntsig rau txiv neej-ua kom muaj zog tiv thaiv kab mob
Kev poob-ntawm-kev hloov pauv ntawm EIJ1 txhim kho kev tiv thaiv rau Pst DC3000 ntawm SA txoj hauv kev
Txhawm rau tshawb xyuas lub luag haujlwm ntawm EIJ1 hauv kev tiv thaiv cov nroj tsuag, peb tau txheeb xyuas qhov Arabidopsis mutant (SALK_142975) uas muaj T-DNA ntxig rau hauv qhov thib peb exon ntawm EIJ1 (Daim duab 3, A thiab B). Quantitative RT-PCR tsom xam pom tias EIJ1 tau hais tsis zoo nyob rau hauv no eij1 T-DNA insertion mutant; yog li ntawd, peb muab lub npe no EIJ1 allele eij{{10}} (Daim duab 3C). Tom qab ntawd, peb tau soj ntsuam cov kab mob tiv thaiv phenotype ntawm 3-lub lim tiam-laus eij1-1 cov nroj tsuag mutant thiab Arabidopsis (A. thaliana) ecotype Columbia (Col-0; wild-type) nroj tsuag los ntawm inoculation nrog PST DC 3000. Kev soj ntsuam ntawm cov nplooj muaj kab mob ntawm 5 hnub tom qab inoculation (dpi) qhia tsawg dua necrotic lesions ntawm eij1-1 nplooj tshaj ntawm Col-0 nplooj (Daim duab 3D). Raws li qhov tshwm sim, eij1-1 nplooj pom cov kab mob qis dua li Col-0 nplooj ntawm 5 dpi, txawm hais tias cov kab mob titer tsis pom qhov txawv ntawm Col-0 thiab eij1-1 tawm ntawm 0 dpi (Daim duab 3E). Tom ntej no, peb tsim eij1-1 EIJ1pro: EIJ1-6HA cov kab sib txuas los ntawm kev hloov cov EIJ1 genomic fragment fused nrog 6xHA tag sequence rau hauv eij1-1 keeb kwm yav dhau. Cov kab sib txuas ua tiav tau cawm cov kab mob tiv thaiv phenotype ntawm eij1-1 (Daim duab 3E), txhawb nqa tias eij1-1 yog qhov poob-ntawm-kev ua haujlwm mutant. Txhawm rau txiav txim siab txog cov txiaj ntsig ntawm EIJ1 ntawm kev tiv thaiv apoplastic, tus kab mob tiv thaiv phenotype ntawm cov kab sib txuas ntxiv no kuj tau soj ntsuam los ntawm kev nkag siab ntawm Pst DC3000, thiab cov txiaj ntsig zoo sib xws tau txais (Cov Duab Ntxiv 4A). Txhawm rau kom paub meej txog kev ua haujlwm ntawm EIJ1, peb mam li txheeb xyuas lwm tus Arabidopsis mutant eij1-2 (WiscDsLox343G09), uas muaj T-DNA tso rau hauv cheeb tsam txhawb nqa ntawm EIJ1 (Cov duab ntxiv 5, A thiab B). Cov ntaub ntawv EIJ1 tsis tshua pom muaj nyob hauv eij1-2 mutant, qhia tias eij1-2 yog null allele ntawm EIJ1. Zoo ib yam li eij1-1, kev soj ntsuam kab mob loj hlob tau qhia tias eij1-2 nyob rau ntawm cov kab mob qis qis dua li Col-0 (Cov duab ntxiv 5, D thiab E). Hauv cov ntsiab lus, cov txiaj ntsig no qhia tau tias EIJ1 ua lub luag haujlwm tseem ceeb hauv kev tiv thaiv cov nroj tsuag.
Daim duab 1 EIJ1 cuam tshuam nrog EDS1 hauv vitro thiab hauv planta. (A) Schematic daim duab qhia txog cov chaw nyob hauv EIJ1, EDS1, thiab lawv cov ntawv txiav tawm. (B) Cov kev ntsuam xyuas poov xab ob-hybrid qhia txog kev sib cuam tshuam ntawm EIJ1, EDS1, thiab lawv cov ntawv luv luv. Hloov pauv cov poov xab tau loj hlob ntawm SD/-Trp/-Leu/-Nws/-Ade thiab SD/-Trp/-Leu nruab nrab. Npua, vector-tsuas yog tswj; AD, activation domain; BD, DNA-binding domain. (C) Pull-down assay qhia txog kev sib cuam tshuam ncaj qha ntawm Nws-EIJ1 thiab GST-EDS1 fusion proteins hauv vitro. Nws-EIJ1 cov proteins tau tsim nrog immobilized GST lossis GST-EDS1 protein. Immunoprecipitated fractions tau kuaj pom los ntawm cov tshuaj tiv thaiv-Nws thiab cov tshuaj tiv thaiv-GST, feem. (D) Co-IP qhia kev sib cuam tshuam ntawm EIJ1 thiab EDS1 hauv nplooj ntawm Arabidopsis. Tag nrho cov proteins muab rho tawm los ntawm 3-lub lim tiam-hnub nyoog 35Spro: EIJ1-6HA (raws li kev tswj genotype) thiab 35Spro: EIJ1-6HA 35Spro: EDS1-3FLAG nplooj tau immunoprecipitated los ntawm xws li anti-FLAG antibody los yog preimmune serum (IgG). Cov coimmunoprecipitated proteins tau kuaj pom siv cov tshuaj tiv thaiv FLAG thiab cov tshuaj tiv thaiv HA, feem. Cov txiaj ntsig sawv cev tau nthuav tawm hauv (B–D) tau rov ua nws tus kheej peb zaug.
Kev tsim cov hydrogen peroxide (H2O2), ib hom reactive oxygen hom, yog lub cim ntawm kev ua tiav ntawm kev paub txog cov kab mob thiab kev ua kom cov nroj tsuag tiv thaiv cov lus teb, suav nrog HR-txog programmed cell tuag (Lam et al., 2001; Torres et al., 2006; Nanda et al., 2010). Yog li ntawd, peb tau soj ntsuam qib H2O2 hauv 3-lub lim tiam-laus kab mob-inoculated Col-0, eij1-1, thiab eij1-1 EIJ1pro: EIJ1-6HA cov nroj tsuag siv 3, 3-diaminobenzidine (DAB) txoj kev staining. Cov txiaj ntsig tau pom tias H2O2 tau nce mus rau qib siab hauv cov nroj tsuag muaj kab mob tab sis tsis pom nyob rau hauv cov nroj tsuag uas tsis muaj tshuaj lom (mock) (Daim duab 3F). Ib qib siab ntawm H2O2 tau kuaj pom hauv eij1-1 nplooj ntau dua li hauv nplooj ntawm Col- 0 thiab cov kab txuas ntxiv inoculated nrog Pst DC3000 (Daim duab 3F). Tsis tas li ntawd, thaum trypan-xiav staining qhia tias kev tuag ntawm tes (microHR, Alvarez li al., 1998) tshwm sim hauv eij1-1 tawm ntawm 24-h post-inoculation (HPI) nrog Pst DC3000, tab sis tsis muaj microHR tau pom nyob rau hauv Col-0 thiab eij1-1 EIJpro1:EIJ1-6HA cov nroj tsuag (Daim duab 3G). Inoculation nrog avirulent Pst DC3000 (AvrRps4) ua rau muaj ntau dua ntawm H2O2 thiab ntau microHR, txawm hais tias tsis pom qhov txawv ntawm peb genotypes (Daim duab 3, F, thiab G). Cov txiaj ntsig no tau hais txog lub luag haujlwm tseem ceeb ntawm EIJ1 hauv basal tiv thaiv kab mob.
