Bacterial Outer Membrane Vesicles Induce A Transcriptional Shift nyob rau hauv Arabidopsis mus rau lub cev tiv thaiv kab mob ua rau kom tshem tawm cov kab mob loj hlob hauv Planta

Abstract

Cov kab mob Gram-negative tsim spherical blebs ntawm lawv cov cell periphery, uas tom qab dissociate los ntawm cov kab mob cell phab ntsa los tsim extracellular vesicles. Cov qauv nano-scale no, hu ua txheej txheej membrane vesicles (OMVs), tau pom los txhawb kev kis kab mob thiab kab mob thiab tuaj yeem ua rau muaj kev tiv thaiv kab mob hauv cov tsiaj thiab cov nroj tsuag. Txhawm rau kom nkag siab zoo dua cov kev hloov pauv dav dav ntawm cov nroj tsuag tau dhau los tom qab raug OMVs, peb tau kho Arabidopsis thaliana (Arabidopsis) seedlings nrog OMVs purified los ntawm Gram-negative nroj tsuag pathogenic kab mob Xanthomonas campestris pv. campestris thiab ua RNA-seq tsom xam ntawm OMV thiab cov nroj tsuag uas kho tau zoo ntawm 2, 6, thiab 24 teev tom qab kev sib tw. Qhov kev hloov pauv hloov tau tshaj tawm tau tshwm sim nyob rau thawj ob lub sijhawm kuaj cov ntsiab lus, raws li tau pom los ntawm tus naj npawb ntawm cov noob sib txawv thiab qhov nruab nrab hloov pauv. OMVs ua rau muaj kev hloov pauv loj ntawm kev ua haujlwm ntawm lub cev tiv thaiv kab mob, txhawb nqa ntau txoj hauv kev tiv thaiv kab mob nrog rau ntau yam kev tiv thaiv kab mob. Sib piv cov lus teb ntawm Arabidopsis rau OMVs thiab rau cov neeg lim dej huv, qhia tias OMVs ua rau muaj cov noob zoo sib xws thiab cov kev ua raws li cov neeg siv khoom ib leeg, txawm li cas los xij, txoj hauv kev tau qhib los ntawm OMVs thiab tsis yog los ntawm lwm cov elicitors tau kuaj pom. Pretreating Arabidopsis cov nroj tsuag nrog OMVs thiab tom qab ntawd kis lawv nrog cov kab mob kab mob ua rau txo qis hauv cov kab mob loj hlob. Kev hloov pauv hauv cov nroj tsuag elongation receptor (EFR), flagellin receptor (FLS2), lossis brassinosteroid-insensitive 1- txuam kinase (BAK1) co-receptor, tsis cuam tshuam rau lub cev tiv thaiv kab mob ntawm OMVs. Ua ke cov txiaj ntsig no qhia tau tias OMVs ua rau muaj kev hloov pauv dav dav hauv Arabidopsis ua rau muaj kev txhim kho ntawm ntau txoj hauv kev tiv thaiv kab mob, thiab tias qhov kev hloov pauv no yuav pab txhawb kev tiv thaiv kab mob.

KEYWORDS

Arabidopsis thaliana, kab mob kab mob, extracellular vesicles, OMVs, txheej membrane vesicles, cog kev tiv thaiv, RNA-seq, Xanthomonas campestris pv. campestris

cistanche cov txiaj ntsig rau txiv neej-ua kom muaj zog tiv thaiv kab mob

1 QHOV TSEEB

Cov nroj tsuag niaj hnub ntsib nrog kev ua phem microbes uas vam meej ntawm lawv cov ntaub so ntswg thiab cuam tshuam kev loj hlob ib txwm. Ib qho kev tiv thaiv zoo yog nyob ntawm kev tshawb pom sai thiab raug thiab txheeb xyuas cov microbe invading. Rau lub hom phiaj no, cov nroj tsuag siv dav cov kab ke soj ntsuam los saib xyuas cov kab mob ntxeem tau (Cook li al., 2015). Nws yog speculated tias thawj kab ntawm cov nroj tsuag soj ntsuam system, los yog thawj cellular interface uas cov nroj tsuag thiab microbes cuam tshuam, yog lub intercellular chaw, lub apoplast. Nyob ntawd, kev lees paub ntawm invading microbes yog kho los ntawm membrane-bound, extracellularly exposed pattern recognition receptors (PRRs) (Boutrot & Zipfel, 2017; Couto & Zipfel, 2016). PRRs lees paub cov kev txiav txim siab microbial uas tau nthuav dav thiab tau txais txiaj ntsig ntawm ntau cov kab mob microbes thiab hu ua, microbe- lossis kab mob sib txuas nrog cov qauv molecular (MAMPs) (Ranf li al., 2016). Txij li cov kab mob microbes tau txais mutagenesis ntawm tus nqi nrawm, evolutionary pab tiv thaiv kab mob receptors tau yoog kom pom cov cheeb tsam tshwj xeeb ntawm cov khoom siv microbial tseem ceeb uas tsis tuaj yeem muab pov tseg yooj yim lossis hloov pauv vim yog tus nqi qoj ib ce hnyav. Piv txwv li, cov kab mob flagellin yog ib qho tseem ceeb hauv lub cev ntawm ntau cov microbes nrog rau cov kab mob, thiab tam sim no yog ib qho ntawm MAMPs kawm zoo tshaj plaws (Felix et al., 1999; Zipfel et al., 2004). Kev nkag siab ntawm flagellin, los yog cov hluavtaws epitope flg22 (suav nrog kev txuag 22 amino acids ntawm N-terminus ntawm flagellum tsev thaiv, flagellin), los ntawm cognate cognate tiv thaiv kab mob receptor flagellin sensing 2 (FLS2), ua rau muaj kev hloov pauv loj. , ua raws li kev tiv thaiv kab mob zoo uas tiv thaiv kab mob (Chinchilla li al., 2007; Gómez-Gómez & Boller, 2000). Ntau tus paub MAMPs muaj feem xyuam nrog microbe lub cell phab ntsa. Piv txwv li, fungal chitin (Fesel & Zuccaro, 2016), kab mob peptidoglycan (PG) (Erbs li al., 2008; Gust li al., 2007), kab mob lipopolysaccharides (LPS) (Dow et al., 2000; al Silipo. ., 2005), flagellin (Boutrot & Zipfel, 2017; Felix li al., 1999), thiab ntau dua. Txawm li cas los xij, nws tsis paub meej tias cov cell-wall-associated Cheebtsam cuam tshuam nrog lawv cov cognate immune receptors hauv planta. Txawm hais tias qhov no tshwm sim vim kev tuag ntawm tes thiab / lossis kev puas tsuaj ntawm phab ntsa ntawm tes, lossis los ntawm kev tso tawm cov khoom siv xws li flagellum, yog lub ntsiab lus xav tau kev tshawb nrhiav ntxiv (Bahar, 2020).

Ib qho piv txwv ntawm kev tso tawm ntawm cov cell-phab ntsa tawg los ntawm cov kab mob Gram-tsis zoo yog kev tshem tawm ntawm cov hlwv extracellular (EVs) uas bleb thiab pinch tawm ntawm daim nyias nyias mus rau ib puag ncig ib puag ncig (Kulp & Kuehn 2010; Théry et al., 2018 ). Cov kab mob EVs no feem ntau hu ua cov hlwv sab nraud (OMVs), thiab peb yuav ua raws li cov npe no (Shwechheimer & Kuehn, 2015). Cov txheej txheem ntawm OMV tso tawm tshwm sim tsis tu ncua thiab nyob rau hauv ntau yam ib puag ncig, nrog rau thaum lub sij hawm tus tswv colonization (Gurung li al., 2011; Ionescu li al., 2014; Jin li al., 2011). Ntxiv rau qhov tseem ceeb ntawm cov khoom siv sab nrauv xws li txheej membrane (OM) cov protein, LPS, thiab lipids, OMVs encapsulate periplasmic fluids, muaj ntau hom molecules xws li proteins, cell phab ntsa degrading enzymes, polysaccharides, thiab nucleic acids (Kuehn & Kesty, 2005). Txij li thaum OMVs raug tso tawm thaum lub sij hawm tus tswv colonization, thiab txij li thaum lawv cov khoom thauj muaj MAMPs, nws yog kev ntxias kom xav tias lawv ua raws li cov neeg nqa khoom ntawm lub cev tiv thaiv kab mob xa cov eliciting molecules nyob ze rau lawv cov cognate tiv thaiv kab mob (Bahar, 2020; Katsir & Bahar, 2017). Tseeb tiag, OMVs tau pom tias yuav ntxias cov tsiaj nyeg thiab cov nroj tsuag tiv thaiv kab mob thaum nthuav tawm rau lawv cov tswv (Bahar li al., 2016; Ellis & Kuehn, 2010; Janda et al., 2021; McMillan et al., 2021). Thaum nyob rau hauv mammalian hlwb ob qho tib si LPS thiab protein Cheebtsam ntawm OMVs ua raws li lub cev tiv thaiv kab mob (Ellis li al., 2010), nyob rau hauv cov nroj tsuag, nws tseem tsis tau paub meej uas OMV molecules yog lub tseem ceeb tiv thaiv elicitors.

