Transgenerational gene silencing causes gain of virulence in a plant pathogen.

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by  Qutob, Dinah

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Received27Sep 2012|Accepted 3Dec 2012 |Published 15 Jan 2013 DOI:10.1038/ncomms2354 OPEN
Transgenerational gene silencing causes gain
of virulence in a plant pathogen
Dinah Qutob1, B. Patrick Chapman1 & Mark Gijzen1
Avirulence(Avr)genesofplant pathogensencodeeffector proteinsthattriggerimmunityin
plantscarryingappropriateresistance(R)genes.ThePhytophthorasojaeAvr3agenedisplays
allelicvariationinmessengerRNAtranscriptlevels.P.sojaestrainswithdetectableAvr3agene
transcripts are avirulent on plants carrying the R-gene Rps3a, whereas strains lacking Avr3a
mRNAescapedetectionbyRps3aandarevirulent.Hereweshownon-Mendelianinteractions
betweennaturallyoccurringAvr3aallelesthatresultintransgenerationalgenesilencing,and
weidentifysmallRNAmoleculesof25nucleotidesthatareabundantingene-silencedstrains
but not in strains with Avr3a mRNA. This example of transgenerational gene silencing is
exceptional because it is naturallyoccurring and results in gain of virulence in a pathogenic
organism.
1AgricultureandAgri-FoodCanada,1391SandfordStreet,London,OntarioN5V4T3,Canada.Correspondenceandrequestsformaterialsshouldbe
addressedtoM.G.(email:[email protected]).
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P
lant pathogens secrete effector proteins to enable disease, (Supplementary Fig. S2). These results demonstrate heritable
but evolution favors host plant immune systems to transgenerational gene silencing of Avr3a in crosses between
recognize these factors as cues for activating rapid defence P. sojae strains ACR10 and P7076.
responses that arrest the infection1. Effector-triggered immunity
in plants is the consequence of signalling events that are
DistinctiveprofileofsmallRNA(sRNA)insilencedstrains.To
determined by resistance (R) gene products interacting, directly
explore the cause of transgenerational silencing of Avr3a, we
or indirectly, with pathogen avirulence (Avr) effectors.
performeddeepsequencingofsRNAfromeachparentalP.sojae
Phytophthorasojaecausesstemandrootrotofsoybeanandis
a major disease problem that plagues this crop2. The P. sojae strain ACR10 and P7076, and from a sample of F2 cultures with
the genotype Avr3aP7076/Avr3aP7076. Results from this analysis
Avr3a gene encodes a predicted protein of 111 amino acids that
show that the profile of sRNA coverage across the Avr3a region
includes a signal peptide, a host-targeting motif and a carboxy-
differsbetweenACR10andP7076(Fig.1b).ThesRNAsequences
terminal effector domain, which are common features of
oomycete Avr effectors3–6. Avr3a resides in a 10.8 kilobase (kb) matching to the Avr3a region are far more abundant in strain
ACR10 compared with P7076 (Table 2). The most prevalent of
DNA segment that displays copy number variation among
P. sojae strains5,6. Four haplotypes of Avr3a are known and these sRNA molecules are 24–26 nucleotides in length (Fig. 1c).
In strain ACR10, there is a nearly continuous coverage of sRNA
gain of virulence on the R-gene Rps3a is caused by messenger
sequences that align to a 3.7kb span of the Avr3a interval
RNA transcript differences between strains. In crosses between
P. sojae strains P6497 (Avr3aP6497/Avr3aP6497) and P7064 includingthe50and30intergenicsegmentsbutnotextendinginto
(Avr3aP7064/Avr3aP7064), avirulence towards Rps3a plants flankinggenes.Incontrast,instrainP7076coverageofmatching
sRNAislimitedtoa0.3-kbsegmentoccurringinthe50intergenic
segregates as a dominant Mendelian trait conditioned by the
presence of Avr3aP6497. Transcripts of Avr3aP6497 but not region. Sequencing of sRNA from homozygous Avr3aP7076/
Avr3aP7064 mRNA are detectable in each of the parental strains Avr3aP7076 F2 cultures resulted in a profile of coverage that is
and their progeny5,6. nearly identical to that from strain ACR10 (Avr3aACR10/
Avr3aACR10) rather than that from P7076 (Avr3aP7076/
Sequencing of Avr3a alleles and analysis of mRNA transcripts
in17differentP.sojaestrainsrevealedfourdifferenthaplotypes6, Avr3aP7076). The sRNA profiles were replicated from indepen-
dentsamplesofACR10,P7076,F andF cultures,andextended
but the inheritance behaviour of each of these haplotypes in 1 2
to include samples from additional P. sojae strains P6497 and
segregating populations has not been investigated. The present
ACR16(Table2;SupplementaryFig.S3).Theresultsconsistently
study was intended to test the inheritance Avr3a in P. sojae
show a pattern of extensive sRNA coverage of Avr3a occurs in
strainswithhaplotypesthataredifferentfromstrainsP6497and
gene-silenced strains.
