Comparative Transcriptome Analysis Reveals Common and Specific Tags for Root Hair and Crack ...

12 Pages·2007·1.6 MB·English

Comparative Transcriptome Analysis Reveals Common
and Specific Tags for Root Hair and Crack-Entry Invasion
in Sesbania rostrata1[W][OA]
Ward Capoen2, Jeroen Den Herder, Stephane Rombauts, Jeroen De Gussem, Annick De Keyser,
Marcelle Holsters*, and Sofie Goormachtig
Department of Plant Systems Biology, Flanders Institute for Biotechnology and Department of Molecular
Genetics, Ghent University, B–9052 Ghent, Belgium
ThetropicallegumeSesbaniarostrataprovidesitsmicrosymbiontAzorhizobiumcaulinodanswithversatileinvasionstrategiesto
allownoduleformationintemporarilyfloodedhabitats.Inaeratedsoils,thebacteriaenterviatheroothaircurlingmechanism.
Submergence prevents this epidermal invasion by accumulation of inhibiting concentrations of ethylene and, under these
conditions, the bacterial colonization occurs via intercellular cortical infection at lateral root bases. The transcriptome of
both invasion ways was compared by cDNA-amplified fragment length polymorphism analysis. Clusters of gene tags were
identified that were specific for either epidermal or cortical invasion or were shared by both. The data provide insight into
mechanismsthatcontrolinfectionandillustratethatentryviatheepidermisaddsalayerofcomplexitytorhizobialinvasion.
Legume plants can thrive in nitrogen-poor habitats for the induction of root hair curling (RHC) and IT
because of their ability to establish a symbiotic inter- formationandfortheinitiationofcelldivision.NFsac-
action with soil bacteria, collectively called rhizobia. tivate an abundance of responses, including ion fluxes,
Bacterial Nod factors (NFs) are key signaling mole- membranedepolarization,phosholipaseactivity,phos-
cules for host specificity and they initiate the nodula- pholipidsignaling,andcalciumspiking(D’Haezeand
tionprocessintheplant(D’HaezeandHolsters,2002; Holsters, 2002; Oldroyd and Downie, 2004, 2006). A
Geurts and Bisseling, 2002). Integration of plant de- study of bacterial and plant mutants led to the hy-
velopmental programs results in bacterial invasion pothesisthat NF perception triggersa signalingpath-
and de novo cortical cell division, eventually leading way, which prepares the plant for bacterial entry and
to functional nodules with differentiated nitrogen- cortical cell division, and an entry pathway for IT
fixingbacteria. formationinthecurledroothair(Ardoureletal.,1994).
Twoinvasionstrategieshavebeenstudiedindetail. The latter response requires stringent NF recognition
The most common involves the curling of susceptible and has been proposed to be solely active within the
root hair cells in the elongation zone I of the root. epidermis that is also the site for nodulation check-
Bacteria are entrapped in the curl and local plant cell points, such as inhibition by ethylene (Geurts et al.,
wall hydrolysis and membrane invagination precede 1997;Oldroyd et al., 2001).
the formation of a tubular infection thread (IT) that Directcorticalcolonization(crackentry)takesplace
guides the rhizobia through epidermis and cortex to- during lateral root base (LRB) nodulation on hydro-
wardtheincipientnodule.NFsignalingisresponsible ponically grown roots of the semiaquatic legume
Sesbania rostrata (Den Herder et al., 2006). In well-
aeratedsoils,S.rostrataisinvadedviatheRHCmodeand
1ThisworkwassupportedbytheResearchFoundation-Flanders
nodules appear in root zone I (Goormachtig et al.,
(grantsnos.G.0066.07andG.0341.04andpredoctoralfellowshipto
2004a, 2004b). While RHC nodulation is inhibited by
J.D.H.)andtheInstituteforthePromotionofInnovationbyScience
ethylene upon submergence, LRB nodulation makes
andTechnologyinFlanders(predoctoralfellowshiptoW.C.).
2Present address: Department of Disease and Stress Biology, active use of ethylene signaling to allow nodule for-
JohnInnesCentre,NorwichNR47UH,UK. mationunderfloodingconditions.Noepidermalentry
*Corresponding author; e-mail [email protected]; stagesareapparentduringthisinvasion.Thebacteria
fax[phone]. gaindirectaccesstothecortexviaepidermalcracksat
Theauthorsresponsiblefordistributionof materialsintegralto the sites of lateral root emergence (Den Herder et al.,
thefindingspresentedinthisarticleinaccordancewiththepolicy 2006).Afewcorticalcells aretriggeredbyNFstodie,
described in the Instructions for Authors (www.plantphysiol.org)
thus forming cavities that are colonized by the bacte-
are: Sofie Goormachtig (sofi[email protected]) and
ria.Theseinfectionpocketsserveasalaunchingpoint
MarcelleHolsters([email protected]).