Ntxiv nrog rau kev hloov kho lub cev tiv thaiv kab mob, OMVs kuj tau pom tias muaj cov xwm txheej phem thiab muaj feem cuam tshuam rau ntau yam txheej txheem. Qhov no suav nrog kev sib txuas lus ntawm tes (Deatheragea & Cooksona, 2012; Mashburn & Whiteley, 2005; Raposo & Stahl, 2019), xa cov co toxins rau lub hom phiaj hlwb (Ellis & Kuehn, 2010; Kadurugamuwa & Beveridge, 1996), biofilm tsim (Schooling & Beveridge, 2006), quenching of antimicrobial compounds (Manning & Kuehn, 2011), teb rau kev ntxhov siab (MacDonald & Kuehn, 2013), kab rov tav hloov mus rau noob (Fulsundar et al., 2014; Velimirov & Ranftler, 2018) (thiab Ell) & Kuehn, 2010; Kunsmann et al., 2015). Txawm hais tias feem ntau ntawm cov piv txwv no los ntawm kev tshawb fawb ntawm cov kab mob mammalian kab mob, cov kev tshawb fawb tsis ntev los no nrog cov kab mob cog qoob loo kuj txhawb tias OMVs txhawb cov kab mob virulence thiab cog colonization. Ionescu et al. (Ionescu et al., 2014) pom tias OMV ntau lawm los ntawm cov nroj tsuag kab mob Xylella fastidiosa thaum lub sij hawm xylem hlab ntsha colonization inhibits kab mob txuas mus rau cov dej ua cov ntsiab lus ntawm cov nroj tsuag (xylem), tilting qhov sib npaug ntawm sessile thiab mobile daim ntawv ntawm lub kab mob mus rau daim ntawv mobile. Daim foos no ntseeg tau tias yuav txhawb nqa cell dispersion hauv xylem, ua rau kom cov nroj tsuag poob sai dua (Ionescu li al., 2014). Lwm qhov kev tshawb fawb tau pom tias muaj kev phom sij xws li hom II-secreted lipases / esterase thiab xylanase, thiab hom III-secreted effectors, yog secreted hauv koom nrog OMVs (Chowdhury & Jagannadham, 2013; Sidhu et al., 2008; Solé et al. ., 2015) qhia tias OMV yuav muaj lub luag haujlwm tseem ceeb hauv cov kab mob virus. Lub molecular complexity ntawm OMVs, nrog rau nws dual thiab tejzaum nws tawm tsam lub luag hauj lwm nyob rau hauv lub tswv tsev (inducing kev tiv thaiv thiab txhawb virulence), ua rau peb kawm txog cov lus teb dav dav ntawm Arabidopsis thaliana (Arabidopsis) cov nroj tsuag rau OMV kev sib tw thiab sim seb qhov kev hloov pauv no. yuav ua rau muaj kev tiv thaiv los yog raug rau cov kab mob tom ntej.

2 Cov Khoom Siv thiab Cov Txheej Txheem

2.1 Cov khoom siv cog thiab kev loj hlob

Arabidopsis thaliana (Arabidopsis) qus-hom Col-0 kab nrog rau cov kab hauv qab no: bak- (Schlesinger et al., 2011) thiab ntog puas tau (Nekrasov et al., 2009) tau siv hauv txoj kev tshawb no. Arabidopsis noob tau nto sterilized thiab sown rau Murashige thiab Skoog (MS) agar daim hlau raws li tau piav qhia (Bahar li al., 2016). Cov phaj tau khaws cia hauv qhov tsaus ntuj ntawm 4 ◦C rau 2-4 hnub thiab tom qab ntawd hloov mus rau 22◦C rau germination rau 5-8 hnub. Cov noob qoob loo uas zoo sib xws tau hloov mus rau hauv 24- cov phiaj zoo (ob lub noob rau ib qhov dej) uas muaj 1 ml ntawm MS nruab nrab nrog 1% (w:v) ntawm sucrose (Duchefa Biochemie) thiab loj hlob rau lwm 8-10 hnub ntawm tib yam kev mob ua ntej sib tw nrog elicitor raws li tau piav qhia hauv qab no. Rau priming assays, noob ntawm Arabidopsis qus hom Col{17}} kab thiab mutant kab ( ntog puas tau thiab rov qab-) tau germinated raws li tau piav saum toj no thiab ces transplanted rau hauv 7 × 7 × 6 cm pots (1 seedling / lauj kaub) muaj mix. av Green #7611 (Evenari, Ashdod, Israel) thiab loj hlob ntawm 9.5 h photoperiod ntawm 22-24˚C. Nroj tsuag tau

2.2 Cov kab mob sab nrauv membrane vesicles purification

Glycerol stocks of Xanthomonas campestris pv. campestris(Xcc) 33913 tau streaked rau Nutrient Agar (Difco, NA, Becton, Dickinson, thiab Company) daim hlau thiab loj hlob rau 2-5 hnub ntawm 28˚C. Ib pawg neeg tau sau tseg thiab siv los ua kom muaj kab mob 3-mL YEB (cov poov xab extract broth) pib muaj 10 ug/ml cephalexin hydrate (Cp, Sigma-Aldrich). Cov pib tau loj hlob thaum hmo ntuj ntawm 28◦C nrog 185–200 rpm co thiab tom qab ntawd siv rau inoculate 500 ml ntawm PSB (peptone sucrose broth) nruab nrab nrog tshuaj tua kab mob (raws li tau piav qhia saum toj no) hauv 2-L Flasks ntawm qhov ratio ntawm ∼1:1000 (v:v). Cov kab lis kev cai tau loj hlob raws li tau piav qhia saum toj no rau OD600 ntawm 0.6-0.8 thiab tom qab ntawd cov kab mob kab mob tau spun thiab OMVs tau muab rho tawm los ntawm supernatant raws li tau piav qhia (Mordukhovich & Bahar, 2017). Kev npaj OMV tsis zoo tom qab ntawd raug rau Optiprep gradient centrifugation kom tau txais OMVs huv si, raws li tau piav qhia (Bahar li al., 2016; Mordukhovich & Bahar, 2017). Txhua OMV batch raug purified los ntawm 1. Purified OMVs tau siv tam sim lossis khaws cia ntawm 4◦C txog 7 hnub ua ntej siv. OMVs loj faib tau ntsuas los ntawm kev siv lub teeb pom kev zoo (Zetasizer Nano ZS, Malvern Panalytical, Worcestershire, UK) thiab muaj qhov nruab nrab ntawm 121.7 ± 55.43 nm (SD). Particle concentration tau ntsuas zoo ib yam thiab tau muab rau hauv txhua qhov kev sim piav qhia hauv qab no.

cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob

2.3 Arabidopsis seedling kev sib tw nrog OMVs

Txhawm rau tshuaj xyuas cov lus teb ntawm Arabidopsis rau OMV kev sib tw, Col-0 cov qoob loo loj hlob hauv 24- cov phiaj xwm zoo li tau piav qhia saum toj no. Hnub ua ntej qhov kev sib tw OMV, MS nruab nrab tau muab tshem tawm ntawm daim hlau thiab hloov nrog 250 ul ntawm sterile dH2O, thiab cov phaj tau tso rau ntawm lub rooj ntev zaum. Thaum sawv ntxov tom qab, 20 ul ntawm OMVs purified (30 ug ib ml sib raug rau 1.44 × 109 particles ib qhov dej), los yog sterile dH2O li mock, tau ntxiv rau txhua qhov dej. Cov noob tau sau 2, 6, thiab 24 teev tom qab kev sib tw, blotted qhuav ntawm daim ntawv, thiab snap-khov nrog kua nitrogen hauv 2-mL Eppendorf Safe-Lock tubes (Hamburg, Lub teb chaws Yelemees). Nyob rau txhua lub sij hawm taw tes, plaub OMV-kho thiab plaub lub qhov dej tsis zoo raug muab sau, sawv cev rau plaub qhov kev hloov pauv roj ntsha rau txhua qhov kev kho mob ntawm txhua lub sijhawm.

