Transcriptional reprogramming by root knot and migratory nematode infection in rice

Transcriptional reprogramming by root knot and migratorynematode infection in rice

Tina Kyndt1, Simon Denil2, Annelies Haegeman1, Geert Trooskens2, Lander Bauters1, Wim Van Criekinge2,3,

Tim De Meyer2 and Godelieve Gheysen1

1Department ofMolecular Biotechnology, Ghent University (UGent), Coupure Links 653, B-9000, Ghent, Belgium; 2Department ofMathematicalModelling, Statistics and Bioinformatics, Ghent

University (UGent), Coupure Links 653, B-9000, Ghent, Belgium; 3NXTGNT, Ghent University, Medical Research Building, Ghent University Hospital, De Pintelaan 185, B-9000, Ghent,

Belgium

Author for correspondence:Godelieve Gheysen

Tel: +32 92645888Email: [email protected]

Received: 12 June 2012

Accepted: 27 July 2012

New Phytologist (2012)doi: 10.1111/j.1469-8137.2012.04311.x

Key words: differential expression,Hirschmanniella oryzae,Meloidogyne

graminicola, mRNA-Seq,Oryza sativa,transcriptome.

Summary

� Rice is one of the most important staple crops worldwide, but its yield is compromised by

different pathogens, including plant-parasitic nematodes. In this study we have characterized

specific and general responses of rice (Oryza sativa) roots challenged with two endoparasitic

nematodes with very different modes of action.� Local transcriptional changes in rice roots upon root knot (Meloidogyne graminicola) and root

rot nematode (RRN, Hirschmanniella oryzae) infection were studied at two time points (3 and

7 d after infection, dai), using mRNA-seq.� Our results confirm that root knot nematodes (RKNs),which feed as sedentary endoparasites,

stimulate metabolic pathways in the root, and enhance nutrient transport towards the induced

root gall. The migratory RRNs, on the other hand, induce programmed cell death and oxidative

stress, and obstruct the normal metabolic activity of the root. While RRN infection causes up-

regulation of biotic stress-related genes early in the infection, the sedentary RKNs suppress the

local defense pathways (e.g. salicylic acid and ethylene pathways). Interestingly, hormone

pathwaysmainly involved in plant developmentwere strongly induced (gibberellin) or repressed

(cytokinin) at 3 dai.� These results uncover previously unrecognized nematode-induced expression profiles related

to their specific infection strategy.

Introduction

Rice is one of the most important crop plants worldwide, and it hasproven to be an excellent model system for monocotyledonousplants. Estimates of annual yield losses as a result of plant-parasiticnematodes on this crop range from 10 to 25%worldwide, with therice root knot nematode (RKN) Meloidogyne graminicola andthe root rot nematode (RRN) Hirschmanniella oryzae being thepredominantnematode species attacking thisplant.M. graminicolais the most important pathogen in Southeast Asian aerobic riceculture, while H. oryzae is mainly found in areas where rice isgrown in submerged conditions (Bridge et al., 2005).

Although they are both endoparasitic nematodes,M. graminicolaand H. oryzae have very different lifestyles. Migratory nematodessuch as the rice RRN H. oryzae penetrate the root, at any zoneexcept the root tip, through a combination of mechanical actionand secretion of cell wall-degrading or -modifying enzymes. Insidethe root,H. oryzae can move freely in the air channels between theradial lamellae of the parenchyma and feeds upon the cortical cells.A few days after entry, the female lays eggs, which hatch in 4–5 dinside the roots. Under suitable conditions the life cycle is

completed in c. 5 wk. Infected cells become necrotic, leading tolarge root lesions. Roots invaded by this nematode turn yellowishbrown and rot, and this root damage causes significant growthretardation to the plant (Bridge et al., 2005). The second-stagejuvenile of the rice RKNM. graminicola invades the plant root inthe root elongation zone and moves towards the root tip, where itpunctures some selected vascular cells with its stylet and injectspharyngeal secretions into it, ultimately leading to the reorgani-zation of these cells into typical feeding structures called giant cells,from which the nematode feeds for the remainder of its sedentarylife cycle (Gheysen & Mitchum, 2011). Already at 1 d afterinfection (dai), swelling of infected root tips can be observed. At3 dai, hyperplasy and hypertrophy of the cells surrounding thegiant cells result in the formation of terminal hook-like galls, atypical characteristic of the rice RKN (Bridge et al., 2005). Afterthree moults, the females lay their eggs inside the galls, while mostother RKNs deposit egg masses at the gall surface, and hatchedjuveniles can reinfect the same or adjacent roots. In well-drainedsoil at 22–29°C the life cycle of M. graminicola is completed in19 d. By contrast to migratory nematodes, RKNs hardly cause anynecrosis, as they migrate in between rather than through the plant

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cells by enzymatic softening of the middle lamella of the root cells(Gheysen & Mitchum, 2011).

During the infection process plant-pathogenic nematodes causemajor changes in plant gene expression (Gheysen & Mitchum,2011). Nematode-induced transcriptome changes have beenmainly identified by microarray analysis on dicotyledonous plantsupon infection with sedentary nematodes (Alkharouf et al., 2006;Ithal et al., 2007a,b; Klink et al., 2007; Szakasits et al., 2009;Barcala et al., 2010). Except for our own work on systemic defensepathways in H. oryzae-infected rice (Kyndt et al., 2012b), no dataon the response of plants upon migratory nematode infection areavailable. The systemic impact on the rice defense system appearedto be quite different between RKN- and RRN-infected plants,exemplifying the different modes of action of these two nematodes(Kyndt et al., 2012b).

While the transcriptome response has been studied in detailfor different plant species in a pathogenic interaction withbacterial and fungal pathogens, far less research has beenperformed on the molecular interaction between plants andnematodes (Gheysen & Mitchum, 2009). The comparativetranscriptome analysis of rice upon infection with sedentary ormigratory nematodes described in this paper provides importantinsights into general and specific defense strategies of themonocotyledonous rice plant upon root infection and aboutgenes that are potentially targeted by effectors of nematodes withtotally different lifestyles.

Materials and Methods

Infection

Oryza sativa L. cv ‘Nipponbare’ (GSOR-100, USDA) wasgerminated for 6 d at 30°C, transferred to synthetic absorbentpolymer (SAP) substrate (Reversat et al., 1999) and further grownat 26°C under a 16 : 8 h light : dark regime. Nematodes werecultured and extracted as described in (Kyndt et al., 2012b).Twelve-day-old plants were inoculated with 250 nematodes perplant for M. graminicola and 400 nematodes per plant forH. oryzae. Control plants were mock-inoculated. One day afterinoculation, the plants were transferred to a hydroponic culturingsystem with Hoagland solution (Reversat et al., 1999) to synchro-nize the infection process. Infected and control tissues werecollected at 3 and 7 dai. In the case of RKN, galls were sampled.Since RKN-induced galls are located at the root tip, uninfectedroot tips, encompassing the meristem and the elongation zone(c. 0.3 mm long), were sampled as control material. For migratoryRRN infection, the whole root tissue without these root tips, wassampled. For each treatment, two or three biological replicates,with each replicate containing a pool of six different plants, wereanalyzed by mRNA-Seq. For quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR) validation, another twoindependent biological replicates were taken. RNA was extractedusing the Qiagen RNeasy Plant Mini Kit (Qiagen, Venlo, theNetherlands), with an additional sonication step after addition ofbuffer RLT (Qiagen). RNA integrity was checked using the AgilentBioAnalyzer 2100 (Agilent, Diegem, Belgium).