Daim duab 2 Kev nthuav qhia tus qauv ntawm EIJ1 mRNA thiab cov protein. (A) Chloroplast thiab nucleus localization ntawm EIJ1. Ib qho EIJ1-mCherry fusion protein tau nthuav tawm nyob rau hauv N. benthamiana nplooj thiab tshuaj xyuas los ntawm confocal microscopy. DAPI fluorescence qhia lub nucleus. Chlorophyll fluorescence yog qhia raws li xim ntsuab. Bar=10 lm. (B) GUS staining qhia cov lus qhia ntawm EIJ1 hauv cov hauv paus hniav, stems, rosette nplooj, paj, siliques, thiab 10-hnub qub seedlings (los ntawm sab laug mus rau sab xis). Bar=1 hli. (C) Kev nthuav qhia qauv ntawm EIJ1 nyob rau hauv ntau cov ntaub so ntswg ntawm Col nroj tsuag nrog rau cov hauv paus hniav, stems, rosette nplooj, paj, thiab siliques, raws li pom los ntawm qRT-PCR. Tus txheeb ze qhia tau suav los ntawm normalizing qib transcription ntawm cov tshuaj ntsuam gene tiv thaiv ntawm ACTIN2 (ib yam li hauv qab no). Cov ntaub ntawv yog qhov nruab nrab ± SD ntawm peb cov khoom siv roj ntsha. Cov tsiaj ntawv me qhia qhov sib txawv tseem ceeb (ib-txoj kev ANOVA, P 5 0.01; Cov Ntaub Ntawv Ntxiv 1). (D) Kev tsom xam ntawm EIJ1 siv 3-lub lim tiam Col nroj tsuag dip-inoculated nrog Pst DC3000 (OD600=0.1) los yog txau nrog 0.05 mM SA. Cov ntaub ntawv yog qhov txhais tau tias ± SD ntawm peb lub bio replicates (Cov ntaub ntawv ntxiv 1). (E-G) GUS staining qhia qhov induction ntawm EIJ1 qhia los ntawm cov kab mob. Peb lub lis piam pEIJ1:EIJ1-GUS cov nroj tsuag tau raug thuam inoculated (10 mM MgCl2; E) los yog dip-inoculated nrog Pst DC3000 (OD600=0.1; F). Bar=1 hli. Daim duab loj qhia tias EIJ1-GUS concentrates ntawm thaj tsam trichome hauv (G). Bar=100 lm. Nplooj ntawm 4 hpi tau sau thiab incubated nrog GUS staining tov. (H) thiab (I) Kev nthuav qhia tus qauv ntawm EIJ1 tom qab Pst DC3000 lossis SA kev kho mob. Tag nrho cov proteins tau muab rho tawm los ntawm 3-lub lim tiam-hnub 35Spro: EIJ1-6HA cov nroj tsuag dip inoculated nrog Pst DC3000 (OD600=0.1; H) los yog txau nrog 0.05 mM SA (I), thiab kuaj pom siv cov tshuaj tiv thaiv HA antibody. Cov vaj huam sib luag hauv qab qhia staining nrog Coomassie Brilliant Blue (CBB) raws li kev tswj kev thauj khoom. Cov tshuaj tiv thaiv immunoblot tau nthuav tawm hauv (H) thiab (I) tau rov ua nws tus kheej peb zaug.
Daim duab 3 Kev txheeb xyuas ntawm Arabidopsis EIJ1 mutated allele eij1-1. (A) Schematic daim duab qhia T-DNA qhov chaw nkag hauv eij1-1 (SALK_142975). Exons yog sawv cev los ntawm lub thawv thiab introns los ntawm kab ntawm lub thawv. Cov thawv dub nthuav qhia thaj tsam EIJ1 coding, thiab lub thawv dawb sawv cev rau thaj tsam tsis tau txhais. Daim duab peb sab qhia qhov chaw nkag ntawm T-DNA. (B) PCR amplification ntawm genomic DNA lees paub tias eij1-1 yog homozygous insertion mutant. Cov primers siv (P1, P2) yog qhia hauv (A). LB qhia txog T-DNA sab laug ciam teb primer thiab M qhia cov cim DNA molecular. (C) qRT-PCR tsom xam ntawm EIJ1 qhia. Tag nrho RNA raug rho tawm los ntawm 3-lub lim tiam-laus eij1 thiab Col nplooj. Cov kev txheeb ze ntawm EIJ1 hauv eij1-1 tau teeb tsa rau 1. Cov ntaub ntawv yog qhov nruab nrab ± SD ntawm peb cov khoom siv roj ntsha (Cov ntaub ntawv ntxiv 1). (D) Tus kab mob tiv thaiv phenotype ntawm Col, eij1-1, thiab ob tus neeg sawv cev EIJ1 ntxiv cov kab transgenic eij1 EIJ1pro: EIJ1-6HA. Cov nroj tsuag muaj hnub nyoog peb lub lis piam tau dip-inoculated nrog Pst DC3000 (OD600=0.05). Cov nplooj pom tau yees duab ntawm 5 dpi. Bars=5 hli. (E) Cov kab mob loj hlob ntawm Pst DC3000 ntawm cov nroj tsuag tau piav qhia hauv (D) ntawm 0 thiab 5 dpi. cfu / g, colony tsim units ib g tshiab hnyav. Cov ntaub ntawv yog qhov nruab nrab ± SD ntawm peb cov khoom siv roj ntsha. Cov tsiaj ntawv me qhia qhov sib txawv tseem ceeb (ib-txoj kev ANOVA, P 5 0.01, Cov Ntaub Ntawv Ntxiv 1). (F) Kev kuaj H2O2 qib hauv nplooj ntawm Col,
EIJ1 kho cov nroj tsuag tiv thaiv kab mob los ntawm kev tawm tsam EDS1 kev ua
Kom nkag siab txog kev sib raug zoo ntawm EDS1 thiab EIJ1, peb tsim eij1-1 eds1-22 ob kab mutant los ntawm hla eij1-1 mutant nrog eds1-22, poob-ntawm- muaj nuj nqi mutant allele ntawm EDS1-90 (AT3G48090; Col{11}} keeb kwm yav dhau), uas nthuav tawm cov kab mob tsis zoo (Yang and Hua, 2004). Tsis muaj qhov sib txawv morphological tau pom ntawm eij1-1, eds1-22, thiab eij1-1 eds1-22 mutant nroj tsuag nyob rau hauv ib txwm loj hlob tej yam kev mob (Daim duab 4, A thiab B). Tom qab inoculation nrog Pst DC3000, lub eij1-1 cov nroj tsuag mutant tso tawm kom muaj zog tiv taus tshaj Col-0 cov nroj tsuag ntawm 5 dpi (Daim duab 4, A thiab B). Txawm li cas los xij, eij1-1 eds1-22 ob chav mutant nthuav tawm cov kab mob necrotic zoo kawg nkaus thiab cov kab mob siab titer, piv nrog eds 1-22 ib leeg mutant, qhia tias eij1-1 allele tsis tuaj yeem los cawm tus kab mob uas raug phenotype ntawm eds1-22 (Daim duab 4, A thiab B). Kev tiv thaiv kab mob kuj tau raug soj ntsuam tom qab lub siab infiltration ntawm Pst DC3000, thiab tau txais cov txiaj ntsig zoo sib xws (Cov duab ntxiv 4B). Tsis tas li ntawd, peb siv EDS1 null allele, eds1-2; Col-0 keeb kwm; Bartsch li al., 2006), los tsim cov eij1-1 eds1-2 ob chav mutant nroj tsuag rau kev tshuaj ntsuam genetic. Zoo ib yam li eij1-1 eds1-22, lub eij1-1 eds1-2 ob chav mutant nroj tsuag kuj pom tau zoo kawg li necrotic lesions thiab cov kab mob siab dua li eij1-1, uas yog piv nrog cov eds 1-2 ib leeg mutant (Cov duab ntxiv 6). Cov pov thawj caj ces no qhia tias EDS1 yog epistatic rau EIJ1. Raws li cov kev soj ntsuam saum toj no, H2O2 