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16.5F: Agrobacterium en Kroongalsiekte - Biologie

16.5F: Agrobacterium en Kroongalsiekte - Biologie


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Argobacterium veroorsaak Kroongalsiekte deur 'n DNS-plasmied na die gasheerplant oor te dra, wat veroorsaak dat die gasheer voedingstowwe daarvoor maak.

Leerdoelwitte

  • Som die simbiotiese verwantskap tussen plante en agrobakterie op

Kern punte

  • Kroongalsiekte word veroorsaak deur Agrobacterium tumefaciens, 'n bakterie wat plante besmet. Die bakterieë veroorsaak gewasse op die stam van sy gasheer.
  • Agrobacterium tumefaciens manipuleer sy gashere deur 'n DNA-plasmied na die selle van sy gasheer oor te dra. Plasmiede word gewoonlik gebruik om DNA van bakterieë na bakterieë oor te dra.
  • Sodra dit in die gasheersel is, integreer die plasmied homself in die gasheerplantsel se genoom en dwing die gasheer om unieke aminosure en ander stowwe te produseer wat die bakterieë voed. Hierdie verbindings is onbruikbaar deur die meeste bakterieë, so Argobacteria kan ander spesies uitkompeteer.

Sleutel terme

  • plasmied: 'n Sirkel van dubbelstring-DNS wat apart is van die chromosome, wat in bakterieë en protosoë voorkom.
  • pilus: 'n Haaragtige aanhangsel wat op die seloppervlak van baie bakterieë gevind word.

Kroongalsiekte word veroorsaak deur 'n bakterie genaamd Agrobacterium tumefaciens. Die siekte manifesteer as 'n tumoragtige groei gewoonlik by die aansluiting van die wortel en loot. A. tumefaciens kan 'n deel van sy DNA na die gasheerplant oordra, deur 'n plasmied - 'n bakteriese DNA-molekule wat onafhanklik van 'n chromosoom is. Die nuwe DNA-segment veroorsaak dat die plant ongewone aminosure en planthormone produseer wat die bakterieë van koolstof en stikstof voorsien.

Bakterieë gebruik gewoonlik plasmiede vir horisontale geenoordrag, sodat hulle gene met verwante bakterieë kan deel om hulle te help om stresvolle omgewings te hanteer. Plasmiede kan byvoorbeeld aan bakterieë die vermoë verleen om stikstof te bind, of om antibiotiese verbindings te weerstaan. Tipies dra bakterieë plasmiede oor deur konjugasie: 'n skenkerbakterie skep 'n buis wat 'n pilus genoem word wat die selwand van die ontvangerbakterieë binnedring en die plasmied-DNS gaan deur die buis. Die ander bakterieë integreer die plasmied óf in sy chromosome, óf dit bly vryswewend in die sitoplasma. In beide gevalle ontvang die ontvanger-bakterieë nuwe genetiese materiaal.

In die geval van kroongalsiekte, A. tumefaciens dra 'n plasmied wat T-DNA bevat oor na die selle van sy gasheerplant deur vervoeging, soos dit sou met 'n ander bakterie. As dit egter eers in die plantsel is, integreer die DNA semi-lukraak in die genoom van die plant en verander die gedrag van die sel.

Die nuwe plasmiedgene word deur die plantselle uitgedruk, en veroorsaak dat hulle ensieme afskei wat die aminosure oktopien of nopalien produseer. Dit dra ook gene vir die biosintese van die planthormone, ouksien en sitokiniene, en vir die biosintese van opiene, wat 'n koolstof- en stikstofbron vir die bakterieë verskaf.

Hierdie menings kan deur baie min ander bakterieë gebruik word en gee A. tumefaciens mededingende voordeel.


Redaksie: 𠇊grobacterium biologie en die toepassing daarvan op transgeniese plantproduksie”


  • 1 Departement Lewenswetenskappe, Nasionale Chung Hsing Universiteit, Taichung, Taiwan
  • 2 Departement Biologiese Wetenskappe, Purdue Universiteit, West Lafayette, IN, VSA
  • 3 Instituut vir Plant- en Mikrobiese Biologie, Academia Sinica, Taipei, Taiwan

Die buitengewone Agrobacterium navorsingsverhaal het begin uit die soeke na die veroorsakende middel van kroongalsiekte meer as 100 jaar gelede. Agrobacterium tumefaciens is eers in 1897 uit wingerdgalle geïsoleer en later in 1907 van Paris Daisy geïsoleer (Cavara, 1897a,b Smith en Townsend, 1907). Die Agrobacterium infeksiemeganisme behels die verwerking en oordrag van 'n spesifieke DNA-fragment (die oorgedra-DNA, T-DNA) vanaf 'n bakteriese tumor-induserende (Ti) plasmied. Oordrag na die plant vind plaas via 'n tipe IV-afskeidingstelsel (T4SS), waarna T-DNS in die plantgasheergenoom geïntegreer word (Gelvin, 2010 Lacroix en Citovsky, 2013). Hierdie interkoninkryk-DNA-oordrag lei tot oorproduksie van die planthormone ouksien en sitokinien, wat lei tot gewasse. Die interkoninkryk DNA oordrag vermoë van Agrobacterium en die moontlikheid om die onkogene in die T-DNS te vervang met gene van belang het gemaak Agrobacterium-bemiddelde transformasie die gewildste tegniek om transgeniese plante te genereer.

Hierdie navorsingsonderwerp bied 'n versameling resensies en oorspronklike navorsingsartikels oor Agrobacterium gene betrokke by bakteriële fisiologie/virulensie en plantgene betrokke by transformasie en verdediging teen Agrobacterium. ’n Resensie deur Kado (2014) verskaf ’n historiese oorsig van hoe A. tumefaciens is eers vasgestel as die oorsaak van kroongalsiekte. In hierdie oorsig beklemtoon Kado sleutel vroeë plantpatologie en mylpaal molekulêre biologie studies wat lei tot die gevolgtrekking dat die uitdrukking van onkogene in inheemse T-DNS die oorsaak van tumorgroei in plante is. Met die vaste fondament van hierdie baanbrekersontdekkings, A. tumefaciens ontwikkel van 'n fitopatogeen tot 'n kragtige genetiese transformasie-instrument vir plantbiologie en biotegnologie-navorsing.

Die eerste volledige genoomvolgorde van 'n Agrobacterium spesie (A. tumefaciens C58) is in 2001 voltooi (Goodner et al., 2001 Wood et al., 2001). Die 5.67-megabasis genoom van hierdie stam dra een sirkelvormige chromosoom, een lineêre chromosoom en twee megaplasmiede: die Ti-plasmied pTiC58 en 'n tweede plasmied, pAtC58. In die resensie deur Platt et al. (2014), die eienskappe, ekologie, evolusie en komplekse interaksies van hierdie twee A. tumefaciens megaplasmiede word bespreek. Die koste en voordele aan A. tumefaciens stamme wat die Ti-plasmied en/of die pAtC58-plasmied dra, word bespreek en aangebied vanuit 'n ekologiese en evolusionêre perspektief. Modelleringsvoorspellings word aangebied vir die relatiewe koste en voordele aan A. tumefaciens stamme wat die Ti- en/of die pAtC58-plasmiede huisves, bepaal deur omgewingshulpbronne. Vervoeging en amplifikasie van die Ti plasmied word gereguleer deur die TraI/TraR kworum-sensing (QS) stelsel en konjugale menings. Lang en Faure (2014) hersien huidige kennis van die genetiese netwerke en molekulêre basis van die A. tumefaciens kworumwaarnemingstelsel. Hierdie skrywers bespreek ook die biologiese en ekologiese impak van die QS-stelsel op Ti-plasmiedvervoeging, kopiegetal en interaksies tussen Agrobacterium en gasheerplante.

