Kev txuag Cysteine-nplua nuj secretory Protein MaCFEM85 cuam tshuam nrog MsWAK16 kom ua kom cov nroj tsuag tiv thaiv
Oct 26, 2023
Abstract: Metarhizium anisopliaeyog cov kab mob entomopathogenic uas tuaj yeem txhim kho cov nroj tsuag kev loj hlob thiab kev tiv thaiv thaum ua raws li endophyte hauv cov nroj tsuag. Txawm li cas los xij, me ntsis paub txog kev sib cuam tshuam ntawm cov protein lossis lawv cov txheej txheem ua kom muaj zog. Feem ntau hauv cov kab mob fungal extracellular membrane (CFEM) cov proteins tau raug txheeb xyuas tias yog cov tshuaj tiv thaiv kab mob tiv thaiv kab mob uas cuam tshuam lossis qhib cov lus teb rau cov nroj tsuag. Ntawm no, peb tau txheeb xyuas CFEM domain-muaj protein ntau, MaCFEM85, uas yog nyob rau hauv lub plasma membrane. Cov poov xab ob-hybrid (Y2H), glutathione-S-transferase (GST) rub-down, thiab bimolecular fluorescence complementation kev soj ntsuam pom tau hais tias MaCFEM85 cuam tshuam nrog lub extracellular domain ntawm aMedicago saib(alfalfa) membrane protein, MsWAK16. Gene qhia kev txheeb xyuas tau pom tias MaCFEM85 thiab MsWAK16 tau raug tswj hwm zoo hauvM. anisopliaethiabM. sativ, raws li, los ntawm 12 mus rau 60 teev tom qab kev sib koom ua ke. Ntxiv cov poov xab ob-hybrid kev soj ntsuam thiab amino acid qhov chaw tshwj xeeb hloov pauv tau qhia tias CFEM domain thiab 52th cysteine tshwj xeeb yog xav tau rau kev sib cuam tshuam ntawm MaCFEM85 nrog MsWAK16. Kev tiv thaiv kev ua haujlwm tau pom tias JA tau nce-tswj, tab sis Botrytis cinerea qhov txhab loj thiab Myzus persicae kev tsim tawm tau raug txwv los ntawm kev nthuav qhia ib ntus ntawm MaCFEM85 thiab MsWAK16 hauv tus qauv tswv cog Nicotiana benthamiana. Ua ke, cov txiaj ntsig no muab kev nkag siab tshiab rau hauv cov txheej txheem molecular hauv qab kev sib cuam tshuam ntawm M. anisopliae nrog cov tswv tsev.
cistanche ntxiv cov txiaj ntsig-nce kev tiv thaiv
Ntsiab lus: phab ntsa-txuas nrog kinase; CFEMs; Metahizium anisopliae; cog kev tiv thaiv; Medicago saib
1. Taw qhia
Kev sib cuam tshuam ntawm cov nroj tsuag thiab microbes yog dav thiab yog qhov tshwm sim ntawm yav dhau los thiab tsis tu ncua co-evolution. Cov kev sib cuam tshuam no tuaj yeem muaj txiaj ntsig, nruab nrab, lossis qhov tshwm sim tsis zoo rau txhua tus neeg koom [1–3]. Cov txiaj ntsig zoo ntawm rhizospheric microbiota tuaj yeem txhim kho cov nroj tsuag kev loj hlob thiab txhim kho kev noj qab haus huv tag nrho los ntawm kev tiv thaiv cov kab mob hauv av lossis txhim kho cov khoom noj khoom haus [4,5]. Muaj ntau yam microbes muaj txiaj ntsig tuaj yeem txhim kho cov peev txheej cog xws li kev noj zaub mov zoo, kev loj hlob, thiab kab mob thiab kab tiv thaiv [6,7]. Metarhizium Sorok¯ın yog ib qho tseem ceeb genus ntawm entomopathogenic fungi nrog lub peev xwm los colonize nroj tsuag [8], tab sis kuj tseem muaj pov thawj qhia tias cov tswv cuab ntawm cov genus tuaj yeem txhim kho cov kab mob tiv thaiv kab mob. Piv txwv li, ib txoj kev tshawb nrhiav pom 60% inhibition ntawm Fusarium solani (Mart.) Sacc. nyob rau hauv lub xub ntiag ntawm Metarhizium robertsii (yav tas los hu ua M. anisopliae) piv nrog cov tswj tsis muaj M. robertsii [9]. Qee cov kab mob Metarhizium muaj peev xwm txhim kho cov nroj tsuag tiv thaiv kab tsuag thiab kab mob los ntawm kev hloov cov kev qhia ntawm cov noob tiv thaiv kab mob. Endophytic colonization nrog M. Roberts activates kev qhia ntawm ob qho tib si jasmonic acid (JA) thiab salicylic acid (SA) tiv thaiv txoj hauv kev hauv pobkws nplooj; Tsis tas li ntawd, xws li colonization yog txuam nrog kev txhawb nqa ntawm cov nroj tsuag kev loj hlob thiab kev tawm tsam ntawm Agrotis epsilon (Hufnagel) larval development [10]. Metarhizium guizhouense qhib lub -1, 3-glucanase thiab chitinase nyob rau hauv cov txiv hmab txiv ntoo tev ntawm Lansium parasiticum ua rau kev loj hlob inhibition ntawm Botrytis sp. thiab Fusarium sp. ntawm cov txiv hmab txiv ntoo ntawm Aglaia dookkoo Griff [11]. Ntxiv mus, thaum M. anisopliae tau siv rau cov txiv laum huab xeeb, ntau yam kev hloov pauv xws li WRKYs, MYCs, TGAs, thiab ethylene-responsive transcription factor tau qhib, thaum nitrate transporters thiab proteins binding dehydration-responsive elements kuj tau qhia txawv [12] . Qhov no qhia tias M. anisopliae tswj cov nroj tsuag tiv thaiv cov noob raws li nws colonizes cov nroj tsuag. Txawm li cas los xij, tsis tshua muaj kev paub txog cov txheej txheem hauv qab ntawm cov nroj tsuag tiv thaiv cov lus teb tom qab kis los ntawm Metarhizium. Effectors yog ib qho txhais tau tias los ntawm cov kab mob microorganisms tswj cov nroj tsuag tsis kam [13]. Kev ua kom cov nroj tsuag tsis kam yog feem ntau tshwm sim los ntawm oxidative tawg thiab induction ntawm cov tshuaj hormones, metabolites, thiab lwm yam teeb liab [13]. Cov nroj tsuag cov tshuaj hormones SA thiab JA ua lub luag haujlwm tseem ceeb hauv kev tsim cov nroj tsuag tsis kam, kho ob txoj kev tiv thaiv kev tiv thaiv [14]. SA txoj kev taw qhia txoj hauv kev ua haujlwm feem ntau hauv cov nroj tsuag kev tsis haum tshuaj thiab hauv cov txheej txheem tau txais kev tiv thaiv rau cov kab mob, tab sis nws kuj tseem tuaj yeem koom nrog hauv kev tiv thaiv cov nroj tsuag tsis ncaj ncees uas raug cuam tshuam los ntawm stinging kab noj [15]. Kev sib sau ntawm SA yog txuam nrog kev nthuav qhia ntawm cov noob encoding lipid hloov cov proteins (LTPs) thiab phenylalanine ammonia-lyase (PAL) [16], uas kho cov synthesis thiab tsub zuj zuj ntawm downstream tshwj xeeb metabolites xws li flavonoids thiab lignin [17]. JA signaling txoj haujlwm ua haujlwm ncaj qha thiab tsis ncaj qha rau cov khoom puas tsuaj, kab mob fungal, thiab kab tsuag. Cov kev tshawb fawb tau pom tias JA signaling muaj kev tswj xyuas cov kev sib txuas ntawm cov metabolites tshwj xeeb xws li terpenoids, phenylpropanoids, thiab alkaloids, uas muaj ntau yam kev ua haujlwm lom neeg [18]. Qee cov metabolites tshwj xeeb tau pom tias muaj nyob hauv cov nroj tsuag cog tom qab kho nrog methylJA (MeJA), suav nrog paclitaxel hauv Taxus hom [19,20], terpenoids hauv Centella Asiatica (L.) Nroog [21], thiab saponins hauv ginseng [22] . Daim ntawv thov ntawm SA thiab chitosan (CHT) raws li elicitors induced lignin tsub zuj zuj thiab tiv thaiv enzyme tshwm sim nyob rau hauv txiv lws suav, uas txo qhov tshwm sim ntawm Ralstonia solanacearum (Smith) Yabuuchi kab mob [23]. Tsis tas li ntawd, JA thiab SA qib hauv cov nplej tau nce zuj zus los ntawm kev siv cov elicitor PeaT, txhim kho cov lus teb tiv thaiv rau oat aphid Sitobion avenae (Fabricius) [24]. Qhov no qhia tau hais tias cov nroj tsuag tiv thaiv kab mob tuaj yeem tswj hwm los ntawm cov nyhuv no los ntawm cov tshuaj hormones cog thiab metabolites.
cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob
Feem ntau hauv cov kab mob fungal extracellular membrane (CFEM) cov proteins muaj nyob rau hauv ntau hom fungi. Lawv encode domains uas feem ntau yog 60 amino acids (aa) ntev thiab muaj yim tus yam ntxwv spaced cysteine [25]. CFEM domains zoo ib yam li epidermal growth factor (EGF)-zoo li domains, uas ua haujlwm li extracellular receptors, teeb liab transducers, los yog adhesion molecules nyob rau hauv host-pathogen kev sib cuam tshuam [26]. Proteins uas muaj CFEM domains tuaj yeem tswj xyuas cov nroj tsuag tiv thaiv teb los ntawm kev ua yeeb yam. Nyob rau hauv cov nplej nplooj xeb fungus Puccinia triticina Erikas, CFEM effector tus neeg sib tw PTTG_08198 ua kom nrawm nrawm ntawm kev tuag ntawm tes thiab txhawb nqa cov pa oxygen reactive (ROS) tsub zuj zuj [27]. Lub anthracnose fungus Colletotrichum graminicola (Ces.) GW Wils muaj tsib effectors (CgCFEM6, 7, 8, 9, thiab 15) uas tau pom tias inhibit BAX-induced programmed cell tuag hauv Nicotiana benthamiana Domin [28]. Cov phytosymbiotic mycorrhizal fungus Laccaria bicolor (Maire) PDOrton tau tshaj tawm kom zais ntau cov CFEM proteins, xws li Lac310796, Lac296573, thiab Lac296572, hauv cov ntaub so ntswg [29]. Txawm hais tias cov haujlwm nyuaj ntawm cov proteins no tsis tau kawm ntxiv hauv mycorrhizal symbiosis, cov txiaj ntsig ua ntej qhia tias CFEM cov proteins tuaj yeem ua haujlwm hauv kev taw qhia ntawm cov kab mob thiab cov nroj tsuag. M. anisopliae tuaj yeem ua haujlwm raws li ib qho entomopathogenic lossis endophytic fungus hauv cov nroj tsuag tswv [30]. Txawm li cas los xij, lub luag haujlwm ntawm CFEM cov proteins tseem tsis tau tshaj tawm. Peb yav dhau los tau txheeb xyuas qhov CFEM domain-muaj protein ntau hauv M. anisopliae, MaCFEM85 (GenBank ID: MZ682609) [31]. Ntawm no, peb tshaj tawm txog kev sib raug zoo ntawm MaCFEM85 thiab Medicago sativa phab ntsa-txuas nrog kinase MsWAK16. Peb tau txheeb xyuas cov txheej txheem MaCFEM85 thiab ua cov kev sim los txiav txim siab seb cov seem twg yog qhov tseem ceeb rau kev cuam tshuam nrog MsWAK16. Thaum kawg, siv N. benthamiana ua peb cov qauv cog, peb tau soj ntsuam cov nroj tsuag tiv thaiv cov lus teb rau Botrytis cinerea thiab aphid Myzus persicae nrog thiab tsis muaj kev nthuav qhia ntawm MaCFEM85 thiab MsWAK16.
Cov txiaj ntsig ntawm cistanche tubulosa- ua kom muaj zog tiv thaiv kab mob
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2. Cov txiaj ntsig
2.1. MaCFEM85 tau ze ze tshaj plaws rau Non-Pathogenic Fungal CFEMs thiab Localized rau Plasma Membrane
Ib tsob ntoo phylogenetic tau tsim los tshuaj xyuas kev sib raug zoo ntawm MaCFEM85 thiab lwm yam CFEM cov proteins los ntawm cov kab mob thiab cov kab mob uas tsis yog cov kab mob fungi. Cov no suav nrog Magnaporthe oryzae, Fusarium oxysporum (Schl)., Neurospora crassa Shear & BODodge, thiab Beauveria bassiana (Bals.) (saib Tshooj 4). MaCFEM85 muaj feem cuam tshuam nrog CFEM cov proteins hauv B. bassiana, thiab yog li ntawd, evolutionarily ze rau non-pathogenic dua li cov kab mob fungi (Daim duab 1a).
Daim duab 1a
2.2. MaCFEM85 tau tswj hwm thaum muaj kev cuam tshuam nrog M. sativa
Phab ntsa-txuas nrog receptor-zoo li kinases (WAKs) sawv cev rau ib pab pawg hauv cov receptor-zoo li cov protein kinases (RLKs) superfamily, thiab suav nrog cov npe extracellular, transmembrane helix, thiab intracellular kinase domain. Lawv ua lub luag haujlwm tseem ceeb hauv kev tswj cov nroj tsuag kev loj hlob, kev loj hlob, kev ntxhov siab, thiab cov kab mob tiv thaiv kab mob [32]. RNA tau muab rho tawm los ntawm M. anisopliae hyphae thiab M. sativa cov hauv paus hniav tom qab co-incubation los tshawb xyuas cov qauv qhia ntawm MaCFEM85 thiab MsWAK16. Ob leeg MaCFEM85 thiab MsWAK16 tau qhia nyob rau theem siab dua thaum sib koom ua ke los ntawm 12 txog 60 h (Daim duab 1c,d). Los ntawm 0 txog 36 h, MaCFEM85 qhia maj mam nce, nce siab ntawm 36 teev. Nws yog × 593 lub sij hawm ntau heev qhia nyob rau hauv kev sib xyaw ntawm M. anisopliae thiab M. sativa piv nrog M. anisopliae loj hlob ib leeg. Txawm hais tias MaCFEM85 kev qhia tau txo qis me ntsis hauv lub sijhawm 48-60 h, nws tseem tau nthuav tawm ntawm qib siab dua li hauv M. anisopliae loj hlob ib leeg. MsWAK16 kuj tseem muaj kev tswj hwm zoo, nrog rau kev qhia txog qhov kawg ntawm 36 teev. Hauv M. sativa tus kab mob M. anisopliae, MsWAK16 yog × 89.06 lub sij hawm nthuav tawm ntau dua li hauv M. sativa yam tsis muaj M. anisopliae. Los ntawm 36 mus rau 60 h, theem ntawm MsWAK16 tau txo qis me ntsis, tab sis nws cov lus qhia tseem muaj ntau dua li cov nroj tsuag uas tsis muaj tshuaj lom.
2.3. MaCFEM85 cuam tshuam nrog MsWAK16 Hauv Vitro thiab Hauv Vivo
Txhawm rau tshawb xyuas lub peev xwm ua haujlwm ntawm MaCFEM85 hauv kev teb rau M. anisopliae kev kho mob, ib qho tshuaj ntsuam poov xab ob-hybrid (Y2H) tau ua los txhawm rau txheeb xyuas tus tswv tsev cov protein uas cuam tshuam nrog MaCFEM85. Kev sib cuam tshuam ntawm extracellular domain ntawm MsWAK16 (MsWAK16-ED) thiab MaCFEM85 tau tshuaj xyuas nrog ib-rau-ib poov xab ob-hybrid assay siv MaCFEM85 hauv pGBKT7 raws li BD vector thiab MsWAK16 hauv pGADT7 raws li AD vector. Tag nrho cov poov xab hloov pauv tau loj hlob zoo ntawm SD-T / L tsis muaj qhov nruab nrab, thiab pawg tswj hwm zoo thiab pab pawg sim tau loj hlob zoo ntawm SD-T / L / H / A + X- -gal deficient nruab nrab. Qhov no tau qhia tias MaCFEM85 tuaj yeem cuam tshuam nrog MsWAK16-ED (Daim duab 2a).
Daim duab 2. Kev lees paub ntawm kev sib cuam tshuam ntawm MaCFEM85 thiab MsWAK16.
Rau hauv vitro validation, glutathione-S-transferase (GST)-tagged MsWAK16-ED (encoding ib yam khoom ntawm 60 kDa) tau muab tso rau hauv pGEX6-P2, thiab polyhistidine (Nws)-tagged MaCFEM85 ( encoding ib yam khoom ntawm 17 kDa) tau muab tso rau hauv pET-21b. Immunoblotting nrog Nws thiab GST cov tshuaj tiv thaiv tau pom tias cov protein sib xyaw ua ke MaCFEM85-Nws cuam tshuam nrog GST-MsWAK16-ED prey tab sis tsis nrog GST ib leeg thiab GST-MsWAK16- ED cuam tshuam nrog Nws -MaCFEM85 (Daim duab 2b). Txhawm rau kom paub meej ntxiv txog kev sib cuam tshuam ntawm MaCFEM85 thiab MsWAK16, peb tau ua qhov kev ntsuam xyuas bimolecular fluorescence complementation (BiFC) nrog MaCFEM85-YFPN thiab MsWAK16-YFPC tsim. Co-expression of MaCFEM85-YFPN thiab MsWAK16-YFPC nyob rau hauv cov nplooj ntawv luam yeeb ua rau lub teeb daj daj, qhia tias MaCFEM85 cuam tshuam nrog MsWAK16 (Daim duab 2c).
2.4. Cov Chaw Tseem Ceeb rau MaCFEM85 thiab MsWAK16 Kev Sib Tham
Txhawm rau txheeb xyuas thaj tsam ntawm MaCFEM85 uas yuav tsum muaj rau kev sib cuam tshuam nrog MsWAK16-ED, protein ntau tau kwv yees nrog online software SMART (Daim duab 3a). MaCFEM85 muaj cov npe CFEM hauv thaj tsam 19-86 aa. Cov qauv tertiary kuj tau kwv yees (Daim duab 3b). Lub yim cysteine nyob rau hauv lub npe no ua rau plaub disulfide daim ntawv cog lus (CYS26 thiab CYS69, CYS30 thiab CYS64, CYS43 thiab CYS50, thiab CYS52 thiab CYS85), tswj kev ruaj ntseg ntawm cov protein (Daim duab 3b). Txhawm rau kom lees paub qhov chaw sib cuam tshuam ntawm qhov chaw hauv MaCFEM85, xya qhov hloov pauv ntawm cov protein tau tsim thiab muab tso rau hauv pGBKT7 raws li cov khoom noj protein. Txhua tus vector recombinant tau co-transfected nrog AD-MsWAK16-ED hauv Y2H kub hom. Cov kab mob nrog CYS52 hloov pauv tsis tau loj hlob ntawm cov xov xwm xaiv (Daim duab 3c, qhia hauv liab), qhia tias CYS52 yuav tsum muaj rau kev sib cuam tshuam ntawm MaCFEM85 nrog MsWAK16-ED.
2.5. Kev sib cuam tshuam ntawm MaCFEM85 nrog MsWAK16 Ua kom cov nroj tsuag tiv thaiv kab mob
2.5.1. Kev Ntsuas Lub Luag Haujlwm ntawm MaCFEM85 thiab MsWAK16 hauv Kev Tiv Thaiv Kab Mob B. cinerea
Txhawm rau tshawb nrhiav qhov muaj peev xwm koom nrog ntawm MaCFEM85 thiab MsWAK16 hauv cov lus teb tiv thaiv kab mob, peb tau tshuaj xyuas seb puas muaj kev cuam tshuam ntau dhau ntawm MaCFEM85, MsWAK16, lossis MaCFEM85 thiab MsWAK16 tuaj yeem ua rau muaj zog tiv thaiv B. cinerea. Peb tau qhia cov vectors nyob rau hauv N. benthamiana nplooj. Kev ntsuam xyuas Western blot pom tau tias MaCFEM85 thiab MsWAK16 tau qhia nyob rau theem sib piv thaum lawv tau hais tawm ib leeg lossis ua ke, uas qhia tau tias MaCFEM85 thiab MsWAK16 tau ua tiav hauv kev luam yeeb (Daim duab 4a). Kev ntsuam xyuas kab mob kuj tau ua tiav siv N. benthamiana infiltrated nrog MaCFEM85, MsWAK16, MaCFEM85+MsWAK16, lossis GFP tswj. Cov kab mob me me (los ntawm ~ 30% ntawm 2 d tom qab inoculation) ntawm nplooj infiltrated nrog MaCFEM85, MsWAK16, los yog MaCFEM85+MsWAK16 piv nrog cov nroj tsuag tswj (Daim duab 4b). Cov ntaub ntawv no qhia tau hais tias ib ntus qhia ntawm MaCFEM85 nyob rau hauv N. benthamiana tau hais kom muaj zog tiv thaiv B. cinerea thiab hais tias MaCFEM85 thiab MsWAK16 zoo tswj cov lus teb tiv thaiv B. cinerea.
