Isolation of pathogenicity genes from xanthomonas species and a study of their regulation

115 Pages·1991·4.1 MB·English

by  Swarup, Sanjay, 1961-

ISOLATIONOFPATHOGENICITYGENESFROMXANTHOMONAS
SPECIESANDASTUDYOFTHEIRREGULATION
By
SANJAYSWARUP
ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL
OFTHEUNIVERSITYOFFLORIDAINPARTIALFULFILLMENT
OFTHEREQUIREMENTSFORTHEDEGREEOF
DOCTOROFPHILOSOPHY
UNIVERSITYOFFLORIDA
1991
ACKNOWLEDGMENTS
I express my profound gratitude and appreciation to Dr. Dean W. Gabriel,
chairmanofmysupervisorycommitteeforhissupport,kindness,andsoundadvice;and
to Drs. D.R. Pring, L.C. Hannah and W.B. Gurley, members ofmy supervisory
committee,fortheirguidance.
I owe special thanks to Dr. R.H. Brlansky for his help with the electron
microscopywork.ThanksarealsoduetoDrs.RobertDeFeyterandMarkT.Kingsley
fortheirstimulatingscientificdiscussionsanddelightfulcompany. Ialsoacknowledge
the help ofDr. Moosa Hojjati in the maintenance ofthe plants in the greenhouse
facilities. IgratefullyacknowledgethetechnicalassistanceofBlanca,Susan,andHolly.
IappreciatemyfriendsinGainesvillewhowerealwaysthereinthegoodtimes
andthebadtimes. ThanksarealsoduetomyfriendsHilisaBartolomeandLeonor
Maia,especiallyfortheirhelpduringpreparationofthismanuscript.
Iwouldliketothankmyparentsandmywife,Nidhi,fortheirpatienceduring
thisperiodofabsencefromhome.
11
TABLEOFCONTENTS
ACKNOWLEDGMENTS ii
ABSTRACT iv
CHAPTERS
1 INTRODUCTION 1
2 APATHOGENICITYLOCUSFROMXANTHOMONAS
CITRIENABLESSTRAINSFROMSEVERAL
PATHOVARSOFX. CAMPESTRISTOFORM
CANKER-LIKELESIONSONCITRUS 20
3 AXANTHOMONASCITRIPATHOGENICITYGENE,
pthA,PLEIOTROPICALLYENCODES
GRATUITOUSAVIRULENCEONNONHOSTS 46
4 STRUCTURALMANIPULATIONSOFpthAFROM
XANTHOMONASCITRISEPARATETHE
PLEIOTROPICFUNCTIONSOF
PATHOGENICITYANDAVIRULENCE 66
5 SUMMARYANDCONCLUSIONS 91
LITERATURECITED 95
BIOGRAPHICALSKETCH 108
AbstractofDissertationPresentedtotheGraduateSchool
oftheUniversityofFloridainPartialFulfillmentofthe
RequirementsfortheDegreeofDoctorofPhilosophy
ISOLATIONOFPATHOGENICITYGENESFROMXANTHOMONAS
SPECIESANDASTUDYOFTHEIRREGULATION
By
SanjaySwamp
August1991
Chairman:DeanW.Gabriel
MajorDepartment:PlantPathology
A "vimlenceenhancement"approachwasdevisedforcloningvimlencegenes
fromhighlyvimlentpathogensintolesservimlentpathogens,compatibleonthesame
host(s). TheapproachwastomovegenomicDNAfromahighlyvimlentdiseaseagent
toalessvimlentagentandassaytransconjugantsforincreasedvimlence.Genepth\from
thehighlyvimlentpathogenXanthomonascitriwasclonedbyvimlenceenhancementof
theopportunisticcitmspathogenX.campestrispv.citrwnelo. TransconjugantsofX.c.
pv.citrumelowithpthAhadtheabilitytoinducecankerouslesionsoncitms. Marker
exchangemutagenesisofX.citrirevealedthatpthAwasessentialforbothpathogenicity
oncitmsandforanon-hosthypersensitiveresponse(HR)elicitationonbean. Lackof
HRinductionbyanX.citripthA"mutantdidnotextendthehostrangeofX.citritobean
IV
V
plants. Thisdemonstratedthattheavirulencefunctionofpth\playednoevidentrolein
limitingthehostrangeofX. citrionbeanandthatthehypersensitiveresponseofbean
to X. citri appeared gratuitous. GenepthA was shown to induce cultivar-specific
avirulencewhenmovedintostrainsof X.c. malvaceanm(Xcm)andinoculatedonto
congeniccottonlines. Structuralcharacterizationofpthkshowedthatitbelongedtoa
XanthomonasavrgenefamilyconsistingofwellcharacterizedgenesfromXcmandX.c.