[W]TheonlineversionofthisarticlecontainsWeb-onlydata. for ITs that grow toward the nodule primordium
[OA]Open Access articles can be viewed online without a sub- (Goormachtiget al., 2004a).
scription. The occurrence of two different invasion strategies
www.plantphysiol.org/cgi/doi/10.1104/pp.107.102178 onthesameplantallowsustocomparethemolecular
1878 Plant Physiology, August 2007, Vol. 144, pp. 1878–1889, www.plantphysiol.org (cid:2) 2007 American Society of Plant Biologists
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Comparative Transcriptomics of Rhizobial Invasion
mechanisms governing both processes. Hydroponic RESULTS
nodulation occurs without intervention of epidermal
Specific Sampling of Responsive Tissues
responses, in contrast to nodulation under aerated
conditionswheretheepidermalroothairsareinvolved S.rostrataseedlingswereplantedinLeonardjarsor
in the initial stages (Goormachtig et al., 2004b). More- in tubes with liquid medium to favor RHC invasion
over,thestructuralrequirementsofNFsaremorestrin- and LRB nodulation, respectively (see‘‘Materials and
gent for RHC than for crack-entry invasion (D’Haeze Methods’’;Goormachtigetal.,2004b).Approximately
etal.,2000;Goormachtigetal.,2004b),supportingthe 900 plants in Leonard jars were inoculated with
hypothesis of an additional checkpoint for epidermal Azorhizobium caulinodans ORS571 (pBBR5-hem-gfp5-
entry (Ardourel et al., 1994). These physiological and S65T) that constitutively expresses a GFP reporter
morphological dissimilarities indicate that each inva- (D’Haezeetal.,2004).Thehydroponicallygrownroots
sion pathway requires a specific set of genes and were inoculated with wild-type ORS571 (pBBR-hem-
functions. gfp-S65T) or with the NF-deficient mutant ORS571-
The advent of high-throughput transcript analysis V44(pBBR-hem-gfp-S65T)thatcontainsaTn5insertion
tools has greatly improved our knowledge of the in thenodA gene (D’Haezeet al., 1998).
molecular mechanisms that regulate nodule initiation To enrich for responsive material, tissues in the
anddevelopment(Lievensetal.,2001;Fedorovaetal., process of being invaded by GFP-labeled rhizobia
2002;PoulsenandPødenphant,2002;Colebatchetal., were harvested under fluorescent stereomicroscopy.
2004; El Yahyaoui et al., 2004; Ku¨ster et al., 2004; For the hydroponic roots, time-based harvesting was
Manthey et al., 2004; Mitra and Long, 2004; Asamizu possible because of the high synchronicity of LRB
et al., 2005; Lohar et al., 2006; Starker et al., 2006). So nodulation.UninoculatedLRBs and LRBs at6,12,24,
far, all studies were focused on one invasion system. 48,and72hpostinoculation(hpi)withORS571(pBBR-
Here, we compared transcript profiles of RHC and hem-gfp-S65T) were collected (Fig. 1, A–D). For the
LRB invasions in S. rostrata by using the PCR-based time course with A. caulinodans ORS571-V44 (pBBR-
methodofcDNA-amplifiedfragmentlengthpolymor- hem-gfp-S65T), uninoculated and LRBs at 24 and 48
phism (AFLP) because of its high sensitivity and re- hpi weretaken.
producibility(Bachemetal.,1996;Breyneetal.,2003). NodulationinLeonardjarsviaRHCinvasionisnot
Approximately7,000geneswerescreened,resultingin synchronized; hence, a morphology-based harvesting
627differentiallyexpressedtags.Statisticalanalysisof procedure was used (Fig. 1, E–I). From uninoculated
thedatasetrevealedacoregroupofgenescommonto roots, the zone directly behind the root apical meri-
bothinvasiontypesaswellasalargenumberofgenes stem (designated rhc0) was collected (Fig. 1E). From
specific forone invasion mode. plants inoculated with ORS571 (pBBR-hem-gfp-S65T),
Figure1. Tissuesamplingandanalysisofdiffer-
entiallyexpressedcDNA-AFLPtags.ForLRBnod-
ulation, plants were inoculated with either A.
caulinodans ORS571-V44, a strain deficient in
NFproduction,orA.caulinodansORS571,both
containingaGFP-expressingplasmidforvisuali-
zationwithfluorescencemicroscopy.A,Asaneg-
ative control, LRBs of uninoculated plants were
harvested. B to D, Representative stages of LRB
nodulation at 6 (B), 24 (C), and 72 h (D) after
inoculation with GFP-labeled A. caulinodans. E
toI,Amorphology-basedapproachusedtocol-
lecttissueforRHCinvasion.Representativestages
are shown. As a negative control, 1.5-cm-long
root segments without apical meristem were
harvested(E).Frominoculatedplants,smallroot
fragmentswithITswereexcised(F;rhc1,624–36
hpi).Latertimepointswererootfragmentswith
ITsandbumpsindicativeforcorticalcelldivisions
(G;rhc2,648–60hpi),primordiabeforetheonset
ofnitrogenfixation(H;rhc3,672–100hpi),and
youngfixingnodules(I;rhc4,6140hpi).J,Plotof
log(CV) values for all tags ordered along the
2
abscissa. RHC samples contain more differen-
tiallyexpressedtagsthanLRBsamples,which,in
turn, have more samples than when inoculated
withORS571-V44.
Plant Physiol. Vol. 144, 2007 1879
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Capoen et al.
we excised small root fragments with IT-containing a predominantly RHC-specific pattern, whereas clus-
root hairs (rhc1, 624–36 hpi; Fig. 1F), root fragments ter6tagswerespecificallyexpressedduringLRBnod-
with ITs and small bumps, indicating cortical cell ulation, accompanied by some very early transient
divisions(rhc2,648–60hpi;Fig.1G),youngprimordia RHCtags.