Daim duab 3. Kev txheeb xyuas qhov chaw sib cuam tshuam ntawm MaCFEM5 thiab MsWAK16.
Daim duab 4. Induction ntawm cov nroj tsuag tsis kam los ntawm kev qhia ib ntus ntawm MaCFEM85 thiab MsWAK16.
2.5.2. Kev Ntsuas Lub Luag Haujlwm ntawm MaCFEM85 thiab MsWAK16 hauv Aphid Defense hauv N. benthamiana
Txhawm rau tshawb xyuas ntxiv txog lub luag haujlwm ntawm MaCFEM85 thiab MsWAK16, N. benthamiana cov nroj tsuag hloov pauv hloov pauv ntawm MaCFEM85 thiab MsWAK16 tau kis nrog M. persicae, thiab cov pej xeem raug soj ntsuam. Rau qhov kev sim no, ib cheeb tsam loj ntawm txhua N. benthamiana nplooj yog agroinfiltrated nrog cov recombinant binary vector pYBA1132 uas muaj MaCFEM85 thiab MsWAK16. GFP tau siv los ua tus tswj. Thaum 12 teev tom qab infiltration, 20 M. pericae cov neeg laus raug kaw rau ntawm txhua nplooj, nthuav tawm thaj chaw nkag mus rau aphids. Hauv peb hnub tom ntej no, cov neeg laus aphid tuag thiab cov nymphs raug kaw; cov nymphs ces muab tshem tawm. Tsis muaj qhov sib txawv tseem ceeb hauv kev pheej hmoo tuag ntawm M. pericae cov neeg noj zaub mov ntawm cov nroj tsuag qhia MaCFEM85, MsWAK16, lossis MaCFEM85+MsWAK16 (χ2=3.65827, DF=3, p < 0.30081) ) (Daim duab 4c). Txawm li cas los xij, ntawm 24, 48, thiab 72 h, qhov nruab nrab ntawm cov xeeb ntxwv uas tsim los ntawm txhua tus neeg laus aphid tau ntau dua ntawm N. benthamiana qhia GFP tswj piv nrog cov uas qhia txog MaCFEM85, MsWAK16, lossis MaCFEM85+MsWAK16 (Daim duab 4d ib).
2.5.3. Kev Ntsuas Lub Luag Haujlwm ntawm MaCFEM85 thiab MsWAK16 hauv Hormone Accumulation thiab Hormone-Related Gene Expression
Txhawm rau txheeb xyuas qhov sib txawv ntawm cov lus teb tiv thaiv ntawm N. benthamiana cov nroj tsuag qhia eGFP, MaCFEM85, MsWAK16, thiab MaCFEM85+MsWAK16, peb ntsuas JA, SA, thiab tag nrho cov qib flavonoid, thiab kev qhia ntawm cov noob muaj feem xyuam. Cov txiaj ntsig tau pom tias JA thiab SA qib sib txawv ntawm cov nroj tsuag qhia eGFP, MaCFEM85, MsWAK16, thiab MaCFEM85+MsWAK16 (Daim duab 5a). Piv nrog rau cov uas qhia eGFP, JA qib tau qis dua hauv cov nroj tsuag qhia MaCFEM85; SA qib tau nce me ntsis, tab sis qhov sib txawv tsis tseem ceeb. Hauv qhov sib piv, cov nroj tsuag qhia MsWAK16 tsis pom qhov sib txawv tseem ceeb hauv JA qib piv nrog eGFP tswj, qhov SA qib tau qis dua hauv kev tswj hwm (Daim duab 5a). JA thiab SA qib tau nce thiab txo qis, feem, hauv cov nroj tsuag qhia txog MaCFEM85+MsWAK16 piv nrog eGFP.
Daim duab 5. Hormone ntau ntau thiab cov tshuaj hormone teb cov noob qhia
Tag nrho cov ntsiab lus flavonoid tau nce siab dua hauv cov nroj tsuag uas qhia txog MaCFEM85+MsWAK16 piv nrog rau lwm pab pawg kho mob (Daim duab 5a). Cov qib biosynthetic gene qhia tau zoo sib xws hauv cov nroj tsuag qhia MsWAK16 thiab MaCFEM85+MsWAK16. Piv txwv li, cov noob muaj feem xyuam rau JA cov lus teb, uas yog COI1, MYC2, thiab PDF1.2, tau raug kho kom zoo dua qub piv nrog cov nroj tsuag qhia GFP (Daim duab 5b). Ib yam li ntawd, SA-txog cov noob NPR1, WRKY70, thiab PR1 tau muaj qhov sib txawv loj hauv cov nroj tsuag uas qhia MsWAK16 thiab MaCFEM85+MsWAK16; NPR1 tau hloov kho, qhov WRKY70 thiab PR1 tau raug txo qis dua piv nrog kev tswj hwm (Daim duab 5b). Peb kuj tau tshuaj xyuas cov kev qhia ntawm cov noob tseem ceeb hauv Shikimic acid thiab phenylpropanoid synthesis txoj hauv kev, uas yog ob qho tib si cuam tshuam nrog flavonoid synthesis. Feem ntau, kev qhia ntawm MaCFEM85 ib leeg tsis ua rau muaj kev qhia ntau dua ntawm cov noob hauv luam yeeb. Txawm li cas los xij, cov noob no tau raug tswj hwm hauv kev haus luam yeeb qhia MsWAK16 lossis MsWAK16+MaCFEM85 piv nrog GFP tswj (Daim duab 5c).
cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob
3. Kev sib tham
3.1. Kev txuag CFEM Protein Motif ua haujlwm ntau yam hauv cov kab mob fungal
CFEM domain yog tshwj xeeb rau fungi thiab feem ntau tshwm sim nyob rau hauv fungal extracellular membrane proteins. Cov npe tau tshwm sim hauv cov poj koob yawm txwv tsis ntev los no ntawm Ascomycota thiab Basidiomycota [33]. Kev tshawb fawb tsis ntev los no tau pom tias CFEM cov proteins hauv cov kab mob fungal tuaj yeem ua raws li cov tshuaj tiv thaiv kab mob, ua rau cov nroj tsuag tiv thaiv kab mob los yog ua kom muaj zog nyob ntawm seb hom kab mob [28,34,35]. Muaj ob peb tsab ntawv ceeb toom ntawm CFEM domain-muaj proteins nyob rau hauv M. anisopliae, tsuas yog nyob rau hauv lub ntsiab lus ntawm evolutionary piv nrog rau lwm yam fungi [36]. Nyob rau hauv txoj kev tshawb no, peb piv M. anisopliae CFEM cov proteins nrog rau lwm cov CFEM cov proteins paub nyob rau hauv ob qho tib si pathogenic thiab non-pathogenic fungi. Peb tau lees paub tias qhov ze tshaj homolog ntawm MaCFEM85 yog Cfem5 pom nyob rau hauv Beauveria bassiana, ib qho ntawm 12 CFEM proteins nyob rau hauv cov hom (Sab ntxiv daim duab S1). BbCfem5 yog qhov tseem ceeb rau kev nrhiav tau hlau [37]. Ntxiv mus, raws li kev kwv yees tertiary qauv, CFEM sau hauv MaCFEM85 zoo ib yam li Surface Antigen Protein 2 (CSA2) pom nyob rau hauv Candida albicans (CPRobin) Berkhout, uas 65 seem (96% ntawm cov kab ke) tau ua qauv nrog 99.8. % kev ntseeg siab [31]. Candida albicans yog cov kab mob tsiaj; CSA2 ua lub luag haujlwm tseem ceeb hauv kev loj hlob thiab pathogenicity los ntawm kev rho tawm heme los ntawm hemoglobin thiab thauj heme los ntawm phab ntsa ntawm tes mus rau plasma [38-40]. Peb xav tias MaCFEM85 tuaj yeem koom nrog hauv M. anisopliae virulence ntawm kab hosts. Cov haujlwm sib xyaw ua ke ntawm MaCFEM85 hauv cov tsiaj txhu kab mob thiab ua kom cov nroj tsuag tiv thaiv kab mob ua rau cov protein ua rau kev tshawb fawb yav tom ntej zoo siab.