vesicatoria. GenepthKwasshowntobe4.5kbinsizeandtohave17nearlyidentical
102bpdirectDNArepeatsinthecentralregion. Invitromanipulationsofthe102bp
repeatsofpthA resultedin separationofthepleiotropicfunctions, pathogenicityand
avirulence. Cloneswereidentifiedwhichhadlostavirulencebutpossessedenhanced
virulenceorhadlostvirulence-enhancementbutstillexpressedavirulence. Threeavr
allelesfromX. citriandonefromX. phaseoli, similartopthA, werealso foundto
encodeforavirulencein Xcm on several cotton lines. Theseobservations strongly
suggestthatelicitationofdiseasesymptoms(normosensitiveresponse;NR)andofthe
diseaseresistanceresponse(hypersensitiveresponse;HR)mayinvolveverysimilarbut
notnecessarilythesamemechanisms.
CHAPTERI
INTRODUCTION
Plantsprovideahighlyspecializedecologicalnicheforanumberofmicrobial
species. Amongthemanyplant-associatedbacteriaknown,thosebelongingtothegenus
Xanthomonasarealwaysplant-associatedandusuallypathogenic. Xanthomonasasa
genusisverywidespread,colonizingmembersofmostmajorplantfamilies. However,
individual strains have host ranges restricted to only certain plant groups. This
phenomenonofhostspecializationwasonceusedasthebasisfortaxonomicspeciation
ofthegenus,butthiswaslaterrevisedandallbutfiveXanthomonasspp.wereplaced
into 125 pathogenic variants (pathovars) ofone species (Bradbury, 1984). Strains
belongingtoonepathovargenerallyareabletoattackthesamesetofplantspeciesand
cause similar disease as other members of the same pathovar. However, several
pathovarshaveoverlappinghostrangesenablingmembersofdifferentpathovarstobe
abletoattackatleastacommonsubsetofhostspecies. Sincepathovarshavenostanding
inbacterialtaxonomy,suggestedstandardsfortheirusearenotrigorouslyenforcedand
oftenheterogeneousstrainswillbeincludedinapathovarbasedsolelyonthe"hostplant
fromwhichfirstisolated"(Starr, 1981)criterion. Themainproblemwiththepathovar
systemisthattheorganismisbeingclassifiedonthebasisofahost(response)phenotype
(SchrothandHildebrand, 1983). Recently,phylogenetically-basedtaxonomiesrelying
onnucleicacidcomparisonsofstrains(CookseyandGraham,1989;Gabrieletal.,1989)
1
2
arehelpingresolvetheconfusionbyclassifyingbacteriaonthebasisoftheirgenotypes.
Oncethe strainsare taxonomicallywell-defined, thealmostexclusiveassociationof
certaingroupsofXanthomonasstrainswithcertaingroupsofplantsandtheirhighlevels
ofhostspecificitymakeXanthomonasspp.amodelsystemtostudythegeneticbasisof
plant-microbeinteractions.
TheplantdiseaseselicitedbyXanthomonasspeciesaremorediversethanthehost
rangesofthecausalagents. Thetypesofsymptomselicitedbyxanthomonadsinclude
leafspotsandstreaks(causedbylocalinfection);wiltsandblights(causedbysystemic
f
orvascularinfection);rots(causedbytissuemaceration);andcankers(causedbyhost
cellproliferation)(Starr, 1981). Intuitively,altogetherdifferentsetsofgenefunctions
mayberequiredfortheinductionofthesedifferentkindsofsymptoms. Therefore, a
comprehensivestudyofthegeneticsofvirulenceofXanthomonasspeciesislikelyto
involve a number ofdifferent experimental strategies appropriate for the kinds of
diseases. Inthischapter,Iattempttobringforthspeculativeconceptsregardinghost
specificityandvirulenceofXanthomonasspp. whichhaveemergedbecauseofsome
recent work. I also re-emphasize some older concepts which have become better
establishedasaresultofrecentobservations.
t
PyramidingofGeneFunctionsinXanthomonasSpecies
Strains ofXanthomonas spp. are non-obligate biotrophic organisms. This
observation has at least three direct implications. Xanthomonas strains are
chemoorganotrophicandtherefore,canbeculturedonsyntheticmediawithrelativeease.