Library preparation and Illumina GAIIx sequencing

Approx. 2 lg of total RNAof each samplewas used formRNA-Seqlibrary construction according to the manufacturer’s recommen-dations (Illumina, San Diego, CA, USA). The denatured librarieswere diluted to a final concentration of 6 pMand loaded on a singleread flow cell. Multiplexing was applied to minimize lane effects.After cluster generation, the multiplexed library was sequenced onan Illumina Genome Analyzer II (76 cycles).

Mapping reads to genome data and annotated transcripts

Reads weremapped to theO. sativa ssp. japonica reference genome(build MSU6.0) in two phases using TopHat version 1.3.1(Trapnell et al., 2009) andCufflinks, version 1.0.3 (Trapnell et al.,2010). A detailed description of the workflow and settings used inthe data analysis is given in Supporting Information, Notes S1.

Identification of novel transcriptionally active regions(nTARs)

The Cufflinks program generates a GTF file, including alltranscripts annotated in MSU6 and putative novel transcriptsderived from the data. All putative nTARsmarked as splice variantsof known genes or located within intronic regions were disregardedand the 4684 remaining nTARs were compared by BLASTN(E < 1e–4) with nTARs from our previous analysis on rice roottissue (Kyndt et al., 2012a). A more detailed description of thisanalysis is given in Notes S1. BLASTx searches were performedagainst the Swiss-Prot and trEMBL and all predicted rice proteins(http://rice.plantbiology.msu.edu/). Homologs of the nTARs inrice expressed sequence tags (ESTs) were searched by tBLASTx(E < 1e–4).

Calculation, normalization and profiling of gene expression

Expression was quantified per sample and per annotated orunannotated locus as the sum of all reads mapped to the respectivegene exons. Splice variants were treated as a single gene. Expressionprofiles were assessed using the R-package ‘baySeq’, version 1.5.1.(Hardcastle&Kelly, 2010).To compensate for artificial differencesin read distributions, the original library sizes were multiplied byadditional normalization factors calculated using the methodsdescribed in Robinson&Oshlack (2010), with standard settings asimplemented in the edgeR package (version 2.0.3). For all furtheranalyses the expression level of each transcript for each conditionwas estimated as the average number of reads detected across thebiological replicates. The fold change (FC) is calculated as the ratioof the average number of reads + 1 (to avoid 0 values) in thedifferent conditions. Log2-transformed values of FC were used.

Gene Ontology and enrichment analyses

GeneOntology (GO) analysis andGOenrichmentwere performedusing agriGO (Du et al., 2010). Parametric analysis of gene setenrichment (PAGE) (Kim & Volsky, 2005), based on differential

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gene expression levels (log2FC), was executed. Benjamini andHochberg false discovery rate (FDR) correction was performedusing the default parameters to adjust the P-value.

In addition, we usedMapMan (Thimm et al., 2004) to visualizethe expression of genes ontometabolic pathways and theWilcoxonSigned Rank (WSR-test (with Benjamini and Hochberg correc-tion) to test the statistical significance of differential expression ofthese pathways.

Validation of mRNA-Seq by qRT-PCR

Based on potential functional importance, 15 genes were selectedper nematode and per time point for validation in independentsamples by qRT-PCR. After removal of transcripts with unspecificamplification, at least 12 genes remained for validation. Chromo-somal location of these transcripts and primer sequences arepresented in Table S1. qRT-PCR was performed and analyzed asdescribed in Kyndt et al. (2012a).

Detection of nematode transcripts in infected tissues

The nature of the sample preparation allowed us not only toexamine the rice transcriptome, but also simultaneously to capturethat of the invading nematodes. Since no reference genomesare available for these nematodes, we used a de novo assemblymethod employing only those reads that could not be mapped inthe reference rice annotation approach (termed ‘unmappedfraction’). First, transcript contigs were assembled using Velvet(v1.1.07, Zerbino & Birney, 2008) with variable k-mer lengths(k = {43,47,53,57}). The contig files from each assembly weremerged per nematode using the multi-K method described inSurget-Groba & Montoya-Burgos (2010). These contigs wereBLASTed against the rice genome (MSU6.0) and against 454-ESTcontigs available in our laboratory (M. graminicola, A. Haegemanet al., unpublished; H. oryzae, L. Bauters et al., unpublished)(E < 1e–15). For more details, see Notes S1.

Results

Root tissueswere collected at two stages after nematode inoculation:3 dai (‘early’) and 7 dai. For simplicity, we call the 7 dai data ‘late’,although a complete life cycle ofM. graminicola takes 2–3 wk and5 wk up to 2 months for H. oryzae. A total of 98 671 971 readswere acquired andmade publicly available (GSE35843). The shortreads were aligned against the whole reference genome sequence ofcv Nipponbare (MSU6.0) and in total 49.20% of the sequencedreads could bemapped (Table 1). The total length ofmapped readswas c. 3.69 billion bases, representing 9.5-fold the rice genome sizeand c. 36-fold coverage of the annotated transcriptome. Theexpression of a total of 42 640 different rice genes was detected inthe analyzed tissues.

nTARs in infected tissue

TopHat andCufflinks were used to detect potential exon junctionsand unannotated transcribed regions. The locations of the resulting

nTARswerematched againstO. sativaMSU6genes. Cufflinks alsodiscovered a series of potential gene extensions (marked with ‘-ext’in data supplements). All nTARs overlapping known exons wereexcluded, resulting in 8290 putative nTARs. After excludingoverlaps in intronic regions, 4684 putative nTARs remained(Table S2). A total of 2070 sequences showed homology to an ESTor cDNA record for ssp. japonica, thus supporting tentativetranscripts not included in the MSU6.0 genome. A total of 2491nTARs have a significant BLAST hit (BLASTn E < 1e–04) withnTARs detected in our previous transcriptome data of healthy riceroot tissues (Kyndt et al., 2012a). Hence, an extra set of 2193nTARs was detected in rice roots challenged by nematodeinfection.

To predict the function of these 4684 putative nTARs, differentdatabase searches were performed. First, the nTAR sequences wereBLASTX-ed against all rice proteins and 1099 nTARs resulted in asignificant transcript hit (Table S2; E < 1e–4); hence these nTARsare potential paralogs of previously annotated loci. A SwissProt/trEMBL search was executed to detect known signatures and, for2062 nTARs, a hit was found with a known protein family (TableS2) (E < 1e–4). For 901 nTARs, a functional annotation could bepredicted based on similarity with known genes from O. sativa orother plants. Based on this homology, putatively coding regionswere found for protein kinases, transcription factors, disease ordrug resistance proteins, cyclins, auxin response factors andlipoxygenases.