before the onset of nitrogen fixation (rhc3, 672–100 The RHC-specific tags (cluster 5) were further sub-
hpi; Fig. 1H), and young fixing nodules (rhc4, 6140 clustered into six groups with distinct expression
hpi;Fig. 1I). patterns (Fig. 3; Supplemental Fig. S1). Tags of sub-
cluster 5A were down-regulated whereas those of
subclusters 5B and 5C were transiently induced dur-
cDNA-AFLP Transcript Profiling and Cluster Analysis
ing RHC invasion. Subclusters 5D to 5F contained
The samples were used for cDNA-AFLP transcript genes whose expression level gradually increased,
profiling to identify genes that were differentially starting at different time points. Subclustering of the
regulatedduringnoduleinitiation inS.rostrataunder LRB-specific tags (cluster 6) provided five groups,
hydroponic or aeroponic conditions (see ‘‘Materials eachrepresentedbyaspecificexpressionpattern(Fig.
and Methods’’). The 128 primer combinations ana- 4; Supplemental Fig. S2). Subcluster 6A grouped tags
lyzed allowed visualization of some 7,000 transcript- thatweretransientlyup-regulatedandfromwhichthe
derivedtags.Todeterminewhatshouldbeconsidered expression level was highest at 24 hpi. Subcluster 6B
differential, approximately 3,000tagswere plotted on containedtagsfromwhichtheexpressionlevelsteadily
agraph,arrangedbyascendinglog coefficientofvar- increased during the course of the experiment. Sub-
2
iance (CV; see ‘‘Materials and Methods’’). A distinct groups 6C and 6D also represented transiently ex-
changeinthecurvewasvisiblearoundlog (CV)50.1 pressedtags,buttheexpressiondidnotreachthesame
2
(Fig. 1J). Hence, a tag was considered differential level as that of subgroup 6A. Subgroups 6C and 6D
when its log (CV) was 0.1 orhigher. Of the 1,600 tags differed with respect to time points at which the ex-
2
with a differential expression pattern in either the pression dropped again. Finally, tags that were tran-
RHC or LRB nodulation systems or in both that were siently repressed during LRB invasion were grouped
sequenced,627gaveasignificantBLASThit(Evalue, in subcluster 6E.
1023)tosequencesinpublicdatabases(Altschuletal., Thelarge-scaleprofilingexperimentwasdoneonly
1997) andwereretained for further analysis. once because of the considerable work load and te-
Forafirstinsightintosimilaritiesbetweenthetran- dious harvesting procedures. Nevertheless, material
script pools of each sample, the experiments were fromabiologicalrepeat(see‘‘MaterialsandMethods’’)
clustered hierarchically with the Pearson correlation was analyzed by quantitative reverse transcription
asastatisticaltool(see‘‘MaterialsandMethods’’;Fig. (qRT)-PCR for 10 tags, overall confirming the expres-
2A).ThebaseofthetreeconsistedoftheV44series;the sion patterns obtained in the cDNA-AFLP (Supple-
inoculations with wild-type A. caulinodans split in mental Fig. S3).
threedifferentclusters. The early LRB stages,crack 6,
12, and 24 h formed a subcluster (Fig. 2A), the rhc1
Functional Classes in the Data Set
grouped separately, and the late time points of both
the RHC and LRB samples, i.e. from 48 h after inoc- The differential tags were assigned to functional
ulationonward, clusteredtogether(Fig. 2A). classes according to the 16 categories used in the Med-
Hierarchical cluster analysis revealed distinct ex- icago EST Navigation System database (El Yahyaoui
pression profiles (Fig. 2B). The tags were separated et al., 2004). Approximately 32% of all tags corre-
into common and noncommon groups, the former sponded to putative genes or genes with unknown
referringtogenessimilarlyregulatedinbothLRBand function;aputativefunctioncouldbeassignedto68%.
RHC nodulation and the latter to genes that were The relative contribution of the different functional
specificallyexpressedduringLRBorRHCnodulation. classes in the RHC-specific, crack-entry-specific, and
By K means analysis, used as a rough clustering commonclusterswasverysimilar(SupplementalFig.
method for the common andnoncommon groups,six S4).However,differentisoformswereoftenexpressed
groups of tags were distinguished. The annotations, inoneortheotherinvasionway.Forinstance,several
along with the data set are available online (Supple- tags encoded peroxidase genes, of which some were
mental Table S1). Clusters 1 to 4 consisted of the 337 common, but one was specific for crack entry, imply-
common gene tags. On average, cluster 1 contained ing different roles and substrates. Similarly, of three
tagsthatweregraduallyup-regulatedinbothinvasion NADPH oxidase-encoding tags,onewas common, one
ways and reached a plateau at later stages; cluster 2 wasspecificforRHC,andoneforcrackentry.
comprisedmostlytagsthatweredown-regulatedinall By means of BLASTN, we compared all S. rostrata
series; tags corresponding to genes that were tran- tags with published expression profiling experiments
siently up-regulated were grouped in cluster 3; and, in Medicago truncatula (El Yahyaoui et al., 2004; Lohar
finally, the common tags, whose transcript level did etal.,2006).OnlytagswithanEvalueunder1023were
notreachaplateauatlatertimepoints,werefoundin withheld for further analysis, resulting in 136 and 84
cluster 4, with a subset of this cluster already ex- common tags with the data sets of Lohar et al. (2006)
pressedattheearliesttimepoints.Cluster5displayed and El Yahyaoui et al. (2004), respectively (Fig. 5).
1880 Plant Physiol. Vol. 144, 2007
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Copyright © 2007 American Society of Plant Biologists. All rights reserved.