tau nce mus rau qib siab hauv eij1-1 nplooj tab sis mus rau qib qis hauv eds1- 22 thiab eij1-1 eds1-22 nplooj, piv nrog Col{{ 53}} nplooj, raws li Pst DC3000 inoculation (Daim duab 4C). Tsis tas li ntawd, microHR hauv eij1-1 tau ua kom pom tseeb los ntawm kev poob-ntawm-kev ua haujlwm ntawm EDS1 ntawm 24 hpi nrog Pst DC3000 lossis avirulent Pst DC3000 (AvrRps4; Daim duab 4D). Raws li qhov tshwm sim no, cov lus qhia theem ntawm ICS1 thiab PR1 hauv eij1-1 eds1-22 thiab eds1-22 cov nroj tsuag tau sib piv tab sis qis dua cov hauv eij1-1 thiab Col{ {71}} cov nroj tsuag (Daim duab 4E). Cov txiaj ntsig no txhawb nqa tias EIJ1- cov lus teb nruab nrab ntawm lub cev tiv thaiv kab mob yog nyob ntawm EDS1. Txhawm rau tshawb xyuas lub luag haujlwm ntawm EIJ1-EDS1 kev cuam tshuam hauv cov nroj tsuag tiv thaiv kab mob, peb tau ua RNA-seq tsom xam ntawm eij1-1, eds1-22, thiab Col-0 tawm ntawm 24 hpi nrog Pst DC3000 ; nrog uninoculated Col-0 nplooj tau siv los ua kev kho mob. Tag nrho ntawm 2762, 923, thiab 622 qhov sib txawv ntawm cov noob (DEGs) raug txheeb xyuas hauv Col_Pst DC3000 piv rau Col_mock, eij1-1_Pst DC3000 piv rau Col_ Pst DC3000, thiab eds1-22_Pst DC3000 piv rau Col_Pst DC3000 kev sib piv. Cov noob no tom qab ntawd raug xa mus rau raws li kab mob teb, EIJ1- tswj hwm, thiab EDS{100}} tswj cov noob, raws li (Cov duab ntxiv 7A thiab Cov Ntaub Ntawv Teeb 2). Ntawm cov noob no, 471 tau tswj hwm los ntawm EIJ1; 396 tau tswj hwm los ntawm EDS1 (Daim duab 4F; Cov Ntaub Ntawv Ntxiv Ntxiv 2); thiab 124 tau raug tswj los ntawm EIJ1 thiab EDS1 (Daim duab 4F). Kev txheeb xyuas pawg pom tau tias 93.5% (116) ntawm cov noob sib koom ua ke (124) tau nce- tswj los ntawm EDS1 thiab qis-tswj los ntawm EIJ1 (Daim duab 4F; Ntxiv Daim duab 7B thiab Cov Ntaub Ntawv Teeb 2), txhawb nqa lub luag haujlwm ntawm EIJ1 ntawm EDS1 ua haujlwm. Gene ontology (GO) tsom xam ntxiv tau qhia tias feem ntau ntawm cov noob tswj kev tiv thaiv tsis zoo los ntawm EIJ1 thiab EDS1 feem ntau koom nrog hauv cov lus teb kev ntxhov siab thiab cov lus teb hauv lub cev tsis muaj zog (Daim duab 4G; Cov lus ntxiv S1). Cov kev ntsuam xyuas no qhia tias EIJ1 thiab EDS1 tswj cov noob caj ces muaj feem cuam tshuam rau qhov sib txawv thaum kis kab mob. Ib feem ntau ntawm EDS{135}} tswj cov noob (272/396) tsis tau tswj hwm los ntawm EIJ1, tej zaum vim yog qhov hloov pauv qis dua (52) lossis EIJ1- ywj siab ua haujlwm ntawm EDS1.
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EIJ1 cuam tshuam nrog EDS1 hauv cytoplasm thiab inhibits kev lag luam nuclear ntawm EDS1
EIJ1 yog nyob rau hauv lub chloroplast thiab nucleus ntawm nplooj hlwb (Daim duab 2A), whereas EDS1 protein muaj ib tug nucleocytoplasmic tis qauv, tshwm sim nyob rau hauv ob qhov chaw (Garcı'a li al., 2010). Txhawm rau tshawb xyuas qhov twg thiab yuav ua li cas EIJ1 cuam tshuam nrog EDS1 hauv planta, peb tau ua qhov kev ntsuam xyuas ib ntus siv Arabidopsis protoplasts. Interestingly, txawm hais tias EIJ1 tuav nws tus qauv subcellular localization qauv nyob rau hauv chloroplast thiab nucleus nyob rau hauv lub xub ntiag ntawm ntsuab fluorescent protein (GFP; tswj), chloroplast-localized EIJ1-mCherry tau diffused nyob rau hauv lub cytoplasm thaum coexpressed nrog EDS{ {9}}GFP nyob rau hauv protoplasts (Daim duab 5A, saib replicates hauv Cov Ntaub Ntawv Ntxiv 2). Qhov tshwm sim no tau lees paub nyob rau hauv N. benthamiana epidermal hlwb, nyob rau hauv uas EIJ1-mCherry tau qhia ib ntus nrog EDS1-GFP lossis GFP tswj (Daim duab ntxiv 8, saib cov ntawv txuas ntxiv hauv Cov Ntaub Ntawv Ntxiv 2) . Cov kev soj ntsuam no qhia tias EDS1 overexpression tuaj yeem ua rau subcellular relocalization ntawm EIJ1. Tom ntej no, txhawm rau txiav txim siab qhov chaw subcellular qhov twg EDS1 thiab EIJ1 cuam tshuam, peb tau ua qhov kev ntsuam xyuas rub tawm siv cov recombinant Glutathione S-Transferase (GST)-EDS1 fusion protein (bait) thiab tag nrho cov protein extract, nuclei-depleted fractions, thiab nuclei- enriched feem ntawm 35Spro: EIJ1-HA transgenic kab (hnyav proteins). Peb cov txiaj ntsig tau pom tias EDS1 immunoprecipitated nrog EIJ1-HA thaum siv tag nrho cov protein extract thiab nuclei-depleted feem ntawm 35Spro: EIJ1-HA nplooj, tab sis nws tsis tau thaum siv cov nuclei-enriched feem (Daim duab 5B), qhia tias kev sib cuam tshuam ntawm EIJ1 thiab EDS1 tshwm sim hauv cytoplasm.
Daim duab 4 EIJ1 thiab EDS1 oppositely tswj cov nroj tsuag tsis kam mus rau cov kab mob. (A) The disease resistance phenotype of Col, eij1-1, eds1-22, and eij1-1 eds1-22. Cov nroj tsuag muaj hnub nyoog peb lub lis piam tau dip-inoculated nrog Pst DC3000 (OD600=0.05). Cov nplooj pom tau yees duab ntawm 5 dpi. Bar=5 hli. (B) Cov kab mob loj hlob ntawm Pst DC3000 ntawm cov nroj tsuag tau piav qhia hauv (A) ntawm 0 thiab 5 dpi. Cov ntaub ntawv yog qhov nruab nrab ± SD ntawm peb cov khoom siv roj ntsha. Cov tsiaj ntawv me qhia qhov sib txawv tseem ceeb (ib-txoj kev ANOVA, P 5 0.01, Cov Ntaub Ntawv Ntxiv 1). (C) Kev kuaj H2O2 qib hauv Col, eij1-1, eds1-22, thiab eij1-1 eds1-22 nroj tsuag. Cov nroj tsuag muaj hnub nyoog peb lub lis piam tau dip-inoculated nrog Pst DC3000 los yog Pst DC3000 (AvrRps4; OD600=0.1), thiab ces stained nrog DAB ntawm 24 hpi. Bar=500 lm. (D) Cell death analysis of Col, eij1-1, eds1-22, and eij1-1 eds1-22 nroj tsuag. Peb-lub lis piam cov nroj tsuag tau dipinoculated nrog Pst DC3000 los yog Pst DC3000 (AvrRps4; OD600=0.1), thiab tom qab ntawd stained nrog trypan xiav ntawm 24 hpi. Bar=500 lm.