Genes responding to infection by RKNs in rice

Different comparative gene expression analyses were performed fornematode-infected tissues and their respective healthy control. Thedifferent comparisons that were made, the number of biologicalreplicates per condition and the number of statistically significant

Table 1 Overview of the obtained sequencing data and mapping of thesesequences on to the rice genome

Totalnumber ofsequencedreads

Total numberofmappings

Number ofreads withat least onemapping

Uninfected roots 3 dai 10 431 413 16 289 011 4 540 703Uninfected root tips 3 dai 12 370 829 83 510 504 7 035 305Uninfected roots 7 dai 8 599 730 70 245 377 4 157 671Uninfected root tips 7 dai 17 737 509 147 909 076 7 990 824Migratory nematode-infected roots 3 dai

11 680 617 201 021 741 5 925 094

Migratory nematode-infected roots 7 dai

7 082 550 151 245 945 4 431 705

Root galls 3 dai 15 351 251 270 299 197 7 300 833Root galls 7 dai 15 418 072 302 814 270 7 184 996Total 98 671 971 1 243 335 121 48 567 131Mapping percentage 49.20%Coverage of the ricegenome

19.28 242.91 9.49

Coverage of the ricetranscriptome

73.02 920.15 35.94

dai, days after infection.

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differentially expressed genes (DEGs) that were identified areshown in Table 2.

Transcriptome changes in young gall tissue (3 dai) At 3 dai, theRKNs have penetrated the root and moved to the tip, where theyinduce root galls. Galls generally contain three to four J2 or J3 stagenematodes (Kyndt et al., 2012b). Gene set enrichment analysis onrelative expression levels (log2FC) of all transcripts in the galls vsnoninfected root tips (Fig. 1a, Table S3) showed that genesinvolved in ‘metabolic process’ were up-regulated at 3 dai, whileGO terms ‘plant-type cell wall organization or biogenesis’,‘transmembrane transport’ and ‘photosynthesis’ were down-regulated in the infected tissue. Pathway mapping with MapManshowed a significant up-regulation of the cell wall-degrading beta-1,3-glucan hydrolase gene family and down-regulation of bioticstress-related genes in 3 dai galls (Fig. 2). For example, a generaldown-regulation of the phenylpropanoid pathway was detected(results not shown).

One hundred and fifty-six transcripts were found to besignificantly differentially expressed (FDR < 0.05) (Tables 2,S4). Twelve of them were independently validated by qRT-PCR,and the expression pattern was confirmed in all cases (Table 3).

Among the strongest down-regulated genes are three thioningenes, a nodulinMtN3 family protein, a lipid phosphatase protein,a MYB family transcription factor and a putative dirigent gene,most probably involved in lignan biosynthesis (Davin & Lewis,2000). Among the genes that are most strongly up-regulated inyoung gall tissue, there were three genes of the lipid transfer protein(LTP)-gene family, the gibberellin (GA) receptor GID1L2, aharpin-induced protein 1 domain-containing protein, an auxinresponse factor, isochorismatase, and many different proteinkinases and chitinases.

Transcriptome changes in mature gall tissue (7 dai) At 7 dai, thegalls have matured, and giant cells within the gall tissue are fullydeveloped (Engler et al., 1999). Nematodes developing withinthese galls are now at the J3 or J4 stage. In 7 dai galls, genes involvedin ‘transcription’, ‘post-translational protein modification’,

‘peptide transport’, and ‘regulation of nitrogen compound meta-bolic process’ were up-regulated (Fig. 1b, Table S3). Genesinvolved in ‘translation’, ‘chromatin organization’, ‘ion transmem-brane transport’, and ‘plant-type cell wall organization’ weregenerally expressed to a lesser degree than in uninfected root tips.MapMan pathway mapping showed a significant modification ofthe ATP-synthesis pathway, light reaction, Calvin cycle, cell wallmodification, andminor CHOmetabolism–trehalose in 7 dai gallsvs noninfected root tips.Whiledown-regulated at 3dai,manygenesinvolved in phenylpropanoid and jasmonate (JA) biosynthesispathways were up-regulated in 7 dai gall tissue (results not shown).

At an FDR cutoff of 0.05, 991DEGswere found in 7 dai galls vshealthy root tips (Tables 2, S4). Twelve of them were indepen-dently analyzed by qRT-PCR and the trend of expression wasconfirmed in all cases (Table 3).

The strongest up-regulation was found for genes encoding, forinstance, a putative abscisic stress-ripening protein, OsLTPL116,OsLTPL124, GA receptor GID1L2, PR-genes chitinase CHIT14,thaumatin and defensin DEF8, and caffeoyl-CoA O-methyltrans-ferase, a key enzyme in the biosynthesis of lignin cell wallprecursors. Among the down-regulated genes there are eight genesbelonging to the thionin gene family, two dirigent genes andcellulose synthase CSLF3.

General response of rice root tips upon RKN infection We alsocompared the gene expression level in all sampled gall tissues (atboth time points) with the six uninfected root tip samples to look atconsistent trends in the root gall transcriptome, regardless of thetime point after infection (Fig. 1c, Table S3).

Five hundred and ten rice loci were detected to be consistentlyand significantly differentially expressed. For instance, a consistenttwofold (log2FC) up-regulation of OsPR10 (LOC_Os03g18850)was detected in the 3 and 7 dai galls vs the uninfected rice root tips.We also detected a significant and consistent activation of genesinvolved in gibberellin metabolism, with the two key oxidaseenzymes that catalyze the penultimate steps inGAbiosynthesis, GA20-oxidase 1 and 2, and the receptor GID1L2 strongly induced ingall tissue at both time points (Fig. 3).

Table 2 Comparative gene expression analyses executed on mRNA-Seq data obtained in the current study, showing the number of biological replicates,differentially expressed genes (DEGs, listed in Tables S4–S6) and the included novel transcriptionally active regions (nTARs)

Biological replicatesper treatment No. of DEGs

No. of DEGsup-regulated

No. of DEGsdown-regulated

RKN response Early (3 dai galls vs 3 dai healthy root tips) 3 156 (16 nTARs) 76 80Late (7 dai galls vs 7 dai healthy root tips) 3 991 (56 nTARs) 935 56General (galls vs healthy root tips) 6 510 (43 nTARs) 374 136RKN-specific response 6 382 (63 nTARs) 269 113

RRN response Early (3 dai roots vs 3 dai healthy roots)a 2 95 (12 nTARs) 80 15Late (7 dai roots vs 7 dai healthy roots)a 2 111 (26 nTARs) 72 39General (infected roots vs healthy roots) 4 114 (12 nTARs) 70 44Root rot nematode-specific response 5 578 (144 nTARs) 578 0

General nematoderesponse

All infected root tissues vs healthy roottissues

10 94 (26 nTARs) 69 24

dai, days after infection; RKN, root knot nematode.aFDR < 0.1



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Genes responding to infection by migratory RRNs in rice

Transcriptome changes early upon RRN infection At 3 dai with400H. oryzae the root system generally contains 20–100migratorynematodes (Kyndt et al., 2012b) and some cells within the roots arenecrotic. Comparing the transcriptome of infected root tissue withnoninfected roots at 3 dai (Table S3, Fig. 1d) revealed that genesinvolved in ‘electron transport’, ‘response to stress’, ‘post-transla-tional protein modification’, ‘plant-type cell wall organization orbiogenesis’, and ‘programmed cell death’ were induced uponinfection. On the other hand, genes involved in ‘photosynthesis’,and ‘generation of precursor metabolites and energy’ are down-regulated at 3 dai after RRN infection. MapManmapping showeda significant change in the phenylpropanoid pathway, as well asflavonoids,Calvin cycle, hemicellulose synthesis, lipid degradation,secondary metabolism-simple phenols, and cell wall degradation.Receptor kinases, the ethylene (ET) and JA metabolism, proteindegradation and post-translational modification were significantlyaffected upon migratory nematode infection. In the JA metabo-lism, different paralogs of the biosynthetic enzymes allene oxidesynthase and lipoxygenase are induced 3 d after RRN inoculation.