2.4 RNA kev ua kom huv

RNA tau muab rho tawm los ntawm Arabidopsis seedlings siv TRIzol reagent (Invitrogen) raws li cov chaw tsim khoom cov lus qhia. RNA tau ua kom huv ntxiv los ntawm kev siv Turbo DNA-free Kit (Ambion, Thermo Fisher Scientific), thiab RNA Clean-Up thiab Concentration Kit (Norgen Biotek) raws li cov chaw tsim khoom cov lus qhia. Purified RNA cov qauv tau raug soj ntsuam zoo thiab kev soj ntsuam zoo siv lub tshuab TapeStation 2200 (Agilent Technologies), RNA Screen Daim kab xev, thiab RNA Screen

2.5 RNA lub tsev qiv ntawv kev tsim kho thiab ua raws

Rau txhua qhov kev kho mob thiab lub sij hawm taw tes, ob qho piv txwv uas qhia qhov purity siab tshaj plaws tau raug xaiv los tshuaj xyuas. TruSeq mRNA cov tsev qiv ntawv (Illumina) nrog PolyA ntes tau npaj los ntawm cov qauv RNA xaiv ntawm Crown Institute of Genomics ntawm Weizmann Institute of Science (Rehovot, Israel). Txhua tus qauv tau tagged, thiab lub pas dej tau npaj los ntawm cov qauv. Lub pas dej no tau muab tso rau hauv ob txoj kab NGS, thiab khiav hauv Illumina HiSeq sequencing tshuab, ntawm cov zis siab khiav hom, nyeem ib zaug (SR) 60 (v4).

2.6 Sequencing nyeem thawj zaug

The raw sequence reads were cleaned with Trimmomatic software v 0.36 (Bolger et al., 2014), removing low-quality reads and remaining adapter sequences. The clean reads were mapped to the reference Arabidopsis TAIR10 reference genome (Lamesch et al., 2012) using bowtie2 (Langmead & Salzberg, 2012) and quantification of gene expression was done using RSEM (Li & Dewey 2011). Principal component analysis and sample correlation matrix were calculated with the function cor() and pre-comp (), respectively, of the R base package version 3.6.1. DEGs were determined using the DESeq2 tool (Love et al., 2014). The FDR (false discovery rate) cutoff chosen was FDR < 0.05. The LogFC (Log of the fold change) cutoff for the up-regulated and the downregulated genes, was > 

2.7 Gene ontologies thiab gene piav qhia 

Gene Ontologies (GO) tau txais los ntawm kev siv TAIR's GO Annotations (http://www.arabidopsis.org/tools/bulk/go/index.jsp). Gene piav qhia, raws li cov qauv gene, tau txais los ntawm kev siv TAIR's gene description search (http://www.arabidopsis. org/tools/bulk/genes/). GO enrichment tau suav nrog AgriGO lub vev xaib (http://bioinfo.cau.edu.cn/agriGO/index.php) siv Arabidopsis genome locus (TAIR10) raws li kev siv

2.8 Quantitative-PCR thiab RNA-seq validation

Txhawm rau kom ntseeg tau cov ntaub ntawv RNA-seq, RNA cov qauv ntawm kev thuam- thiab OMV-kev sib tw cog qoob loo tau siv rau cDNA synthesis, tom qab ntawd los ntawm quantitative-PCR (qPCR) siv cov gene-specific primers (Supp. Table S4), raws li tau piav qhia (Bahar li al. , 2016). Zuag qhia tag nrho, 17 DEGs los ntawm RNA-seq cov ntaub ntawv tau sim, siv plaub qhov kev hloov pauv roj ntsha ntawm RNA ntawm OMV- lossis cov nroj tsuag uas raug kho. Cov txheeb ze qhia ntawm cov noob kuaj tau muab piv nrog ubiquitin qhia, siv 7500 Fast real-time PCR tshuab (Applied Biosystems), raws li tau piav qhia (Bahar li al., 2016).

2.9 Sib piv Arabidopsis transcriptional teb rau MAMPs thiab rau OMVs 

Peb OMV-induced dataset tau muab piv nrog cov ntawv sau tseg ntawm Arabidopsis sib tw nrog flg22, (Denoux li al., 2008) elf26 (Zipfel et al., 2006), PGN (Willmann et al., 2011),son OGet (Daal. , 2017) thiab LPS (Livaja li al., 2008). Rau kev sib piv cov ntaub ntawv, GO cov ntsiab lus enrichment tau ua thiab cov induced GOs tau pom los ntawm Venn daim duab raws li tau piav saum toj no.

2.10 Arabidopsis priming kev sim

Txhawm rau kuaj cov nyhuv priming ntawm Xcc OMVs ntawm Arabidopsis kis nrog Pseudomonas syringae pv. lws suav DC3000 (Pst), peb ua raws li cov txheej txheem piav qhia los ntawm Zipfel li al. (2004). Nyob rau hauv luv luv, nplooj ntawm 6-8 lub lis piam-laus Arabidopsis nroj tsuag, zus raws li tau piav saum toj no, tau infiltrated nrog 50-100 ul ntawm purified Xcc OMVs (30 ug / ml, sib xws rau 3.6-7.2 × 109 hais. ib nplooj), 1 μM flg22, los yog dej siv ib rab koob-tsawg koob. Rau txhua qhov kev kho mob, 5 nplooj / tsob ntoo thiab peb tsob nroj replicates tau siv. Pst inoculum tau npaj los ntawm kev cog cov kab mob ntawm King's B nruab nrab daim hlau (20 g / L peptone, 1.5 g / L MgSO4 x7H2O, 10 ml / L glycerol, thiab 15 g / L agar) ntawm 28˚C rau 2-3 hnub, thiab tom qab ntawd resuspending colonies nrog dej thiab kho cov inoculum concentration rau 105 CFU / ml. Pst inoculum tau nkag mus rau hauv cov nplooj ntoo hauv 24 teev tom qab priming, siv lub koob txhaj tshuaj tsawg dua (kwv yees li 100 ul tau nkag mus rau hauv txhua nplooj). Kev loj hlob ntawm cov kab mob tau txiav txim siab ntawm 0 (1 h tom qab inoculation) thiab 2 hnub tom qab inoculation (dpi) los ntawm kev sau thiab hnyav cov nplooj inoculated, macerating lawv nyob rau hauv 1 ml ntawm 10 mM MgCl2 thiab plating 10-fold serial dilutions ntawm King's B agar daim hlau. Tus naj npawb ntawm CFU ntawm txhua phaj tau txiav txim siab 2 hnub tom qab thiab suav rau ib g nplooj.

FIGURE 1 Arabidopsis transcriptional teb rau OMVs ntawm 2, 6 thiab 24 tom qab kev sib tw. Thawj qhov kev tsom xam (A) thiab cov qauv sib txheeb matrix (B) ntawm Arabidopsis seedlings transcriptional teb rau OMV, los yog thuam, ntawm 2, 6 thiab 24 teev tom qab kev sib tw

2.11 Kev ntsuam xyuas kev loj hlob ntawm cov kab mob hauv vitro 

Txhawm rau ntsuas cov txiaj ntsig ntawm purified Xcc OMVs ntawm Pst kev loj hlob hauv vitro, Pststarters tau loj hlob hauv King's B kua nruab nrab rau 24 h ntawm 28˚C thiab tom qab ntawd siv los inoculate peb kab lis kev cai sib txawv uas muaj 12 ml ntawm King's B nruab nrab txhua, hauv {{3 }}mL Falcon raj ntawm qhov sib piv ntawm 1:100. Cov kab mob kab mob tau hloov kho nrog 30 ug / ml OMVs (1: 50 lossis 1: 100, sib haum rau 1.44 × 109 lossis 7.2 × 108 particles ib ml, raws li), lossis PBS raws li kev tswj hwm, thiab incubated rau 20 h ntawm 28˚C. Cov kab mob loj hlob tau raug ntsuas los ntawm kev siv lub spectrophotometer (Amersham Biosciences) ntawm qhov muag pom (OD) ntawm 600 nm tshaj 22 teev.