Muab hais tias EIJ1 tsis zoo tswj cov nroj tsuag tiv thaiv kab mob hauv EDS1-txoj kev nyob ntawm (Daim duab 4), tom ntej no peb tau soj ntsuam cov txiaj ntsig ntawm cov kab mob ntawm kev sib cuam tshuam ntawm EIJ1 thiab EDS1. Kuj ceeb tias, Pst DC3000 tus kab mob txhim kho EDS1-EIJ1 kev sib cuam tshuam hauv cytoplasm es tsis yog nyob rau hauv ob qho tib si chloroplast thiab nucleus (Daim duab 5C, saib replicates hauv Supplemental File 2). Qhov kev tshawb pom no, ua ke nrog cov qauv nucleo-cytoplasmic faib ntawm EDS1 thiab lub luag haujlwm ntawm EDS1 nuclear localization nyob rau hauv kev txhawb zog ntawm cov kab mob tiv thaiv ntawm qib transcriptional (Garcı'a li al., 2010), yuam peb los tshawb xyuas lub luag haujlwm ntawm EIJ1 hauv subcellular lag luam ntawm EDS1 thaum lub sij hawm kab mob. Thaum peb tau hais tawm ib ntus EDS1-3FLAG hauv Arabidopsis protoplasts cais tawm ntawm eij1-1, EIJ1 overexpression, thiab Col-0 cov nroj tsuag, EIJ1 tshwj xeeb tshaj yog txwv kev lag luam ntawm EDS1 rau hauv lub nucleus (Cov duab ntxiv 9) . Tsis tas li ntawd, kev tshuaj ntsuam immunoblot qhia me ntsis qhov txawv ntawm qhov ntau ntawm tag nrho EDS1 protein ntawm eds1-22 EDS1pro: EDS1-3FLAG thiab eij1-1 eds1-22 EDS1pro: EDS{{30} }FLAG transgenic nroj tsuag (Daim duab 5D, saib replicates hauv Supplemental File 2). Txawm li cas los xij, piv nrog eds1-22 EDS1pro: EDS1-3FLAG transgenic nroj tsuag, Pst DC3000- inoculated eij1-1 eds1-22 EDS1pro: EDS13FLAG nroj tsuag pom tau tias muaj kev nce ntxiv nyob rau hauv cov theem ntawm nuclei-enriched EDS1 protein thiab txo cov theem ntawm cytoplasm-localized EDS1 (Daim duab 5D, saib replicates hauv Supplemental File 2), lees paub lub luag hauj lwm ntawm EIJ1 hauv cytoplasm-rau nucleus kev lag luam ntawm EDS1. Tom ntej no, peb tau sim seb qhov kev faib tawm tshiab no puas tau kho los ntawm EIJ1-EDS1 kev cuam tshuam cuam tshuam rau kev hloov pauv ntawm cov noob muaj feem cuam tshuam hauv cov nroj tsuag. Lub 35Spro: EIJ1-mCherry thiab 35Spro: EDS1-3FLAG effector constructs tau hloov ib tus zuj zus los yog ua ke nrog cov neeg sau xov xwm tsim ua N. benthamiana nplooj epidermal hlwb (Daim duab 5E). Ntawm 24 hpi nrog Pst DC3000, EDS1 tau qhib qhov kev qhia ntawm PR1 thiab ICS1 (Daim duab 5F), thaum EIJ1 tau txwv tsis pub EDS1-induced gene expression (Daim duab 5F). Tsis zoo li tag nrho-ntev EIJ1, N-terminal kawg ntawm EIJ1 protein, uas tsis muaj kev cuam tshuam hauv eij1-1, tsis tuaj yeem cuam tshuam EDS1- induced PR1 qhia (Cov duab ntxiv 10, A thiab B. ), qhia tau hais tias cov qauv ntawm cov protein tsis zoo ntawm EIJ1 yog qhov tsim nyog rau nws txoj kev ua haujlwm tsis zoo. Ua ke, cov txiaj ntsig no txhawb nqa tias EIJ1 ua haujlwm raws li qhov tseem ceeb ntawm kev tawm tsam hauv EDS1- tawm tsam kev tawm tsam los ntawm kev tswj hwm kev lag luam nucleocytoplasmic ntawm EDS1. Vim tias cov protein EIJ1 tau maj mam degraded hauv Pst DC3000-inculated los yog SA-kho cov nroj tsuag tom qab ib tug sai pib induction (Daim duab 2, H thiab kuv), peb tau soj ntsuam seb EDS1, nyob rau hauv lem, ua rau degradation ntawm EIJ1 ntawm ib protein-protein kev sib raug zoo. Cell-free degradation assays and in vivo protein detection assays using eds1-22 35Spro: EIJ1-HA transgenic nroj tsuag qhia tias tsis muaj qhov tsis muaj lossis tsis muaj EDS1 cuam tshuam rau EIJ1 degradation (Cov duab ntxiv 11), qhia tias EDS1 tsis koom nrog cov kab mob ua rau kev puas tsuaj ntawm EIJ1.
Cov txiaj ntsig ntawm cistanche tubulosa- ua kom muaj zog tiv thaiv kab mob
Kev kis kab mob hloov pauv subcellular localization ntawm EIJ1
Kev txhim kho kev sib raug zoo ntawm EIJ1 thiab EDS1 hauv cytoplasm raws li kab mob inoculation (Daim duab 5, B, thiab C) qhia txog qhov ua tau ntawm cov kab mob tshwm sim ntawm EIJ1 hauv cov nroj tsuag. Txhawm rau ntsuas qhov muaj peev xwm no, peb tau tshuaj xyuas cov subcellular localization ntawm EIJ1 hauv N. benthamiana nplooj inoculated nrog Pst DC3000. Nyob rau hauv cov xwm txheej tsis muaj kab mob, EIJ1-mCherry tau nyob hauv thaj chaw rau chloroplast thiab nucleus. Txawm li cas los xij, ntawm 24 dpi, cov teeb liab fluorescence tau txo qis hauv cov chloroplasts thiab tau txhim kho hauv cytoplasm (Daim duab 6A). Txhawm rau txiav txim siab ntxiv seb cov kab mob ua rau kev tsis sib haum xeeb ntawm EIJ1 los ntawm chloroplast mus rau cytoplasm hauv nplooj, immunoblot tsom xam ntawm EIJ1 protein tau ua los ntawm 35Spro: EIJ1-6HA transgenic nroj tsuag. Tag nrho cov proteins, chloroplast proteins, thiab cytoplasmic proteins raug cais los ntawm 35Spro: EIJ1-6HA transgenic nroj tsuag ntawm 0 thiab 4 hpi nrog Pst DC3000. Qhov kev nplua nuj ntawm EIJ1 tau nce ntau ntxiv hauv cytoplasmic feem tab sis txo qis hauv chloroplast feem ntawm 24 dpi (Daim duab 6B, saib cov ntawv luam hauv Cov Ntaub Ntawv Ntxiv 2), qhia txog qhov tsis zoo ntawm EIJ1 cov protein ntau hauv chloroplast thiab cytoplasm.