3.2. Disulfide Bonds yog cov qauv tseem ceeb rau kev ua haujlwm ntawm cov protein
Lub conformational kev ncaj ncees ntawm cov qauv protein yog ncaj qha ntsig txog nws lub peev xwm ua haujlwm. Cov nyiaj disulfide tsim los ntawm cysteine khub txhawb nqa protein folding thiab conformational stability thiab raug txiav txim siab los tsim cov chaw tseem ceeb rau kev lees paub thiab khi ntawm cov receptors tshwj xeeb lossis ligands [41]. Piv txwv li, cuam tshuam ib qho ntawm peb qhov kev khaws cia disulfide daim ntawv cog lus hauv cov nroj tsuag kab mob Cladosporium fulvum effector protein AVR4 ua rau muaj kev cuam tshuam ntawm protease thiab txo qis chitin-binding peev xwm [42]. Hauv peb lwm C. fulvum proteins (ECP1, ECP2, thiab ECP5), ib qho kev hloov pauv ntawm alanines rau cysteine dampen lub txiv lws suav hypersensitive teb, qhia tias cov cysteine yuav yog ib qho tseem ceeb rau kev tswj kev ruaj ntseg thiab kev ua haujlwm siab heev [43]. Tsis tas li ntawd, nyob rau hauv mature protein MC69 ntawm Magnaporthe oryzae, mutagenesis ntawm ob qhov chaw khaws cia cysteine residues (Cys36 thiab Cys46) tuaj yeem cuam tshuam MC69 kev ua haujlwm yam tsis muaj kev cuam tshuam rau kev zais cia, qhia txog qhov tseem ceeb ntawm cov nyiaj disulfide tshwj xeeb hauv cov kab mob ntawm MC69 [44]. CFEM domain zoo nkaus li zoo sib xws hauv qhov loj thiab nyob rau hauv cov qauv ntawm cysteine residues rau EGF-zoo li domains, uas muaj peb los yog plaub khub ntawm disulfide bonds. EGF proteins tuaj yeem ua haujlwm raws li cov cell-nto receptors, teeb liab transducers, los yog adhesion molecules nyob rau hauv host-pathogen kev sib cuam tshuam [45]. Nyob rau hauv txoj kev tshawb no, peb tsis tsuas pom tias CFEM domain ntawm MaCFEM85 muaj yim conserved cysteines uas tsim plaub khub ntawm disulfide bonds (Daim duab 3b), tab sis peb kuj validated tias CFEM yog lub tseem ceeb domain rau kev sib cuam tshuam ntawm MaCFEM85 thiab MsWAK16. Tsis tas li ntawd, los ntawm qhov chaw qhia kev hloov pauv ntawm cysteine residues rau alanine, peb pom tias cysteine residue ntawm txoj hauj lwm 52 yog qhov chaw tseem ceeb uas yuav tsum muaj rau kev sib cuam tshuam (Daim duab 3c). Cov txiaj ntsig no muab lub hauv paus rau kev tshawb fawb ntxiv ntawm kev ua haujlwm ntawm lub cev ntawm CFEM85-WAK16 kev sib cuam tshuam.
3.3. Kev sib cuam tshuam ntawm MaCFEM85 nrog MsWAK16 Activated Plant Defenses
Hauv kev sib cuam tshuam ntawm microbial-cog, cov lus teb tiv thaiv cov nroj tsuag feem ntau yog qhib los ntawm microbial effector proteins. Induced tiv thaiv yog dhau los ua ib qho cuab yeej tseem ceeb hauv kev tiv thaiv kab mob lom los txhawb kev tiv thaiv. CFEM cov proteins tau raug txheeb xyuas tias yog cov tshuaj cuam tshuam nrog kev tswj hwm ntawm cov nroj tsuag tiv thaiv kab mob lossis inhibition [28,46]. Txawm li cas los xij, muaj qhov tsis txaus ntseeg ntawm kev tshawb fawb luam tawm txuas cov kev cai ntawm cov kab mob tiv thaiv kab mob no rau lawv lub luag haujlwm tshwj xeeb hauv cov kab mob thiab kab mob. Hauv txoj kev tshawb no, peb tau siv cov kab mob entomopathogenic thiab endophytic fungus, M. anisopliae, txhawm rau txheeb xyuas qhov kev sib cuam tshuam ntawm MaCFEM85 thiab cell phab ntsa-txuas nrog kinase, MsWAK16, hauv M. sativa. Cov txiaj ntsig tau pom tias qhov kev sib cuam tshuam no txo qis qhov mob sib kis tom qab inoculation nrog B. cinerea (Daim duab 4b) thiab txo cov pejxeem kev loj hlob ntawm M. persicae (Daim duab 4d), qhia txog kev sib cuam tshuam ntawm MaCFEM85 thiab MsWAK16 nce M. sativa tiv thaiv aphids. Kev hloov pauv hauv qib JA, SA, thiab ethylene (ET) tuaj yeem siv los ua cov cim los ntsuas qhov induction ntawm cov nroj tsuag tsis kam [47,48]. Ntawm no, peb tau pom tias MaCFEM85 cuam tshuam nrog MsWAK16 los cuam tshuam cov nroj tsuag tiv thaiv los ntawm kev tswj hwm hormonal thiab inhibit qhov kev loj hlob ntawm M. persicae (Daim duab 4d). Cov kev tshawb fawb zoo sib xws tau tshaj tawm yav dhau los. Piv txwv li, Brevibacillus laterosporus effector protein PeBL1 tau pom tias yuav ua rau JA thiab SA tsub zuj zuj hauv cov txiv lws suav thiab txo qis kev loj hlob ntawm cov neeg thib ob thiab thib peb ntawm M. persicae uas pub rau cov nroj tsuag. Tsis tas li ntawd, cov txiv lws suav tshuaj tsuag nrog cov tshuaj muaj txiaj ntsig zoo rau M. persicae [49]. Kev siv cov elicitor protein PeBC1 rau cov taum ntau (Phaseolus vulgaris L.) ua rau muaj kev tshaj tawm thiab tseem ceeb cuam tshuam rau cov txiv duaj ntsuab aphids. Cov nroj tsuag kho nrog PeBC1 qhia txog kev txhim kho tseem ceeb ntawm cov noob ntsig txog JA thiab SA [50]. Lub Beauveria bassiana elicitor PeBb1 txo cov fecundity ntawm M. persicae ntawm haus luam yeeb thiab induces kev qhia ntawm JA thiab ET cov noob caj noob ces [51]. Hauv txoj kev tshawb fawb tam sim no, kev nthuav qhia ib ntus ntawm MaCFEM85 thiab MsWAK16 hauv kev haus luam yeeb tau txhawb nqa JA cov lus teb ntsig txog cov noob COI1, MYC2, thiab PDF1.2 (Daim duab 5b) thiab nce JA thiab tag nrho cov ntsiab lus flavonoid (Daim duab 5a), uas yog piv rau cov txiaj ntsig. nyob rau hauv cov ntaub ntawv raws li tau piav saum toj no. Txawm li cas los xij, peb tsis tau kuaj pom cov qib siab ntawm SA hauv cov luam yeeb 12 teev tom qab nkag mus, thiab cov nroj tsuag hloov pauv hloov pauv MsWAK16 lossis MaCFEM85+MsWAK16 tau pom tias txo qis SA qib thiab txo qis ntawm cov noob koom nrog hauv SA teb (Daim duab 5a,b) . Qhov no qhia tau hais tias stimulation ntawm cov nroj tsuag sib txawv cov tshuaj hormones tuaj yeem ua tsis tau tsuas yog rau kev sib koom ua ke, tab sis kuj rau kev sib tham thiab kev tawm tswv yim, tso cai rau cov nroj tsuag teb kom tsim nyog rau cov stimuli sib txawv. Nce qib JA tab sis qis SA qib tau tshaj tawm yav dhau los hauv cov nroj tsuag. Piv txwv li, hauv A. thaliana tsis zoo rau SA tsub zuj zuj, JA qib tau 25-fold siab dua hauv hom tsiaj qus A. thaliana, thiab JA- teb cov noob tau qhib [52]. Nws kuj tau raug tshaj tawm tias qee cov kab mob tuaj yeem nce JA qib hauv cov nroj tsuag kom inhibit SA tsub zuj zuj, zam kev puas tsuaj los ntawm SA. Piv txwv li, Pseudomonas syringae tsom rau COI1 receptor los ntawm cov tshuaj lom, coronatine, uas tswj tsis zoo rau JA txoj kev [53]. Qhov no txhawb nqa MYC2-induced upregulation of the transcription factors ANAC019, ANAC055, and ANAC072 [54], inhibiting the transcription of ICS1 (ib tug tseem ceeb noob rau SA synthesis) thiab yog li downregulating SA ntau lawm thiab teeb liab transduction. Hauv txoj kev tshawb fawb tam sim no, kev sib cuam tshuam ntawm MaCFEM85 thiab MsWAK16 tau ua rau muaj kev nce ntxiv ntawm JA thiab kev txhawb nqa ntawm JA-responsive genes tab sis inhibition ntawm SA tsub zuj zuj thiab kev hloov pauv ntawm SA cov noob caj noob ces. Qhov no tau qhia tias kev sib cuam tshuam ntawm MaCFEM85 thiab MsWAK16 induced JA, yog li txhim kho cov nroj tsuag tsis kam mus rau aphids.
Hauv N. benthamiana transiently nthuav qhia MaCFEM85 thiab MsWAK16, JA qib tau nce thiab B. cinerea qhov txhab loj tau txo qis (Daim duab 4b), zoo ib yam nrog cov kev tshawb fawb yav dhau los. JA koom nrog cov nroj tsuag tiv thaiv kab thiab necrotrophic pathogens [52,55]. Raws li cov kab mob necrotrophic, B. cinerea tau inhibited los ntawm JA thiab ET tsub zuj zuj [56]. Nyob rau hauv A. thaliana ET-deficient mutant ein2-1 thiab JA-response mutant coi1-1, theem ntawm kev tiv thaiv cov noob, PDF1.2 tau txo qis thiab rhiab heev rau B. cinerea tau txhim kho [ 57] ib. Peb pom ntawm no tias JA tsub zuj zuj thiab kev hloov pauv ntawm JA-txog cov noob tau nce ntau hauv N. benthamiana ib ntus qhia txog MaCFEM85 thiab MsWAK16, qhov sib sau ntawm SA thiab kev hloov pauv ntawm SA-txog noob caj noob ces tau inhibited. MaCFEM85 thiab MsWAK16 qhia kuj pom tias muaj kev cuam tshuam rau B. cinerea. Qhov no tau qhia tias JA tau qhib los ntawm MaCFEM85 thiab MsWAK16 thiab ua lub luag haujlwm tseem ceeb hauv cov nroj tsuag tiv thaiv B. cinerea.