3
Thisindicatesthatatleastapartofthegenefunctionsoftheseorganismsisdirected
towardstheirmaintenanceoutsidetheplantenvironmentandthatadditionalfunctionsare
requiredforhostcolonizationandparasitism(Fig. 1-1), Secondly, beingbiotrophic,
Xanthomonasstrainsdonotkillthehostcellsinadvanceoftheirinvasion,asnecrotrophs
do. Geneticmechanismsinvolvedintoxinproductionare,notsurprisingly,relatively
uncommon as major or general virulence factors in Xanthomonas-cz\xs&A diseases.
AlthoughcarboxylicacidsfromX.campestrispv.malvacearumandX.c.pv.oryzaehave
beenreportedasblight-inducingtoxins,ithasbeensuggestedthattheirphytotoxicitymay
besolelyduetotheiracidicnature(RobesonandCook, 1985). Thegenesinvolvedin
conditioningthevirulenceofbiotrophicparasitesmustberelativelyunobtrusiveintheir
effects. ThethirdcharacteristicofbiotrophslikeXanthomonasspeciesisthattheyare
welladaptedtotheirhostsand,therefore,haverelativelynarrowhostranges(Gabriel,
1989). Sincethesemicrobesarepredominantlyplantassociated, itisexpected that
xanthomonadswouldpossess, inadditiontoconservedgeneralvirulencegenes, host-
specificvirulencegenes.
AllXanthomonasstrainsappeartobeplantassociated,aseitherendophytesor
epiphytes. MostbutnotallnaturallyoccurringendophyticXanthomonas strainsare
pathogenicbutanumberofstudieshavereportedisolationofendophyticxanthomonads
fromasymptomatictissues. Forexample,anon-pathogenicendophytic Xanthomonas
straincapableofmultiplyingandspreadinginplantawasfoundtobeassociatedwitha
numberofapplecultivars(Maasetal., 1985). Suchstrainsaredifficulttodetectin
naturebecausetheycausenosymptoms. Thexanthomonadsthathavebeenmoststudied
4
HYPERSENSITIVE
HOST
RESPONSE
SYMPTOM
ELICITATION NORMOSENSITIVE
RESPONSE
PARASITIC HOVSITRUSLPEENCICFEICj/ n,SV, P.SP
GROWTHINPLANTA VGIERNUELREANCLE// hrp, vi^r, nod
(BasicMetabolism)
Figure 1-1. Pyramidingofgene functionsinXanthomonas spp. Referto text for
explanationsofavr,hrp,hsn,nod,andpth.
5
arethosewhichwereisolatedpreciselybecausetheycausedmajordetrimentaleffects.
ThesemaybeunrepresentativeofXanthomonasecologically. Recently,DNAprobes
(derived from general virulence genes) have been used to detect opportunistic
Xanthomonasstrainsassociatedwithvarioushosts(StallandMinsavage, 1990). The
ability ofnon-pathogenicendophytic strains to parasitize (i.e., colonize) host tissue
suggests that functions related to pathogenicity—i.e., eliciting a recognizable host
response,ornormosensitiveresponse(NR)(Klement,1982)-aresuperimposedonthose
requiredforbasiccompatibility. Pathogenicitygenesaddanotherlevelinthepyramiding
ofgenefunctionsinbiotrophs,suchasXanthomonasspp.(Fig. 1-1). Agenefunction
highlyrelatedtopathogenicitymaybeavirulence; avirulencegenesareknowntoelicit
thehypersensitiveresponse(HR),itselfthoughttobequantitativelybutnotqualitatively
differentfromtheNR(Klement, 1982).
GeneralVirulenceFunctions
Several general virulencegenes havebeenidentified invariousXanthomonas
speciesbybothchemical(Bonasetal.,1989)andtransposon(KamounandKado,1990)
mutagenesis. Mutationsinsuchgenesareidentifiedbyalossoftheabilityofthemutant
straintoinduceadefensehypersensitiveresponse(HR)innon-hostplantsandtobe
pathogeniconthehomologoushost;suchgenesare,therefore,termedhrpgenes(Willis
etal., 1991). Hypersensitive reaction referstotherapid localizedplantcelldeath;
characteristicoftheinteractionwhenanincompatiblepathogenisintroducedintoplant
tissue(Klement, 1982). StrainswithHRP'phenotypeareinvariablyimpairedintheir