3 TSEEM CEEB

3.1 RNA-seq tsom xam qhia txog ntau pawg ntawm Arabidopsis genes sib txawv qhia nyob rau hauv teb rau OMV kev sib tw

Txhawm rau kawm txog kev hloov pauv hauv Arabidopsis tom qab OMV kev sib tw, peb tau kho Arabidopsis seedlings nrog OMVs purified los ntawm cov kab mob pathogen Xanthomonas campestris pv. campestris 33913 (Xcc) thiab sau cov nroj tsuag RNA ntawm 2, 6 thiab 24 teev tom qab kev sib tw (hpc). RNA los ntawm OMV- thiab cov qauv kuaj tau raug kho raws li tau hais tseg thiab tshuaj xyuas raws li tau piav qhia hauv Cov Khoom Siv thiab Cov Txheej Txheem. Kev sib raug zoo ntawm tus thawj xib fwb (Daim duab 1A) thiab cov qauv kev sib raug zoo ntawm kev txheeb xyuas (Daim duab 1B) qhia tau hais tias cov khoom siv roj ntsha hauv txhua qhov kev kho mob sib raug zoo ua ke, ib qho kev qhia ntawm tag nrho cov lus teb zoo sib xws ntawm cov khoom siv roj ntsha. OMV tau kho cov qauv ntawm 2 thiab 6 hpc pawg ua ke, qhia tias OMV kev kho mob muaj txiaj ntsig zoo rau cov lus teb ntawm cov lus teb ntau dua li lub sijhawm ua piv txwv (Daim duab 1B). Ntawm qhov tod tes, OMV tau kho cov qauv ua ke nrog lawv cov kev kho mob sib txawv ntawm 24 hpc, qhia tias qhov kev hloov pauv hloov los ntawm OMVs ntawm lub sijhawm no tau txo qis thiab zoo ib yam li cov nroj tsuag uas tau kho.

cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob

Txhua lub sij hawm taw tes kuaj, OMV-kho cov noob tau muab piv nrog cov nroj tsuag uas kho tau zoo thiab qhov txawv ntawm cov noob (DEGs) tau muab rho tawm. Nyob rau txhua lub sij hawm cov ntsiab lus ua ke, tag nrho ntawm 984 thiab 175 noob tau pom tias muaj qhov cuam tshuam loj heev (Cog hloov pauv> 1 lossis ← 1, p tus nqi thiab FDR < 0.05) nce- lossis txo qis, raws li, teb rau OMV kev sib tw (Daim duab 2A; Supp. Table S1). Gene qhia Log fold-hloov (LogFC) ranged los ntawm qhov siab tshaj plaws ntawm 9.08 (AT1G26410, 6 hpc), uas sib raug rau ntau dua 500-fold hloov, mus rau -5.73 (AT3G17520, 24 hpc). Qhov ntau tshaj ntawm DEGs tau pom nyob rau ntawm 2 thiab 6 hpc, qhov twg tag nrho ntawm 647 thiab 876 DEGs, raws li, tau txheeb xyuas (nce- thiab down-regulated ua ke). Ntawm 24 hpc, 121 DEGs tau pom. Ntau tshaj 50% ntawm kev tswj hwm cov noob ntawm 2 thiab 6 teev tom qab OMV kev sib tw tau sib koom ntawm lawv (Daim duab 2B). Txhawm rau tshuaj xyuas qhov hloov pauv ntawm lub cev ntawm lub cev, peb tau tshem tawm tag nrho cov kev tswj hwm DEGs uas tau pom nyob rau txhua lub sijhawm (37 genes) thiab muab piv rau lawv qhov hloov pauv raws sijhawm (Daim duab 2C). Qhov kev hloov pauv ntawm cov noob no txawv txawv ntawm cov sijhawm sib txawv (ib txoj kev ANOVA; F2,108=8.1633, p=0.0005). Ib qho kev sib piv post hoc siv Tukey Kramer HSD qhov ntsuas tau qhia tias LogFC ntawm ob qho tib si 2 (M=3.45, SD=1.72) thiab 6 hpc (M=3.87, SD=1.98) tau siab dua ntawm 24 hpc (M=2.32, SD=1.31) (p=0.0145 thiab p=0) .0005, ntsig txog) (Daim duab 2D). Thaum qhov txhais tau tias LogFC qhia ntawm 6 hpc yog siab dua ntawm 2 hpc, nws tsis yog qhov sib txawv (p=0.5413). Li no, thaum txiav txim siab tus naj npawb ntawm DEGs thiab tag nrho cov noob LogFC ntawm peb lub sijhawm kuaj, peb tuaj yeem xaus tias qhov kev hloov pauv tseem ceeb tshaj plaws tshwm sim ntawm 2 thiab 6 h tom qab OMV kev sib tw. Txhawm rau tshuaj xyuas qhov siv tau ntawm RNA-seq cov txiaj ntsig, qhov kev qhia ntawm 17 cov noob tau tswj hwm tau txiav txim siab siv ntau PCR (qPCR) nrog cov primers tshwj xeeb. Kaum plaub ntawm cov noob tshuaj ntsuam xyuas pom zoo ib yam li hauv RNA-seq tsom xam thiab tau txais kev tswj hwm zoo dua piv nrog kev thuam. Peb ntawm cov noob caj noob ces uas tau kuaj pom muaj kev txheeb ze dua tab sis tsis txawv ntawm kev thuam los ntawm txoj kev no (Supp. Figure S1).

FIGURE 2 Arabidopsis sib txawv qhia cov noob hauv kev teb rau OMV kev sib tw. (A) Tag nrho tus naj npawb ntawm cov noob sib txawv (DEGs) (nce- lossis down-regulated, LogFC> 1 lossis ← 1, p-value thiab FDR < 0.05) ntawm 2, 6, thiab 24 teev tom qab kev sib tw. (B) Sib tshooj ntawm DEGs ntawm lub sijhawm sib txawv (sab laug, nce- tswj; txoj cai, qis- tswj). (C) Up-regulated genes pom nyob rau hauv tag nrho peb-lub sij hawm cov ntsiab lus tau npaj rau ntawm LogFC qhia graph, qhia gene qhia lub sij hawm. (D) LogFC qhov nruab nrab ntawm tag nrho DEGs ntawm lub sijhawm sib txawv. Cov ntawv sib txawv qhia txog qhov sib txawv ntawm p <0.05 los ntawm Tukey-Kramer HSD test

3.2 Arabidopsis teb rau OMVs nrog kev hloov pauv hloov mus rau kev ua kom lub cev tiv thaiv kab mob

Txhawm rau txheeb xyuas Arabidopsis txoj hauv kev cuam tshuam los ntawm OMV kev sib tw, peb siv AgriGO lub vev xaib (Du li al., 2010; Tian et al., 2017). Peb tau txheeb xyuas 333 thiab 55 qhov tseem ceeb (FDR < 0.05) nce- thiab qis-tswj cov noob caj noob ces ontology (GO) cov ntsiab lus, raws li txhua lub sijhawm cov ntsiab lus ua ke hauv kev teb rau OMV kev sib tw. Ze li ntawm 25% ntawm kev tswj hwm GOs tau cuam tshuam nrog cov lus teb cog rau kev txhawb nqa (Daim duab 3A). Nyob rau hauv 'teb lus teb rau stimulus' qeb, cov ntsiab lus GO tseem ceeb tshaj plaws tau cuam tshuam nrog kev ntxhov siab, rau biotic stimulus, tshuaj, thiab endogenous stimulus (Daim duab 3B; Supp. Table S2). Cov cuab yeej AgriGO kuj tau txheeb xyuas 103 qhov ua tau zoo ntawm kev tswj hwm molecular xws li 'transferase kev ua haujlwm', kinase kev ua haujlwm', 'kev ua haujlwm hloov pauv', 'ion thiab hlau ion khi', 'carbohydrate binding', 'protein binding', 'catalytic activation' , 'Adenyl nucleotide binding', 'transmembrane receptor kev ua' thiab ntau txoj haujlwm khi (Supp. Table S2). Lub cellular qhov chaw ntawm cov ntsiab lus tseem ceeb yog nyob rau hauv sib txawv compartments ntawm lub cell nrog rau lub nucleus, vacuole, thiab endomembrane system, tab sis yog feem ntau notably txuam nrog lub cell periphery thiab suav nrog 'plasma membrane', 'extracellular cheeb tsam', 'cell phab ntsa' thiab 'apoplast' ontologies (Supp. Table S2). Cov kev tswj hwm tsis zoo, ntawm qhov tod tes, suav nrog cov ntsiab lus 'toxin catabolic thiab metabolic txheej txheem', ' teb rau dej thiab dej deprivation', 'lipid thauj thiab localization', thiab lwm yam (Supp. Table S2). GOs muaj feem xyuam rau cov lus teb rau biotic stimulus tsis enriched nyob rau hauv lub down-regulated noob. Qhov tseem ceeb ntawm kev tswj hwm molecular muaj xws li ntau cov ntsiab lus redox xws li 'oxidoreductase kev ua haujlwm', 'heme binding', 'hlau ion khi', 'lipid binding', 'oxygen binding', thiab ntau dua oxygen ntsig txog kev ua haujlwm (Supp. Table S2) . Down-Regulated cov ntsiab lus kuj nyob rau hauv cheeb tsam extracellular. Txhawm rau txiav txim siab seb GO cov ntsiab lus twg DEGs nrog cov lus qhia siab tshaj plaws yog, peb tau lim cov npe DEGs thawj zaug los ntawm kev xaiv cov noob uas muaj LogFC siab dua 4, lossis tsawg dua -4 (xws li 16-fold sib txawv) . Peb tau txheeb xyuas 117 cov noob uas ua tau raws li cov qauv no, ntawm 115 tau nce-tswj thiab 2 txo qis-tswj txhua lub sijhawm ua ke. Vim tias tsawg tsawg ntawm cov noob tswj hwm nrog LogFC tsawg dua -4, tsis muaj kev txwv tsis pub GOs tau pom. Ntawm qhov tod tes, 72 qhov tseem ceeb tshaj-tswj GOs tau txheeb xyuas, ntawm cov uas tseem ceeb tshaj plaws yog cuam tshuam nrog 'teb teb rau lwm cov kab mob biotic stimulus', 'teb rau lwm yam kab mob', 'cellular teb rau qib oxygen', 'tiv thaiv teb' , ' teb rau kev ntxhov siab ' thiab ntau lub cev tiv thaiv kab mob GOs (Daim duab 3C).