Daim duab 5 EIJ1 cuam tshuam nrog EDS1 hauv cytoplasm los kho cov kev lag luam nuclear ntawm EDS1. (A) Kev soj ntsuam kev hloov pauv hloov pauv qhia qhov chaw ntawm EDS1 thiab EIJ1 hauv Arabidopsis protoplasts. Bar=10 lm. (B) Pull-down assay ntawm kev sib cuam tshuam ntawm EIJ1 thiab EDS1. Tag nrho cov proteins, nuclei-depleted fractions, thiab nuclei-enriched fractions rho tawm los ntawm 3-lub lim tiam-laus eij1-1 35Spro: EIJ1-6HA transgenic nroj tsuag tau incubated nrog immobilized GST-EDS1 los yog GST proteins , raws. IPs raug kuaj pom siv cov tshuaj tiv thaiv HA lossis anti-GST cov tshuaj tiv thaiv. PEPC thiab histone H3 tau siv los ua cov cim cytoplasmic thiab nuclear, feem. Asterisks qhia cov kab ntawm GST-EDS1 proteins. (C) Pst DC3000 kab mob txhim kho cov protein sib cuam tshuam ntawm EDS1 thiab EIJ1 hauv cytoplasm. Tag nrho cov proteins, nuclei-depleted fractions, nuclei-enriched fractions, thiab chloroplast proteins raug rho tawm los ntawm 3-lub lim tiam EIJ1pro: EIJ1-6HA 35Spro: EDS1-3FLAG nroj tsuag dip-inoculated nrog Pst DC3000 (OD600=0.1) los yog mock-inoculated ntawm 4 hpi, thiab immunoprecipitated siv cov tshuaj tiv thaiv FLAG antibody los yog preimmune serum (IgG). Cov co-immunoprecipitated proteins tau kuaj pom siv cov tshuaj tiv thaiv HA thiab anti-FLAG antibodies. N-depleted, nuclei-depleted feem; N-enriched, nuclei-enriched feem. PEPC thiab histone H3 tau siv los ua cov cim cytoplasmic thiab nuclear, feem. (D) Immunoblot tsom xam qhia tau hais tias EIJ1 kho cov cytoplasmic piv rau nuclear kev faib tawm ntawm EDS1. Tag nrho cov proteins, nuclei-depleted fractions, thiab nuclei-enriched fractions raug rho tawm los ntawm 3-lub lim tiam-laus eds1-22 EDS1pro: EDS1- 3FLAG thiab eij1-1 eds{{59 }} EDS1pro: EDS1-3FLAG transgenic nroj tsuag dip-inoculated nrog Pst DC3000 (OD600=0.1) los yog mock-inoculated ntawm 4 hpi, thiab kuaj pom siv anti-FLAG antibody. Qhov txheeb ze ntawm cov proteins los ntawm eds1-22 EDS1pro: EDS1-3FLAG nrog cov kev kho mob mock inoculation teem rau 1. M, thuam; P, Pst DC 3000. PEPC thiab histone H3 tau siv los ua cov cim cytoplasmic thiab nuclear, feem. Cov txiaj ntsig tau nthuav tawm hauv (A), (C), thiab (D) tau rov ua nws tus kheej peb zaug (Cov Ntaub Ntawv Ntxiv 2). (E) thiab (F) Kev soj ntsuam ib ntus ntawm PR1 thiab ICS1 kev txhawb nqa kev ua haujlwm tswj hwm los ntawm EIJ1 thiab EDS1. Ntau yam kev tsim siv nyob rau hauv qhov kev ntsuam xyuas kev hloov pauv tau qhia hauv (E). PR1pro: GUS los yog ICS1pro: GUS tau cotransformed nrog effectors los yog lub khoob vector (Control) rau hauv N. benthamiana nplooj spayed nrog Pst DC3000 (OD600=0.1) rau 24 teev. Kev txheeb ze GUS kev ua (GUS/Luciferase) uas qhia txog theem ntawm PR1 thiab ICS1 tswj hwm los ntawm ntau yam cuam tshuam tau qhia hauv (F). Cov ntaub ntawv yog qhov txhais tau tias ± SD ntawm peb lub bio replicates (Cov ntaub ntawv ntxiv 1). Cov tsiaj ntawv me qhia qhov sib txawv tseem ceeb (ib-txoj kev ANOVA, P 5 0.01).
Pathogen-triggered EIJ1 dissociation los ntawm chloroplast qhia tias muaj peev xwm ua haujlwm ntawm EIJ1 hauv cytoplasm. Txhawm rau kuaj qhov muaj peev xwm no, peb immobilized EIJ1 protein rau chloroplasts los ntawm kev txuas cov chloroplast-targeting sequence (CTS; Lee li al., 2008) mus rau N-terminal kawg ntawm EIJ1-mCherry reporter gene. CTS-EIJ1-mCherry fusion tau tshwm sim nyob rau hauv N. benthamiana nplooj nyob rau hauv kev tswj ntawm haiv neeg txhawb nqa. Fluorescence tau kuaj pom hauv cov chloroplasts nkaus xwb, tab sis tsis nyob hauv cytosol thiab nucleus ntawm Pst DC3000-inoculated N. benthamiana nplooj (Cov duab ntxiv 12A). Qhov kev soj ntsuam no tau lees paub tias CTStagged EIJ1 tau khaws cia hauv chloroplast thiab tsis raug yuam mus rau hauv cytoplasm los ntawm kab mob. Peb tom qab tsim EIJ1pro: CTS-EIJ1-6HA transgenic Arabidopsis kab los soj ntsuam cov kev ua haujlwm lom neeg ntawm CTS-EIJ1 hauv cov nroj tsuag tiv thaiv kab mob. Raws li qhov xav tau, tsis zoo li eij1-1 EIJ1pro: EIJ1-6HA, the eij1-1 EIJ1pro: CTS-EIJ1-6HA kab qhia tsis muaj qhov sib txawv tseem ceeb hauv kev tiv thaiv kab mob piv nrog eij1-1, qhia tias EIJ1 immobilized hauv chloroplast tsis tuaj yeem cawm tus kab mob tiv thaiv phenotype ntawm eij1-1 (Daim duab 6C). Raws li qhov kev soj ntsuam no, cov lus qhia ntawm PR1 tau khaws cia nyob rau theem siab hauv eij1-1 EIJ1pro: CTSEIJ1-6HA transgenic nroj tsuag, zoo ib yam li eij1-1 (Cov duab ntxiv 12B). Tsis tas li ntawd, CTS-EIJ1 pom muaj peev xwm txo qis EDS1 kev hloov pauv kev ua haujlwm dua li EIJ1 (Daim duab 6, D, thiab E). Cov txiaj ntsig no tau ua rau peb txiav txim siab tias tus tswv teb thaum ntxov rau cov kab mob kis tau ua rau subcellular relocalization ntawm EIJ1, uas nyob rau hauv lem modulates lub cev tiv thaiv kab mob los ntawm repressing kev lag luam ntawm EDS1 rau hauv lub nucleus.
EIJ1 tswj cov kev ua phem ua phem rau cov lus teb
Lub AvrRps4 effector protein ntawm P. syringae secreted los ntawm hom III secretion system tau lees paub los ntawm TIR-NB-LRR receptor RPS4 los pib ETI, uas yog nyob ntawm EDS1 (Gassmann li al., 1999; Wirthmueller li al., 2007; Heidrich et al., 2012). Peb inoculated eij1-1 tawm nrog avirulent Pst DC3000 (AvrRps4) lossis virulent Pst DC3000 los ntsuas seb EIJ1 puas koom nrog ETI. Cov eij1-1 nplooj tau pom muaj zog tiv thaiv kab mob Pst DC3000 ntau dua li Col-0 nplooj (Daim duab 7A). Txawm li cas los xij, tsis muaj qhov sib txawv tseem ceeb ntawm Col-0 thiab eij{18}} tawm hauv kev tawm tsam rau avirulent Pst DC3000 (AvrRps4), txawm hais tias ob qho tib si genotypes pom muaj zog tiv thaiv avirulent Pst DC3000 (AvrRps4) dua li Pst DC3000 ( Daim duab 3, F thiab G; Daim duab 7, A), qhia tias EIJ1 yuav tsis tsim nyog rau RPS4- ntxias ETI. Peb kuj tau txiav txim siab qhov kev tiv thaiv ntawm cov nroj tsuag rau Pst DC3000 hrcC, uas ua rau muaj qhov tsis zoo ntawm hom III zais cia thiab tsis tuaj yeem txhaj cov tshuaj tiv thaiv rau hauv cov hlwb los tiv thaiv PTI (Gassmann li al., 1999; Gloggnitzer li al., 2014). Ob lub eij1-1 mutant thiab Col-0 nplooj tau pom zoo sib xws rau Pst DC3000 hrcC (Cov duab ntxiv 13), qhia tias EIJ1 tsis koom nrog PTI.