Comparative Transcriptomics of Rhizobial Invasion
Figure 2. Cluster analysis. A, Hierarchical clustering of sample expression profiles visualizing the similarities in expression
patternsatlatetimepoints.Differencesbetweensamplesaremorepronouncedbefore48h.B,Hierarchicalclusteringofall627
differentiallyexpressedtags.Genetagswithalog(CV)ofatleast0.1(correspondingtoa2-foldchangeinexpression)were
2
sequenced and expression profiles of the corresponding genes were clustered. Blue and yellow indicate down- and up-
regulation,respectively.C,Kmeansclusteringofdifferentiallyexpressedgenetags.Clusters1to4areconsideredcommon,
cluster 5 harbors the RHC-specific genes, and cluster 6 contains tags specific for LRB invasion. From left to right: v,
Hydroponicallygrownrootsuninoculated(v0)orinfectedwithA.caulinodansORS571-V44at24dpi(v1)and48dpi(v2);C,
hydroponicallygrownroots,uninoculated(c0)andinfectedwithA.caulinodansORS571at6(c1),12(c2),24(c3),48(c4),and
72h(c5)postinoculation;r,aeratedroots,uninoculated(r0)andcorrespondingtorhc1(r1),rhc2(r2),rhc3(r3),andrhc4(r4).The
topsectionsdepictexpressionpatternsthatarerepresentativeformosttagsfoundinclusters1to6.
Plant Physiol. Vol. 144, 2007 1881
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Copyright © 2007 American Society of Plant Biologists. All rights reserved.
Capoen et al.
Subsequently, expression patterns of these common FECTIONS1(MtDMI1),MtDMI2,MtDMI3,theLysM-
tags were found to be similar in 61.0% (83 from 136) RLKs NFP and Lyk3 and their orthologs, and the
and60.7%(51from84)ofthecases,respectively.Nine transcription factors (TFs) NSP1 and NSP2 (Endre
tags had a similar expression pattern in the three et al., 2002; Stracke et al., 2002; Limpens et al., 2003;
differentexperiments. Madsen et al., 2003; Radutoiu et al., 2003; Ane´ et al.,
2004; Le´vy et al., 2004; Mitra and Long, 2004; Kalo´
SrLyr3, a LysM-Receptor-Like Kinase, Is Expressed etal.,2005;Smitetal.,2005;Heckmannetal.,2006).As
during RHC and LRB Nodulation no wide-scale genetic or genomic tools are available
for S. rostrata, we have to resort to reverse genetics
Many differential tags corresponded to putative
strategies to unravel the role of some of the key
kinases, receptor-like kinases (RLKs), and protein
components in LRB nodulation (Capoen et al., 2005).
phosphatases, and might be important players in
Anotherapproachtocollectdataonrelevantfunctions
nodulation. One tag from a common up-regulated
for alternative nodulation processes in a nonmodel
clusterwashomologoustogenescodingfortheLysM
legumeconsistsinalarge-scaletranscriptomeanalysis
domain-containing RLKs, the family to which the
tohuntfordifferentiallyexpressedgenes(Goormachtig
putative NF receptors belong (Limpens et al., 2003;
etal.,1995;Lievensetal.,2001;Schroeyersetal.,2004).
Madsenetal.,2003;Radutoiuetal.,2003;Arrighietal.,
AcomparativecDNA-AFLPtranscriptomeanalysis
2006; Fig. 6A). The full-length cDNA clone was iso-
of the two nodulation modes of S. rostrata allowed
lated with RACE (see ‘‘Materials and Methods’’) and
allocation of 627 tags in six clusters with differential
designated SrLyr3, because BLAST searches identi-
expression profiles. The onset of each invasion strat-
fied MtLyr3 of M. truncatula as the closest homolog
egywascharacterized byspecific geneexpression.At
(Arrighi et al., 2006). The corresponding amino acid
later stages corresponding to primordium formation
sequenceofSrLyr3is85%similartothatofMtLyr3and
and nodule differentiation, many tags were common
63%totheproteinencodedbyArabidopsis(Arabidopsis
between the RHC and LRB nodulation series. The
thaliana) At2g23770. These LysM-RLKs belong to the
potential relevance of these differential tags will be
same clade II as the NF receptor NFP, but they are
discussedintheversatileS.rostratasymbiosiscontext
located in a separate branch (Fig. 6B; Arrighi et al.,
andin the general contextof legume nodulation.
2006). The dissimilarity to NFP is demonstrated by
differences in the kinase domain,such asthe presence
of an activation loop. However, the kinase domain Common Genes Related to Early Signaling Events
mightnotbeactivebecausesomeimportantsequences,
LRBandRHCnodulationsdependbothonNFsand
such as the P loop, the DFG motif,and the Ser/Throf
genes involved in NF perception are expected to be
theactivationloopareabsentorhavebeensubstituted
up-regulatedinbothinvasionmodesfromearlystages
(Arrighietal.,2006;Rielyetal.,2006;Zhuetal.,2006).
on. Several tags with such an expression pattern are
Confirmation that the gene is up-regulated in both
reminiscentofregulatorymechanisms,suchasubiquitin-
invasion ways was obtained by qRT-PCR analysis on
dependentproteindegradation(M12-180.8),transcrip-
RNAfromabiologicalrepeatexperiment.DuringLRB
tionalcontrol(M12-288.3),chromosomereorganization
nodulation, SrLyr3 transcripts started to accumulate
(M21-459.1 and M33-349.4), and hormone perception.
between 12 and 24 hpi and increased further at later
Tag M22-259.3 is related to MtRR4 (TC 103991), a
timepoints(Fig.6C).IntheRHCseries,thetranscript
response regulator involved in cytokinin signaling
level was already up-regulated at the stage of curled
that is up-regulated during nodule development in
root hairs and remained more or less similar at later
M.truncatula(Gonzalez-Rizzoetal.,2006).
stages(Fig. 6C).