P7064. To our surprise, we discovered that genetic outcrosses
between P. sojae strains ACR10 and P7076 resulted in
transgenerational gene silencing of Avr3a and gain of virulence Discussion
on soybean plants carrying the Rps3a gene. Previous studies have shown that transformation of organisms,
including species of Phytophthora, with recombinant DNA or
RNA molecules can cause internuclear or trangenerational gene
Results silencing that resembles our observations of naturally occurring
Transgenerational gene silencing of Avr3a. To determine the silencing of Avr3a (refs7,8). Moreover, results fromcomparative
inheritance of other Avr3a alleles, we created crosses between genome sequencing of species within the Phytophthora infestans
P. sojae strains P7076 (Avr3aP7076/Avr3aP7076) and ACR10 clade suggest a role for epigenetic mechanisms in the rapid
(Avr3aACR10/Avr3aACR10).ThestrainP7076isavirulent,whereas evolution of Phytophthora species9. Thus, we propose that
strainACR10isvirulentonRps3aplants,andmRNAtranscripts transgenerational gene silencing of Avr3a in P. sojae is the
are detectable for Avr3aP7076 but not Avr3aACR10, as we have resultofepigeneticchangesthatmodulatetheexpressionstateof
previouslyshown5,6(Fig.1a).Toselecthybridprogenyandfollow this gene, and that are transmitted to progeny in a non-
segregation of Avr3a alleles we used co-dominant DNA markers Mendelian fashion.
derived from cleaved amplified polymorphic (CAP) fragments, Given the emerging functions of sRNAs in controlling
becauseAvr3aP7076andAvr3aACR10differinsequence5,6.Atotal epigenetic marks and gene expression states in numerous
of 28 F hybrid (Avr3aP7076/Avr3aACR10) progeny were isolated, experimental systems, it is noteworthy that we found an
1
scoredforvirulenceonRps3aplantsandassessedforthepresence association between sRNAs and gene silencing at the Avr3a
of Avr3a mRNA transcripts by reverse transcriptase polymerase locus. In plants, sRNA of 24–26 nucleotides are involved in
chainreaction(RT–PCR)(Table1,Fig.2,SupplementaryFig.S1). directing chromatin changes including DNA methylation,
All28F progenywerevirulentonRps3aplantsandwerelacking whereas shorter ones of 21–22 nucleotides participate in mRNA
1
Avr3a mRNA transcripts. This result was unexpected because degradation10. Heterochromatic silencing in yeast is initiated by
heretofore avirulent alleles of Avr3a that accumulate mRNA an interaction of sRNA and RNA polymerase II, which guides
transcriptsweredominanttovirulentallelesthatlacktranscripts, histone modifications that control chromatin structure and gene
such as in crosses between P. sojae strains P6497 and P7064. To expression11. Thus, the evidence suggests a role for sRNA in
further explore the segregation of Avr3a alleles, virulence and directing the transgenerational gene expression changes of the
mRNA transcript accumulation in this cross, we generated three P. sojae Avr3a gene.
different F populations from separate F individuals. In all, 139 Our observations of transgenerational gene silencing of Avr3a
2 1
F progeny were tested for segregation of Avr3a alleles and provide a remarkable example of pathogen adaptation to host
2
mRNAtranscriptsandscoredforvirulenceonRps3aplants.The immune surveillance. The interactions between pathogens and
Avr3aP7076andAvr3aACR10allelessegregatednormallyineachF their hosts are often interpreted as an evolutionary race, where
2
population but remarkably all 139 F progeny lacked Avr3a pathogen virulence and host resistance are at the forefront12.