Tydens die aanvanklike interaksie tussen Agrobacterium en plantselle, bakterieë waarneem verskeie plant-afgeleide seine in die risosfeer met behulp van Ti plasmied-gekodeerde virulensie geen (vir geen) en chromosomale virulensie geen (chv geen) produkte. Die huidige kennis van hoe A. tumefaciens sintuie en reageer op verskillende plant-afgeleide seine word opgesom in die oorsigartikel deur Subramoni et al. (2014), wat ook die meganismes bespreek van hoe die planthormone ouksien, salisielsuur en etileen bakteriële virulensie beïnvloed. Ten slotte bespreek hierdie resensie die kompleksiteit en ingewikkeldheid van Agrobacterium seinpaaie en die onderliggende regulatoriese meganismes tydens die aanvanklike gasheerselherkenning om daaropvolgende suksesvolle infeksie te maksimeer. In die oorspronklike navorsingsartikel deur Lin et al. (2014), word die meganistiese regulering van die membraansensor VirA-proteïen verder gedissekteer. VirA histidienkinase en die sitoplasmiese reaksie reguleerder VirG proteïen speel saam 'n sentrale rol in regulering vir geenuitdrukking in reaksie op fenole. Gebaseer op 'n homologie-model van die VirA-skakelstreek, is verskeie mutante en chimeriese VirA-proteïene gegenereer en ondersoek vir hul vermoë om te induseer VirB promotoraktiwiteit. Die vermoë van VirA om drie afsonderlike insetseine, fenole, suikers en omgewings-pH waar te neem en daarop te reageer, speel 'n beduidende rol in die beveiliging van suksesvolle infeksie.

Agrobacterium aanhegting aan plantselle is 'n belangrike vroeë stap in die vordering van kroongalsiekte. Beweeglike bakterieë swem na gasheerselle en tree dan fisies met gasheerselle in om aggregate te vorm en 'n meersellige bakteriese gemeenskap bekend as 'n biofilm te vestig. Verskeie genetiese en omgewingsfaktore wat affekteer Agrobacterium aanhegting en biofilmvorming word in die artikel deur Heindl et al. (2014). Die funksies van verskillende tipes eksopolisakkariede wat die biofilm uitmaak en onderliggende meganismes wat behels hoe die tweede boodskapper sikliese-di-GMP, die ChvG/ChvI sisteem, fosforvlakke en suurstofspanning bakteriese aanhegting en virulensie beïnvloed, word ook opgesom. In die oorsigartikel deur Matthysse (2014) word vroeë studies en huidige kennis van die meganismes van polêre en laterale bakteriële aanhegting opgesom. Hierdie twee meganismes dra albei by tot bakteriese aanhegting. Wanneer die omgewings kalsium- en fosfaatvlakke en pH-waardes laag is, oorheers polêre aanhegting. Daarbenewens dra die fosfolipiede (PL's), fosfatidielcholien (PC) en fosfaatvrye lipied ornithine lipiede (OL's) by tot Agrobacterium virulensie. In die resensie deur Aktas et al. (2014), word die biosintetiese weë en die fisiologiese rolle van hierdie membraanlipiede opgesom. Die tipiese eukariotiese membraan lipied PC word nie gereeld in bakterieë aangetref nie, maar dit maak byna 22% van die Agrobacterium membraanlipied. Interessant genoeg kan rekenaars en OL's teenoorgestelde rolle speel in Agrobacterium virulensie. Die vermindering van tumorvorming in 'n PC-tekort Agrobacterium mutant kan voortspruit uit verswakte vir geenuitdrukkings wat deur VirA/VirG beheer word. Die afwesigheid van OL's in A. tumefaciens kan gasheerverdedigingsreaksies verminder en dus vroeër en groter tumorvorming veroorsaak.

Plantselle het 'n verskeidenheid reseptore wat sogenaamde mikrobe- of patogeen-geassosieerde molekulêre patrone (MAMPs of PAMPs) herken en vervolgens plantverdedigingsreaksies aktiveer, 'n proses wat bekend staan ​​as Patroonherkenningreseptor-Geactiveerde Immuniteit (PTI) (Boller en Felix, 2009 Boller en He, 2009). Agrobacterium kan effektors gebruik om plantstelsels te kaap en plantverdedigingsreaksies te ontduik. Pitzschke (2013) hersien strategieë wat gebruik word deur Agrobacterium om plantverdedigingsreaksies tot sy eie voordeel te draai. Besmette plantselle begin 'n mitogeen-geaktiveerde proteïenkinase seinkaskade wat VIP1 (Agrobacterium VirE2-interaksie proteïen 1) fosforilering en translokasie na die plantkern om verdedigingsgeen-uitdrukking te induseer. Aan die ander kant, Agrobacterium kan VIP1 kaap om T-DNA te help om die plantkern binne te gaan. Gebaseer op die huidige kennis van plantverdedigingsreaksies teen Agrobacterium infeksie bespreek Pitzschke (2013) verskeie biotegnologiese benaderings om transformasiedoeltreffendheid te verhoog. In 'n ander resensie deur Gohlke en Deeken (2014), vroeë plantreaksies op Agrobacterium, insluitend verskeie verdedigingsreaksies, hipersensitiewe reaksies en fitohormoonvlakveranderings word bespreek. Die veranderinge in plantmorfologie, voedingstoftranslokasie en metabolisme wat deur kroongal tumorvorming veroorsaak word, word ook hersien. Die skrywers som belangrike genomiese, epigenomiese, transkriptomiese en metabolomiese studies op wat epigenetiese veranderinge wat verband hou met T-DNA-integrasie en galontwikkeling openbaar. Vervolgens het Hwang et al. (2015) hersien belangrike patogeniese opwekkers, gasheerselreseptormolekules en hul stroomaf seintransduksiepaaie in gasheerplante tydens die PAMP-geaktiveerde immuunrespons. Hulle beklemtoon onlangse ontdekkings wat plantimmuniteit verbind met endomembraanhandel en aktiendinamiese veranderinge. Effekte van beide die gasheerfisiologie, insluitend hormoonvlakke, sirkadiese klok, ontwikkelingstadia en omgewingsfaktore, insluitend ligblootstellingslengtes en temperatuur, op plantverdedigingsreaksies en bakteriële virulensie word hersien en bespreek.

In die natuur, bewyse van antieke horisontale geenoordragte (HGT) van Agrobacterium aan plante is in die genera waargeneem Nicotiana en Linaria. Reekse homoloog aan mikimopien-tipe Agrobacterium rhizogenes pRiA4 T-DNA is die eerste keer ontdek in die genoom van ongetransformeerde boomtabak, Nicotiana glauca, en genoem �llular T-DNA” (cT-DNA White et al., 1983). Matveeva en Lutova (2014) hersien cT-DNA-organisasie, verspreiding, uitdrukkingsregulering en 'n moontlike korrelasie met genetiese tumorvorming in Nicotiana spesies. Hulle hersien ook onlangse bevindings van cT-DNA in die genome van Linaria spesies en in ander tweesaadlobbige families. Die skrywers stel voor dat plante wat cT-DNA in hul genome handhaaf, moontlik mikroörganismes in die risosfeer kan bevoordeel deur opiene in die wortelsone af te skei. Hulle stel ook voor dat voetspore van antieke pRi T-DNA-invoegings in die plantgenoom selektiewe voordeel aan hierdie plante kan bied.