One of the earliest root hair responses to NFs is a
rhythmiccalciumoscillationthatensueswithin10min
DISCUSSION of NF addition (Oldroyd and Downie, 2006). Several
tags related to calmodulin-like proteins as well as
Commonalities and Differences
a Ca21-dependent protein kinase, were up-regulated
BothLRBandRHCnodulationonS.rostratadepend in the two invasion modes(Fig.7A).Calcium spiking
onperceptionofbacterialNFsanddownstreamevents might also occur in cortical cells (Miwa et al., 2006),
(D’Haezeetal.,2003;Goormachtigetal.,2004a,2004b). andinhibitorsofCa21spikinginhibitLRBnodulation
The overall outcome of the processes is the same: the (W. Capoen, W. D’Haeze, and M. Holsters, unpub-
formationoffunctionalnodules.Thedifferencesrelate lished data). Hence, calcium signaling is presumably
to the invasion mode, with NF perception linked to important in LRB as well as in RHC nodulations.
eithercorticalcelldeathortoinvertedtipgrowthina One tag homologous to phosphatidyl inositol
roothair,andtothelocalphysiologyofthenodulation 3-kinase, three tags for putative inositol polyphos-
zones.ForRHCnodulation,asstudiedinM.truncatula phate 5-phosphatases, and one tag similar to an ino-
and Lotus japonicus, several essential components of sitol 4-methyltransferasewereweaklyandtransiently
the NF perception, signal transduction, and response up-regulated at early stages, followed by a down-
cascade have been identified. Examples are the regulationatlaterstages(Fig.7B).Althoughphospho-
M. trucatula genes coding for DOES NOT MAKE IN- lipidspresumablyplayaroleduringearlyNFsignaling,
1882 Plant Physiol. Vol. 144, 2007
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Comparative Transcriptomics of Rhizobial Invasion
Figure 3. RHC-specific gene tags. Average expres-
sion patterns of the six RHC-specific subclusters.
Hierarchicalclusterscorresponding tothesegroups
are available as Supplemental Figure S1. The same
timepointswereusedasthosementionedinFigure2.
basedonpharmacologicalevidence(denHartogetal., up-regulated, some repressed (Fig. 7C). Among the
2003;Charronetal.,2004),ourexpressionanalysissug- latter were the GRAS protein GAI (a negative regula-
geststhattranscriptsforphospholipidsignalingfunc- tor of GA action), confirming the prediction for tight
tions arerepressed at later time points. regulation of GA during nodule initiation (Lievens
A common early up-regulated tag corresponded to et al., 2005), two Myb1 like, two AP2 domain, and
SrLyr3,theS.rostrataorthologofMtLyr3(Arrighietal., two WRKY TFs, related to the Arabidopsis orthologs
2006),arecentlydescribedmemberoftheM.truncatula WRKY28 and WRKY69 that are differentially up-
LysM-RLK family. To this family belong MtLyk3 and regulated in biotic interactions (www.genevestigator.
MtNFP,theLysMdomainproteinsthatplayakeyrole ethz.ch).
innodulation, presumablyby binding NFs as ligands Remarkably, several differential tags were homolo-
(Madsen et al., 2003; Radutoiu et al., 2003; Limpens gous to genes involved in growth and differentiation
etal.,2005;Mulderetal.,2006).SrLyr3andMtLyr3have ofshoots,flowers,andfruits(Fig.7D),suchasPETAL
aclosehomologinArabidopsis,aplantthatisunable LOSS, the MADS-box protein FRUITFULL, Nam-like
toestablishsymbioses(Fig.6B).Apreliminaryexpres- proteins 10 and 14, CONSTANS, the KNAT3 homeo-
sionanalysissuggeststhattheseLysM-RLKsarelikely box gene, GIGANTEA, CYCLOIDEA, LEUNIG, IRKI,
candidatestoperceiveendogenoussignalsinvolvedin aninteractoroftheinflorescenceandrootapicesRLK
plantdevelopmentalprogramsorinpathogendefense (Hattan et al., 2004), and DEM, defective embryo and
(W.CapoenandM.Holsters,unpublisheddata). meristem (Irish, 1999; Ferra´ndiz et al., 2000; Kieffer
and Davies, 2001; Komeda, 2004; Veit, 2004). Nodula-
tion might have features in common with shoot api-
Common Genes Related to Nodule Development
cal meristem development, as suggested previously
Plant development is controlled by specific TFs. (Szczyglowski and Amyot, 2003). However, differen-
Numerous TF tags werepresent in the data set, some tiationprocessesidentifiedasshootspecificmightalso
Figure 4. LRB-specific gene tags. Average expression patterns of all five LRB-specific subclusters. Hierarchical clusters
correspondingtothesegroupsareavailableasSupplementalFigureS2.Fortheusedtimepoints,seeFigure2.
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Capoen et al.
suchcross-linkingofglycoproteins(Brewin,2004).The
expression profiles of the two amine oxidases have
beenconfirmedbyRT-PCRandanalysisoftheirfunc-
tion in nodulation is ongoing (J. Den Herder and M.
Holsters, unpublished data).