4. Cov ntaub ntawv thiab cov txheej txheem
4.1. Cov kab mob fungal, cov khoom siv cog, thiab kev coj noj coj ua
Metarhizium anisopliae isolate hom Ma 9 tau coj los ntawm qos yaj ywm-qab zib-agar (PSA) nruab nrab (200 g peeled qos yaj ywm extract boiled hauv dej, 20 g sucrose, thiab 15 g agar / L). Cov hmoov sporangium tshiab tau sau tom qab 10 d. Nicotiana benthamiana cov nroj tsuag tau loj hlob nyob rau hauv ib qho kev nyab xeeb chamber nrog 14/10 h lub teeb / tsaus voj voog (27/25 ◦C). Rau kev hloov pauv noob caj noob ces ntawm Agrobacterium-mediated transformation, Agrobacterium tumefaciens strain GV3101 tau coj mus rau hauv Luria Broth (LB) nruab nrab (10 g tryptone, 5 g poov xab extract, thiab 10 g NaCl / L). Poov xab strain kub (OE Biotech Co., Ltd., Shanghai, Suav teb) yog kab lis kev cai ntawm poov xab extract peptone dextrose (YPDA) nruab nrab (10 g poov xab extract, 20 g peptone, 20 g qabzib, thiab 0.03 g adenine hemisulfate / L). Rau txhua tus kab mob thiab kab mob, cov tshuaj tua kab mob tsim nyog tau siv, uas yog rifampin, kanamycin, lossis ampicillin (25, 50, lossis 50 µg / mL, raws li). Cov kab mob thiab cov plasmids siv hauv txoj kev tshawb no tau teev nyob rau hauv Cov Lus Qhia Ntxiv S1. Medicago sativa noob tau nto sterilized nrog 75% ethanol rau 1 min, tom qab ntawd 50% NaClO (5.5%) rau 15 min. Cov noob tau muab sib xyaw kom huv si thiab tom qab ntawd ntxuav hauv dej tsis huv peb zaug rau 5 feeb txhua. Cov noob tau muab tso rau ntawm qhov kub ntawm 4 ◦C hauv qhov tsaus rau ntau tshaj 24 teev, tom qab ntawd germinated ntawm 1% dej agar daim hlau ntawm chav tsev kub thaum hmo ntuj. Peb hnub tom qab germination, cov yub loj hlob tau hloov mus rau lim ntawv ntawm 9-cm sterile petri tais diav, nrog 20 seedlings ib phaj. Cov kev kho mob thiab kev tswj tau muab faib rau 15 lub tais txhua, raws li. Tom qab ntawd cov nroj tsuag tau irrigated nrog 10 mL ntawm M. anisopliae spore suspension muaj 107 / mL, thiab tswj tau irrigated nrog 10 mL sterile dej. Thaum 0, 12, 24, 48, thiab 60 h, xaiv random ntawm seedlings raug coj los ntawm peb petri tais diav rau txhua lub sij hawm. Cov qauv no tau khov rau hauv cov kua nitrogen thiab khaws cia ntawm -80 ◦C.
4.2. qRT-PCR thiab Plasmid Construction
Tag Nrho RNA tau muab rho tawm los ntawm M. anisopliae (hyphae) thiab M. sativa (roots) nrog TRIzol reagent (Invitrogen, CA, USA) ua raws li cov chaw tsim khoom cov lus qhia. Qhov zoo thiab kev nplua nuj ntawm cov txiaj ntsig RNA raug ntsuas nrog NanoPhotometer® (Implen, Münich, Lub Tebchaws Yelemees). Thawj-strand cDNA synthesis (txog 2ug RNA) tau ua tiav siv 5 × All-In-One RT Master Mix (Applied Biological Materials Inc., Vancouver, BC, Canada) ua raws li cov chaw tsim khoom cov lus qhia. qRT-PCR tau ua tiav siv 2 × SYBR Green qPCR Master Mix los ntawm US EVER BRIGHT ® INC. thiab ABI QuantStudio 5 system ua raws li cov chaw tsim khoom cov lus qhia. Cov primers siv rau txhua tus noob tau teev nyob rau hauv Cov Lus Ntxiv S2. Maury [58], Msactin [59], thiab Nbactin [60] tau siv los ua cov genes sab hauv rau normalization ntawm cov ntaub ntawv qhia. Cov tshuaj tiv thaiv tau ua raws li cov xwm txheej hauv qab no: 5 feeb ntawm 95 ◦C, ua raws li 40 lub voj voog ntawm 95 ◦C rau 15 s thiab ntawm 60 ◦C rau 40 s. Muaj peb qhov kev rov ua dua tshiab rau txhua tus qauv. Tus txheeb ze qhia tau suav nrog txoj kev 2−∆∆Ct [61]. Kev txheeb xyuas qhov tseem ceeb tau txiav txim siab siv ib txoj kev tsom xam ntawm qhov sib txawv (ANOVA) thiab Duncan qhov kev sim ntau yam hauv SPSS v20.0 (SPSS).
4.3. Transient Expression of Proteins hauv N. benthamiana
MaCFEM85 coding sequence (CDS) tau nthuav dav nrog ultra-fidelity DNA polymerase siv cDNA ua tus qauv. Rau kev ntsuam xyuas subcellular localization, PCR cov khoom uas muaj CDS rau MaCFEM85 tau cloned rau hauv pYBA1132-eGFP (digested nrog EcoRI thiab SalI) thiab pcambia1300-cherry (EcoRI thiab SacI), feem. Tag nrho cov constructs tau validated nrog sequencing (Tsingke Biotechnology Co., Ltd. Beijing, Tuam Tshoj). Cov primers siv nyob rau hauv txoj kev tshawb no yog teev nyob rau hauv Supplementary Table S2. Rau kev nthuav qhia ib ntus ntawm MaCFEM85-eGFP thiab MaCFEM85-mCherry hauv N. benthamiana, A. tumefaciens hom GV3101 tau hloov pauv nrog txhua plasmid thiab txheeb xyuas nrog PCR. Cov kab mob Agrobacterium tau coj mus rau hmo ntuj ntawm 28 ◦C nrog kev co. Cov hlwb raug sau los ntawm 5 min 5000 rpm centrifugation nyob rau hauv chav tsev kub, ntxuav peb zaug nrog sterile ob-distilled dej, thiab resuspended nyob rau hauv 10 mM MgCl2 tsis (muaj 10 mM MES thiab 10 mM acetosyringone, pH 5.7). Lub cell ncua kev kawm ntawv tau hloov kho rau OD600 ntawm 0.5, tom qab ntawd nkag mus rau hauv qab ntawm 4- mus rau 5-lub lim tiam-laus N. benthamiana nplooj nrog 1-mL syringe. Txhua nplooj yog infiltrated nrog 50 µL A. tumefaciens; peb nplooj raug kho rau ib tsob nroj thiab peb tsob ntoo nyob hauv txhua pab pawg kho mob. Cov nplooj uas kho tau raug sau tom qab 30 teev thiab pom nrog Zeiss LSM980 confocal microscope (Carl Zeiss, Lub teb chaws Yelemees) los txiav txim qhov sub-cellular localization.
cistanche cov txiaj ntsig rau cov txiv neej-ua kom muaj zog tiv thaiv kab mob
4.4. Phylogogenetic Analysis
Ib tsob ntoo phylogenetic tau tsim los siv txoj hauv kev sib koom ua ke hauv MEGA X. 1000 bootstrap replicates tau siv tus qauv P-distance. Cov amino acid sequences siv los tsim tsob ntoo tau txais nrog BLASTP tshawb nrhiav tawm tsam NCBI database ntawm cov kab mob ntsig txog (piv txwv li, Magnaporthe oryzae, Botrytis cinerea, Fusarium graminearum, Colletotrichum graminicola, Fusarium oxysporum, Neurospora crassa, Aspergillus the fumigatusligillus fumigatus. , Trichoderma harzianum, Beauveria bassiana, thiab Paecilomyces lilacinus) siv MaCFEM85 raws li cov lus nug.
4.5. Poov Ob-Hybrid Assays
Lub Matchmaker Kub Poov Ob-Hybrid System (Clontech Laboratories, Inc.; tam sim no Takara Bio USA, Inc.) tau siv los txheeb xyuas qhov kev cuam tshuam ntawm MaCFEM85 thiab MsWAK16. MaCFEM85 yam tsis muaj lub teeb liab peptide tau nkag mus rau hauv pGBKT7 raws li cov kab nuv ntses thiab cov khoom siv ntxiv ntawm MsWAK16 (MsWAK16-ED) tau muab tso rau hauv pGADT7 raws li cov neeg raug tsim txom. Kev npaj thiab hloov pauv ntawm cov poov xab muaj peev xwm tau ua tiav siv Frozen-EZ Poov Hloov Hloov II Cov Khoom Siv ™ (ZYMO Research, CA, USA) ua raws li cov chaw tsim khoom cov lus qhia. Cov bait thiab prey plasmids tau sib koom ua ke rau hauv cov poov xab kub. Protein-protein kev sib cuam tshuam tau txheeb xyuas raws li kev loj hlob ntawm SD ob qhov kev tso tawm nruab nrab (DDO, SD / -Trp-Leu) thiab SD plaub plaub qhov kev tso tawm nruab nrab (QDO, SD / -Trp-Leu-His-Ade) daim hlau.
4.6. BiFC Assay
Txhawm rau tsim cov BiFC tsim, pUC-SPYNE thiab pUC-SPYCE vectors tau linearized los ntawm kev zom nrog BamHI. Cov CDSs tag nrho ntawm MaCFEM85 thiab MsWAK16 yog txhua tus cloned thiab muab tso rau hauv cov plasmids linearized siv cov enzyme recombinant kom tau txais MaCFEM85-YFPN thiab MsWAK16-YFPC tsim. Cov plasmids uas muaj MaCFEM85 thiab MsWAK16 tau sib koom ua ke nrog cov vectors khoob raws li kev tswj tsis zoo (MaCFEM85-YFPN + pUC-SPYCE, MsWAK16-YFPC + pUC-SPYNE, thiab pUC-SPYNE + pUC-SPYCE) . Tag nrho cov vectors tau nkag mus rau hauv N. benthamiana ntawm Agrobacterium-mediated transformation raws li tau piav qhia saum toj no. Fluorescence signals tau pom nyob rau hauv nplooj epidermal hlwb siv Zeiss LSM980 confocal microscope (Carl Zeiss, Lub teb chaws Yelemees). Cov primers siv rau kev tsim kho vector yog teev nyob rau hauv Ntxiv Table S2.