FIGURE 3 Arabidopsis gene ontology (GO) cov ntsiab lus txhawb ntxiv rau cov lus teb rau Xcc OMV kev sib tw. Daim ntawv qhia ncuav qab zib sawv cev ntawm qeb kev faib khoom hauv cov txheej txheem kev noj qab haus huv (A) thiab Cov Lus Teb rau stimulus (B) GO cov ntsiab lus. (C) Cov npe ntawm Arabidopsis noob nrog LogFC ntawm> 4 hauv kev teb rau OMV kev sib tw. Cov noob tau raug lim los ntawm cov ntaub ntawv tiav ntawm DEGs thiab siv los txheeb xyuas cov ntsiab lus GO enriched siv AgriGo webtool. FDR txiav tawm < 0.05

3.2 OMV kev sib tw ua rau muaj kev tswj hwm ntawm lub cev tiv thaiv kab mob

MAMP sensing thiab cog lus teb rau MAMPs feem ntau kho los ntawm membrane-bound PRRs uas kho cov kab mob kev nkag siab thiab kev txo qis ntawm kev kis kab mob. PRRs feem ntau muab faib ua ob pawg receptor kinases (RKs) thiab receptor-like proteins (RLPs) (Boutrot & Zipfel, 2017). Txhawm rau txheeb xyuas cov PRRs uas tau hais txawv txawv hauv cov lus teb rau OMVs, peb muab peb cov DEG teeb tsa nrog cov npe tau tsim ua ntej ntawm Arabidopsis RKs (Kemmerling li al., 2011; Mott et al., 2016) thiab RLPs (Wang et al., 2008). Peb tau txheeb xyuas nyob rau hauv peb dataset 33 thiab 10 up-regulated RKs thiab RLPs, ntsig txog, txhua lub sij hawm cov ntsiab lus ua ke (Table 1). Tsis muaj RKs lossis RLPs tau pom nyob rau hauv peb cov npe ntawm 175 cov noob caj noob ces. Kemerling et al. (2011) tau txhais cov npe ntawm 49 RKs, uas nws cov lus qhia tau cuam tshuam los ntawm MAMPs xws li flg22 thiab NLP (necrosis thiab ethylene -inducing peptide 1-zoo li protein), lossis kev kho kab mob. Peb piv cov npe no nrog cov tswj hwm RLKs los ntawm peb qhov kev sim thiab pom tias 45% ntawm RKs txhais los ntawm Kemmerling li al. (2011) kuj raug ntxias los teb rau OMVs. Ntawm cov, tseem ceeb yog FRK1, SOBIR1, SERK4, RLK/IKU2, PSKR1, HAESA, EFR, BIR thiab IOS1 (Table 1). Ntawm cov pab pawg RK, FRK1 muaj qhov qhia siab tshaj plaws ntawm 2 thiab 6 hpc nrog LogFC ntawm 7.11 thiab 5.2, raws li, thaum qhov nruab nrab LogFC ntawm tag nrho RKs yog 2.11 thiab 2.13 ntawm 2 thiab 6 hpc, feem. Thaum daim nyias nyias-bound RKs thiab RLPs feem ntau kho kom haum rau cov cellular sensing ntawm invading microbes, nucleotide binding site-leucine-nplua nuj rov (LRR) receptors (NLRs), yog intracellular immune receptors. Peb pom 7 qhov sib txawv NLR genes up-regulated nyob rau hauv teb rau OMV-kev sib tw ntawm 2 thiab 6 hpc, tsis muaj leej twg pom ntawm 24 hpc (NLR daim ntawv tau muab rho tawm los ntawm TAIR, 102 noob) (Table 1).

cistanche cov txiaj ntsig rau txiv neej-ua kom muaj zog tiv thaiv kab mob

3.3 OMVs induce qhov kev qhia ntawm ntau yam WRKY transcription yam

WRKY transcription factors (TFs) tau pom los ua lub luag haujlwm hauv cov tshuaj tiv thaiv kab mob, koom nrog hauv MAMP-triggered immunity (MTI) thiab effector-triggered immunity (ETI) cov lus teb (Birkenbihl et al., 2018; Rushton et al., 2010 ). OMV kev sib tw ua rau muaj kev tswj hwm ntawm 20 qhov sib txawv WRKY TFs (cov npe muab rho tawm los ntawm TAIR, 70 noob) tsuas yog ntawm 2 thiab 6 HPC (Table 1). WRKY TFs tsis tuaj yeem los ntawm peb cov kev tswj hwm cov noob caj noob ces. Lwm tsev neeg ntawm TFs cuam tshuam los ntawm OMV cov kev sib tw yog MYB domain-muaj cov proteins (Tsuda & Somssich, 2015), paub tias muaj kev koom tes hauv ntau cov txheej txheem suav nrog biotic thiab abiotic stresses (Ambawat et al., 2013). Zuag qhia tag nrho, 9 qhov sib txawv MYB TFs (cov npe tau muab rho tawm los ntawm TAIR, 211 noob) tau hais txawv txawv hauv kev teb rau OMV kev sib tw, 5 nce- tswj thiab 4 down-regulated (Table 1). Cov chav kawm ntxiv ntawm qhov sib txawv ntawm TFs uas tau kuaj pom muaj nyob hauv Table 1.

3.4 Sib piv Arabidopsis transcriptional teb rau OMV nrog cov lus teb rau purified MAMPs

Txhawm rau kawm txog qhov sib txawv ntawm Arabidopsis cov lus teb rau kev ntxuav cov elicitor piv rau cov qauv tsim thiab molecularly complex - OMVs, peb piv peb cov ntaub ntawv RNA-seq nrog cov ntaub ntawv transcriptomic uas twb muaj lawm ntawm Arabidopsis cov lus teb rau MAMPs paub suav nrog flg22, (Denoux et al., 2008) elf2 (Zipfel et al., 2006), PGN (Willmann et al., 2011), OGs (Davidsson et al., 2017) and LPS (Livaja et al., 2008). Enriched GOs tau muab rho tawm los ntawm cov ntaub ntawv hais saum toj no raws li tau piav qhia saum toj no (Supp. Table S3) thiab muab piv nrog GOs enriched tom qab qhov kev sib tw OMV. Feem ntau, Arabidopsis GO cov ntsiab lus raug ntxias los ntawm OMVs zoo ib yam li cov neeg raug ntxias los ntawm ib leeg, proteinaceous, thiab tsis-proteinaceous MAMPs, sib koom 56, 51 thiab 47% ntawm OMV-induced GOs nrog GOs induced los ntawm flg22, elf26 thiab PGN, feem. Ntawm qhov tod tes, qhov sib tshooj qis dua hauv kev ntxias GOs tau pom nrog LPS thiab OGs, sib koom 24 thiab 33%, feem, nrog OMV-induced GOs (Daim duab 4A). Qhov tseem ceeb, cov kab mob ntsig txog 1 (PR1) gene (At2g14610), lub ntsiab lus ntawm LPS-induced immune responses (Silipo et al., 2005, 2008), tsis tuaj ntawm OMV-induced gene list ntawm txhua lub sijhawm kuaj. Plaub caug-ib lub GOs tau pom tias raug ntxias los ntawm OMV thiab tsis yog los ntawm lwm yam MAMPs tau sim ntawm no (Daim duab 4B). Cov npe no suav nrog GOs cuam tshuam txog 'apoptosis', ' teb rau tshuaj', 'kev thauj tshuaj thiab 'kev thauj tshuaj ntau', thiab 'lipase kev ua haujlwm' (Supp. Table S3, qhia los ntawm asterisks thiab bold font).