2
mRNA transcripts and were virulent on Rps3a plants (Table 1). Transgenerational gene silencing of an Avr effector locus
A sampling of eight F individuals, derived from homozygous represents a novel method of host immune evasion that offers
3
Avr3aP7076/Avr3aP7076 F cultures, indicates that gene silencing the ability to spread quickly in pathogen populations, but yet is
2
is maintained and meiotically stable in these inbred lines potentially reversible as the sequence of the Avr gene itself
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5,000
Avr3a
4,000
Allele mRNA Rps3a
Avr3aP6497 + A tnuo 3,000
c
e
c
Avr3aACR10 – V ne
uq 2,000
e
S
Avr3aP7076 + A
1,000
Avr3aP7064 – V
0
21 22 23 24 25 26 27 28
Length of small RNA (nt)
256 ACR10
Avr3aACR10/Avr3aACR10
16
1
s
da 0
e
7r 0 256 P7076
1 / e 16 Avr3aP7076/Avr3aP7076
g
a 1
re
v
o
c
fo h 0
tpeD 21566 FA2v rp3oaoPl7076/Avr3aP7076
1
0
Avr3a
10.8 kb
Figure1|CharacteristicsofP.sojaeAvr3aallelesanddeepsequencingofsRNA.(a)AschematicillustrationofAvr3aallelesfromfourdifferentstrainsof
P.sojae.ThesegmentofDNAharbouringAvr3aandfourotherpredictedgenesisshown.Predictedopenreadingframesareindicatedbyboldarrows.
ShownforeachalleleisthecopynumberoftheDNAsegment,presence(þ)orabsence((cid:2))ofAvr3amRNAtranscriptsanddiseaseoutcomeonRps3a
plantsasvirulent(V)oravirulent(A).(b)ProfilesofsRNAfromP.sojaestrainsACR10andP7076,andfromapoolofF culturesselectedfortheir
2
Avr3aP7076/Avr3aP7076genotype.Averagedepthofcoverageacrossthe10.8-kbgenomesegmentcontainingAvr3awasnormalizedtothetotalnumberof
trimmedreadsforeachsample.(c)SizeofsRNAaligningtothe10.8-kbDNAsegment.ThetotalnumberofcountsofRNAsequencereadsandthesizein
nucleotides(nt)isshownforP.sojaestrainACR10.
Table1| Segregationof Avr3a alleles andvirulence phenotypes inF andF populations (ACR10 (cid:3) P7076) of Phytophthora
1 2
sojae.
Population n Avr3aallelegenotype P Virulence mRNA
Avr3aACR10 Avr3aACR10 Avr3aP7076 A V (þ) ((cid:2))
Avr3aACR10 Avr3aP7076 Avr3aP7076
F 28 0 28 0 1.0 0 28 0 28
1
F -1 51 16 26 9 0.38 0 51 0 51
2
F -2 44 14 16 14 0.19 0 44 0 44
2
F -3 44 10 22 12 0.91 0 44 0 44
2
Thenumberofprogeny(n)ineachpopulationandthePvaluefromaw2-testforMendeliansegregationofgenotypesisshown.Virulent(V)andavirulent(A)phenotypeswerescoredbyinoculationof
plants,andthepresence(þ)orabsence((cid:2))ofmRNAtranscriptsofAvr3aasdeterminedbyRT–PCR.
remains unchanged. We predict that other eukaryotic pathogens Methods
of plants, humans and animals will employ similar mechanisms Phytophthorasojaestrainsandcrosses.TheoriginofP.sojaestrainsand
to escape detection by host immune systems. methodsofculture,propagationandoosporeisolationhavebeendescribed5,6,13–15.