Muab hais tias lub nuclear tsub zuj zuj ntawm EDS1 yog indispensable rau TIR-NB-LRR-induced reprogramming ntawm kev tiv thaiv noob qhia thiab tsis kam (Garcı'a li al., 2010), peb tau tshuaj xyuas seb EIJ1 tswj cov subcellular faib. ntawm EDS1 teb rau ETI. Nplooj ntawm 3-lub lim tiam-laus eds1-22 EDS1pro: EDS1-3FLAG thiab eij1-1 eds1-22 EDS1pro: EDS1-3FLAG nroj tsuag tau inoculated nrog Pst DC3000 lossis Pst DC3000 (AvrRps4), thiab cov nuclei-depleted thiab nuclei-enriched fractions raug rho tawm ntawm 0 thiab 4 hp. Raws li txoj kev tshawb fawb yav dhau los (Garcı'a li al., 2010), qhov nce hauv nuclear localization ntawm EDS1 tau pom nyob rau hauv eds1-22 EDS1pro: EDS1-3FLAG nroj tsuag inoculated nrog Pst DC3000 (AvrRps4), tab sis tsis nyob rau hauv cov inoculated nrog Pst DC3000. Txawm li cas los xij, qhov poob-ntawm-kev ua haujlwm ntawm EIJ1 tsis cuam tshuam rau ETI-induced nuclear tsub zuj zuj ntawm EDS1 (Daim duab 7B; saib replicates hauv Supplemental File 2). Qhov no qhia tau hais tias tus avirulent Pst DC3000 (AvrRps4) ua rau muaj kev lag luam ntawm EDS1 rau hauv lub nucleus los ntawm kev hla EIJ1 lossis tshem tawm txoj haujlwm ntawm EIJ1 yam tsis paub. Txhawm rau ntsuas qhov muaj peev xwm no, peb tau tshuaj xyuas cov txiaj ntsig ntawm avirulent Pst DC3000 (AvrRps4) ntawm qib EIJ1. Zoo ib yam li cov txiaj ntsig tau txais kev siv EIJ1 cov kab ntau dhau (Daim duab 2H), lub eij1-1 EIJ1pro: EIJ1-6HA cov nroj tsuag inoculated nrog Pst DC3000 pom tias nce EIJ1 tsub zuj zuj ntawm theem pib ntawm tus kab mob, txawm tias EIJ1 tau maj mam. degraded tom qab 8 hpi (Daim duab 7C; saib replicates hauv Supplemental File 2). Kev sib sau ntawm EIJ1 txaus los tiv thaiv Pst DC3000- ua rau muaj kev lag luam ntawm EDS1 rau hauv lub nucleus thaum pib kis kab mob (Daim duab 5, D, thiab 7, B). Raws li qhov tshwm sim no, qhov poob-ntawm-kev ua haujlwm ntawm EIJ1 tau nrawm rau kev hloov pauv ntawm lub zog ntawm EDS1 hauv cov nroj tsuag muaj kab mob (Cov duab ntxiv 14). Hauv qhov sib piv, Pst DC3000 (AvrRps4) tshem tawm EIJ1 kev ua haujlwm los ntawm kev ua kom EIJ1 protein degradation sai sai, tab sis tsis yog los ntawm kev tawm tsam EIJ1 cov noob hloov pauv (Daim duab 7C; Cov duab ntxiv 15), yog li txhawb nqa ntau dua nuclear kev hloov pauv ntawm EDS1 thaum ntxov ntawm kev kis kab mob (Daim duab 7C; ). Qhov kev soj ntsuam uas eij1-1 cov nroj tsuag inoculated nrog Pst DC3000 nthuav tawm tsis kam piv rau Col-0 thiab eij1-1 cov nroj tsuag inoculated nrog avirulent Pst DC3000 (AvrRps4) txhawb qhov kev xav no. Qhov no yuav piav qhia, yam tsawg kawg rau qee qhov, yog vim li cas tus kab mob avirulent, tab sis tsis yog tus kab mob phem, tuaj yeem txhim kho qhov kev tiv thaiv ntxiv los ua rau ETI cov lus teb. Yog li, cov kev soj ntsuam no qhia tias EIJ1 muaj feem cuam tshuam nrog kev ua phem ua rau cov lus teb hauv cov tswv tsev.
Daim duab 6 Relocalization ntawm EIJ1 mus rau cytoplasm yog tsim nyog rau EDS1- nyob ntawm qhov tsis kam. (A) Pst DC3000 tus kab mob hloov pauv subcellular localization ntawm EIJ1. EIJ1-mCherry tau qhia ib ntus hauv N. benthamiana nplooj spayed nrog Pst DC3000 (OD600=0.1) lossis mock-inoculated rau 24 teev. Bar=10 lm. (B) Pst DC3000 tus kab mob ua rau muaj kev cuam tshuam ntawm EIJ1 los ntawm chloroplast. Tag nrho cov proteins, cytoplasmic proteins, thiab chloroplastic proteins tau muab rho tawm los ntawm 3-lub lim tiam EIJ1pro: EIJ1-6HA cov nroj tsuag dip-inoculated nrog Pst DC3000 (OD600=0.1) los yog mock-inoculated ntawm 4 hpi thiab kuaj pom los ntawm cov tshuaj tiv thaiv HA antibody. PEPC, histone H3, thiab RbcL tau siv los ua cytoplasmic, nuclear, thiab chloroplastic markers, ntsig txog. Cov txheeb ze ntawm cov proteins nrog kev kho mob mock yog teem rau 1. Cov txiaj ntsig tau nthuav tawm hauv (A) thiab (B) tau rov ua nws tus kheej peb zaug (Cov Ntaub Ntawv Ntxiv 2). (C) Immobilized EIJ1 nyob rau hauv chloroplasts tsis tuaj yeem kho qhov tsis kam ntawm eij1-1. Cov nroj tsuag muaj hnub nyoog peb lub lis piam tau dip-inoculated nrog Pst DC3000 (OD600=0.05). Cov kab mob titer ntawm Pst DC3000 ntawm 0 thiab 5 dpi tau txiav txim siab. CTS, chloroplast localization signals. Cov ntaub ntawv yog qhov txhais tau tias ± SD ntawm peb lub bio replicates (Cov ntaub ntawv ntxiv 1). (D) thiab (E) Kev soj ntsuam ib ntus ntawm PR txhawb kev ua haujlwm tswj hwm los ntawm EIJ1, CTS-EIJ1, thiab EDS1. Ntau yam kev tsim siv nyob rau hauv qhov kev ntsuam xyuas kev hloov pauv tau qhia hauv (D). PR1pro: GUS tau co-transformed nrog effectors lossis lub khoob vector (Control) rau hauv N. benthamiana nplooj spayed nrog Pst DC3000 (OD600=0.1) rau 24 teev. Kev txheeb ze GUS kev ua (GUS/Luciferase) uas qhia txog theem qhia ntawm PR1 tswj los ntawm ntau yam cuam tshuam yog qhia hauv (E). Cov kab yuam kev sawv cev rau tus qauv sib txawv ntawm cov qauv kev ywj pheej triplicate. Cov ntaub ntawv yog qhov nruab nrab ± SD ntawm peb cov khoom siv roj ntsha. Cov tsiaj ntawv me qhia qhov sib txawv tseem ceeb (ib-txoj kev ANOVA, P 5 0.01, Cov Ntaub Ntawv Ntxiv 1).