3.5 OMVs induce Arabidopsis tiv thaiv kab mob

Ntawm no thiab yav dhau los (Bahar et al., 2016), peb tau muab pov thawj qhia tias lub cev tiv thaiv kab mob Arabidopsis raug ntxias los ntawm OMV kev sib tw. Txhawm rau tshuaj xyuas seb qhov OMV-mediated immune induction no txhais tau li cas rau hauv lub cev tiv thaiv kab mob, peb siv cov kab mob loj hlob ntawm cov nroj tsuag (Zipfel li al., 2004) uas Arabidopsis nroj tsuag tau pretreated nrog OMVs ua raws li cov kab mob inoculation. Ib qho kev txo qis ntawm ntau dua 10-fold hauv Pseudomonas syringae pv. txiv lws suav DC3000 (Pst) CFU/g nplooj tau pom nyob rau hauv ob qho tib si OMV- thiab flg22-pretreated nroj tsuag piv nrog mock pretreatment, ob hnub tom qab inoculation (Daim duab 5A). Hauv vitro kev loj hlob ntawm Pst tsis cuam tshuam los ntawm kev sib ntxiv ntawm OMVs rau nruab nrab, qhia tias qhov txo qis Pst loj hlob hauv planta cuam tshuam nrog cov nyhuv priming ntawm OMV, thiab tsis yog qhov cuam tshuam ncaj qha ntawm OMVs rau cov kab mob (Supp. S2). Txhawm rau kuaj seb OMV-induced tsis kam rau Pst yog kho los ntawm FLS2, EFR, lossis BAK1 peb rov ua qhov kev sim no nrog Col-0, bak-mutant, thiab ob kab mutant poob efr. Nrog rau ob txoj kab mutant, OMV pretreatment ua rau txo qis hauv Pst CFU / g nplooj piv nrog cov nroj tsuag uas tau kho (Daim duab 5B-C). Raws li qhov xav tau, fls efr thiab bak-mutant kab kho nrog flg22 muaj cov Pst titers zoo ib yam li cov nroj tsuag tsis kho raws li lawv paub tias tsis teb rau flg22. Txhawm rau sib piv cov txheeb ze txo qis hauv cov kab mob titer hauv Col-0 tiv thaiv kab mob tiv thaiv kab mob mutant poob puas tau thiab bak hauv cov nroj tsuag primed, peb suav qhov sib txawv ntawm Pst titer hauv OMV- thiab cov nroj tsuag uas kho tau hauv peb qhov kev sim ywj pheej (Supp . Daim duab S3). Qhov nruab nrab txo qis hauv Pst titer hauv OMV-pretreated rov qab cov nroj tsuag me dua li qhov pom hauv Col-0 cov nroj tsuag (0.89 vs.1.14 Log CFU/gr nplooj txo rau bak thiab Col-0, feem, ib- txoj kev ANOVA; F2, 4=4.3781, p=0.0523). Peb tsis pom qhov txo qis zoo sib xws nrog fls efr mutant kab (1.26 vs. 1.32 Log CFU txo rau fls efr thiab Col-0, raws li, ib-txoj kev ANOVA: F1,4=0.0352, p=0.5698) (Daim duab 5D-E).

TABLE 1 OMV-induced RKs/RLPs thiab kev tiv thaiv kev tiv thaiv kab mob hauv cov qoob loo Arabidopsis

TAB 1 (Ntxiv mus)

TAB 1 (Ntxiv mus)

4 Kev sib tham

Kab mob sab nrauv vesicles (OMVs) yog cov nanostructures nyuaj uas tshwm sim los ntawm cov kab mob sab nrauv thiab muaj ntau pua cov proteins thiab lwm cov phab ntsa ntawm tes. Nws tau pom yav dhau los tias cov nroj tsuag Arabidopsis teb rau OMV kev sib tw los ntawm kev ua kom lub cev tiv thaiv kab mob xws li ROS tawg, kab mob tiv thaiv kab mob, thiab nruab nrab alkalization (Bahar li al., 2016). Hauv txoj kev tshawb no, peb tau tshuaj xyuas cov lus teb dav dav ntawm Arabidopsis rau cov kab mob OMVs thiab nws cov txiaj ntsig ntawm kev kis kab mob tom ntej. Qhov kev txiav txim siab dhau los ntawm RNA-seq cov ntaub ntawv txheeb xyuas tau ua nyob rau hauv txoj kev tshawb no yog tias Arabidopsis lub cev tiv thaiv kab mob yog primed tom qab raug rau Xanthomonas campestris pv.campestris (Xcc) OMVs. Qhov kev txiav txim siab no tau txais kev txhawb nqa los ntawm qhov sib txawv thiab kev tshuaj xyuas ntxiv. Ua ntej, gene ontology (GO) enrichment nyob rau hauv cov nroj tsuag raug rau Xcc OMVs kom meej meej qhia tau hais tias OMVs yog perceived los ntawm Arabidopsis raws li stressors. Lub cellular qhov chaw ntawm cov nroj tsuag teb tau cuam tshuam feem ntau nrog lub cell periphery tawm tswv yim sab cellular xaav ntawm cov khoom nyuaj, OMVs. Qhov no muab kev txhawb nqa ntxiv rau qhov kev xav tias OMVs thiab lawv cov neeg koom nrog tau hnov ​​​​los ntawm extracellular receptors, zoo ib yam li ntau tus paub MAMPs. Qhov thib ob, peb pom cov chav loj ntawm RKs thiab RLPs tau raug tswj hwm los teb rau OMV kev sib tw. Ntau ntawm cov receptors no tau paub los kho cov kab mob kev nkag siab lossis yav dhau los qhia tias koom nrog cov tshuaj tiv thaiv kab mob. FLG22-induced receptor-like kinase 1 (FRK1) yog qhov induced receptor ntau tshaj. Qhov no yog qhov nthuav vim peb tsis tuaj yeem ntes flagellin hauv peb Xcc OMV proteomics tsom xam (cov ntaub ntawv tsis qhia). Nws paub tias FRK1 tseem raug ntxias los ntawm lwm cov tshuaj tiv thaiv kab mob, tab sis nws yog qhov xav tsis thoob vim li cas nws cov lus qhia ntau dua li lwm tus RKs tau tswj hwm ntawm no. Elongation factor receptor (EFR) kev qhia, ntawm qhov tod tes, tsuas yog nce- tswj hwm ntawm 2 h lub sijhawm taw tes, thiab muaj LogFC ntawm 1.12, txawm tias EF-Tu pom hauv Xcc OMVs (Bahar li al., 2016. ). Peb kuj pom ob peb NLR genes up-regulated nyob rau hauv teb rau OMV kev sib tw, ib nrab ntawm cov uas yog annotated raws li cov kab mob tiv thaiv cov proteins, tab sis lawv cov hauj lwm nyob rau hauv cov nroj tsuag tiv thaiv tsis tau piav. Thaum peb tsis xav tias NLRs tau koom ncaj qha rau OMV kev nkag siab, lawv tuaj yeem ua rau qis qis ntawm RK / RLPs sensing ntawm OMV molecules raws li tau pom nyob rau hauv cov lus teb rau purified MAMPs xws li flg22, elf18, thiab LPS (Denoux li al., 2008 ; Livaja et al., 2008; Zipfel et al., 2006). Thib peb, ntau yam kev tiv thaiv kab mob tiv thaiv kab mob, WRKY, MYB, thiab lwm yam, tau tswj hwm los ntawm OMVs (Bjornson li al., 2021). Hauv peb txoj kev tshawb fawb, qhov kev hloov pauv tseem ceeb hauv kev teb rau OMV tshwm sim ntawm thawj ob lub ntsiab lus (2 thiab 6 hpc). Qhov no tau piav qhia los ntawm ob qho tib si loj dua ntawm cov caj ces sib txawv (DEGs) thiab ntau dua Log foldchange (LogFC) ntawm 2 thiab 6 hpc. Txawm li cas los xij, tawm ntawm tag nrho ntawm 121 DEGs pom ntawm 24 hpc, yuav luag ib nrab (52) tsis pom ntawm 2 lossis 6 hpc. Qhov no qhia tau hais tias ib nrab ntawm DEGs ntawm 24 hpc yog cov noob lig-tswj, uas nws cov lus qhia tau nce- lossis qis- tswj tom qab 6 hpc. Tseeb tiag, Arabidopsis noob nrog cov kev qhia sib txawv raws li cov kev sib tw elicitor tau txheeb xyuas yav dhau los (Bjornson li al., 2021).

FIGURE 4 Kev sib piv ntawm cov Gene ontology (GO) cov ntsiab lus hauv kev teb rau OMV thiab rau ib qho purified MAMPs. Arabidopsis qhia cov ntaub ntawv nyob rau hauv cov lus teb rau MAMP kev sib tw (elf26, flg22, OGs, PGN, thiab LPS; saib cov khoom thiab cov txheej txheem rau cov neeg siv) tau siv los rho tawm GOs uas muaj txiaj ntsig siv AgriGo lub vev xaib. Enriched GO pawg ntawm txhua MAMP tau muab piv nrog cov npe GO enriched nyob rau hauv teb rau OMVs siv Venny (A, thiab Supp. Table S3). Cov ntsiab lus GO enriched tsuas yog nyob rau hauv OMV datasets yog qhia nyob rau hauv (B) txheeb los ntawm lawv FDR tus nqi.