Met hierdie navorsingsonderwerp bied ons 'n platform vir wetenskaplikes om hul begrip van te deel Agrobacterium biologie en hoe Agrobacterium transformeer plante. Hierdie bydraes demonstreer hoe 'n hoogs aktiewe navorsingsgemeenskap in plant- en mikrobiese wetenskappe belangrike patogenesevrae kan verduidelik. Toekomstige navorsing oor Agrobacteium sal voortgaan om ons begrip van plant-patogeen-interaksies te bevorder, en nuwe insigte verskaf wat nuttig is vir plantgenetiese ingenieurswese.


Gevorderd

Wetenskaplike naam
Rhizobium vitis (voorheen genoem Agrobacterium vitis )

Identifikasie
Huidige seisoen galle

  • Die eerste keer in die vroeë somer sigbaar as swellings op die stam
  • Sagte, kronkelende, eeltagtige weefsel, romerig van kleur, wat deur die baslaag uitbars naby beseerde plekke van die wingerdstok
  • By jong wingerdstokke word galvorming dikwels net bokant die entverbinding gesien
  • Teen die laat somer word die galle donkerder en word kurkagtige tekstuur met 'n growwe oppervlak en hou dit vir 'n paar jaar
  • Dooie galle kan van die wingerdstok afskilfer
  • Jong galle kan dikwels aan die periferie van ou galle vorm
  • Tipies gesien vanaf die grondlyn tot by die eerste draad
  • Gestremde besmette wingerde word dikwels tydens lae temperatuur episodes in die winter doodgemaak

Word dikwels verwar met:
Oormatige eelt van kwekerye onder nat toestande: eelt word nie kurkerig en skil af nie.

Biologie
Die kroongalbakterie oorleef binne galle en sistematies besmette wingerde. Die bakterie bly binne-in die wingerdstok, sonder om simptome te veroorsaak, totdat daar 'n besering aan die stam is en dring dan eers die buitenste deel van die stam binne waar dit vinnige selvermeerdering en vervorming van weefselproduserende galle veroorsaak. Die kroongalbakterie kan ook in wingerdgronde in wingerdrommel oorleef. Daar word geglo dat die meerderheid infeksies die gevolg is van simptoomlose besmette plantmateriaal. Oor die algemeen is die voorkoms van kroongal gekorreleer met koue vatbaarheid minder koue verdraagsame variëteite met 'n hoër voorkoms van kroongal infeksie.

Tydperk van Aktiwiteit
Vroeë somer, veral nadat winterbesering in koue-sensitiewe variëteite plaasgevind het.

Scouting Notes
Galle word meestal op die onderste stam aangetref, vanaf die grondlyn tot by die eerste draad, maar luggalle kan meer as een meter op die traliewerk ontwikkel. Monitor hierdie areas van die stam vir galle wat vroeg in die somer begin. Erg siek wingerde toon gewoonlik aansienlike verlagings in opbrengs en groeikragtigheid, wat hulle vatbaar maak vir winterdood.

Drempel
Daar is geen drempel nie. Stamme met kroongalsimptome sal verswak en sterf. Ander simptoomlose stamme op dieselfde wingerdstok, terwyl dit besmet is, kan egter vir baie jare voortgaan om oes te produseer. As die gal by die ent-unie is en geen suiers ontwikkel nie, sal die wingerdstok sterf.

Bestuursaantekeninge
Bestuurspraktyke wat besering verminder, is belangrik in die bestuur van hierdie siekte, aangesien die uitdrukking van kroongal nou verband hou met die voorkoms van besering.

Voor plant

Verliese aan druiweplante as gevolg van kroongal kan met sekere oorwegings tot die minimum beperk word voor wingerdterrein seleksie of plant.

  • Kies plekke met goeie grond- en lugdreinering, vermy rypgevoelige areas
  • Kies onderstokke wat bestand is teen kroongal soos Courderc 3309, 101-14 Mgt, en Riparia Gloire,
  • Kies geharde, koue verdraagsame variëteite waar moontlik
  • Moenie ou wingerdgebiede waar kroongal aanwesig was minder as 2 jaar nadat wingerde verwyder is, herplant nie. Kroongalbakterieë kan in die oorblyfsels van die ou druiweplante oorleef totdat die puin ontbind. Wanneer siek wingerde verwyder word, verwyder soveel as moontlik van die wortelstelsel.
  • Koop wingerdstokke van 'n betroubare bron. Latent besmette kwekerye is die hoofbron van kroongalsiekte in wingerde.
  • Warmwaterbehandeling van wingerdstokke is effektief om kroongalinfeksievlakke in plantmateriaal te verminder.

Na plant

Daar is min wat gedoen kan word om hierdie siekte te beheer sodra dit in die wingerd gevestig is, anders as om beserings aan wingerdstokke (winter, meganies en menslik) te vermy wat die siekte sal aktiveer.


Gevorderd

Wetenskaplike naam
Agrobacterium tumefaciens

Identifikasie

  • Galle op wortels, krone, en soms stamme en steiers
  • Galle is bolvormig, klonterig en grof, wissel van 1 tot meer as 10 cm in deursnee
  • Galle is aanvanklik sag en glad, maar word donker, hard, grof, houtagtig en gekraak soos hulle vergroot en verouder
  • Galle kom gewoonlik net aan die een kant van die wortel voor
  • Jong bome kan redelik vinnig deur kroongalle omgord en doodgemaak word. Galle is gewoonlik nie ernstig op ouer bome nie, tensy hulle deur houtbederfswamme binnegedring word

Word dikwels verwar met
Knopwortelaalwurm wat op wortels slaan - swelling vind plaas oor die hele deursnee van die wortel eerder as net aan die een kant

Biologie
Die patogeen affekteer 'n wye reeks breëblaar, houtagtige plante, insluitend steenvrugte. Bakterieë word in die grond vrygestel wanneer galle nat is of wanneer ouer galweefsel disintegreer. Die bakterie kan in die afwesigheid van gasheerweefsel vir ten minste 1 jaar in die grond oorleef. Gevestigde bome word slegs deur wonde besmet, soos dié wat veroorsaak word deur groeikrake, snoei, skade deur verbouingstoerusting of vriesbesering. Saailinge kan tydens ontkieming besmet word indien in besmette grond geplant word. Die galle belemmer die normale vloei van water en voedingstowwe. Jong bome kan doodgemaak word terwyl ouer bome verminderde groei en groeikrag ervaar.

Bakterieë betree die wortels en kroon deur wonde wat geproduseer word tydens die versorging en hantering van die kwekeryvoorraad. Hulle kan ook ingaan deur wonde wat deur wortelvoedende insekte gemaak is. Na infeksie dring kroongalbakterieë die gasheerweefsel binne en vermeerder tussen gasheerselle. ’n Gedeelte van die bakterieë se genetiese materiaal word by dié van die gasheerselle opgeneem, wat veroorsaak dat hulle vermeerder en ongewone aminosure produseer wat as ’n voedselbron vir die bakterieë dien. Die proliferasie van hierdie selle lei tot galvorming.

Simptome kan binne 'n paar weke by matige temperature ontwikkel of die bakterie kan vir 2-5 jaar latent bly voordat simptome geproduseer word. As kroongal in die kwekery voorkom, is simptome gewoonlik goed ontwikkel op voltooide bome ten tye van grawe.