4.7. GST Pull-Down Assay
Rau GST rub-down assays, MsWAK16-ED tau muab tso rau hauv pGEX-6P-2 vector thiab MaCFEM85 tau muab tso rau hauv pET-21b. Cov purified fusion proteins (GST- MsWAK16- ED) thiab pGEX-6P-2 tsis-load protein (GST) tau siv los ua cov khoom noj protein thiab purified pET-21 b-MaCFEM85 fusion protein (His-MaCFEM85) tau siv los ua cov tsiaj txhu. GST pulldown kev soj ntsuam tau ua nrog Mag-Beads GST Fusion protein purification system (Sangon Biotech, Shanghai, Tuam Tshoj) ua raws li cov chaw tsim khoom cov lus qhia. Luv luv, Mag-Beads tau ntxuav tsib zaug nrog 1 × phosphate-buffered saline (PBS, pH 7.4) kom tso cov cawv tiv thaiv thiab 10 mL ntawm GST lossis GST-MsWAK16-ED tau ntxiv. Cov hlaws dai tau sib xyaw los ntawm inversion ntawm chav tsev kub rau 30 min. Tom qab tshem tawm cov supernatant, Mag-Beads raug ntxuav tsib zaug nrog 1 × PBS. Nws-MaCFEM85 tau ntxiv rau Mag-Beads twb khi nrog GST, thiab qhov sib tov tau ua rau hmo ntuj ntawm 4 ◦C nrog kev sib hloov. Cov hlaws tom qab tau ntxuav tsib zaug nrog 1 × PBS kom tshem tawm cov proteins uas tsis muaj zog. Tom qab ntawd, cov protein immobilized ntawm cov hlaws dai tau muab cais los ntawm SDS-PAGE, hloov mus rau ib daim nyias nyias ntawm nitrocellulose (100 V, 1 h), thiab soj ntsuam ntawm Western blot. Cov daim nyias nyias tau ntxuav peb zaug rau 10 feeb txhua nrog PBS + Tween (PBST). Cov membranes raug thaiv rau 2 h ntawm chav tsev kub nrog 5% skimmed mis nyuj, ces incubated nrog ProteinFind anti-Nws nas monoclonal antibody (TransGen Biotech, Beijing, Tuam Tshoj) (diluted 1: 3000) los yog ProteinFind anti-GST nas monoclonal antibody rau 2 h ntawm 4 ◦C. Cov daim nyias nyias tau muab tso rau hauv ProteinFind tshis tiv thaiv luav IgG (H + L) (HRP) antibody (TransGen Biotech, Beijing, Tuam Tshoj) (diluted 1: 5000) rau 1 h ntawm chav tsev kub. Cov daim nyias nyias tau pom nrog EasySee® Western Blot Kit (TransGen Biotech, Beijing, Suav) ua raws li cov chaw tsim khoom cov lus qhia.
4.8. Kev txheeb xyuas qhov chaw tseem ceeb hauv MaCFEM85
Txhawm rau txiav txim siab qhov loj me uas yuav tsum tau muaj rau kev sib cuam tshuam ntawm MaCFEM85 nrog MsWAK16, PCR amplification tau ua los tsim ntau daim ntawv txiav tawm ntawm MaCFEM85. CFEM domain (MaCFEM85-CFEM; aa residues 19–86) thiab C davhlau ya nyob twg yam tsis muaj CFEM domain (MaCFEM85-C, aa residues 87–170) tau muab tso rau hauv vector pGBKT7 raws li cov khoom noj protein (Sab Ntxiv Table S1). Lwm tsib qhov sib txawv tau tsim los siv polypeptide synthesis los hloov cysteine rau alanine ntawm txoj haujlwm 26, 30, 43, 52, thiab 26/30/43/50/52/64/69/85 (∆CFEM8526, ∆CFEM83530, ∆CFEM83530, ∆CFEM83530 CFEM8552, thiab ∆CFEM858 tag nrho, ntsig txog). Cov qauv no tau siv los ua Y2H kev sim nrog MsWAK16-ED. Cov hlwb hloov pauv tau raug soj ntsuam rau kev loj hlob ntawm cov khoom hluav taws xob tso tawm SD/- Trp-Leu daim hlau thiab SD/-Trp-Leu-His-Ade daim hlau uas muaj X- -Galactosidase (X- -Gal).
4.9. Cog Resistance Assay
Myzus persicae tau txais los ntawm Henan Quanying Biological Co., Ltd. Ib lub lis piam ua ntej pib bioassays, 150 tus neeg laus aphids tau muab tso rau ntawm peb N. benthamiana nroj tsuag (50 aphids ib tsob nroj). Tom qab 72 teev, tag nrho cov neeg laus raug tshem tawm nrog ib tug mos mos txhuam, thiab cov nymphs raug tso cai noj rau plaub hnub ntxiv ua ntej yuav raug xa mus rau N. benthamiana rau kev soj ntsuam aphid.
4.10. Aphid Performance Assay
Nicotiana benthamiana Domin. (Solanales: Solanaceae) tau siv los ntsuas M. persicae kev ua tau zoo, cog kab mob tiv thaiv B. cinerea, thiab qhia txog cov tshuaj hormones ntsig txog cov noob. Agrobacterium nqa pYBA-eGFP, pYBA-MaCFEM85, pYBA-MsWAK16, lossis pYBA-MaCFEM85+pYBA-MsWAK16 tau loj hlob hauv LB ntxiv nrog cov tshuaj tua kab mob tsim nyog rau 36 teev ntawm 28 ◦C. Cov hlwb raug ntxuav peb zaug, tom qab ntawd rov muab tso rau hauv infiltration buffer (10 mM MgCl2, 10 mM MES, thiab 100 µM acetosyringone, pH 5.6) mus rau OD600 ntawm 0.6. Rau kev sib kis kab mob, cov kab mob nqa MaCFEM85 thiab MsWAK16 tau hloov kho rau OD600 ntawm 1.2, thiab tom qab ntawd sib xyaw ua ke sib npaug. Cov nplooj nthuav dav tau nkag mus nrog cov kab mob siv 1-mL koob txhaj tshuaj tsis muaj koob. Peb nplooj tau nkag mus rau ntawm txhua tsob ntoo thiab txhua qhov kev kho mob tau siv rau peb tsob nroj rau tag nrho ntawm 12 nroj tsuag ib qho kev sim.
Rau kev ntsuas kev ua haujlwm ntawm aphid, 20 tus neeg laus aphids tau siv rau thaj tsam infiltrated ntawm txhua nplooj 12 teev tom qab siv Agrobacterium. Cov aphids raug kaw nrog 5 mm txoj kab uas hla clip tawb. Txhua qhov kev kho mob tau rov ua dua tsib zaug. Cov neeg laus aphid tuag thiab tus naj npawb ntawm cov nymphs tshiab tau kaw txhua hnub rau peb hnub. B. cinerea raug coj mus kuaj rau tsib hnub rau PDAY. Tom qab 12 teev tom qab Agrobacterium infiltration, B. cinerea tshiab tau muab tso rau hauv ib lub ncuav mog qab zib 5-mm fungus, thiab mycelium loj hlob nto tau txuas mus rau thaj tsam infiltration ntawm nplooj nplooj. Txhawm rau txhawm rau txhim kho kab mob, cov nroj tsuag tau khaws cia los ntawm kev npog lawv nrog cov yas zaj duab xis hauv cov tais ntawm 22 ◦C txhawm rau pab txhawb kev kis kab mob. Tom qab 48 teev, kab mob kev loj hlob tau kwv yees nyob rau hauv inoculated nplooj los ntawm kev ntsuas qhov loj ntawm qhov txhab. Txhawm rau txheeb xyuas qhov txheeb ze ntawm cov noob muaj feem cuam tshuam, peb nplooj infiltrated tau sau los ntawm txhua tsob ntoo 12 teev tom qab nkag mus, tom qab ntawd khov rau hauv cov kua nitrogen thiab khaws cia ntawm -80 degree. Tag nrho RNA rho tawm, cDNA synthesis, thiab qRT-PCR tau ua raws li tau piav qhia hauv Tshooj 3.2. Salicylic acid (SA), jasmonic acid (JA), thiab flavonoid qib tau ntsuas hauv 15 hauv-filtrated N. benthamiana nplooj ib txoj kev kho. Cov nplooj tau sau, khov rau hauv cov kua nitrogen, tom qab ntawd khaws cia ntawm -80 degree. Cov tshuaj hormones cog tau suav nrog siv Plant Salicyclic Acid thiab Plant Jasmonic Acid ELISA Cov Khoom Siv (Beijing WeLab Scientific Co., Ltd.), thiab flavonoids tau txheeb xyuas siv Micro Plant Flavonoids Assay Kit (Beijing Solarbio Science & Technology Co., Ltd.) hauv qab no cov neeg tsim khoom cov lus qhia.
4.11. Kev txheeb cais
Cov ntaub ntawv tau txheeb xyuas hauv SPSS v20.0. Qhov sib txawv tseem ceeb hauv kev qhia qib ntawm MaCFEM85 thiab MsWAK16 tau txiav txim siab siv tus tub ntxhais kawm qhov t-test. Qhov sib txawv tseem ceeb hauv SA, JA, thiab flavonoid qib tau txiav txim siab siv ib-txoj kev ANOVA thiab Duncan qhov kev sim ntau yam nrog rau qhov pib ntawm p <0.05.
5. Cov lus xaus
Nyob rau hauv txoj kev tshawb no, peb tau txheeb xyuas thiab qhia txog ib tug tshiab secreted protein, MaCFEM85, nyob rau hauv M. anisopliae. Nws tau pom tias yog ib qho kev tiv thaiv kev tiv thaiv uas tuaj yeem cuam tshuam nrog MsWAK16 thiab qhib N. benthamiana tiv thaiv teb. Peb tau pom tias CFEM domain thiab cysteine residue ntawm txoj hauj lwm 52 hauv MaCFEM85 yog qhov tseem ceeb rau kev sib cuam tshuam. Qhov kev sib cuam tshuam no tuaj yeem ua rau JA ntsig txog lub cev tiv thaiv kab mob thiab cov kab mob-tiv taus thiab kab-tiv taus hauv cov nroj tsuag.