Only 25 genes were found to be significantly differentiallyexpressed at a cutoff of FDR < 0.05. As this dataset contained

fewer biological replicates (Table 2) and hence less statisticalpower, we decided to set the FDR cutoff at < 0.1. At this cutoff, 95DEGs were found (Tables 2, S4). Twelve genes were validated byqRT-PCR and the trend of expression was confirmed in all but onecase (Table 3).

OsLTPL124 (LOC_Os04g52260) was 5.3-fold (Log2FC)down-regulated at 3 d after migratory nematode infection. A geneencoding a CLAVATA1 (CLV1) protein kinase (LOC_Os05g51740) was 1.5-fold (log2FC) down-regulated in the infectedroot tissue. Transcription factors OsWRKY62, OsWRKY70 andOsWRKY11 were strongly up-regulated at 3 dai after migratorynematode infection. An induction of oxidative stress is exemplifiedby the up-regulation of three peroxidase precursors and twoglutathione S-transferases.

Transcriptome changes late upon RRN infection Seven daysafter migratory RRN infection, the roots show large necroticlesions, and nematodes have laid eggs within the root tissue.When comparing the transcriptome of infected vs uninfected roottissues, (Table S3, Fig. 1e) we detected an up-regulation of‘primary metabolic process’, ‘protein metabolic process’ and‘defense response’, while ‘photosynthesis’ and ‘porphyrin meta-bolic processes’ were down-regulated in the infected roots.

(a)

(b)

(c)

(d)

(e)

(f)

Fig. 1 Results of parametric analysis of gene set enrichment of transcriptome data of root knot nematode (RKN;Meloidogyne graminicola)- or migratoryroot rot nematode (Hirschmanniella oryzae)-infected rice (Oryza sativa) roots at 3 (early) or 7 d (late) after infection (dai). (a) 3 dai with RKNs; (b) 7 dai withRKNs; (c) general response upon RKN infection; (d) 3 dai with root rot nematodes; (e) 7 dai with root rot nematodes; (f) general response upon root rotnematode infection.Z-scores of all secondary levelGeneOntology (GO) terms are shown.Darkgray bars,GO terms that are up-regulated in the infected tissuevs the corresponding control; light gray bars, GO terms that are down-regulated in the infected tissue vs the corresponding control.



MapMan mapping showed a general down-regulation of genesinvolved in the Calvin cycle and light reactions, while mitochon-drial electron transport was up-regulated at 7 d after migratorynematode infection. The JA pathway and phenylpropanoidpathways did not respond as strongly as at 3 dai (results notshown).

At a cutoff of FDR < 0.05, 38 DEGs were found at 7 d aftermigratory nematode infection. Considering that only two biolog-ical replicates per treatment were analyzed for these tissues(Table 2), the statistical analysis was also performed with lessstringent conditions (FDR < 0.1), resulting in 111 DEGs(Tables 2, S4). The trend of expression was confirmed by qRT-PCR in 11 out of 14 cases (Table 3).

Among the strongest down-regulated genes, for instance, arethose coding for phosphoribulokinase, glyceraldehyde-3-phos-phate dehydrogenase and fructose-bisphospate aldolase, allinvolved in primary plant metabolism (glycolysis and Calvincycle). Transcripts coding for OsWRKY62 and OsWRKY77 arehighly up-regulated upon infection, as well as a nucleotide-bindingsite–leucine-rich repeat (NBS-LRR) disease resistance protein(LOC_Os09g34150) and patatin (LOC_Os08g28880).

General response of rice upon RRN infection The gene expres-sion profile in all RRN-infected rice root samples was comparedwith the transcriptome of uninfected root samples to detectconsistent transcriptome changes regardless of the time point afterinfection (Table S3, Fig. 1f).

Differential expression analysis detected 114 loci to be signif-icantly differentially expressed at both time points (FDR < 0.05)upon RRN infection in rice roots (Table 2). Three genes involvedin carbohydrate metabolism, coding for fructose-1,6-bisphospha-

tase (LOC_Os03g16050), glyceraldehyde 3-phosphate dehydroge-nase (LOC_Os04g38600) and fructose-bisphospate aldolase(LOC_Os11g07020), are strongly and consistently down-regulatedin the infected roots compared with the uninfected root tissue. Asignificant differential expression was found for the WRKY familytranscription factors and DUF26 and wall-associated receptorkinases. Among them, for instance, a strong up-regulation wasdetected in the transcripts coding for OsWRKY62 (LOC_Os09g25070) andOsWRKY59 (LOC_Os01g51690) in the infectedroots. Further,OsSub13, a subtilisin-like serine proteinase belongingto a class of pathogenesis-relatedproteins (Jorda&Vera, 2000), and aprotein-disulfide isomerase (PDI), involved in the biosynthesis of agroup of plant defense peptides called cyclotides (Jennings et al.,2001), were strongly up-regulated.

General response of rice upon infection with nematodes

Differential gene expression analysis identified 94 loci to besignificantly and consistently differentially expressed in rice roottissue upon infection with either migratory RRNs or RKNs(FDR < 0.05; Table S4). Among these genes that are generallyresponding upon root challenge with nematodes is OsWRKY62,which is 3.4-fold up-regulated in infected vs noninfected tissue.Peng et al. (2008) showed that overexpression of OsWRKY62suppresses the activation of defense-related genes, implying thatthis gene functions as a negative regulator of innate immunityin rice. Seven of these general nematode-responsive DEGswere arbitrarily chosen to independently analyze their expres-sion in infected and uninfected tissue at 3 dai by qRT-PCR.The trend of expression was confirmed in all but one case(Table 3).

Fig. 2 MapMan visualization of biotic stress-related genes, and genes involved in auxin and cytokinin pathways. The visualization shows the observeddifferential expression patterns, based on the log2 fold changes of mRNA levels, in root knot (Meloidogyne graminicola)- or migratory root rot nematode(Hirschmanniella oryzae)-infected rice (Oryza sativa) roots at 3 (early) or 7 d (late) after infection (dai). Red dots, gene is up-regulated in infected tissue vs thecorresponding healthy control; blue, down-regulation.



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Table 3 Differential expression patterns (log2FC) of a selected set of rice genes in root knot nematode (RKN)- or root rot nematode (RRN)-infected root tissuesat 3 and 7 d after infection (dai), as detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR) andmRNA-Seq data (down-regulatedgenes, green; up-regulated genes, red)



Comparison between the response of rice upon RKN andRRN infection

A comparison between the biotic stress responses in rice root tissueafter infection with RKNs vs RRNs provides insights intomechanisms that are specifically related to the different infectionstrategy of each nematode. In Table 4, an overview of the generaltrends observed at 3 dai with RKNs or RRNs is shown.

To detect specific RKN-responsive genes, the RRN-infectedtissue was also used as control tissue next to the uninfected roottissues, and vice versa.