Hauv peb txoj kev tshawb fawb, qhov kev hloov pauv tseem ceeb hauv kev teb rau OMV tshwm sim ntawm thawj ob lub ntsiab lus (2 thiab 6 hpc). Qhov no tau piav qhia los ntawm ob qho tib si loj dua ntawm cov caj ces sib txawv (DEGs) thiab ntau dua Log fold hloov (LogFC) ntawm 2 thiab 6 hpc. Txawm li cas los xij, tawm ntawm tag nrho ntawm 121 DEGs pom ntawm 24 hpc, yuav luag ib nrab (52) tsis pom ntawm 2 lossis 6 hpc. Qhov no qhia tau hais tias ib nrab ntawm DEGs ntawm 24 hpc yog cov noob lig-tswj, uas nws cov lus qhia tau nce- lossis qis- tswj tom qab 6 hpc. Tseeb tiag, Arabidopsis noob nrog cov kev qhia sib txawv raws li qhov kev sib tw elicitor tau txheeb xyuas yav dhau los (Bjornson li al., 2021). Kev nthuav qhia sai thiab feem ntau ntawm cov noob caj noob ces peb tau pom ntawm no yog ua raws li lwm cov kev tshawb fawb uas tau sim cov lus teb ntawm lub cev ntawm Arabidopsis rau MAMPs. Piv txwv li, Denoux et al. (2008) thiab Bjornson et al. (2021) tau pom tias qhov kev hloov pauv ntawm Arabidopsis hauv kev teb rau ntau yam MAMPs tshwm sim nyob rau hauv feeb mus rau teev, thiab feem ntau, DEGs rov qab mus rau theem pib ∼ hauv 24 teev tom qab cog kev sib tw. Tsis zoo li kev sib cuam tshuam ntawm cov nroj tsuag thiab cov kab mob, qhov kev sib cuam tshuam yog qhov muaj zog thiab tsis tu ncua thaum sib tw nrog cov qauv tsis muaj sia nyob, xws li purified MAMP los yog nrog OMVs- nws tuaj yeem cia siab tias cov nroj tsuag teb, tsawg kawg ntawm qib transcriptional, yuav ua tau ib ntus thiab tsis txhawb nqa ntev hnub. Kev tshawb fawb hnyav nyob rau hauv peb lub xyoos dhau los tau nthuav tawm ntau cov nroj tsuag tiv thaiv kab mob ua lub luag haujlwm rau kev lees paub microbe. Ntau yam ntawm cov receptors no muaj peev xwm los txheeb xyuas cov kab mob microbial nkaus xwb thiab tab tom kawm kom paub meej kom nkag siab zoo dua cov kab mob, kev tiv thaiv kab mob, thiab cov lus teb ntawm cov qauv thiab cov qoob loo cog qoob loo. Txawm li cas los xij, cov nroj tsuag ib txhij raug rau ntau yam kab mob microbial los ntawm ntau qhov chaw, uas ntxiv qhov nyuaj rau kev tiv thaiv kab mob thiab cov lus teb. Peb tau txaus siab los tshuaj xyuas qhov sib txawv ntawm cov lus teb ntawm Arabidopsis rau cov lus purified MAMPs piv rau OMVs, uas sawv cev rau cov qauv ntawm cov microbial ntau dua, tab sis ib qib ntawm qhov nyuaj tshem tawm ntawm microbe nws tus kheej. OMVs muaj cov yam ntxwv tsis zoo, degradative enzymes, co toxins, thiab lwm yam biomolecules uas tuaj yeem muaj lub luag haujlwm rau kev loj hlob ntawm cov kab mob hauv cov nroj tsuag, thiab yog li nws yog qhov txaus siab los sim yog tias OMV kev sib tw ua rau cov GOs tshwj xeeb uas tsis raug ntxias los ntawm hluavtaws MAMPs.

FIGURE 5 Pretreating Arabidopsis nplooj nrog OMVs induces tiv thaiv kab mob tom ntej. Col-0 cov nroj tsuag (A) tau pretreated nrog OMVs, dej (mock), los yog flg22 raws li kev tswj, thiab 24 teev tom qab inoculated nrog ib tug 105 CFU/ml ncua kev kawm ntawv ntawm Pst DC3000 siv needleless syringe infiltration . Pst DC3000 cell titer nyob rau hauv inoculated nplooj tau txiav txim siab 48 h tom qab inoculation los ntawm serial dilution platings. Arabidopsis Col-0 thiab fls efr (B), lossis bak (C) cov nroj tsuag raug sim hauv qhov kev sim zoo li tau piav qhia hauv (A). Qhov nruab nrab Log Pst DC3000 CFU/gr txo qis tom qab OMV pretreatment (piv nrog cov nroj tsuag tsis kho) hauv Col-0 thiab ntog efr (D), thiab Col-0 thiab rov qab (E), tau muab piv. Txhua lub bar sawv cev rau qhov txhais tau tias Log Pst DC3000 CFU/gr txo los ntawm peb qhov kev sim ywj pheej (cov ntaub ntawv ntawm cov kev sim ywj pheej tau nthuav tawm hauv Supp. Fig. S3). Qhov sib txawv tsis yog qhov tseem ceeb (Cov tub ntxhais kawm ob tus tub-ntxhais kawm ntawv t-test. p qhov txiaj ntsig tau qhia saum toj ntawm cov kab kos). Kev sim A, B, thiab C tau ua tsawg kawg peb zaug nrog cov txiaj ntsig zoo sib xws (3 tsob nroj / rov ua dua ib qho kev kho mob hauv txhua qhov kev sim). Asterisks (**) qhia qhov sib txawv tseem ceeb piv nrog kev thuam (Dunnet's test p <0.001)

Rau peb cov kev sib piv transcriptomic, peb tau sau cov ntaub ntawv los ntawm cov kev tshawb fawb nrog cov kev sim zoo sib xws, piv txwv li, hauv cov nroj tsuag thiab cov ntsiab lus zoo sib xws. Ib qho tseem ceeb sib tshooj hauv GO enrichment tau pom hauv Arabidopsis teb rau OMV thiab rau flg22, elf26, thiab PGN. Qhov no tsis yog qhov xav tsis thoob vim nws paub tias ntau txoj hauv kev tiv thaiv tau qhib los ntawm kev paub txog kab mob zoo sib xws, tsis hais tus elicitor tshwj xeeb lossis nws qhov chaw (Bjornson li al., 2021; Zipfel li al., 2006). Txawm li cas los xij, qee qhov tshwj xeeb GOs tau pom tias tau tswj hwm los ntawm OMVs thiab tsis yog los ntawm lwm tus MAMPs peb tau soj ntsuam. Ntawm cov no yog GOs cuam tshuam txog kev puas tsuaj ntawm phab ntsa ntawm tes xws li 'lipase kev ua haujlwm' thiab 'hydrolase kev ua haujlwm ntawm glycosyl bonds', uas yuav qhia tau tias cov nroj tsuag tiv thaiv kab mob yog tsom rau OMV degradation. Interestingly, peb GOs cuam tshuam txog kev thauj yeeb tshuaj kuj tau pom tias muaj kev tswj hwm tshwj xeeb los ntawm OMV kev sib tw. Qhov no yuav qhia tau tias cov nroj tsuag tau ntsib nrog cov tshuaj lom neeg raug xa mus rau hauv lawv lub hlwb, tej zaum los ntawm OMV-mediated xa. Tsis zoo li Arabidopsis cov lus teb rau kev tiv thaiv kab mob peptides thiab PGN, peb tau pom me ntsis sib tshooj ntawm Arabidopsis cov lus teb rau OMVs OGs, thiab LPS. Qhov kev sib tshooj me me no, tshwj xeeb tshaj yog nrog LPS, yog qhov xav tsis thoob, txiav txim siab tias nyob rau hauv cov tsiaj txhu, LPS tau lees paub zoo tias yog cov neeg muaj peev xwm pab txhawb lub cev tiv thaiv kab mob los ntawm OMVs (Ellis li al., 2010). Tsis tas li ntawd, qhov tseeb peb tsis tuaj yeem nrhiav kev txhim kho ntawm LPS lub cev tiv thaiv kab mob PR1 (Silipo li al., 2005), tuaj yeem qhia tias LPS tsis yog ib qho tseem ceeb ntawm cov nroj tsuag tiv thaiv kab mob nrog cov kab mob OMVs. Txawm li cas los xij, qhov no tseem yuav tau tshuaj xyuas kom meej. OMVs tau pom tias yuav pab txhawb rau cov kab mob colonization ntawm cov tsiaj nyeg thiab cov nroj tsuag thiab qee qhov ua rau cov kab mob virulence. Ntawm qhov tod tes, OMVs qhib lub tswv tsev tiv thaiv kab mob li no, ua raws li rab ntaj ob sab, txhawb cov kab mob muaj sia nyob thiab kev ua phem ntawm ib sab, thiab pub rau tus tswv tsev saib xyuas thiab ua kom muaj kev tiv thaiv tus tswv ntawm lwm tus (McMillan & Kuehn, 2021 ). Peb qhov kev ntsuam xyuas thawj zaug tau pom tias qhov kev sib tw OMV ua rau muaj kev cuam tshuam tseem ceeb ntawm Pseudomonas syringae pv. txiv lws suav DC3000 (Pst) kev loj hlob hauv planta, zoo ib yam li cov nyhuv priming pom nrog hluavtaws MAMPs (Jung et al., 2009). Li no, hauv qhov piv txwv no, kev tswj hwm ua ntej ntawm OMVs rau cov ntaub so ntswg inoculated tsis txhawb cov kab mob colonization tab sis theej primed cov nroj tsuag kom induce ib tug zoo tiv thaiv kab mob uas inhibited pathogen loj hlob. Cov txiaj ntsig no yog nyob rau hauv txoj kab nrog peb cov ntaub ntawv sau tseg thiab nrog peb txoj kev tshawb fawb yav dhau los, txhawb kev xav tias OMVs ua rau muaj kev tiv thaiv tiv thaiv kab mob hauv Arabidopsis. Ob txoj kev tshawb fawb tsis ntev los no kuj tau qhia tias Arabidopsis pretreatment nrog OMVs los ntawm cov kab mob lossis cov kab mob Pseudomonas commensal, tiv thaiv kab mob Pst tom ntej (Janda li al., 2021; McMillan li al., 2021). Cov txiaj ntsig no suav nrog qhia tias nyob rau hauv cov xwm txheej tau sim, OMV infiltration tsis pab txhawb kev kis kab mob. Hauv kev tshawb fawb dhau los, peb tau pom tias ntau lub cev tiv thaiv kab mob tiv thaiv kab mob tswj hwm WT cov lus teb rau Xcc OMVs. Cov mutants no suav nrog PRRs paub, paub txog cov proteinaceous (FLS2, EFR, RLPReMAX) lossis non-proteinaceous MAMPs (LYM1/LYM3) (Bahar li al., 2016). Interestingly, Janda et al. (2021) tau tshaj tawm tias thaum FLS2 receptor mutant kab ntawm Arabidopsis (flu) tau sib tw nrog OMVs los ntawm Pst, FRK1 qhia tsis hloov pauv thiab zoo ib yam li cov nroj tsuag uas tau kho, qhia tias FLS2 kho cov lus teb rau Pst OMVs. Nws yog qhov ua tau tias flagellin muaj ntau nyob rau hauv Pst OMV kev npaj ntau dua li nws yog nyob rau hauv Xcc 33913 OMVs, thiab yog li ntawd qhov kev tshem tawm ntawm flagellin receptor muaj ib tug pronounced ntxim rau cov nroj tsuag teb rau Pst tshaj rau Xcc OMVs.