Benewens primêre galle, ontwikkel sekondêre galle soms op 'n afstand van die aanvanklike infeksie. Hierdie galle kan op ongewonde weefsel ontwikkel en die bakterieë kan nie met hulle geassosieer word nie.

Tydperk van Aktiwiteit
Simptome kan binne 'n paar weke by matige temperature ontwikkel of die bakterie kan vir 2-5 jaar latent bly voordat simptome geproduseer word.

Drempels
Daar is geen verdraagsaamheid vir kroongalbesmette kwekerybome nie.

Scouting Notes
Inspekteer kwekerybome vir tekens van galle voor plant. Monitor jong bome vir tekens van ineenstorting en ouer bome vir verlies aan groeikragtigheid. Gaan wortels, krone, stamme en steiers na vir kroongal.

Bestuursaantekeninge
Plant in goed gedreineerde landerye en roteer besmette veldterreine met nie-gasheerplante soos grasse of korrels.

In die kwekery moet 'n steriele plantmedium gebruik word.

Gebruik slegs kroongalvrye kwekeryvoorraad van 'n betroubare kwekery. Inspekteer kwekeryvoorraad sorgvuldig voor plant en gee die hele lot terug indien simptomatiese bome gevind word. Plant saailinge met min of geen hak in.

Hanteer jong bome om so veel as moontlik besering te vermy, beide tydens plant en gedurende die lewe van die boom in die boord.

Verwyder bome wat gevind word met groot galle wat die krone omring wanneer die bome onproduktief word.

Wanneer 'n voorheen geaffekteerde terrein herplant word, verwyder soveel as moontlik van die ou boomwortels, groei 'n grasrotasie-gewas om oorblywende gasheermateriaal te help afbreek en patogeenvlakke te verminder, en verreken die nuwe bome van die vorige boomspasiëring om kontak met gesonde nuwe wortels met enige besmette wortels wat mag oorbly.


16.5F: Agrobacterium en Kroongalsiekte - Biologie

Agrobacterium tumefaciens
Deur Alyssa Collins
'n Klasprojek vir
PP728 Grondgedraagde plantpatogene
Noord-Carolina Staatsuniversiteit
Departement Plantpatologie

Agrobacterium tumefaciens, die oorsaak van die ekonomies belangrike siekte, kroongal, is ook vir jare bestudeer vanweë die merkwaardige biologie daarvan. Die meganisme wat hierdie bakterie gebruik om plantweefsel te parasiteer behels die integrasie van van sy eie DNA in die gasheergenoom wat lei tot onooglike gewasse en veranderinge in plantmetabolisme. A. tumefaciens het die eerste suksesvolle ontwikkeling van 'n biologiese beheermiddel aangespoor en word nou gebruik as 'n hulpmiddel om gewenste gene in plante te manipuleer.

Gasheerreeks en verspreiding

Agrobacterium tumefaciens is kosmopolities in verspreiding, wat tweesaadlobbige plante in meer as 60 verskillende plantfamilies affekteer. Kroongal kom die meeste voor op steenvrugte en kernbome, sowel as braambome en verskeie spesies sierplante.

Agrobacterium tumefaciens is 'n lid van die familie Rhizobiaceae. Hierdie bakterieë is Gram-negatief en groei aërobies, sonder om endospore te vorm. Die selle is staafvormig en beweeglik, met een tot ses peritrichous flagella. Selle is 0,6-1,0 m m by 1,5- 3,0 m m en kan alleen of in pare bestaan. In kultuur op koolhidraatbevattende media produseer selle groot hoeveelhede ekstrasellulêre polisakkariede, wat kolonies 'n volumineuse, slymerige voorkoms gee.

Onlangs is 'n herklassifikasie van die spesie van Agrobacterium is onderneem deur gebruik te maak van ribosomale RNA-volgordebepaling as 'n taksonomiese hulpmiddel. Die gevolglike nomenklatuur plaas die voormalige spesie, A. tumefacians biovar 1, A. radiobakter biovar 1, en A. rhizogenes biovar 1, binne die nuwe takson: Agrobacterium tumefaciens.
Isolasie
A. tumefaciens kan effektief geïsoleer word vir identifikasie vanaf galweefsel, grond of water. Optimale galweefsel vir isolasie is wit of roomkleurig van 'n jong, aktief groeiende gal. Die gal moet gewas of oppervlak gesteriliseer word met 20% huishoudelike bleikmiddel, en verskeie kere in steriele water afgespoel word. Sny 'n paar monsters uit verskillende dele van die wit weefsel van die gal, en verdeel monsters verder in klein stukkies. Plaas hierdie stukke in 'n kultuurbuis wat steriele gedistilleerde water of buffer bevat, vortex en laat staan ​​vir ten minste 30 minute. Gebruik 'n inokulasielus, strooi hierdie suspensie op Medium 1A (Schaad et al., 2001), en inkubeer by 25-27 ° C. Verskillende stamme sal teen verskillende tempo's groei. 'n Mens kan ook hierdie selektiewe medium gebruik om op te spoor A. tumefaciens in grondverdunnings of besproeiingswater.

Daar moet egter op gelet word dat die teenwoordigheid van A. tumefaciens selle in 'n monster dikteer nie noodwendig die bestaan ​​van die kroongal-aanhitsende stam in die monster nie. Slegs selle wat 'n spesifieke plasmied bevat (die Tekplasmied) kan siektes veroorsaak. A. tumefaciens stamme wat nie die plasmied het nie, leef as risosfeer-bewonende bakterieë sonder om siektes te veroorsaak.

Simptome

Kroongal manifesteer hom aanvanklik as klein swelsels op die wortel of stam naby die grondlyn, en soms op luggedeeltes van die plant. Jong gewasse, wat dikwels lyk soos die eeltweefsel wat voortspruit uit wonde, is sag, ietwat bolvormig en wit tot roomkleurig. Soos gewasse ouer word, word hul vorm redelik onreëlmatig, en hulle word bruin of swart. Tumore kan slegs deur 'n smal stukkie weefsel aan die gasheeroppervlak verbind word, of kan voorkom as 'n swelling van die stam, nie duidelik apart nie. Die weefsel kan sponserig en verkrummel dwarsdeur die gal wees of kan houtagtig en knoopagtig wees. Verskeie gewasse kan op dieselfde plant voorkom en kan heeltemal of gedeeltelik van die oppervlak van die plant af vrot, wat moontlik seisoen na seisoen herhaaldelik in dieselfde area ontwikkel. Bykomende simptome sluit in verdwerging, chlorotiese blare, en plante kan meer vatbaar wees vir ongunstige omgewingstoestande en sekondêre infeksie.