ROS production and metabolism are also compo-
nentsofdefenseresponses.Severaltagscorresponding
to defense-related genes were up- or down-regulated
during nodulation (Fig. 7E). Previous transcript pro-
filing experiments in M. truncatula have revealed a
similarbehaviorofdefense-relatedgenes(ElYahyaoui
et al., 2004; Manthey et al., 2004). Rhizobial infection
probablyrequiresactiverepressionofcertaindefenses
(Mitho¨fer,2002).Amongthedown-regulatedgeneswere
a TIR domain-containing protein TSDC, an Hs1pro1
homolog (nematode resistance), and a polygalacturo-
nase inhibitor. To the up-regulated clusters belonged
tags homologous to genes coding for a putative avr9
elicitorresponseprotein,aproteinwithhomologytoa
Figure 5. Data set comparison with existing nodulation transcript
XA21-like receptor kinase, and several cytochrome
profiles.Graphicalrepresentationofsimilaritiesbetweenthisstudyand
P450 (Fig. 7E). Defense functions might restrict bacte-
twosimilararray-basedexpressionprofilingexperimentsinM.trunca-
rial invasion because approximately 90% of all infec-
tula(El-Yahyaouietal.,2004;Loharetal.,2006).Thenumbersofgenes
tion events abort prematurely in M. truncatula (Pauly
with similar expression patterns between the different data sets are
shownintheoverlapswithintheVenndiagram. et al., 2006), or they might protect the nodule that is
richinnutrientsandanattractivetargetforpathogens.
Several tags correspond to functions involved in
underlierootformationorhaveparallelprogramsfor vesicletransportandvesicletargeting:twokinesins,a
root development. Indeed, M. truncatula ESTs with microtubule-associated protein MAP65-1c, a-soluble
closest homology to the S. rostrata tags were found in NSFattachmentprotein,andthesmallGproteinROP9
roottissuesinthedatasetofTheInstituteforGenome (Fig.7F).ROPshavebeenimplicatedintipgrowthand
Research.Moreover,inArabidopsis,aCONSTANS-like H O production and might be involved in IT pro-
2 2
3 gene controlled lateral root development, a process gression (Yang et al., 1994; Maunoury et al., 2007). In
that has much in common with nodule formation M.truncatula,aspecificsyntaxinMtSyP132waslocal-
(Dattaetal.,2006).AlsoKNAT3expressionoccurredin ized to the plasma membrane surrounding ITs and
young Arabidopsis root tissue and was specifically infection droplets, illustrating the need for targeted
excluded from lateral root primordia (Truernit et al., exocytosis of IT development and growth (Catalano
2006).Similarly,theKNAT3genewasrepressedduring et al., 2007). The specific down-regulation of a SNAP
noduleformation. protein in our data set (M34-231.6) might reflect a
change in the nature of vesicles targeted to the site of
infection (Fig. 7F).
Common Genes Related to Invasion
As both invasion ways make use of cortical ITs for
RHC Nodulation-Specific Genes
bacterial penetration, tags that code for functions for
IT growth might be shared. IT progression involves Of the RHC-specific tags, 68 were strongly down-
cell wall modifications and expression of specific regulated and 122 up-regulated. Among the latter
matrixproteins(Brewin,2004).Tagsputativelycoding were tags corresponding to the nodulin genes MtN6
for (hydroxy) Pro-rich proteins, a lignin biosynthesis and MtN21 of M. truncatula. MtN6 expression pre-
enzyme (caffeic acid O-methyltransferase), and a cel- cedesinfectionandhasbeenproposedtoplayarolein
lulasearecontinuouslyup-regulatedthroughoutnod- the preparation of cells for IT passage (Mathis et al.,
ulation.Sixtagsdisplayhomologytofunctionsinvolved 1999). Also LRB nodulation comes with IT formation,
in reactive oxygen species (ROS) production and me- but differences in physiological conditions and hor-
tabolism,suchastwoperoxidases,twodistinctamine mone landscapes between LRBs and zone I might ac-
oxidases, a respiratory burst oxidase (NADPH oxi- count for the specific involvement of this tag in RHC
dase),andanascorbateoxidase(Fig.7E;Supplemental invasion.
TableS1).ROScomponentshavebeenlocalizedinITs AtaghomologoustotheauxineffluxcarrierPIN2is
andhydrogenperoxide(H O )-drivencross-linkingof specific for RHC invasion. In M.truncatula, anorthol-
2 2
root nodule extensins has been put forward as a ogoustagisup-regulatedduringnodulation(Schnabel
driving force for IT progression (Brewin, 2004; Den andFrugoli,2004).AtagencodinganADP-ribosylation
Herder et al., 2007). Amine oxidases might provide a factorhasasimilarexpressionpattern.ADP-ribosylation
source of peroxide used by peroxidases to perform factorshavebeenimplicatedinthecorrecttargetingof
1884 Plant Physiol. Vol. 144, 2007
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Copyright © 2007 American Society of Plant Biologists. All rights reserved.
Comparative Transcriptomics of Rhizobial Invasion
Figure6. SrLyr3sequence,expressionprofile,andphylogeneticanalysis.A,AlignmentofLYR3orthologsinS.rostrata(SrLYR3),
M.truncatula(MtLYR3),andL.japonicus(LjLYR3).B,PhylogeneticanalysisofknownM.truncatula,L.japonicus,S.rostrata,and
ArabidopsisLysMdomain-containingRLKs.Thecompletesequencewastakenforcomparison.C,qRT-PCRanalysisofSrLyr3
duringhydroponic(crack)andaeroponic(rhc)nodulation.
both PIN2 and a ROP GTPase in growing root hair thosepreexistingatLRBs,suggestingaroleforPIN2in
cells(XuandScheres,2005),implyingthatthesethree thisprocess(Mathesiusetal.,2000;vanNoordenetal.,
proteinsmightpolarizeinvertedtipgrowthintheroot 2006). Therefore,the RHCnodulation-specific expres-
hair. sion pattern of an S. rostrata PIN2 tag surely needs
The number of RHC-specific tags is approximately further investigation.