Daim duab 7 EIJ1 thiab EDS1 modulate cog basal kuj. (A) Cov kab mob loj hlob ntawm Col thiab eij1-1 mutant. Cov nroj tsuag muaj hnub nyoog peb lub lis piam tau dip-inoculated nrog Pst DC3000 lossis Pst DC3000 (AvrRps4; OD600=0.05). Cov kab mob titer ntawm Pst DC3000 ntawm 0 thiab 5 dpi tau txiav txim siab. Cov ntaub ntawv yog qhov nruab nrab ± SD ntawm peb cov khoom siv roj ntsha. Cov tsiaj ntawv me qhia qhov sib txawv tseem ceeb (ib-txoj kev ANOVA, P 5 0.01, Cov Ntaub Ntawv Ntxiv 1). (B) EIJ1 tsis tshua muaj kev cuam tshuam rau Pst DC3000 (AvrRps4)-ua rau tsub zuj zuj ntawm EDS1 hauv nuclei. Nuclei-depleted fractions thiab nuclei-enriched fractions raug muab rho tawm los ntawm 3-lub lim tiam-eds1-22 EDS1pro: EDS1-3FLAG thiab eij1-1 eds1-22 EDS1pro: EDS1-3FLAG transgenic nroj tsuag dip inoculated nrog Pst DC3000 (OD600=0.1), Pst DC3000 (AvrRps4; OD600=0.1), los yog mock inoculated ntawm 4 hpi, thiab kuaj pom siv ib qho anti-FLAG antibody. PEPC thiab histone H3 tau siv los ua cov cim cytoplasmic thiab nuclear, feem. Tus txheeb ze siv cov proteins los ntawm cov nroj tsuag nrog kev kho mob mock yog teem rau 1. M, mock; P, Pst DC3000; R, Pst DC3000 (AvrRps4). (C) Pst DC3000 (AvrRps4) kab mob txhawb kev degradation ntawm EIJ1 protein. Tag nrho cov proteins tau muab rho tawm los ntawm 3-lub lim tiam eij1-1 EIJ1pro: EIJ1-6HA cov nroj tsuag dip-inoculated nrog Pst DC3000 lossis Pst DC3000 (AvrRps4; OD600=0.1) , thiab kuaj pom siv cov tshuaj tiv thaiv HA antibody. Lub vaj huam sib luag hauv qab qhia tau staining nrog Coomassie Brilliant Blue (CBB) raws li kev tswj kev thauj khoom. Cov txiaj ntsig ntawm tus neeg sawv cev tau nthuav tawm hauv (B) thiab (C) tau rov ua nws tus kheej ob zaug ntxiv thiab soj ntsuam nrog cov tshuaj tiv thaiv ACTIN antibody raws li kev tswj hwm kev thauj khoom (Cov Ntaub Ntawv Ntxiv 2). (D) Hypothetical qauv ntawm EIJ1 regulating EDS1-dependent plant basal resistance. Thaum cov nroj tsuag raug cuam tshuam los ntawm cov kab mob, EIJ1 raug tso tawm sai sai ntawm chloroplast mus rau hauv cytoplasm qhov twg nws cuam tshuam nrog EDS1. Kev sib cuam tshuam EIJ1-EDS1 tiv thaiv kev kaw ntawm EDS1 los ntawm cytoplasm mus rau hauv lub nucleus thiab yog li txwv tsis pub muaj kev tawm tsam ntawm EDS1, tso cai rau kev hloov pauv ntawm EDS1- cov lus teb tiv thaiv kab mob. Arrowed thiab blunted kab qhia ua kom muaj zog thiab kev tsim txom, feem.
Kev sib tham
Txhawm rau kom muaj sia nyob kom muaj sia nyob, cov nroj tsuag tau hloov kho cov txheej txheem meej rau kev txhim kho kev loj hlob thiab tiv thaiv cov kab mob nkag mus, txawm hais tias ntau cov ntsiab lus ntawm cov txheej txheem no tseem tsis meej. EDS1 tuav lub luag haujlwm tseem ceeb hauv cov nroj tsuag lub cev tiv thaiv kab mob hauv lub cev, vim nws tswj cov kab mob biotrophic thiab hemibiotrophic (Wiermer li al., 2005). Hauv txoj kev tshawb no, peb tau txheeb xyuas cov noob, EIJ1, uas encodes chaperone DnaJ protein, thiab pom tias EIJ1 teb rau cov kab mob thaum ntxov thiab ua lub luag haujlwm hauv kev tiv thaiv kab mob hauv lub cev los ntawm kev cuam tshuam ncaj qha nrog EDS1. Raws li cov kev tshawb pom no, peb muab cov qauv kev cai rau EIJ1 (Daim duab 7D). Thaum cov nroj tsuag raug cuam tshuam los ntawm cov kab mob, EIJ1 raug tso tawm sai sai ntawm chloroplast mus rau cytoplasm, qhov twg nws cuam tshuam nrog EDS1. Qhov kev sib cuam tshuam ntawm EIJ1-EDS1 no tiv thaiv kev kaw ntawm EDS1 los ntawm cytoplasm mus rau hauv lub nucleus, thiab qhov no txwv tsis pub muaj kev tiv thaiv nuclear ntawm EDS1, tso cai rau kev hloov pauv ntawm EDS1-cov lus teb hauv lub cev xws li induction ntawm SA biosynthesis thiab qhia tawm ntawm kev tawm tsam. -Cov noob caj noob ces. Thaum cov nroj tsuag raug kev txom nyem tsis tu ncua los ntawm cov kab mob invading, EIJ1 yog maj mam degraded, ua rau muaj zog nuclear tsub zuj zuj ntawm EDS1 rau kev tiv thaiv ntxiv. Cov kev tshawb pom no qhia txog lub luag haujlwm tseem ceeb ntawm EIJ1 thaum lub sij hawm tus tswv teb thaum ntxov rau cov kab mob ntxeem tau, ua kom qhov sib txawv hauv peb txoj kev nkag siab txog yuav ua li cas subcellular localization ntawm EDS1 yog tswj kom muaj kev tiv thaiv.
Kev soj ntsuam ib ntus qhia tias EIJ1 yog DnaJ protein. Arabidopsis genome encodes xya hom classical ntawm DnaJ proteins (Pulido thiab Leister, 2018). Cov kev tshawb fawb yav dhau los qhia tau hais tias DnaJ cov proteins ua lub luag haujlwm tseem ceeb thiab sib txawv hauv kev tiv thaiv cov nroj tsuag. Piv txwv li, taum pauv (Glycine max) GmHSP40 zoo tswj cov cell tuag thiab kab mob tiv thaiv (Liu thiab Whitham, 2013). Nyob rau hauv sib piv, cov mov (Oryza sativa L.) Os-DjA6 protein tsis zoo tswj cov nroj tsuag innate tiv thaiv los ntawm ubiquitination-mediated proteasome degradation txoj kev (Zhong li al., 2018). P. syringae-specific Hopl1 virulence effector, uas muaj J domain, yog tsom mus rau chloroplast, uas nws cuam tshuam nrog cov nroj tsuag kev ntxhov siab- teb cov tshuab (Jelenska li al., 2007). Cov kev tshawb fawb no qhia tau hais tias DnaJ cov proteins ua lub luag haujlwm sib txawv hauv cov lus teb tiv thaiv cov nroj tsuag. Tsip1, N. tabacum DnaJ protein homologous to EIJ1, regulates Tsi1-mediated transcriptional activation of genes in response to stress (Ham et al., 2006). Tsi1 encodes APETALA2 (AP2)-type transcription factor uas ua haujlwm raws li tus tswj hwm tseem ceeb hauv ob qho tib si biotic thiab abiotic kev ntxhov siab hauv N. tabacum (Park et al., 2001). SA ua rau muaj kev hloov pauv ntawm Tsip1 los ntawm chloroplast mus rau cytoplasm, uas pab txhawb kev sib cuam tshuam ntawm Tsip1 thiab Tsi1 thiab kev nkag mus tom ntej ntawm Tsip1-Tsi1 complex rau hauv lub nucleus rau kev ua kom muaj zog ntawm cov noob muaj kev ntxhov siab (Ham et al., 2006) . Tsis zoo li Tsip1, EIJ1 tau nyob hauv thaj chaw rau chloroplast thiab nucleus nyob rau hauv ib qho xwm txheej hauv txoj kev tshawb no. Peb tau pom tias cov kab mob kis tau ua rau muaj kev hloov pauv ntawm EIJ1 los ntawm chloroplast mus rau cytoplasm thiab tias kev sib cuam tshuam ntawm EIJ1 thiab EDS1 hauv cytoplasm inhibits EDS1-kev sib haum xeeb nuclear. Raws li qhov kev tshawb pom no, qhov poob-ntawm-function eij1mutant nthuav tawm muaj zog tiv thaiv kab mob ntau dua li cov tsiaj qus (Col-0). Qhov no tau lees paub tias EIJ1 ua lub luag haujlwm tsis zoo hauv cov nroj tsuag innate tiv thaiv. Raws li cov kev tshawb fawb yav dhau los, kev sib koom tes ntawm nuclear thiab cytoplasmic kev ua ub no ntawm EDS1 yog qhov tseem ceeb rau cov nroj tsuag kom ua tiav lub cev tiv thaiv kab mob rau cov kab mob (Garcı'a li al., 2010; Heidrich et al., 2011). Lub chaw nuclear ntawm EDS1 yog xav tau rau TNL-induced resistance transcriptional reprogramming los ntxias SA-txog thiab lwm yam kev tiv thaiv cog lus teb (Wirthmueller li al., 2007). Lub pas dej cytoplasmic EDS1 kuj tseem khaws cia thaum lub sijhawm kis kab mob kom ua tiav cov kab mob tiv thaiv (Garcı'a li al., 2010). Cov kev soj ntsuam no ua rau muaj kev txhawj xeeb tseem ceeb txog yuav ua li cas cov nroj tsuag tau tswj hwm kev faib tawm ntawm EDS1 ntawm cytoplasm thiab nucleus rau kev tiv thaiv cov lus teb kom raug thaum muaj kev sib tw los ntawm cov kab mob. Ntawm no, peb qhia tau hais tias qhov tseeb faib ntawm EDS1 rau cov subcellular compartments yog tswj nyob rau hauv ib feem los ntawm EIJ1 ntawm protein-protein sib cuam tshuam. Qhov poob-ntawm-kev hloov pauv ntawm EIJ1 tseem ceeb txhawb kev lag luam ntawm EDS1 rau lub hauv paus (Figures 5, D, thiab 7, B), ua rau muaj kev tiv thaiv kab mob zoo dua.