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ACR10 P7076
Avr3aACR10/Avr3aACR10 Avr3aP7076/Avr3aP7076
s
n
ia
rts
la
tn
e
ra
P
rps3a Rps3a rps3a Rps3a
F -1 F -2 F -3
1 1 1
Avr3aACR10/Avr3aP7076 Avr3aACR10/Avr3aP7076 Avr3aACR10/Avr3aP7076
1
F
rps3a Rps3a rps3a Rps3a rps3a Rps3a
F -1 progeny
1
Avr3aACR10/Avr3aACR10 Avr3aACR10/Avr3aP7076 Avr3aP7076/Avr3aP7076
rps3a Rps3a rps3a Rps3a rps3a Rps3a
F -2 progeny
1
Avr3aACR10/Avr3aACR10 Avr3aACR10/Avr3aP7076 Avr3aP7076/Avr3aP7076
2
F
rps3a Rps3a rps3a Rps3a rps3a Rps3a
F -3 progeny
1
Avr3aACR10/Avr3aACR10 Avr3aACR10/Avr3aP7076 Avr3aP7076/Avr3aP7076
rps3a Rps3a rps3a Rps3a rps3a Rps3a
Figure2|PhotographsofP.sojae-infectedsoybeanplantsshowingdiseaseoutcomes.VirulenceassaysofP.sojaecultureswereperformedon
greenhouse-grownseedlingsofsoybeancultivarWilliams(rps3a)andtheisolineL83-570(Rps3a)byhypocotylinoculationwithP.sojaemycelia.Shown
arediseaseoutcomes6daysafterinoculationofplantswiththeparentalstrains,theF hybridsusedtoconstructeachofthethreeF populations,orwith
1 2
representativeF individualsillustratingeachAvr3agenotypefromthethreeF populationstested.Potsare10cmindiameter.
2 2
P.sojaeisanoomyceteanddiploidmicroorganismthatreproducessexually thereforeoosporescandevelopfromself-fertilizationorfromout-crossingwhen
throughthedevelopmentofoogonia(femalegametophyte)andantheridia(male differentstrainsoftheorganismaregrowntogether2.Toperformsexualcrossesof
gametophyte).P.sojaeishomothallicandisnotknowntohavematingtypes, P.sojae,parentalstrainsareco-cultivatedandoosporesareisolatedfromculture
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Table2| Deep sequencingofsRNAfrom Phytophthora sojae.*
Samplew Avr3agenotype Avr3amRNAz Virulenceon Totalnumberof MatchestoAvr3a Normalizedmatches
Rps3a trimmedsRNA segmenty per106reads
sequencereads
Parentalstrains
P7076(rep1) Avr3aP7076/Avr3aP7076 þ A 4,640,922 306 65
P7076(rep2) Avr3aP7076/Avr3aP7076 þ A 13,040,733 659 50
ACR10(rep1) Avr3aACR10/Avr3aACR10 (cid:2) V 3,338,276 1,715 513
ACR10(rep2) Avr3aACR10/Avr3aACR10 (cid:2) V 13,164,585 9,488 720
Progeny
F1-2 Avr3aACR10/Avr3aP7076 (cid:2) V 48,505,836 10,603 218
F2-2-14 Avr3aACR10/Avr3aACR10 (cid:2) V 50,286,928 30,809 612
F2-2-26 Avr3aACR10/Avr3aP7076 (cid:2) V 45,921,916 17,812 387
F2-2-35 Avr3aP7076/Avr3aP7076 (cid:2) V 45,141,247 12,883 285
F2pool(rep1) Avr3aP7076/Avr3aP7076 (cid:2) V 14,712,321 4,411 299
F2pool(rep2) Avr3aP7076/Avr3aP7076 (cid:2) V 20,495,465 6,204 302
Otherstrains
P6497 Avr3aP6497/Avr3aP6497 þ A 17,919,740 345 19
ACR16 Avr3aACR16/Avr3aACR16 (cid:2) V 20,450,286 3,338 163
*SamplesofsRNAwerepurifiedfrommyceliaculturesofP.sojaeforlibraryconstructionandsequencing,asdescribedinMethodssection.
wSourceofallP.sojaestrainsandsequenceofAvr3aalleleshaspreviouslybeendescribed5,6.
zPresence(þ)orabsence((cid:2))ofAvr3amRNA,asdeterminedbyRT–PCR.
yMatchesofsRNAsequencesto10.8kbAvr3asegment,asshowninFig.1.