3-fold higher than that of crack-entry-specific tags,
suggesting that the passage of the epidermis adds a
LRB Nodulation-Specific Genes
layerofcomplexityandspecificitytonoduleinitiation,
as already illustrated by the more stringent NF struc- LRB nodulation involves intercellular invasion and
turalrequirements(D’Haezeetal.,2000;Goormachtig induction of local cell death for infection pocket for-
et al., 2004a). However, crossing the epidermis alone mation.Ethylene,GA,andH O areimportantplayers
2 2
does not account for all the RHC nodulation-specific in the process (D’Haeze et al., 2003; Lievens et al.,
gene expression because the expression of more than 2005). Tags in subclusters 6A, 6C, and 6D (Fig. 4;
50% of the tags is modulated at time points when the Supplemental Fig. S2) are good candidates to be in-
ITs reach the primordium cells. Also, many RHC volved in cell death execution because they are tran-
nodulation-specific tags are similar to genes coding siently induced and specific to crack entry. One tag
for general metabolic functions, such as amino acid (M34-318.5) was similar to superoxide dismutases in-
biosynthesis (Fig. 3; Supplemental Fig. S1). Thus, volved in H O generation. Moreover, tag M21-618.0
2 2
differences in tissue physiology between hydroponic was homologous to a gene coding for a hexose trans-
LRBs and root zone I might contribute to the high porter(SupplementalFig.S2).Ahexosetransporterof
number of RHC-specific genes. LRBs might, to a Arabidopsis has been implicated in induction of pro-
certain extent, be predisposed for nodule formation, grammedcelldeath(Nørholmetal.,2006).Apapain-
forinstancebytheprevailinghormoneconcentrations. like Cys protease had a similar transient expression
Indeed, in the root zone I of clover (Trifolium repens), pattern(M33-163.9;SupplementalFig.S2).Papain-like
NFs induced auxin landscapes that were similar to proteases are involved in cell death-related processes
Plant Physiol. Vol. 144, 2007 1885
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Copyright © 2007 American Society of Plant Biologists. All rights reserved.
Capoen et al.
Figure7. Hierarchicalclusteranal-
ysisofaselectedgroupoftagsac-
cordingtotheprocessinwhichthey
mightbeinvolved.AtoF,Clustered
tags involved in calcium signaling
(A), phosphoinositol signaling (B),
transcriptional regulation (C), meri-
stem signaling (D), defense re-
sponses (E), and targeted vesicle
trafficking(F).
(Beers et al., 2000). Additionally, a GPI anchor trans- Finally, tag M34-622.0 corresponded to a basic helix-
amidase, a type 2A protein phosphatase, a 7-trans- loop-helix Arabidopsis TF (At4g37850) that was up-
membrane protein, and a His kinase were present in regulated upon Pseudomonas syringae infection, salt
subcluster 6D (Supplemental Fig. S2). Tag M43-408.5 stress, and jasmonate treatment (www.genevestigator.
was similar to an Arabidopsis hydrolase (At2g14110) ethz.ch).IncomparisontotheRHCclusters,veryfew
thatwasslightlydifferentiallyexpresseduponosmotic of the LRB-specific tags were highly up-regulated or
stressandsenescence-inducedprogrammedcelldeath. down-regulatednordidtheirexpressionlevelincrease
1886 Plant Physiol. Vol. 144, 2007
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Copyright © 2007 American Society of Plant Biologists. All rights reserved.
Comparative Transcriptomics of Rhizobial Invasion
ordecreaseduringthefullcourseofnodulation(com- eluate,5mLwasusedasatemplateforsubsequentreamplification.AfterPCR
parenumberoftagsinsubclusters5A,5D,5E,and5F reactionswiththecorresponding12/12primercombinations,theresulting
amplicons were sequenced directly. Low quality sequences were removed
[Supplemental Fig. S1] and in subcluster 6B [Supple-
fromthedataset.
mental Fig. S2]). The establishment of a nodule in the
Allsequenceswerecomparedtothenonredundantproteindatabaseatthe
physiological context of the hydroponic LRBs seems EuropeanMolecularBiologyLaboratoryandtotheM.truncatulaGeneIndex
simpler than that in the zone-I cortex of developing atTheInstituteforGenomeResearchwithBLASTXandBLASTNalgorithms,
root hairs. respectively (Altschul et al., 1997). Tags homologous (E value , 1023) to
accessionsineitherdatabasewerewithheldinthefinaldatabase.
CONCLUSION qRT-PCR Analysis
In summary, LRB nodulation shows less stringent TissuesobtainedfromabiologicalrepeatwereanalyzedbyqRT-PCRas
described(Vliegheetal.,2005).First-strandcDNAwassynthesizedwiththe
NF structure requirements and fewer transcriptional
SuperscriptRTIIcDNAsynthesiskit(Invitrogen).qRT-PCRswererunona
changes than RHC nodulation in the same host. A
iCycler iQ (Bio-Rad) with a kit containing SYBR Green (Eurogentec). A
number of plant functions have been identified that ubiquitingenewasusedasaconstitutivecontrol(forprimersequences,see
are potentially involved in preparing the root cortex Supplemental Table S2; Corich et al., 1998). All reactions were done in
for bacterial colonization. These tags will be helpful triplicate, averaged, and normalized with the 22DDCT method (Livak and
Schmittgen,2001).
tools to further investigate the molecular characteris-
tics of intercellular invasion in Sesbania and, by com-
parison,inotherlegumespeciesthatallowcrack-entry In Silico Analysis
invasionof symbiotic rhizobia.