In RRN-infected roots, biotic stress-related genes are stronglyup-regulated at 3 dai, while this response is attenuated at thelater time point after infection (Fig. 2). For RKN-infected galls,many genes involved in biotic stress response are down-regulatedat 3 dai, but up-regulated at 7 dai. This up-regulation at 7 dai,however, is not as strong as is seen in the 3 dai RRN-infectedroots.

Differential gene expression analysis identified 382 loci that areonly significantly differentially expressed uponRKN infection, and

not upon H. oryzae infection (FDR < 0.05; Table S5). Amongthem, for instance, the AP2-like transcription factor PLETHORA2 (LOC_Os07g03250) is highly up-regulated in gall tissue. Twotranscripts coding for WKRY-transcription factors (OsWRKY34and OsWRKY36), two genes annotated as growth-regulatingfactors (LOC_Os02g47280 and LOC_Os04g51190) and onecoding for osmotin (LOC_Os12g38150) are specifically inducedin gall tissue. On the other hand, five loci coding for dirigentproteins (LOC_Os01g25030, LOC_Os07g44250, LOC_Os07g44280, LOC_Os07g44450 and LOC_Os10g18760), major regu-lators of lignan biosynthesis (Davin&Lewis, 2000), are specificallydown-regulated in the gall tissue.

Five hundred and seventy-eight loci were detected to besignificantly differentially expressed in migratory nematode-infected root tissue, but not affected in gall tissue (FDR < 0.05;Table S6). For instance, seven transcription factors of the WRKY-superfamily were up-regulated in H. oryzae-infected roots,WKRY11, 45, 51, 59, 66, 77 and an unclassified WRKY gene.Also, nine genes annotated as ‘disease-resistance proteins’ were up-regulated upon H. oryzae infection but not upon RKN infection.

Fig. 3 MapMan visualization of the gibberellic acid biosynthesis pathway. The visualization shows the observed differential expression patterns, based onthe log2 fold changes of mRNA levels, in root knot (Meloidogyne graminicola)-infected rice (Oryza sativa) galls vs healthy root tips. Red dots, gene isup-regulated in infected tissue vs the corresponding healthy control; blue, down-regulation.



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Three loci coding for flavonol synthase genes and one for flavonol-3-O-glycoside-7-O-glucosyltransferase 1 were found to be specif-ically more transcribed in RRN-infected root tissue, as well as fourloci coding for peroxidases.

Detection of nematode transcripts in infected root tissue

As 51.8%of sequenced reads (Table 1) could not bemapped to therice genome (the ‘unmapped fraction’), we evaluatedwhether someof these originated from the infecting nematodes. After de novoassembly, contigs were BLASTed against the rice genome(MSU6.0) again and against EST data from the nematodes usedin this study.

In all, 3856 of the contigs assembled from gall tissue matchedEST data fromM. graminicola and another 137matched EST datafrom H. oryzae. Among the contigs assembled from migratorynematode-infected roots, 3744 matched ESTs fromH. oryzae, andanother 1291matched ESTs fromM. graminicola. (Table 5). Hitswith ESTs from the other nematode are most likely highlyconserved genes that are present in both nematodes but whichmight not have been picked up during EST-sequencing of bothnematodes. A SwissProt/trEMBL search was performed on thecontigs with a nematode hit, and for 88% (M. graminicola) and57% (H. oryzae) of them, a potential function can be predicted onthis basis. Table 5 shows that the majority of these transcriptspotentially code for housekeeping genes, but a few putativenematode secreted proteins were also found.

Discussion

Using mRNA-Seq, we studied transcriptome changes induced in acompatible interaction between plant-parasitic nematodes andO. sativa. We acquired almost 100 million reads from infectedand healthy root tissues, thereby obtaining count data for more than40 000 rice transcripts. A good consistency (91%) between expres-sion levels identified by mRNA-seq and qRT-PCR analyses wasestablished (Table 3). The comparison between the transcriptome ofthe plant upon infection with two types of nematodes, exhibitingdistinct feedingbehaviors, provides anunprecedented insight into thecompatible interaction between nematodes and the plant root.

Plants generally respond to nematode infection by differentialexpression of genes involved in stress and defense responses, cellwall alteration, metabolism and nutrient allocation, signal trans-duction and phytohormone action (Li et al., 2008; Escobar et al.,2011). In accordance with these previous observations, genesinvolved in those categories were differentially expressed in thecurrent study. However, when comparing the response of rice rootsupon infection with migratory and sedentary endoparasiticnematodes, many discrepancies arise. General trends, as deducedfrom the data presented in this paper, are shown in Table 4. Someof themore remarkable differences and similarities will be discussedin the following sections.

For the RKN analysis, whole gall tissues were used and thus geneexpression differences reflect a combination of changes occurring ingiant cells and surrounding gall tissues. The study of Barcala et al.(2010) showed that, although some genes have similar regulation inboth galls and giant cells, the majority had different expressionpatterns.

Metabolic activity and nutrient transport

Feeding sites of sedentary endoparasitic nematodes, such asMeloidogyne spp. and cyst nematodes, are the only source ofnutrients for these root parasites throughout their lives (Jammeset al., 2005; Szakasits et al., 2009). Hofmann et al. (2010) showedthat cyst nematodes cause a major reprogramming of the primarymetabolism in local and systemic tissues upon cyst nematodeinfection. The current study confirmed that phenomenon in rootgalls of rice roots (Fig. 1, Table S3). This observation was strongestat 3 dai, when genes involved in protein and sucrose biosynthesiswere highly induced, and is in concordance with the view that theRKNs stimulate the cells within the gall to be more active andproduce nutrients to the benefit of the nematode. An activation ofthe trehalose metabolism was observed in both galls and syncytia(Hofmann et al., 2010).Trehalose hasmultiple functions in plants,not only in carbohydrate storage andmetabolism, but also as a stressprotectant, and as ametabolic signalingmolecule involved inmanyplant–pathogen interactions (Fernandez et al., 2010) and cell wallmodification (Bae et al., 2005).

The nematode manipulates the plant to redirect nutrients tothis new nutrient sink, mainly during the initial phase of giantcell formation, while at later stages, intracellular and transmem-brane transport of lipids and proteins is also strongly repro-grammed (Hofmann et al., 2010). For instance, in gall tissue

Table 4 Overview and comparison of the local physiological transcriptionalresponse in rice root tissue upon infection with eitherMeloidogyne

graminicola or Hirschmanniella oryzae

M. graminicola-infected galltissue (3 dai)

H. oryzae-infected roottissue (3 dai)

Metabolism ↑ ↘Nutrient transport ↑ ↘Death ↘ ↑Antioxidant activity � ↑Plant cell wall organizationand biogenesis

↓ ↑

Biotic stress ↓ ↑

Hormone pathways mainly involved in plant defenseSalicylic acid pathway ↓ ↑Jasmonate pathway ↓ and ↑ ↑Ethylene pathway ↓ ↑

Hormone pathways mainly involved in plant developmentAuxin pathway ↓ and ↑ ↑Cytokinin pathway ↓ �GA pathway ↑ �BR pathway ↑ ↑

dai, days after infection; BR, brassinosteroid.↑, generally induced in nematode-infected tissue; ↓, generally repressedin nematode-infected tissue; ↘, slightly repressed in nematode-infectedtissue; �, generally unchanged in nematode-infected tissue; ↓ and ↑, bothstrongly induced and strongly repressed genes detected in nematode-infected tissue.



there was a significant up-regulation of transcripts encodingthe nuclear export protein exportin 1, amino acid and peptidetransporters, transmembrane nucleobase-ascorbate transporters,and plant lipid transfer proteins (LTPs, Tables S3 and S4).LTPs are a family of peptides that facilitate lipid exchangebetween cell membranes (D’Angelo et al., 2008), but for whicha possible defense role in plants has also been suggested (Kimet al., 2006).