Cov ntaub ntawv
1. Paulucci, NS; Anta, GG; Gallarato, LA; Vicario, JC; Cesari, AB; Reguera, YB; Kilmurray, C.; Bueno, MA; García, MB; Dardanelli, MS Plant-microbe kev sib koom tes: Kev cuam tshuam rau kev loj hlob thiab kev noj qab haus huv ntawm cov nroj tsuag. Hauv Cog Microbe Symbiosis: Cov Ntsiab Lus thiab Kev Ua Ntej; Springer: Berlin/Heidelberg, Lub teb chaws Yelemees, 2013; ib., 105–117. [CrossRef]
2. Schenk, PM; Carvalhais, LC; Kazan, K. Unraveling cog-microbe kev sib cuam tshuam: Ntau hom kev sau ntawv tuaj yeem pab tau? Trends Biotechnol. 2012, 30, 177–184. [CrossRef]
3. Xas, J.; Martinoia, E.; Northen, T. Pub Koj Cov Phooj Ywg: Puas Tsob Ntoo Exudates zoo li hauv paus Microbiome? Trends Plant Sci. 2018, 23, 25–41. [CrossRef]
4. Lugtenberg, B.; Kamilova, F. Cog-loj hlob-txhim kho rhizobacteria. Annu. Rev. Microbiol. 2009, 63, 541–556. [CrossRef]
5. Shoresh, M.; Harman, GE; Mastouri, F. Induced systemic resistance thiab cog lus teb rau fungal biocontrol agents. Annu. Rev. Phytopathol. 2010, 48, 21–43. [CrossRef]
6. van de Mortel, JE; de Vos, RC; Dekkers, E.; Pineda, A.; Guillod, L.; Bouwmeester, KJ; van Loon, J.; Dickes, M.; Raaijmakers, JM Metabolic thiab transcriptomic kev hloov pauv tshwm sim hauv Arabidopsis los ntawm rhizobacterium Pseudomonas fluorescens SS101. Nroj Physiol. 2012, 160, 2173–2188. [CrossRef]
7. Lareen, A.; Burton, F.; ib. Schafer, P. Cog cag-microbe kev sib txuas lus hauv shaping cag microbiomes. Cog Mol. Biol. Xyoo 2016, 90, 575–587. [CrossRef]
8. Sasan, RK; Bidochka, MJ Cov kab-pathogenic fungus Metarhizium Roberts (Clavicipitaceae) kuj yog ib qho endophyte uas txhawb kev loj hlob ntawm cov nroj tsuag. Am. J. Bot. 2012, 99, 101–107. [CrossRef]
9. Sasan, RK; Bidochka, MJ Antagonism ntawm endophytic kab kab mob fungus Metarhizium robertsii tiv thaiv taum cog kab mob Fusarium solanif. sp. Phasoli. Ua tau. J. Plant Pathol. Xyoo 2013, 35, 288–293. [CrossRef]
10. Ahmad, Ib.; Jiménez-Gasco, MdM; Luthe, DS; Shakeel, SN; Barbercheck, ME Endophytic Metarhizium robertsii txhawb txoj kev loj hlob ntawm pob kws, suppresses kab loj hlob, thiab hloov cov nroj tsuag tiv thaiv noob qhia. Biol. Tswj 2020, 144, 104167. [CrossRef]
11. Chairin, T.; Petcharat, V. Induction ntawm kev tiv thaiv teb nyob rau hauv Hongkong txiv hmab txiv ntoo (Aglaia dookkoo Griff.) tiv thaiv txiv hmab txiv ntoo rot fungi los ntawm Metarhizium guizhouense. Biol. Tswj 2017, 111, 40–44. [CrossRef]
12. Hao, K.; Wang, F.; Nuj, X.; McNeill, MR. Liu, S.; Wang, G.; Cai, G.; Zhang, Z. Teb cov txiv laum huab xeeb Arachis hypogaea cov hauv paus hniav mus rau lub xub ntiag ntawm cov txiaj ntsig thiab cov kab mob fungi los ntawm kev txheeb xyuas transcriptome. Sci. Rep. 2017, 7, 964. [CrossRef] [PubMed]
13. Patel, ZM; Mahapatra, R.; Jampala, SSM Tshooj 11—Lub luag hauj lwm ntawm fungal elicitors nyob rau hauv cov nroj tsuag tiv thaiv mechanism. Nyob rau hauv Molecular nam ntawm tsob nroj Beneficial Microbes hauv kev ua liaj ua teb; Sharma, V., Salwan, R., Al-Ani, L., Eds.; Kev Kawm Xov Xwm: Cambridge, MA, USA, 2020; ib., 143–158. [CrossRef]
14. Dong, X.; Sa, JA Ethylene, thiab kab mob tiv thaiv hauv cov nroj tsuag. Curr. Opin. Tsob Ntoo Biol. 1998, 1, 316–323. [CrossRef] [PubMed]
15. Thoma, BP; Eggermont, K.; Penninckx, IA; Mau-Mani, B.; Vogelsang, R.; Cammue, BP; Broekaert, WF Separate jasmonate dependent thiab salicylate-dependent tiv thaiv- teb txoj hauv kev hauv Arabidopsis yog qhov tseem ceeb rau kev tiv thaiv cov kab mob sib txawv. Proc. Natl. Acad. Sci. USA 1998, 95, 15107–15111. [CrossRef] [PubMed]
16. Khawm, SR; Irigoyen, S.; Liu, J.; Bedr, R.; Christensen, SA; Schmelz, EA; Sedbrook, JC; Scholthof, K.-BG; Mandadi, KK Brachypodium Phenylalanine ammonia lyase (PAL) Txhawb kev tiv thaiv kab mob tiv thaiv kab mob Panicum mosaic thiab nws cov satellites. mBio 2021, 12, e03518-20. [CrossRef]
17. Liu, R.; Xu, S.; Li, J.; Hu, Y.; Lin, Z. Kev nthuav qhia profile ntawm PAL noob los ntawm Astragalus membranaceus var. Mongholicus thiab nws lub luag haujlwm tseem ceeb hauv flux rau hauv flavonoid biosynthesis. Plant Cell Rep. 2006, 25, 705–710. [CrossRef]
18. De Geyter, N.; Gholami, A.; Goormachtig, S.; Goossens, A. Transcriptional machineries nyob rau hauv jasmonate-elicited cog theem nrab metabolism. Trends Plant Sci. 2012, 17, 349–359. [CrossRef]
19. Mirjalili, N.; Linden, JC Gas theem muaj pes tsawg leeg cuam tshuam rau kev ncua kev coj noj coj ua ntawm Taxus cuspidata. Biotechnol. Bioeng. 1995, 48, 123–132, ib. [CrossRef]
20. Li, ST; Zhang, P. Zhang, M. Fu, CH; Zhou, CF; Dong, YS; Guo, AW; Yu, LJ Transcriptional profile ntawm taxus chinensis hlwb teb rau methyl jasmonate. BMC Genom. 2012, 13, 295. [CrossRef]
21. Tugizimana, F.; Ncube, EN; Steenkamp, PA; Dubery, IA Metabolomics-muab kev nkag siab rau hauv kev tswj hwm ntawm terpenoid synthesis hauv Centella asiatica hlwb los ntawm methyl jasmonate. Tsob Ntoo Biotechnol. Rep. 2015, 9, 125–136. [CrossRef]
22. Lu, M.; Wong, H.; Teng, W. Qhov cuam tshuam ntawm kev tshem tawm ntawm kev tsim cov saponin hauv cell kab lis kev cai ntawm Panax ginseng. Plant Cell Rep. 2001, 20, 674–677. [CrossRef]
23. Mandal, S.; Kar, ib.; Mukherjee, AK; Acharya, P. Elicitor-induced defense teb nyob rau hauv Solanum lycopersicum tawm tsam Ralstonia solanacearum. Sci. Ntiaj teb J. 2013, 25, 561056. [CrossRef]
24. Li, L.; Vang, S.; Yang, X.; Francis, F.; Qiu, D. Protein elicitor PeaT1 enhanced resistance tiv thaiv aphid (Sitobion avenae) hauv nplej. Pest Manag. Sci. 2020, 76, 236–243. [CrossRef] [PubMed]
25. Bujalowski, W. Expanding physiological luag hauj lwm ntawm hexameric DnaB helicase. Trends Biochem. Sci. 2003, 28, 116–118. [CrossRef]
26. Vaknin, Y.; Shadkchan, Y.; Levdansky, E.; Morozov, M.; Romano, J.; Osherov, N. Peb Aspergillus fumigatus CFEM-domain GPI-anchored proteins (CfmA-C) cuam tshuam rau cell phab ntsa stability tab sis tsis ua lub luag hauj lwm hauv fungal virulence. Kab mob. Genet. Biol. 2014, 63, 55–64. [CrossRef]
27. Zhao, S.; Shang, X.; Ib, W.; yus, X.; Liu, D.; Kang, Z.; Wang, X.; Wang, X. Genome-Wide Kev txheeb xyuas cov neeg sib tw effector nrog kev txuag motifs los ntawm cov nplej nplooj xeb fungus Puccinia triticina. Pem hauv ntej. Microbiol. 2020, 11, 1188. [CrossRef]
28. Kob, A.; Jing, Z.; Zhang, K.; Tan, Q.; Wang, G.; Liu, W. Bioinformatic tsom xam thiab functional characterization ntawm CFEM proteins nyob rau hauv pob kws anthracnose fungus Colletotrichum graminicola. J. Integr. Agric. Xyoo 2020, 19, 541–550. [CrossRef]
29. Martin, F.; Aws, A.; Ahren, D.; Brun, UA; Danchin, EG; Duchaussoy, F.; Gibon, J.; Kohler, UA; Lindquist, E.; Pereda, V.; ua al. Lub genome ntawm Laccaria bicolor muab kev nkag siab rau hauv mycorrhizal symbiosis. Xwm Txheej 2008, 452, 88–92. [CrossRef]
30. Stefan, V.; Lara, RJ Entomopathogenic fungi li endophytes: Cog-endophyte-herbivore kev sib cuam tshuam thiab kev cia siab rau kev siv tshuaj lom neeg. Curr. Sci. 2015, 109, 46–54.