With the S. rostrata LYR3 tag as a query, both the genomic sequence
(www.ncbi.nlm.nih.gov)andtheMedicagoEST(www.tigr.org)databaseswere
searchedwithBLASTXandtBLASTN(Altschuletal.,1997).Ofthesequences
MATERIALS AND METHODS retrieved, redundant ones were removed by one-on-one alignments with
ClustalWandtheresultingnonredundantsetwassubjectedtophylogenetic
Plant Material and Bacterial Strains analysis.Sequenceswerealignedwiththefreeworkbenchsoftware(http://
www.clcbio.com). Neighbor-joiningtreeswereconstructed and1,000itera-
Sesbania rostrata Brem seedlings were germinated and grown in tubes tionsweredonetotestnodesignificanceforbootstrapanalysis.
containingliquidmediumorinLeonardjarsasdescribed(Goormachtigetal., Gene expression of this experiment was compared with that of the
1995;Ferna´ndez-Lo´pezetal.,1998).PlantswereinoculatedwithAzorhizobium microarraydatafromLoharetal. (2006) andElYahyaoui etal.(2004). To
caulinodansORS571(pBBR5-hem-gfp5-S65T)orORS571-V44(pBBR5-hem-gfp5- linkthetagstotheESTsspotted onbotharrays,wedownloadedtheEST
S65T;VandenEedeetal.,1987;D’Haezeetal.,2004). sequencesfromtheGeneExpressionOmnibus(GEOGSE3441)oftheNational
Center for Bioinformatics Information and received the corresponding se-
quencesdirectlyfromElYahyaouietal.(2004).ThroughBLASTN,tagscould
RNA Extraction and cDNA-AFLP Experiments
belinkedtoESTsfrombothexperimentsandgeneralexpressiontrendscould
becompared.NospecificE-valuethresholdwasusedbecausethelengthof
SampleswerefrozeninliquidnitrogenuntilRNApurification.RNAwas
thetagsvariedalot.Therefore,forcorrectassignmentofthetagstoESTs,they
extractedasdescribed(Kieferetal.,2000).TheprotocolforcDNA-AFLPwas
were curated manually. The whole data sheet resuming the results for
applied according to Breyne et al. (2002, 2003) with the most selective
individualtagsarealsoavailableforqueryingathttp://www.psb.ugent.be/
oligonucleotides(12/12),resultingin128primercombinationsthatmaxi-
supplementary-databyclickingonthecorrespondingarticle.
mallyreducedtemplatecomplexity,thussimplifyinglateranalysisandband
isolation.GelswerescoredwiththeAFLP-QuantarTM-Pro(Keygene),analyzed
withaMicrosoftAccess-basedsoftwareapplication(ArrayAN;Vandenabeele Isolation of Full-Length Sequences
etal.,2003),andnormalizedontheintensitylevelsofconstitutivebandsin
eachprimercombination.Acorrectionfactorwasintroducedtoaccountfor Thefull-lengthcDNAsequenceofSrLyr3wasobtainedby5#and3#RACE
differencesinlaneintensitybecauseofloadingdifferencesandwascalculated by means of the Smart RACE cDNA amplification kit (CLONTECH). The
bydividingthesumofallindividualbandintensitieswithinonelanebythe fragments were cloned in the pCRII-TOPO vector (Invitrogen). The full-
average of all sums within the respective primer combination. Each band lengthcDNAsequenceofSrLyr3couldbeamplifiedfromS.rostratacDNA
intensityvaluewasdividedbythiscorrectionfactor.ACVwascalculatedas withprimersSrLYR3FULLS(5#-CCTTCCTGTGCATCTGCAAAAAC-3#)and
theSDonallvaluesofthetimecoursedividedbytheaverageexpressionover SrLYR3FULLAS(5#-GGCTGGTATCTCATTCACAACCC-3#).
thetimecourse.
Withineachprimercombination,5%ofthegeneswiththelowestCVvalue SequencedataforSrLyr3fromthisarticlecanbefoundintheGenBank/
weremarkedasconstitutivelyexpressed.Perlane(timepoint),theintensities EMBLdatalibrariesunderaccessionnumberEF408056.
of these bands were summed and divided by the average of the sum,
generatingasecondcorrectionfactorusedtonormalizetherawexpression
Supplemental Data
datageneratedbyAFLP-QuantarTM-Pro.TheCVwasagaincalculatedon
these normalized data for each gene and used as a selection criterion for
Thefollowingmaterialsareavailableintheonlineversionofthisarticle.
differential expression (Vandenabeele et al., 2003) with a cutoff value of
log(CV) 5 0.1. Hierarchical clustering of the data was obtained with the SupplementalFigureS1.HierarchicalclusteringofthesixRHC-specific
2
MultipleExperimentViewer(TheInstituteforGenomeResearch)software subgroups.
(Saeed et al., 2003). The K means clustering was done with the Euclidian
SupplementalFigureS2.Hierarchicalclusteringofthefivecrack-entry-
distance.Kmeanswerecalculatedwithamaximumof50iterationsandthe
specificsubgroups.
numberofclustersmentioned;theresultinghierarchicalclustersweremade
withtheEuclideandistanceandaveragelinkageclustering. SupplementalFigureS3.qRT-PCRverificationofasubsetofdifferential
tags.
Fragment Isolation, Sequencing, and Identification SupplementalFigureS4.Representationofalltagsinfunctionalclasses.
SupplementalTableS1.Completedatasetofidentifiedtags.
Fragmentswereexcisedbysuperimposingthedriedgelwithanautora-
diographandelutedbyincubationin100mLdistilledwaterfor1h.Ofthe SupplementalTableS2.ListofqRT-PCRprimersused.
Plant Physiol. Vol. 144, 2007 1887
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