Patogeniese stamme van A. tumefaciens kan vir tot twee jaar saprofities in grond leef. Wanneer 'n nabygeleë gasheerplant naby die grondlyn deur insekvoeding, oorplantingsbesering of enige ander manier verwond word, beweeg die bakterie chemotakties in die wondplek en tussen gasheerselle in. Hierdie bakterieë stimuleer dan die omliggende gasheerselle om vinnig en onreëlmatig te verdeel. Die bakterie bewerkstellig dit deur 'n stukkie van sy eie DNA in die gasheerselle se chromosome in te voeg, wat oorproduksie van sitokiniene en ouksiene veroorsaak wat plantgroeireguleerders is, en opiene wat as voedingstowwe vir die patogeen dien. Die resulterende weefsel is ongedifferensieer met 'n wit of roomkleur, en selle kan een of meer kerne hê. Hierdie weefsel gaan voort om te vergroot en 'n gewas word op die wortel of stam van die plant gevorm, afhangende van die oorspronklike wondplek. Die bakterieë beset die intersellulêre spasies rondom die periferie van die gal en word nie in die middel van die vergrote gewas gevind nie. Die gewas word nie deur 'n epidermis beskerm nie, wat die weefsel vatbaar maak vir sekondêre patogene, insekte en saprofiete. Degradasie van die gewas deur sekondêre indringers veroorsaak bruin of swart verkleuring en vrystellings A. tumefaciens selle terug in die grond in om met grond of water weggedra te word, of bly in die grond tot die volgende groeiseisoen. In meerjarige plante kan 'n deel van die besmette weefsel lewendig bly en deur bewoon word A. tumefaciens, wat, selfs al het die gewas afgeneem, kan aanhou om 'n nuwe gewas die volgende seisoen op dieselfde plek te veroorsaak.

Bekendstelling van patogene A. tumefaciens stamme kan vermy word deur deeglike inspeksie van kwekeryvoorraad vir kroongalsimptome. Vatbare variëteite moet nie geplant word in gronde wat bekend is dat dit met die patogeen besmet is nie. Hierdie gronde moet vir 'n paar jaar in 'n eensaadlobbige gewas soos mielies of koring geplant word. Kwekeryvee moet kroongalvry gesertifiseer wees en moet eerder gebot as geënt word. As die bedreiging van kroongal bestaan, moet alle praktyke dat wondweefsel vermy en kouende insekte beheer word.

Voorkomende behandeling van sade of oorplantings met die nie-patogene biobeheer organisme Agrobacterium radiobacter is 'n relatief goedkoop en doeltreffende manier om die ontwikkeling van kroongal in kommersiële bedrywighede te bestuur. Toediening van hierdie antagonis deur saad te week of oorplantings te doop kan infeksie deur die meeste stamme van A. tumefaciens as gevolg van die produksie van die antibiotika agrocin 84 deur stam K84 van A. radiobakter. Sommige genesende eienskappe word vertoon deur 'n kommersieel beskikbare mengsel van 2,4-xilenol en metakresol in 'n olie-water-emulsie wanneer dit direk op gevestigde gewasse geverf word. Maar dit word selde gebruik as gevolg van arbeid en tydsbeperkings.

Agrios, G.N. 1988. Plantpatologie, 3de Uitg. Academic Press Inc., Londen. pp. 558-565.

Horst, R.K. 1983. Kompendium van Roosiektes. APS Press, St. Paul, MN. pp 23-25.

Schaad, N.W., J.B. Jones en W. Chun. 2001. Laboratoriumgids vir Identifikasie van Plantpatogene Bakterieë, 3de Uitg. APS Press, St. Paul, MN. pp. 17-35.

Skakels na ander werwe met inligting oor Agrobacterium tumefaciens


Kroongalsiekte van kwekerygewasse

Let op al die galle langs die stam aan die regterkant. Baie het begin om wonde te snoei.

L.W. Moore (oorlede), bakterioloog en plantpatoloog, OSU

Opgedateer deur M. L. Putnam, diagnostikus en plantpatoloog, OSU

Kroongal bly 'n groot probleem vir die kwekerybedryf, beide in houtagtige en kruidagtige plante. Die patogeen wat tradisioneel bekend is om kroongal in die meeste plante te veroorsaak, is Agrobacterium tumefaciens (Rhizobium radiobacter). Die patogeennaam is al dekades lank onder dispuut, en dit is bekend dat A. tumefaciens 'n spesiekompleks is wat uit ten minste 11 verskillende genomospesies bestaan. Hier sal ons verwys na die bakterieë wat kroongal veroorsaak as tumorigeniese agrobakterieë. Ander spesies Agrobacterium kan ook galle veroorsaak: A. rubi is baie minder algemeen vernoem na die gasheer waarin dit die eerste keer gevind is ( Rubus spp.), dit is sedertdien in galle op roos gevind en sal waarskynlik in ander plante gevind word met tyd. Agrobacterium vitis (= Allorhizobium vitis ) veroorsaak gal op wingerde. A. larrymoorei veroorsaak gal van Ficus benjamina, en is onlangs in roosgalle gevind. 'n Nuwe galvormende spesie, ook geïsoleer van roos, is onlangs beskryf en A. rosae genoem. Dit is waarskynlik dat addisionele spesies in die komende jare genoem sal word, aangesien bakterieë wat met galle geassosieer word, nader ondersoek word deur moderne molekulêre tegnieke te gebruik. Al hierdie spesies het 'n soortgelyke biologie. Hierdie bespreking dek die biologie, gasheerreeks, simptome en hantering van die siekte.

Kroongal is 'n tumor-vormende siekte van plante wat veroorsaak word deur tumorigeniese agrobakterieë, waarvan baie vermoedelik in die meeste landbougrond voorkom. Die patogene, in grond of op besmette plante, word versprei deur spatreën, besproeiingswater, hakplante met gesonde plante, plaasmasjinerie, snoeigereedskap, wind en plantdele wat vir voortplanting gebruik word. Wonde is nodig vir die patogeen om 'n plant te besmet. Wonde word gemaak deur snoei en bewerking, opkoms van sywortels, rypbesering, en insek- en aalwurmvoeding. Die patogeen koloniseer die wond, heg stewig aan beseerde plantselle en dra 'n deel van sy DNA oor na die DNA van die plant. Galle verskyn in 'n kwessie van weke by temperature bo 70 ° F op kruidagtige plante houtagtige plante soos rose mag nie gal toon tot maande of jare na blootstelling nie. Latente infeksies ontwikkel tipies in 'n later groeiseisoen in galle. Patogeniese bakterieë kan uit die gal in die omliggende grond of water gestort word waar hulle koloniseer of nuwe plantweefsels besmet.

Alhoewel dit algemeen gerapporteer word dat dit 'n gasheerreeks van honderde het, is hierdie inligting gebaseer op kunsmatige inentings, dikwels van net 'n enkele isolaat. Op 'n praktiese basis is baie minder plante natuurlik vatbaar. (Voorbeelde van gasheerplante wat deur Agrobacterium besmet is, word in Tabel 3 gelys.) Die wortelstelsels van nie-gasheerplante soos onkruide, grasse en graan kan egter die patogeen huisves en as 'n reservoir van inokulum in natuurlike omgewings dien.

Die siekte word kroongal genoem, maar gal kan gevind word aan die basis van steggies, op wortels, krone of op stingels, stokke, wingerde of blare. Blaargalle word gewoonlik aangetref op kruidagtige plante wat 'n sistemiese infeksie het. (Kruidagtige sierplante wat vatbaar is vir kroongal word in Tabel 1 getoon.) Galle kom dikwels by snoeiwonde voor. Galle is gewoonlik afgerond en kan glad of tekstuur wees soos 'n blomkoolkop. Op houtagtige meerjarige plante word galle meer houtagtig en gebars met ouderdom, wat soms 'n deursnee van 6 duim bereik en die stam omgord. Galle op wingerde, bloubessie en braambosvrugte is gewoonlik langwerpige, geweefde rante van weefsel wat deur die buitenste stamweefsels bars.