In the primary metabolism of the gall, a remarkable phenom-enon is observed, which might seem counterintuitive: photosyn-thesis-related genes are highly up-regulated in 7dai galls (Table S3),while roots are generally seen as photosynthetically inactive tissue.However, under light induction, roots can develop chloroplasts(Flores et al., 1993) and we have recently demonstrated (Kyndt

et al., 2012a) that photosynthesis-related genes are activated in theroot tissue in a hydroponic growth system even with minor lightinfluence. Nevertheless, when infected with nematodes, onlyRKN-infected tissues showed this photosynthetic capacity, whileRRN-infested roots revealed a strong suppression of photosynthe-sis (Table S3). This observation is in accordance with theobservations of Szakasits et al. (2009) which highlighted theup-regulation of chloroplast genes in syncytia of cyst nematodes,but induction of photosynthesis has not been described beforein galls.

In RRN-infected plants, on the other hand, the metabolicactivity of the root is suppressed, while many transcripts attributedto cell death are induced, in line with the root necrosis caused byfeeding of the migratory nematode.

Table 5 Overview of results obtained from the unmapped fraction of mRNA-Seq reads

Contigs obtained from unmapped fraction of

RKN-infected root tips Root rot nematode-infected roots

Total number of contigs in the unmapped fraction 10 105 9376No. of contigs with BLAST-hit inOryza sativaMSU6.0 2260 779EST data ofMeloidogyne graminicola 3856 3744EST data of Hirschmanniella oryzae 137 1291Hits with unknown function 471 54

Nematode housekeeping genesCuticle collagen 121 8Actin 9 26Tubulin 42 12Ribosomal proteins 285 165Eukaryotic translation initiation factors 36 3Elongation factors 69 17Heat shock proteins 42 13

Predicted annotation of a selection of putativenematode-secreted proteins

32 kDa beta-galactoside-binding lectinOS = Haemonchus contortus

32 kDa beta-galactoside-binding lectinOS = Haemonchus contortus

Peroxiredoxin OS = Ascaris suum Peroxiredoxin OS = Ascaris suum

Superoxide dismutase (Cu-Zn) OS = Brugia

pahangi

Ancylostoma secreted proteinOS = Ancylostoma caninum

Polyprotein ABA-1 (fragment) OS = Ascaris

suum

Polyprotein ABA-1 (fragment) OS = Ascaris

suum

Putative cathepsin L proteaseOS = Meloidogyne incognita

Glucuronoxylanase xynC OS = Bacillus subtilis

Fatty-acid and retinol-binding protein 1OS = Onchocerca gutturosa

Pectate lyase OS = Meloidogyne javanica

Glutathione peroxidase OS = Schistosoma

mansoni

Beta-1,4-endoglucanase OS = Pratylenchus

penetransProgrammed cell death protein 6 OS = Mus

musculus

Beta-glucanase OS = Rhodothermus marinus

Endoglucanase celAOS = Streptomyces lividansEndoglucanase OS = Clostridium

saccharobutylicum

Endoglucanase Z OS = Dickeya dadantii

Expansin B2 (fragment) OS = Globoderarostochiensis

Expansin B1 protein OS = Globodera

rostochiensis

Expansin-like protein OS = Bursaphelenchusxylophilus

EST, expressed sequence tag; OS, original species; RKN, root knot nematode.



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Cell wall biosynthesis, degradation and modification

The plant cell wall, composed of cellulose microfibrils, hemicel-luloses and pectin, represents a structural barrier to infection by awide range of plant pathogens. Endoparasitic nematodes haveevolved sophisticatedmechanisms to breach this barrier and secretea battery of cell wall-modifying enzymes into the plant root(Gheysen & Mitchum, 2009; Davis et al., 2011). The processesthat give rise to feeding site formation also involve recruitment ofplant genes encoding cell wall-modifying proteins. Our studyrevealed that the biosynthesis of new cell walls is up-regulated inmigratory nematode-infected roots, most probably as a plantdefense strategy. Conversely, cell wall organization and biosynthe-sis are repressed by RKN infection (Table S3).

While cell wall biosynthesis is reduced, plant genes coding forcell wall-degrading enzymes are activated in root galls (reviewed inGheysen & Mitchum, 2009). In rice root galls, endo-b-1,4-glucanases belonging to the glycosyl hydrolase family 17 werestrongly induced. By contrast, there was a down-regulation ofxyloglucan endotransglycosylases (XETs), enzymes that show aspecific activity on xyloglucans, the major component of hemicel-lulose. Cell expansion, which is required for the development ofnematode feeding sites, is generally associated with structuralchanges in cell wall xyloglucans,whichplay an important role in cellwall elasticity and rigidity (Scheller & Ulvskov, 2010). Similar toour observations, transcriptome data from cyst nematode-infectedsoybean and Arabidopsis roots revealed suppression of XETs in thelocally infected tissue (Puthoff et al., 2003; Ithal et al., 2007a;Barcala et al., 2010), while they have been described to be locallyup-regulated in isolated syncytia (Ithal et al., 2007b) and giant cells(Barcala et al., 2010). As previously stated, the galls analyzed in thisstudy were composed of giant cells and their surrounding cells, andwe hypothesize that suppression of cell wall biosynthesis and XETsoccurs mainly in these surrounding cells. Differences in cellwall composition of monocots vs dicots and the fact thatM. graminicola-induced galls are located at the root tip, and hencemust have some effect on cell multiplication in the root meristem,probably also play a role here. To further elucidate the importanceand localization of these observations, studies on isolated giant cellsfrom rice roots are needed.

Plant hormone pathways and defense

The expression of plant defense proteins and changes in plantdevelopment are mainly regulated by phytohormones. Nematodeinfection in plants results in a change in hormone homeostasis,through an interplay between active manipulation by nematodeeffectors secreted in the plant tissue, to promote plant susceptibil-ity, and defense responses activated by the plant to reduce pathogeninfection (Goverse & Bird, 2011).

In contrast to galls, the migratory nematode-infected rootsshowed an induction of many genes known to be responsive toexternal stimuli such as general, biotic and abiotic stress factors(Figs 1, 2). Microarray data from Arabidopsis and soybean haveshown before that the plant defense response is up-regulated in cystnematode-infected tissues (Alkharouf et al., 2006; Ithal et al.,

2007a), but this and other studies (Jammes et al., 2005; Barcalaet al., 2010) show instead an early down-regulation of defensepathways upon RKN infection.

Many genes involved in the phenylpropanoid pathway, respon-sible for the biosynthesis of different metabolites, such as ligninprecursors, flavonoids and hydroxycinnamic acid esters andsalicylic acid (SA; Boudet, 2000), are strongly suppressed in 3 daigalls in rice (this study) and Arabidopsis (Barcala et al., 2010),whereas they were reported to be induced at early time points aftermigratory nematode infection (this study) and cyst nematodeinfection in soybean (Ithal et al., 2007a). The fact that this responseis attenuated in RKN-infected tissue further demonstrates thesubtleness and manipulative power of the RKNs.