31. Cai, N.; Liu, R.; Yan, D.; Zhang, N.; Zhu, K.; Zhang, D.; Nuj, X.; Tu, X.; Zhang, Z.; Wang, G. Bioinformatics tsom xam thiab functional characterization ntawm CFEM proteins ntawm Metarhizium anisopliae. J. Fung. 2022, 8, 661. [CrossRef]
32. Stephens, C.; Hammond-Kosack, KE; thiab Kanyuka, K. WAKsing cog kev tiv thaiv, kab mob waning. J. Exp. 2022, 73, 22–37. [CrossRef]
33. Zhang, ZN; Wu, QY; Zhang, ZPO; Zhu, YY; Murphy, RW; Liu, Z.; Zou, CG Systematic tsom xam qhia qhov txawv thiab keeb kwm ntawm CFEM domain hauv fungi. Sci. Rep. 2015, 5, 13032. [CrossRef] [PubMed]
34. Fang, A.; Gao, H.; Zhang, N.; Zheng, X.; Qias, S.; Li, Y.; Zhou, S.; Koj, F.; Sun, W. Ib tug tshiab effector gene SCRE2 pab txhawb rau tag nrho virulence ntawm Ustilaginoidea virens rau mov. Pem hauv ntej. Microbiol. 2019, 10, 845. [CrossRef] [PubMed]
35. Zhou, W.; Wei, W.; Wu, Y.; Zhou, Y.; Peng, F.; Zhang, S.; Chen, P.; Xu, X. BcCFEM1, CFEM domain-muaj protein ntau nrog putative GPI-anchored site, muaj kev koom tes nyob rau hauv pathogenicity, conidial ntau lawm, thiab kev nyuaj siab kam rau ua nyob rau hauv Botrytis cinerea. Pem hauv ntej. Microbiol. 2017, 8, 1807. [CrossRef]
36. Xu, X.; Li, G.; Li, L.; Sau, Z.; Chen, C. Genome-wide comparative analysis of putative Pth11-related G protein-coupled receptors in fungi belongs to Pezizomycotina. BMC Microbiol. 2017, 17, 166. [CrossRef] [PubMed]
37. Peng, YJ; Hu, J.; Zhang, H. Li, JH; Lin, HY; Ding, JL; Feng, MG; Ying, SH Systematic contributions ntawm CFEM domain uas muaj cov proteins kom tau hlau yog qhov tseem ceeb rau kev sib cuam tshuam ntawm cov kab mob filamentous pathogenic fungus Beauveria bassiana. Ib puag ncig. Microbiol. 2022, 24, 3693–3704. [CrossRef] [PubMed]
38. Roy, UA; Kornitzer, D. Heme-hlau acquisition in fungi. Curr. Opin. Microbiol. Xyoo 2019, 52, 77–83. [CrossRef] [PubMed]
39. Okamoto-Shibayama, K.; Kikuchi, Y.; Koob, E.; Saib, Y.; Ishihara, K. Csa2, ib tug tswv cuab ntawm tsev neeg Rbt5 protein, koom nrog kev siv hlau los ntawm tib neeg hemoglobin thaum Candida albicans hyphal loj hlob. FEMS Yeast Res. 2014, 14, 674–677. [CrossRef]
40. Perez, A.; Pedros, IB; Murgui, A.; Casanova, M.; Lopez-Ribot, JL; Martinez, JP Biofilm tsim los ntawm Candida albicans mutants rau cov noob coding fungal proteins nthuav tawm yim-cysteine-muaj CFEM domain. FEMS Yeast Res. 2006, 6, 1074–1084. [CrossRef]
41. Hogg, PJ Disulfide bonds li keyboards rau protein ua haujlwm. Trends Biochem. Sci. 2003, 28, 210–214. [CrossRef]
42. van den Burg, HA; Westerin, N.; Francoijs, KJ; Rau, R.; Woestenenk, E.; Boeren, S.; de Wit, PJ; Joosten, MH; Vervoort, J. Natural disulfide daim ntawv cog lus-tshuaj cuam tshuam mutants ntawm AVR4 ntawm cov txiv lws suav pathogen Cladosporium fulvum yog rhiab heev rau proteolysis, hla Cf-4- kho kom haum xeeb, tab sis khaws lawv cov chitin binding peev xwm. J. Biol. Chem. 2003, 278, 27340–27346. [CrossRef] 43. Hu, K.; Cai, J.; Zhang, J.; Xia, F.; Kev, Y.; Zhang, H. Xie, W.; Li, H.; Koj, Y.; Cai, Y.; ua al. Kev txhim kho ntawm ntau yam agronomic zoo los ntawm cov kab mob tiv thaiv kab mob ntawm cov phab ntsa ntawm tes. Nat. Nroj tsuag 2017, 3, 17009. [CrossRef] [PubMed]
44. Saitoh, H.; Fujisawa, S.; Mitsuoka, C.; Ib, A.; Hirabuchi, A.; Ikeda, K.; Irieda, H.; Yoshino, K.; Yoshida, K.; Matsumura, H.; ua al. Kev cuam tshuam loj ntawm noob caj noob ces hauv Magnaporthe oryzae txheeb xyuas MC69, cov protein zais cia uas xav tau rau kev kis kab mob los ntawm monocot thiab dicot fungal pathogens. PLoS Pathog. Xyoo 2012, 8, e1002711. [CrossRef] [PubMed]
45. Kulkarni, RD; Kelkar, HS; Dean, R. Ib yim-cysteine-muaj CFEM sau tshwj xeeb rau ib pawg ntawm cov kab mob fungal membrane proteins. Trends Biochem. Sci. 2013, 28, 118–121. [CrossRef] [PubMed]
46. Peng, J.; Wu, L.; Zhang, W. Zhang, Q.; Xing, Q.; Wang, X.; Li, X.; Yan, J. Systemic identification thiab functional characterization of common in fungal extracellular membrane proteins in Lasiodiplodia theobromae. Pem hauv ntej. Tsob Ntoo Sci. 2021, 12, 804696. [CrossRef]
47. Ib., R.; Jones, JD Lub luag haujlwm ntawm cov tshuaj hormones cog hauv cov lus teb cog lus. Cog Mol. Biol. Xyoo 2009, 69, 473–488. [CrossRef]
48. Verma, V.; Ravindran, P. Kumar, PP Plant hormone-mediated kev tswj ntawm kev ntxhov siab. BMC cog Biol. Xyoo 2016, 16, 86. [CrossRef]
49. Javed, K.; Qiu, D. Protein elicitor PeBL1 ntawm Brevibacillus laterosporus txhim khu kev tiv thaiv Myzus persicae hauv Txiv lws suav. Cov kab mob 2020, 9, 57. [CrossRef]
50. Basit, A.; Hanan, UA; Nazir, T.; Majeed, MZ; Qiu, D. Molecular thiab functional characterization ntawm elicitor PeBC1 muab rho tawm los ntawm Botrytis cinerea koom nrog induction ntawm kuj tiv thaiv ntsuab peach aphid (Myzus persicae) nyob rau hauv ntau taum (Phaseolus vulgaris L.). Kab 2019, 10, 35. [CrossRef]
51. Nazir, T.; Hanan, UA; Taub, A.; Majeed, MZ; Anwar, T.; Nawaz, I.; Qiu, D. Putative lub luag hauj lwm ntawm ib tug tseem uncharacterized protein elicitor PeBb1 Tau los ntawm Beauveria bassiana ARSEF 2860 Strain tawm tsam Myzus persicae (Homoptera: Aphididae) nyob rau hauv Brassica rapa ssp. pekinensis. Cov kab mob 2020, 9, 111. [CrossRef]
52. Spoel, SH; Koornneef, A.; Claessens, SM; Korzelius, JP; Van Pelt, JA; Mueller, MJ; Buchala, AJ; Metraux, JP; Brown, R.; Kazan, K.; ua al. NPR1 modulates cross-hais lus ntawm salicylate- thiab jasmonate-dependent tiv thaiv txoj hauv kev los ntawm kev ua haujlwm tshiab hauv cytosol. Plant Cell 2003, 15, 760–770. [CrossRef]
53. Xin, XF; He, SY Pseudomonas syringae pv. Txiv lws suav DC3000: Ib qho qauv kab mob rau kev soj ntsuam cov kab mob susceptibility thiab cov tshuaj hormone signaling hauv cov nroj tsuag. Annu. Rev. Phytopathol. 2013, 51, 473–498. [CrossRef] [PubMed]
54. Zhang, X.; Wang, C.; Zhang, YJ; Sun, Y.; Mou, Z. Arabidopsis mediator complex subunit16 zoo tswj salicylate mediated systemic acquired resistance thiab jasmonate/ethylene-induced defense txoj kev. Plant Cell 2012, 24, 4294–4309. [CrossRef] [PubMed]
55. Doares, SH; Narvaez-Vasquez, J.; Conconi, UA; Ryan, CA Salicylic acid inhibits synthesis ntawm proteinase inhibitors nyob rau hauv txiv lws suav nplooj induced los ntawm systemin thiab jasmonic acid. Nroj Physiol. 1995, 108, 1741–1746. [CrossRef] [PubMed]
56. Pieterse, CM; Leon-Reyes, A.; Van der Ent, S.; Van Wees, SC Networking los ntawm cov tshuaj hormones me me hauv cov nroj tsuag tiv thaiv kab mob. Nat. Chem. Biol. 2009, 5, 308–316. [CrossRef]
57. Penninckx, IA; Thomas, BP; Buchala, UA; Métraux, JP; Broekaert, WF Concomitant activation ntawm jasmonate thiab ethylene teb txoj kev yog yuav tsum tau rau induction ntawm ib tsob nroj defensin noob nyob rau hauv Arabidopsis. Plant Cell 1998, 10, 2103–2113. [CrossRef]
58. Fang, W.; Bidochka, MJ Kev nthuav qhia ntawm cov noob koom nrog hauv kev cog qoob loo, conidiogenesis thiab pathogenesis hauv Metarhizium anisopliae siv ntau lub sijhawm RT-PCR. Mycol. Res. 2006, 110, 1165–1171. [CrossRef]
59. Guerriero, G.; Loj, S.; Hausman, JF Alfalfa Cellulose synthase gene qhia nyob rau hauv abiotic stress: Hitchhiker phau ntawv qhia rau RT-qPCR normalization. PLoS IB 2014, 9, e103808. [CrossRef]
60. Yaj, Y.; Zhang, YJ; Li, B.; Yang, X.; Dong, Y.; Qiu, D. A Verticillium dahliae pectate lyase induces cog kev tiv thaiv kab mob thiab pab txhawb rau virulence. Pem hauv ntej. Tsob Ntoo Sci. 2018, 9, 1271. [CrossRef]
61. Livak, KJ; Schmittgen, TD Kev tshuaj xyuas ntawm cov txheeb ze cov ntaub ntawv qhia cov ntaub ntawv siv lub sijhawm tiag tiag ntawm PCR thiab 2 (-Delta Delta C(T)) Txoj Kev. Methods 2001, 25, 402–408. [CrossRef]