Houtagtige plante wat die eerste jaar wat hulle uitgeplant word besmet is, word ernstiger beskadig. (Houtagtige plante wat vatbaar is vir kroongal word in Tabel 2 getoon.) Jong plante wat erg gegal is, is verswak, verdwerg en onproduktief en vrek soms weens 'n minderwaardige wortelstelsel. Literatuurverslae van kroongalskade is teenstrydig, dit wissel van goedaardig tot aftakelend tot dodelik.

Simptome word duidelik 2 tot 4 weke na infeksie as temperature by of bo 68 ° F is, wat gewoonlik saamval met warmer grondtemperature in Mei of Junie. Aanvanklik lyk die galle soos eeltuitgroeisels maar neem dan vinnig toe in grootte en aantal. Simptoomontwikkeling vertraag baie onder 58 ° F en stop onder 50 ° F. Infeksie word onder 92 ° F tot 95 ° F geïnhibeer. Latente infeksies is simptoomloos en kom gewoonlik voor wanneer grond koel is. Gall symptoms typically develop at the infected wound the following season on rare occasions galls don’t appear until the third growing season.

Some problems can look like crown gall but are not pathogenic. Aerial burr knot on apple tree trunks and branches is a cushion-like assemblage of adventitious roots its cause is thought to be genetic rather than an infectious agent.

Small galls require careful diagnosis because they may be confused with excessive wound callus. Detection using molecular methods specific to plasmid gene regions involved with virulence, or isolation of bacteria later identified as pathogenic is necessary to confirm a crown gall diagnosis. Nonpathogenic Agrobacterium cells are often prevalent in these same tissues and can reach high populations. That makes diagnosis difficult, especially in galls on apple, blueberry, and grapevines where non-pathogens can constitute over 99% of the Agrobacterium population.

Disease Management – Woody Nursery Stock

Pathogen-free plants grown in uninfested soil will not develop crown gall. This emphasizes the importance of planting clean propagating material in clean soil. Good sanitation and cultural practices are important deterrents to crown gall. Discard all nursery stock showing symptoms to avoid contaminating healthy plants and storage facilities. At harvest, leave noticeably galled plants in the field for later pickup and destruction. If possible, choose a rootstock that is less susceptible, avoid planting sites heavily infested by root-attacking insects and nematodes, disinfect pruning equipment between trees, and adopt management practices that minimize wounding. Avoid planting into heavy, wet soil. Don’t plant trees deeper than they grew in the nursery. If possible, incubate dormant seedling roots at 73°F to 76°F for 10 to 14 days to heal wounds and reduce susceptibility to tumorigenic agrobacteria before planting them in wet soil. Use irrigation water from wells, if possible. Avoid planting where galled plants grew in the last 4 to 5 years choose fields that were planted recently to vegetables or grain. In summary, think prevention —avoid exposing plants to tumorigenic agrobacteria at any stage of plant production.

Crown gall is generally much more prevalent in heavy soils or in soil where water stands for a day or so. In New York, crown gall incidence was highest on a heavy clay knoll (15 ft elevation) from which water drained toward flat, loamy portions of the field. In Oregon, gall incidence on an Old Home x Farmingdale pear rootstock selection was severe (495 of 500 trees infected) in a heavy, wet soil, but in the same field only 1 of 500 trees was galled outside the wet area.

Cropping history can influence crown gall incidence. Budded apple trees became badly galled in fields where a previous nursery crop such as grape, peach, raspberry, and rose had been heavily infected. This situation isn’t repeated at every site, but we still recommend avoiding fields with a recent history of crown gall.

Reports of resistance in plants normally susceptible to crown gall are limited and depend on the strains of bacteria present in a given location. There are no reliable lists of cultivars with resistance that hold up in all geographic locations. It is better to select plants that are not susceptible in the first place if crown gall is a chronic problem in a particular field.

Using A. radiobacter K84, a biological control agent, has been very effective against crown gall on a number of hosts, but exceptions exist. Strain K84 produces a toxin against some tumorigenic strains of agrobacteria. This biological control is solely preventive, not curative application timing is critical to properly protect plant wounds caused at harvest or by pruning. Htay and Kerr recommend seed and root treatment with K84 for best results. Not all strains of tumorigenic agrobacteria are sensitive to K84. For example, most agrobacteria isolated from grape tumors are A. vitis , which are insensitive to K84. If K84 has been used properly and galling persists, its use should be discontinued since it is likely the bacteria present are not sensitive to the product.

An improved, genetically engineered strain of K84 called K1026 is available. Its use is preferable, since the K1026 bacteria are not capable of transferring to other bacteria the genes that produce the toxin.

Biological control is compatible with a few pesticides such as metalaxy (Ridomil), thiram and thiophanate-methyl (Topsin) but not with captan, etridiazole alone (Truban), etridiazole plus thiophanate-methyl (Banrot or Zyban), mancozeb, PCNB or streptomycin. It is also not compatible with chlorinated water.

No registered chemicals that effectively control crown gall are currently available in the United States. In general, chemical preplant dips or soil drenches have been ineffective.

Fumigation to rid soil of Agrobacterium generally has been ineffective, and in some cases, growers reported more disease after fumigation.

Heat therapy has been tried in cherry and plum seedlings, and in dormant grape cuttings. Although these measures can reduce the incidence of disease, there will still be a small percentage of plants that remain infected. Time and temperatures needed for effective heat therapy has not been determined for many plants, and injury to the plant material can occur when temperatures are too high. Although promising, heat therapy is not commonly used due to these difficulties.

In solarization, a thin plastic film is stretched over moist soil to capture energy from the sun and heat the soil to temperatures that kill pathogenic microbes. Populations of tumorigenic agrobacteria could not be detected in a solarized sandy loam soil, but solarization did not work in the heavier silty-loam. Mazzard cherry seedlings planted later in solarized and in nonsolarized control plots developed crown gall only in the nonsolarized plots.

Following is a summary of the best practices for managing crown gall. They include experimental results and grower observations. Understandably, physical and economic constraints occasionally may impede applying all these practices. But for best results, follow or adapt the procedures as closely as possible to fit your management plan.

Best Practices for Managing Crown Gall

  • Discard diseased plants as soon as noticed to avoid cross-contaminating other plants, equipment, or storage facilities.
  • Don’t heel-in galled plants with healthy plants.
  • Use good sanitation in handling planting stock.
  • Minimize wounding disinfect pruning tools between plants.
  • Plant only disease-free stock.
  • Plant in clean soil.
  • Avoid fields with a recent history of high crown gall infestation.
  • Avoid fields with heavy infestations of root-attacking insects and nematodes.
  • Select well-drained soils tile heavy soils.
  • Field-fallowing is helpful but may be impractical west of the Cascade Range.
  • Rotate susceptible crops with small grains.
  • Plant when soil is below 50°F.
  • Solarize lighter soils.
  • Avoid mechanical injury from tillage, hoeing.
  • Irrigate with deep-well water or sanitized pond water.
  • Keep grafts and buds above soil line.
  • Avoid high nitrogen and irrigation late in the growing season.

The following are specific procedures for commonly grown plants that can be used in addition to the above general procedures.

Stone Fruit, Nut Crops, Roses: Dip or spray with the biocontrol agent K84 or K1026. Apply to seed, bare roots, and aboveground grafts.

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Agtergrond

Crown gall disease was identified long ago as a bacterial plant disease [1], and its pathogenic bacterium is Agrobacterium tumefaciens, which mainly infects dicots. This disease often results in severe economic losses to the production of cherry and other fruit trees [2,3,4]. Crown gall disease starts with the attachment of A. tumefaciens to plant cell. And then the transfer DNA, a portion of the Ti plasmid, will be integrated into the plant genome. Finally, the symptomatic tumors form and grow [5].