A,avirulent;rep,replicate;V,virulent.
homogenatesbyaseriesofpurificationsteps13–15.Purifiedoosporesarediluted andtheisolineL83-570(Rps3a),accordingtostandardmethods5,6.Soybeanseeds
andplatedoutonwateragartogerminate.Individualgerminatingoosporesare (20–30seedsperpot)weresownin10cmpotsandgrownfor7daysbeforedisease
pickedunderadissectingmicroscopeandtransferredtorichgrowthmedium. testing.Forinoculation,P.sojaeculturesweregrownfor5–7dayson0.9%(v/v)V8
SamplesofpurifiedDNAfromtheresultingculturesaretestedusingco-dominant agarplates.Inoculumwaspreparedbyexcising1cmsegmentsofagarcutfromthe
DNAmarkerspolymorphicbetweentheparents,todeterminewhetherthe growingedgeofmyceliacoloniesandmaceratingtheculturesthrougha3-ml
individualsresultfromself-fertilizationorfromout-crossingeventsbetweenthe syringeattachedtoan18-gaugeneedle.Soybeanplantswereinoculatedinthemid-
parentalstrains.SuchmarkersdesignedtodistinguishAvr3aACR10andAvr3aP7076 sectionofeachhypocotylbymakingasmallincisionforinjectionofthemycelial
alleles(describedbelow)wereusedtoidentifyF progenyincrossesbetweenstrain slurry.Inoculatedplantswerecoveredwithplasticbagstomaintainhumidityfor
1
ACR10andP7076.TheF populationsweregeneratedbyisolatingoosporesfrom 2days.Diseasesymptomswereallowedtodevelopforanadditional4daysbefore
2
self-fertilizedF individuals. phenotypeswerescored.Aminimumofthreeindependentreplicatesofthedisease
1
assaywereperformedforeachP.sojaeculturetested.
PurificationofnucleicacidsandscoringofmarkersandtranscriptsforAvr3a.
DeepsequencingofP.sojaesRNA.ToprofilesRNAmoleculesofP.sojae
MyceliaculturesofP.sojaefornucleicacidisolationweregrownonvegetablejuice
cultures,samplesofRNAwerepurifiedfrommyceliaculturesasdescribedabove.
(V8)agarmediaoverlaidwithcellophane(BioRad).Aftergrowthindarknessfor
7daysat251C,mycelia-growncellophanediskswerepeeledoffthemedia,frozen ThesizeselectionofsRNA,attachmentofadaptormoleculesandlibrarycon-
at (cid:2)801CandgroundtoafinepowderinliquidN usingamortarandpestle. structionwasperformedusingpreparedsuppliesandreagents(Illuminasmall
2 RNAv1.5),followinginstructionsprovidedbythemanufacturer.Sequencingwas
GenomicDNAwaspurifiedusingconventionalphenol–chloroformextraction
proceduresfollowedbyprecipitationwithisopropanol16.PurfiedDNApelletswere performedonaflowcellinstrument(IlluminaGAII).Aftertrimmingtoremove
adaptorsequencesandlow-qualitysequences,thedepthofsRNAcoverageofthe
washedwith70%(v/v)ethanol,driedandresuspendedinasolutionof10mMTris
Avr3aregionwasdeterminedbyalignment,andnormalizedtothenumberofreads
HCl,pH7.4,0.1mMEDTA.CAPmarkerswereusedtotracksegregationofthe
Avr3aACR10andAvr3aP7076alleles.ForCAPsanalysis,PCRamplificationwas perlibrary.Averagedepthofcoverageacrossthe10.8-kbgenomesegmentcon-
performedwith5ngofgenomicDNAastemplateinavolumeof25mlunderthe tainingAvr3awasdeterminedusinga50-basepairmovingintervalandvalues
followingconditions:0.2mMdNTPs,1.5mMMgCl,0.4mMofeachprimer normalizedtothetotalnumberoftrimmedreadsforeachsample.Thenumberof
(forwardprimer50-GCTGCTTCCTTCCTGGTTGC-230andreverseprimer50-GCT trimmedreadsobtainedforeachofthesequencedsRNAlibrariesisprovedin
GCTGCCTTTTGCTTCTC-30),1.25UTaqDNApolymeraseand1(cid:3) PCRbuffer Table2.
asrecommendedbythesupplier(Invitrogen,LifeTechnologies).Theamplification
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Acknowledgements
WethankTheCentreforAppliedGenomics(Toronto)andtheBCCancerAgency This work is licensed under a Creative Commons Attribution-
(Vancouver)forsRNAsequencing;AldonaGaidauskas-Scott,IrinaMutiu,KuflomKuflu NonCommercial-ShareAlike3.0UnportedLicense.Toviewacopyof
andAlexMolnarforcontributionstothework;andBrettTylerandKrzysztof thislicense,visithttp://creativecommons.org/licenses/by-nc-sa/3.0/
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