The JA and ET pathways are known to play synergistic roles inplant innate immunity (Pieterse et al., 2009). In the case ofmigratory nematode-infected roots, a trendof coregulation of genesinvolved in these pathways was also observed, with them generallybeing up-regulated, similar to their systemic induction upon RRNinfection (Kyndt et al., 2012b). AlthoughEThas been suggested tobe critical for syncytium formation during cyst-nematode infec-tions in Arabidopsis (Goverse et al., 2000), RKNs seem to suppressET pathways, both locally (Barcala et al., 2010;Nahar et al., 2011;and this study) and systemically (Kyndt et al., 2012b). Ithal et al.(2007b), detected a strong and consistent suppression of the JApathway in isolated syncytia after cyst nematode infection insoybean, but in RKN galls on rice, no such trend was observed(Nahar et al., 2011; this study). This is an interesting observa-tion in view of the fact that the JA pathway, modulated by ET, isa key player in systemically induced defence against RKNs in rice(Nahar et al., 2011). From these results, we might deduce thatthe RKN does not specifically target the JA pathway, but ratherfocuses on down-regulation of the SA and ET pathways. Thismay explain why activating the JA pathway is the most effectivestrategy when trying to induce systemic defense against RKNs(Cooper et al., 2005; Nahar et al., 2011).

In galls, GA biosynthesis genes and theGID1L2GA receptor arestrongly induced (Fig. 3). GA plays an important role in stimu-lating cell division and elongation (Richards et al., 2001).Similarly, Bar-Or et al. (2005) and Klink et al. (2007) noticedthe up-regulation of GA biosynthesis genes in tomato galls andsyncytia on soybean.

Brassinosteroids (BRs) play important roles in physiological anddevelopmental processes, and their impact on plant defense has alsobeen demonstrated (Belkhadir et al., 2012). De Vleesschauweret al. (2012) showed that the BR pathway is involved in rice rootsusceptibility to Pythium graminicola, by down-regulating the SApathway. Similarly, BRs suppress rice defense against RKNsthrough antagonism to the JA pathway (Nahar et al., 2012). Inlight of this knowledge, it is interesting to notice that the BRpathway is generally slightly induced in nematode-infected tissues,and even significantly at 7 dai in RKN gall tissue (Table S4). TheBR biosynthesis gene OsDWARF (LOC_Os03g40540) anddifferent paralogs coding for the BRASSINOSTEROID INSEN-SITIVE 1-associated receptor kinase 1 (BAK1) are more highlyexpressed in nematode-infected rice roots. BAK1 is involved in BRsignaling, together with BRASSINOSTEROID INSENSITIVE 1



(BRI1), for which the different homologs in the rice genome arealso generally induced in nematode-infected vs uninfected rootsamples. An activation of the BR pathway might be important forthe nematode to overcome root defense.

The auxin pathway is responsible for root initiation, develop-ment, and lateral root formation (De Smet et al., 2010). Thetranscript encoding indole-3-acetic acid-amido synthetase(OsGH3.1), which conjugates IAA to amino acids, was found tobe strongly and consistently suppressed in rice roots infected witheither RKNs or RRNs. This potentially results in higher auxinconcentrations in the infected roots, which can lead to improvedcell division and expansion and cell wall loosening, but probablyalso plays a part in plant defense suppression through itsantagonism with the SA pathway (Domingo et al., 2009). Auxinmanipulation is known to be an important process during initiationand development of the feeding sites of sedentary plant-parasiticnematodes (Grunewald et al., 2009b). Our observations indicatethat not only sedentary nematodes, but also migratory nematodes,manipulate auxin homeostasis in the root (Fig. 2).OsYUCCA1, theonly rice gene for which the role in IAA biosynthesis has beendescribed in detail (LOC_Os01g46290; Yamamoto et al., 2007), isstrongly up-regulated in root galls at 3 dai and also slightly at 7 dai,corroborating previous evidence of induced auxin concentrationsin nematode feeding sites (Karczmarek et al., 2004; Grunewaldet al., 2009a).

Auxin and cytokinin (CK) have contrasting roles in rootdevelopment (Dello Ioio et al., 2007). CKs play an inhibitory rolein root meristem size, root elongation and lateral root formation inArabidopsis (Chapman & Estelle, 2009). In migratory nematodeinfected roots, the CK pathway was not strongly influenced. But incontrast to the enhanced CK amounts generally observed in leavesearly upon infection with biotrophic fungi (Walters &McRoberts,2006), a strong suppression of the CK pathway was detected in 3dai root galls (Fig. 2). At 7 dai, however, the CK pathway isinduced in galls, confirming previous observations of Lohar et al.(Lohar et al., 2004), who proved the requirement of CK toestablish mature galls (7 dai) in Lotus japonicus. Interference withCK concentrations and signaling is probably important for theenlargement of the root tip during early root gall formation.Next toswelling of the rootmeristem,CK suppression and auxin inductionwill probably also lead to the induction of lateral root formation, aphenomenon which is frequently observed in the vicinity of gallsand syncytia (Goverse et al., 2000; Karczmarek et al., 2004). Nextto their developmental function, CK concentrations have also beenshown to repress transporters of macronutriens in rice (Hiroseet al., 2007), and reducing CK concentrations might also benecessary for the conversion of galls into nutrient sinks. Furtherresearch into the exact role of and interplay between the auxin, CKand other hormone pathways in root gall development and plantdefense against nematodes will certainly prove to be worthwhile.

Acknowledgements

Weacknowledge the financial and infrastructural support ofGhentUniversity (‘Bioinformatics: from nucleotides to networks’,BOF_01GA0805, and Stevin Supercomputer Infrastructure).

Nematodes were collected and kindly provided by Prof. Dirk DeWaele (Catholic University Leuven, Belgium) and Zin Thu ZarMaung (Plant Protection Division, Yangon,Myanmar).We thankJean-Pierre Renard, Sarah De Keulenaer and Lien De Smet forexcellent technical assistance. TinaKyndt, AnneliesHaegeman andTim De Meyer (in part) are supported by a FWO postdoctoralfellowship. Simon Denil is supported by an IWT doctoral grant.

References

Alkharouf NW, Klink VP, Chouikha IB, Beard HS, MacDonald MH, Meyer S,

Knap HT, Khan R, Matthews BF. 2006. Timecourse microarray analyses reveal

global changes in gene expression of susceptible Glycine max (soybean) rootsduring infection by Heterodera glycines (soybean cyst nematode). Planta 224:838–852.

Bae HH, Herman E, Bailey B, Bae HJ, Sicher R. 2005. Exogenous trehalose alters

Arabidopsis transcripts involved in cell wall modification, abiotic stress, nitrogen

metabolism, and plant defense. Physiologia Plantarum 125: 114–126.BarcalaM, Garcia A, Cabrera J, Casson S, Lindsey K, Favery B, Garcia-Casado G,

Solano R, Fenoll C, Escobar C. 2010. Early transcriptomic events in

microdissected Arabidopsis nematode-induced giant cells.Plant Journal 61: 698–712.