Crown gall disease affects many fruit trees and causes extensive economic losses in nurseries. In a previous study, 11 tree species were surveyed. The highest disease incidence was found in peach (Prunus persica [L.] Batsch), almond (P. dulcis D Webb), cherry (P. avium L.), apple (Malus sylvestris Mill) and olive (Olea europaea L.) [6]. It was also found the rootstock of peach, cherry, apple and pear (Pyrus communis L.) trees was a influence factor contributing to the significant differences in the frequency of galled plants.

Plants are often exposed to many various bacterial, viral, and fungal pathogens but have evolved potent defense systems to protect themselves [7]. In defense responses of plants, the identification of microbial pathogens plays a key role, as it “turns on” the signal transduction pathway which activates the expression of numerous pathogen-responsive genes [8, 9]. These disease resistance genes are crucial for identifying the effector proteins during the process of pathogen infection [7].

Many biotechnological strategies have been developed and applied in the attempt to control crown gall disease. In transformation experiments, the truncated genes involved in T-DNA transfer have been used to induce plant resistance to crown gall disease [10, 11], and inactivating the oncogenes could prevent tumor formation [12]. Therefore, to obtain plants that are resistant to crown gall disease, much research has been devoted to producing sense and antisense strands of the oncogene sequence by placing these sequences between opposing strong constitutive promoters [13], or to silencing the involved bacterial oncogenes by using premature stop codons [14]. The study of Niemeyer et al. (2014) demonstrated a successful reprogramming of the viral N gene response against crown gall disease [9]. In recent years, Rosalia Deeken’s group has been working on the molecular mechanism between crown gall disease and A. tumefaciens in Arabidopsis thaliana [8, 15,16,17,18]. Pathogen infection always induces response of plant hormones. Lee et al. (2009) explored the physiological changes and adaptations on the aspect of SA, JA, ethylene (ET), and auxin (indole-3-acetic acid, IAA) with changes in the Arabidopsis thaliana transcriptome during tumor development [5].

At present, planting resistant cultivars and developing biological antagonists both are effective measures to control crown gall disease in orchards [3]. The existing biological antagonists are mainly used for prevention but they act poorly as a treatment. So the crown gall-resistant cultivars in agriculture were in need [19]. Previous studies have reported crown gall-resistant cultivars for apple, peach, plum, grapevine, aspen, and roses [20,21,22,23,24,25,26,27]. Crown gall resistance has been assessed in accessions of 20 Prunus species [21]. And it was found that when the strains K12 and C58 of A. tumefaciens were used to infect the main stems or lateral branches of seedlings, the incidence of resistance was up to 30% in some accessions of P. mahaleb. The cherry breeding resource plant P. mahaleb is a cosmopolitan cherry rootstock. In northwest China, it has become one of the main sweet cherry rootstocks because of its excellent biological traits, such as strong resistance to crown gall disease, dwarfing ability and salinity among other desirable traits [28]. By systematic classification of cherry species, P. mahaleb belongs to the III. Cerasus subgenus, Section 5 Mahaleb Focke [29]. It is a deciduous tree or large shrub, growing to 2–10 m (rarely up to 12 m) tall with a trunk up to 40 cm diameter. In most cherry growing countries, mahaleb cherry is used to be rootstock of sweet and sour cherries [28]. This rootstock showed strong resistance to crown gall disease in cherry production, but little is known about its mechanism of crown gall resistance. Furthermore, the actual genes (without modification) underpinning resistance to crown gall have not yet been reported.

In this study, we focused on cherry rootstock ‘CDR-1’ (P. mahaleb), the natural hybrid cultivar of P. mahaleb. The objective of our study was to investigate the resistance mechanism of ‘CDR-1’ to crown gall disease. Here, we carried out morphological observations, physiological and biochemical analyses, gene expression analysis and transcriptomic analysis in ‘CDR-1’, and conducted transient expression and transgenic verification in tobacco. Our results provide evidence that the crown gall resistance of ‘CDR-1’ is likely related to the lignin biosynthetic pathway.


Eksperimentele prosedures

Plantmateriaal

Arabidopsis thaliana ecotype Col-0 was used for the seedling transformation assay, transcriptome assay and Agrobacterium inoculation assay. GS mutants in the Col-0 background, including myb28/myb29 (SALK_136312 x GABI_868E02), cyp81F2-1 (SALK_073776), cyp81F2-2 (SALK_123882), myb51-1 (SM_3_16332), myb51-2 (SALK_059765), cyp79B2/cyp79B3 (Zhao et al., 2002 ), pen2-1 (Lipka et al., 2005 ) and pen2-2 (GABI-KAT 134C04), the camalexin mutants, including cyp71A12 (GABI-KAT 127-H03), cyp71A13-1 (SALK_105136), cyp71A13-3 (SALK_128994) and pad3-1 (CS3805), and the cyp79b2/B3/myb28/29 quadruple (qko) mutant, completely free of GSs and camalexin, were used in the transient seedling transformation assay as described.

Agrobacterium transformation of Arabidopsis seedlings and GUS assays

The virulent A. tumefaciens wild-type strain C58 was used for the infection of Col-0 seedlings. Seeds were germinated in 2 mL of half-strength Murashige and Skoog (MS) (Basal Salt Mixture, PhytoTechnology Laboratories, Kansas City, Kansas, USA) liquid medium [half-strength MS salt supplemented with 0.5% sucrose (w/v), pH 5.7] in each well of a six-well plate. Germination and growth took place in a growth room at 22 °C under a 16-h/8-h light–dark cycle (100 µmol/m 2 /s). Virulence of A. tumefaciens was pre-induced by 200 µ m acetosyringone in AB-MES (AB Minimal Medium plus MES salt, pH 5.5) (Wu et al., 2014 ) at 25 °C for 16 h prior to infection. Die Arabidopsis seedlings were infected with pre-induced A. tumefaciens C58 cells at an optical density at 600 nm (OD600) = 0.02 in half-strength MS medium. If the removal of agrobacterial cells was necessary, co-cultivation medium was removed after the chosen infection time and replaced with 2 mL of freshly prepared half-strength MS medium containing 100 µ m timentin, and incubated for recovery before analysis.

For the monitoring of the transient transformation efficiency, the T-DNA vector pBISN1 carrying the gusA-intron genes (Narasimhulu et al., 1996 ) was transformed into A. tumefaciens strain C58 for infection of Arabidopsis seedlings. GUS staining and activity assays were carried out as described at the chosen infection time (Salinas and Sánchez-Serrano, 2006 Wu et al., 2014). In brief, seedlings were stained by incubation in GUS staining solution containing 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc), and incubated at 37 °C in the dark overnight, followed by destaining in 90% ethanol (EtOH). For the GUS activity assay, liquid nitrogen-frozen seedlings from each well were ground into a fine powder to extract total protein. The GUS activity in 20 µg of protein per 200-μL reaction was quantified with the fluorescence substrate 4-methylumbelliferyl-β- d -glucuronide (MUG). The fluorescence intensity (excitation, 365 nm emission, 455 nm filter at 430 nm) was measured using a Microplate Reader (BioTek, Taipei, Taiwan) at 37 °C for 1 h. GUS activity was normalized to the protein amount and 4-methylumbelliferone standard curve. For statistical analysis, one-way analysis of variance (ANOVA) with Dunnett's test was performed. To determine the effects of GS-derived metabolites and camalexin on GUS enzyme activity in vitro, the selected compounds and DMSO control were each incubated with 5 ng of recombinant GUS protein (Sigma-Aldrich, St. Louis, MO, USA) in GUS extraction buffer containing 1 m m MUG. The reaction mixture was measured for GUS activity at 37 °C for 1 h.