Vim tias EIJ1 tau raug ntxias sai sai thaum ntxov ntawm kev kis kab mob thiab tom qab ntawd degraded thaum lub sij hawm kev loj hlob ntawm tus kab mob, peb xav tias EIJ1 yog ib qho tseem ceeb tsis zoo regulator ntawm kev tiv thaiv kab mob uas tso cai rau cov nroj tsuag los kho lawv cov kev tiv thaiv zoo. EIJ1-kev sib haum xeeb kev tawm tsam ntawm EDS1 kev ua haujlwm tuaj yeem tiv thaiv kev tshem tawm cov tshuaj tiv thaiv kab mob tsis tsim nyog rau lub sijhawm luv luv xws li kev tawm tsam qee zaus los ntawm cov kab mob pathogenic. Xwb, cov kab mob pathogenic tuaj yeem tswj hwm kev tiv thaiv kab mob los ntawm kev ua kom tsis sib haum xeeb ntawm EIJ1 los ntawm chloroplast, tej zaum los ntawm secreting virulent effectors. Peb tsis tuaj yeem tshem tawm qhov ua tau tias EIJ1 ua haujlwm tsis paub txog hauv chloroplast. Raws li peb paub, SA, cov tshuaj hormones cog thiab cov teeb liab molecule cuam tshuam rau kev tiv thaiv cov kab mob biotrophic, yog biosynthesized feem ntau hauv chloroplast thiab tom qab ntawd xa tawm mus rau hauv cytoplasm los ntawm EDS5, multidrug thiab toxin transporter (Serrano li al., 2013). EIJ1 yog qhov zoo li qhov tsis zoo ntawm SA tsub zuj zuj, raws li nws txo qis kev qhia ntawm SA biosynthetic gene ICS1 raws li cov kab mob inoculation (Daim duab 4E). Ntxiv nrog rau EIJ1-kev hloov pauv hloov pauv hloov pauv los ntawm nws qhov kev cuam tshuam nrog EDS1, nws muaj peev xwm tias EIJ1 tseem ua lub luag haujlwm ncaj qha hauv SA biosynthesis lossis SA thauj los ntawm chloroplast hauv qhov tsis paub meej. Nws yuav tsis xav tsis thoob yog tias EIJ1 thiab SA cov txheej txheem biosynthesis tau pom muaj kev sib txuas hauv kev kawm yav tom ntej. Interestingly, EIJ1 kuj nyob rau hauv lub nucleus. Txawm hais tias EIJ1 tsis cuam tshuam nrog EDS1 hauv cov nucleus, tej zaum vim tias tsis muaj cov cofactors tseem ceeb lossis kev hloov kho, kev ua haujlwm ntawm nucleus-localized EIJ1 yog qhov tsim nyog rau kev tshawb nrhiav ntxiv.
cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob
Cov kab mob pathogenic tsim cov nyhuv intracellular los cuam tshuam PTI ntawm tus tswv tsev los ntawm kev tsom cov kab mob tiv thaiv kab mob xws li PRRs, thaum cog cytoplasmic NOD-zoo li receptors paub cov nyhuv no los pib ETI (Dou and Zhou, 2012; Khan et al., 2016). Cov kev tshawb fawb yav dhau los thiab peb cov txiaj ntsig tau pom tias avirulent Pst DC3000 (AvrRps4) txhawb nqa kev lag luam ntawm cytoplasmic EDS1 rau hauv lub nucleus (Garcı'a li al., 2010). Tsis tas li ntawd, peb cov txiaj ntsig tau pom tias tsis muaj qhov sib txawv tseem ceeb hauv Pst DC3000 (AvrRps4) - ua rau muaj kev sib txuas ntawm cov hluav taws xob ntawm EDS1 thiab cov kab mob tom qab ntawm Col-0 thiab eij1-1 cov nroj tsuag mutant (Daim duab 7, A, thiab B) . Tsis tas li ntawd, qhov kev tawm tsam nthuav tawm los ntawm eij1-1 mutant tau muab piv rau qhov uas tau nthuav tawm los ntawm Col-0 cov nroj tsuag inoculated nrog hom III secretion system-defective Pst DC3000 hrcC strain (Cov duab ntxiv 12). Tsis tas li ntawd, qhov sib xyaw ntawm EIJ1 tau qis dua hauv cov nroj tsuag inoculated nrog avirulent Pst DC3000 (AvrRps4) dua li hauv cov nroj tsuag inoculated nrog virulent Pst DC3000 (Daim duab 7C), qhia tias EIJ1 yog ib qho tseem ceeb regulator ntawm virulence immune-triggered hlwb. Vim tias Pst DC 3000-inoculated eij1-1 cov nroj tsuag ua raws li cov phenotype tsis kam ntawm Pst DC3000 (AvrRps4)-inoculated Col-0 cov nroj tsuag (Figures 3, F, G, 4, C, D, thiab 7, A), EIJ1 tuaj yeem ua haujlwm los tiv thaiv kev cuam tshuam ntawm ETI los ntawm cov kab mob phem. Hauv kev xaus, peb tau txheeb xyuas EIJ1, tus kws kho mob DnaJ protein, thiab pom tias nws tau koom nrog hauv kev tiv thaiv kab mob thaum ntxov rau cov kab mob cog. Kev sib cuam tshuam ntawm EIJ1 thiab EDS1 txwv tsis pub muaj kev lag luam nuclear ntawm EDS1, tej zaum yog los tiv thaiv lub sij hawm luv luv los ntawm cov tshuaj tiv thaiv kab mob tsis tsim nyog, txhawm rau pab txhawb kev cuam tshuam ntawm cov kab mob los ntawm kev siv tshuab, lossis ob qho tib si. Tsis hais txog ntawm lub hauv paus txheej txheem, EIJ1 allele tuaj yeem ua haujlwm raws li lub hom phiaj muaj txiaj ntsig zoo rau kev hloov kho cov nroj tsuag hauv lub cev tiv thaiv kab mob hauv kev yug me nyuam. Vim tias EIJ1 tsis cuam tshuam rau kev loj hlob ib txwm ntawm cov nroj tsuag, attenuation ntawm EIJ1 muaj nuj nqi ntawm genome kho yuav prominently nce qib ntawm cov kab mob tsis kam ntawm cov qoob loo nrog me ntsis rau kev loj hlob.
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