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Nws tau raug pom tias cov tshuaj tiv thaiv kab mob sib txawv tuaj yeem tsim cov txiaj ntsig sib txawv, ua rau muaj kev sib cav sib ceg. Piv txwv li, peb tau pom tias nyob rau hauv ib tug nplooj-disc ROS burst assay, cov lus teb ntawm Arabidopsis rau OMVs yog nyob ntawm tus EFR receptor, txawm li cas los xij, nyob rau hauv lub cev tiv thaiv kab mob gene qhia kev soj ntsuam nrog Arabidopsis seedlings, puas tau mutant kab tau teb rau OMVs li. lub WT. McMillan et al. (2021) tau qhia tias kev kho lub cev sib txawv siv rau OMVs tshem tawm qee yam haujlwm xws li kev tiv thaiv kev loj hlob ntawm cov noob. Txawm li cas los xij, nws tsis tau hloov lwm yam kev tiv thaiv kab mob xws li cog priming. Yog li ntawd, nws yog ib qho tseem ceeb uas yuav tau muab ntau yam kev ntsuam xyuas los ntsuam xyuas cov kev tiv thaiv kab mob sib txawv kom tau txais kev pom dav npaum li qhov ua tau ntawm cov nroj tsuag tiv thaiv kab mob teb rau ib qho elicitor muab. Txhawm rau tshuaj xyuas ntxiv txog kev koom tes ntawm qee qhov paub txog PRRs thiab co-receptors, peb tau sim qhov kev poob puas thiab cov kab mutant siv cov nroj tsuag priming assay. Peb cov txiaj ntsig tau pom tias Arabidopsis ob kab mutant ntog tau zoo ib yam los ntawm Xcc OMV raws li WT cov nroj tsuag, txhawb kev xav tias MAMPs uas tsis yog flagellin thiab EF-Tu kuj tseem muaj nyob hauv Xcc 33913 OMVs. Raws li kev tiv thaiv kab mob qog noj ntshav, peb yav dhau los tau hais tias BAK1 co-receptor koom nrog OMV kev nkag siab thiab / lossis cov lus teb (Bahar li al., 2016). Hauv qhov kev tshawb fawb no, peb rov mus xyuas qhov kev pom zoo no siv qhov kev ntsuam xyuas priming. Ntawm no, bak mutant kab yog primed los ntawm OMV pretreatment, tab sis rau ib tug me ntsis tsawg tshaj li WT Col-0 nroj tsuag. Txawm hais tias cov paj no tsis yog qhov tseem ceeb nws tau ntau dua qhov pom nrog ob lub fls puas tau mutant kab. Qhov txiaj ntsig no kuj yog nyob rau hauv txoj kab nrog kev tshawb fawb tsis ntev los no uas tau pom tias kab bak mutant tau teb rau OMV li WT cov nroj tsuag hauv kev sim tshuaj tiv thaiv kab mob (Tran li al., 2021). Zuag qhia tag nrho, qhov no yuav qhia tau tias thaum BAK1 koom nrog hauv OMV kev nkag siab, lwm yam kev tiv thaiv kab mob thiab kev taw qhia txoj hauv kev tau pib los ntawm OMV, ua rau muaj kev tiv thaiv kab mob zoo thiab tiv thaiv kev loj hlob ntawm cov kab mob. Kev koom tes ntawm cov co-receptors BAK1 thiab SOBIR1 hauv kev teb rau OMVs (Bahar li al., 2016) ua rau peb xav tias ntau lub cev tiv thaiv kab mob, zoo li PRRs, koom nrog OMV kev nkag siab. Txawm li cas los xij, ib txoj kev tshawb fawb tsis ntev los no tau qhia tias cog kev tiv thaiv kab mob los ntawm OMVs tuaj yeem yog MAMP-ywj siab thiab tshwm sim los ntawm physio-chemical hloov pauv hauv cov nroj tsuag plasma membrane tshwm sim los ntawm OMV kev koom ua ke (Tran li al., 2021). Qhov no yog ib qho kev xav tsis txaus ntseeg uas tseem yuav tau hais ntxiv. Interestingly, McMillan et al. (2021) tau tshaj tawm tias OMVs kho nrog proteinase K khaws lawv lub cev tiv thaiv kab mob priming, qhia tias qhov haujlwm no yuav tsis muaj kev ywj pheej ntawm OMV proteinaceous cargo. Thaum qhov txiaj ntsig no tuaj yeem txhawb nqa MAMP-kev ywj pheej ntawm kev tiv thaiv kev tiv thaiv kab mob ntawm Tran li al. (2021), lwm yam, tsis yog-proteinaceous MAMPs tam sim no hauv OMVs xws li LPS thiab PGN tuaj yeem qhib MTI (Bahar li al., 2016; McMillan li al., 2021). Tsis tas li ntawd, proteinase K-kho OMVs khaws lawv lub peev xwm los txhawb kev loj hlob ntawm cov noob, qhia tias kev loj hlob inhibition yog nyob ntawm cov proteinaceous cargo ntawm OMVs (McMillan li al., 2021). Ua ke, cov txiaj ntsig no ntxiv hais txog qhov nyuaj ntawm cov nroj tsuag teb rau OMVs thiab qhov tseem ceeb ntawm kev siv ntau yam khoom siv los ntsuas kev koom tes ntawm ib tus neeg elicitor tshwj xeeb hauv txoj hauv kev tshwj xeeb.

Thaum xyoo 2021, plaub qhov kev tshawb fawb ywj pheej suav nrog qhov no (uas yuav tshwm sim ib txhij), tau tshaj tawm tias cov kab mob OMVs hloov kho cov nroj tsuag tiv thaiv kab mob thiab ua rau muaj kev cuam tshuam zoo tiv thaiv kab mob (Janda li al., 2021; McMillan li al., 2021; Tran et al., 2021). Cov txiaj ntsig zoo siab no tso OMVs ua tus tshiab thiab tseem ceeb hauv kev sib cuam tshuam ntawm cov nroj tsuag-microbe, qhov uas tseem muaj ntau yam yuav tsum tau kawm. Hauv txoj kev tshawb no, peb muab kev pom dav dav ntawm cov lus teb ntawm Arabidopsis rau Xcc OMV. Cov kev tshawb fawb ntxiv yuav xav tau kom nkag siab ntxiv txog cov khoom thiab cov txheej txheem koom nrog hauv kev nkag siab ntawm OMVs.

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