Transcriptome analysis

For gene expression profiling of Agrobacterium-infected seedlings, the shoots and roots of Col-0 seedlings (infected or mock control) were separated by cutting with a micro-scissor and immediately frozen in liquid nitrogen. Total RNA was extracted according to the phenol (pH 4.5)/chloroform protocol, followed by gene expression analysis with Affymetrix ATH1 chips (Affymetrix, Santa Clara, CA, USA). The chips of three biological repeats were normalized by the MAS5.0 algorithm using GeneSpring software (Agilent Technologies, Santa Clara, CA, USA), and the genes with an intensity higher than the background value (value > 75), which passed the asymptotic unpaired t-test with Benjamini–Hochberg test correction (FDR, P < 0.05), were selected for further analysis. The fold changes were determined from the signals of infected plant tissues versus mock infection controls under the same conditions, and two-fold changes were used as cut-off to determine Agrobacterium-responsiewe gene. GOBU software (Lin et al., 2006 ) was used to analyse GO. The significant GO items were calculated with elim Fisher's exact test (P < 0.01) based on gene counts (Alexa et al., 2006 ).

Crown gall growth assay

For the crown gall growth assay, the A. thaliana wild-type Col-0 and mutants were cultivated in growth cabinets (Percival, CLF, Wertingen, Germany) under short-day conditions at 22 °C (8 h of 80–100 µmol/m 2 /s light Osram 400 W, Power Star HQI-E 400W/DV, 380–780 nm) (Wuerzburg, Germany) and 16 °C during the dark period (16 h) with a relative humidity of 50%–60%. Tumour development was induced by streaking virulent A. tumefaciens strain C58 into a wound of 1.5 cm in length, scratched into the base of young 5-cm-long inflorescence stalks. Tumour tissue was harvested 28 days after infection using a scalpel and a binocular. Wounded, but uninfected, tumour-free inflorescence stalk sections of the same age served as reference tissues.

GS and camalexin analysis in Arabidopsis saailinge

Extraction and analysis of seedling GSs and camalexin were performed and modified as described previously (Glauser et al., 2012 Zandalinas et al., 2012). In total, 100 mg FW of Arabidopsis seedlings were homogenized and dissolved in 1 mL of 70% high-performance liquid chromatography (HPLC)-grade methanol containing 12.5 ng/μL sinalbin (4-hydroxybenzyl GS) as an internal standard. The supernatants obtained were heated at 80 °C for 20 min and subjected to a UPLC-Synapt G1 high-definition mass spectrometry (HDMS) system (Waters, Taipei, Taiwan). GSs were separated on an Acquity CSH C18 column (length, 100 mm 2.1 mm i.d. 1.7 μm Waters) at a flow rate of 400 μL/min. The GSs were eluted by solvent A (2% acetonitrile and 0.05% formic acid) and solvent B (100% acetonitrile and 0.05% formic acid) for 8 min in 1%–45% solvent B and 1 min in 45%–100% solvent B. The fractions were injected for MS analysis, and negative ion data were recorded in MS1 mode. The peak area was calculated by MassLynx software (Waters), and then normalized to nanomoles for GSs or micrograms for camalexin per gram FW. The GSs were quantified with the given references, including I3M for iGSs, 4MTB for methylthioalkyl GSs and 4MSOB for methylsulfinylalkyl GSs, purchased from AppliChem (Darmstadt, Germany). Camalexin was quantified with pure camalexin (Sigma-Aldrich, St. Louis, MO, USA).

GS and camalexin analysis in Arabidopsis inflorescence stalks

For GS analysis of infected Arabidopsis inflorescence stalks, 100 mg (FW) were lyophilized, thoroughly homogenized and extracted three times with 1 mL of 80% (v/v) methanol. For the first extraction step, benzyl GS (Phytoplan, Heidelberg, Germany) was added to each sample as internal standard. GSs were desulfonated as described previously (Agerbirk et al., 2001 ), and separated on a Grom-Sil 80 ODS 7 pH column (length, 60 mm 4 mm i.d. 4 μm Alltech) (Wuerzburg, Germany) by HPLC (Agilent 1200, Waldbronn, Germany flow rate, 0.25 mL/min). The desulfo GSs were eluted as follows: 0.3 min in 0%–5% solvent A (water), 7 min with 1.2 min hold in 5%–95% solvent B (methanol) and 3.5 min in 95%–5% solvent B. Desulfo-GSs were determined via UV diode array detection (229 nm), identified and quantified using particular response factors (aGSs, 1 iGSs, 0.26) (Gonzáles-Megías and Müller, 2010 ).

Camalexin was extracted from lyophilized tissue (50 mg FW) by the addition of 400 μL of 85% methanol. The samples were thoroughly homogenized with a metal ball in a Mixer Mill 301 (Retsch, Haan, Germany) for 1.5 min at a frequency of 30 Hz. The extract was incubated at 42 °C for 60 min with addition of 0.3 μg/μL camalexin as an external standard. For the identification and quantification of camalexin, HPLC was applied as described by Mikkelsen et al. ( 2009 ).

GS derived metabolites and camalexin treatment for transient transformation assays and Agrobacterium cell counts

The selected aGS-ITCs (LKT Laboratories, St Paul, MN, USA) and camalexin were dissolved in DMSO, and I3M was dissolved in methanol. These compounds were added to the seedling co-cultivation medium for Agrobacterium infection and GUS assays, as described above.

For the measurement of the viable Agrobacterium cell number, the bacterial cells (C58 strain carrying pBISNI) in co-cultivation medium and associated with seedlings at 1 and 3 dpi were collected. Six seedlings per well were washed by 2 mL of double-distilled H2O to remove unbound bacteria and ground by a mortar in 1 mL of 0.9% NaCl solution. The bacterial cells in medium or associated with seedlings were 10× serially diluted and then plated on 523 medium (Kado and Heskett, 1970 ) containing kanamycin, and incubated at 25 ºC for 2 days to obtain colony-forming units (CFUs). The seedling-associated Agrobacterium cell number was further normalized to the plant fresh weight.

Myrosinase activity

Myrosinase activity was determined from 50–200 mg of frozen plant material, which was purified from internal substrate. Activity was measured by the photometric quantification of the released glucose from standardized amounts of externally added substrate according to the protocol developed by Travers-Martin et al. ( 2008 ).

Callus induction assay

Callus induction assay was performed and modified as described previously (Hwang and Gelvin, 2004 ). Col-0 and the tested mutants were grown on half-strength MS agar plates for 3 weeks, and the roots were cut into ∼1-cm segments. About 60 root explants were transferred to agar plates containing callus induction medium (CIM), further incubated for 4 weeks, followed by counting of the number of developing calli and calculation of the rate of callus induction.


Agrobacterium tumefaciens and Crown Gall Disease - PowerPoint PPT Presentation

Antibiotic treatment against bacteria that allow the plant to survive are useful . In Situ Transfer of Antibiotic Resistant Genes from Transgenic (Transplastomic) . &ndash PowerPoint PPT presentation

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