Bar-Or C, Kapulnik Y, Koltai H. 2005. A broad characterization of the

transcriptional profile of the compatible tomato response to the plant parasitic

root knot nematodeMeloidogyne javanica. European Journal of Plant Pathology111: 181–192.

Belkhadir Y, Jaillais Y, Epple P, Balsemao-Pires E, Dangl JL, Chory J. 2012.

Brassinosteroids modulate the efficiency of plant immune responses to microbe-

associatedmolecular patterns.Proceedings of theNational Academy of Sciences,USA109: 297–302.

Boudet AM. 2000. Lignins and lignification: selected issues. Plant Physiology andBiochemistry 38: 81–96.

Bridge J, Plowright RA, Peng D 2005.Nematode parasites of rice. In: Luc M,

Sikora RA, Bridge J, eds. Plant-parasitic nematodes in subtropical and tropicalagriculture. Wallingford, UK: CAB International, 87–130.

ChapmanEJ, EstelleM. 2009.Cytokinin and auxin intersection in rootmeristems.

Genome Biology 10: 210.CooperWR, Jia L, Goggin L. 2005.Effects of jasmonate-induced defenses on root-

knot nematode infection of resistant and susceptible tomato cultivars. Journal ofChemical Ecology 31: 1953–1967.

D’Angelo G, Vicinanza M, De Matteis MA. 2008. Lipid-transfer proteins in

biosynthetic pathways. Current Opinion in Cell Biology 20: 360–370.Davin LB, LewisNG. 2000.Dirigent proteins and dirigent sites explain themystery

of specificity of radical precursor coupling in lignan and lignin biosynthesis. PlantPhysiology 123: 453–461.

Davis EL, Haegeman A, Kikuchi T 2011. Degradation of the plant cell wall by

nematodes. In: Jones J, GheysenG, Fenoll C, eds.Genomics andmolecular geneticsof plant–nematode interactions. Dordrecht, the Netherlands: Springer, 255–272.

DeSmet I, Lau S, VossU,Vanneste S, BenjaminsR,Rademacher EH, SchlerethA,

De Rybel B, Vassileva V, Grunewald W et al. 2010. Bimodular auxin response

controls organogenesis in Arabidopsis. Proceedings of the National Academy ofSciences, USA 107: 2705–2710.

De Vleesschauwer D, Van Buyten E, Satoh K, Balidion J, Mauleon R, Choi IR,

Vera-Cruz C, Kikuchi S, Hofte M. 2012. Brassinosteroids antagonize

gibberellin- and salicylate-mediated root immunity in rice. Plant Physiology 158:1833–1846.

Dello Ioio R, Linhares FS, Scacchi E, Casamitjana-Martinez E, Heidstra R,

Costantino P, Sabatini S. 2007. Cytokinins determine Arabidopsis root-

meristem size by controlling cell differentiation. Current Biology 17: 678–682.Domingo C, Andres F, Tharreau D, Iglesias DJ, Talon M. 2009. Constitutive

expression ofOsGH3.1 reduces auxin content and enhances defense response and

resistance to a fungal pathogen in rice.Molecular Plant–Microbe Interactions 22:201–210.

Du Z, Zhou X, Ling Y, Zhang ZH, Su Z. 2010. agriGO: a GO analysis toolkit for

the agricultural community. Nucleic Acids Research 38: W64–W70.



Research

NewPhytologist12

Engler JD,De Vleesschauwer V, Burssens S, Celenza JL, InzeD, VanMontaguM,

EnglerG,GheysenG. 1999.Molecularmarkers and cell cycle inhibitors show the

importance of cell cycle progression innematode-induced galls and syncytia.PlantCell 11: 793–807.

Escobar C, Brown S, MitchumMG 2011. Transcriptomic and proteomic analysis

of the plant response to nematode infection. In: Jones J,GheysenG, Fenoll C, eds.

Genomics and Molecular Genetics of Plant-Nematode Interactions. Berlin,Germany: Springer, 157–174.

FernandezO, Bethencourt L, Quero A, Sangwan RS, Clement C. 2010.Trehalose

and plant stress responses: friend or foe? Trends in Plant Science 15: 409–417.Flores HE, Dai YR, Cuello JL, Maldonadomendoza IE, Loyolavargas VM. 1993.

Green roots –photosynthesis and photoautotrophy in an underground plant

organ. Plant Physiology 101: 363–371.Gheysen G, Mitchum MG. 2009.Molecular insights in the susceptible plant

response to nematode infection. Plant Cell Monogram 15: 45–81.Gheysen G, MitchumMG. 2011.How nematodes manipulate plant development

pathways for infection. Current Opinion in Plant Biology 14: 415–421.Goverse A, Bird D 2011. The role of plant hormones in nematode feeding cell

formation. In: Jones J,GheysenG, FenollC, eds.Genomics andMolecularGeneticsof Plant-Nematode Interactions. Dordrecht, the Netherlands: Springer, 325–348.

Goverse A, Overmars H, Engelbertink J, Schots A, Bakker J, Helder J. 2000. Both

induction and morphogenesis of cyst nematode feeding cells are mediated by

auxin.Molecular Plant–Microbe Interactions 13: 1121–1129.Grunewald W, Cannoot B, Friml J, Gheysen G. 2009a. Parasitic nematodes

modulate PIN-mediated auxin transport to facilitate infection. PLoS Pathogens 5:e1000266.

Grunewald W, van Noorden G, van Isterdael G, Beeckman T, Gheysen G,

Mathesius U. 2009b.Manipulation of auxin rransport in plant roots during

Rhizobium symbiosis and nematode parasitism. Plant Cell 21: 2553–2562.HardcastleTJ,KellyKA.2010.baySeq:Empirical Bayesianmethods for identifying

differential expression in sequence count data. BMC Bioinformatics 11: 422.Hirose N, Makita N, Kojima M, Kamada-Nobusada T, Sakakibara H. 2007.

Overexpression of a type-A response regulator alters rice morphology and

cytokinin metabolism. Plant and Cell Physiology 48: 523–539.Hofmann J, El Ashry A, Anwar S, Erban A, Kopka J, Grundler F. 2010.Metabolic

profiling reveals local and systemic responses of host plants to nematode

parasitism. Plant Journal 62: 1058–1071.Ithal N, Recknor J, Nettleton D, Hearne L, Maier T, Baum TJ, Mitchum MG.

2007a. Parallel genome-wide expression profiling of host and pathogen during

soybean cyst nematode infectionof soybean.MolecularPlant–Microbe Interactions20: 293–305.

Ithal N, Recknor J, Nettleton D, Maier T, Baum TJ, MitchumMG. 2007b.

Developmental transcript profiling of cyst nematode feeding cells in soybean

roots.Molecular Plant–Microbe Interactions 20: 510–525.Jammes F, Lecomte P, Almeida-Engler J, Bitton F,Martin-MagnietteML,

Renou JP,AbadP, FaveryB. 2005.Genome-wide expressionprofiling of the host

response to root-knot nematode infection in Arabidopsis. Plant Journal 44: 447–458.

Jennings C, West J, Waine C, Craik D, Anderson M. 2001. Biosynthesis and

insecticidal properties of plant cyclotides: the cyclic knotted proteins from

Oldenlandia affinis. Proceedings of the National Academy of Sciences, USA 98:

10614–10619.Jorda L, Vera P. 2000. Local and systemic induction of two defense-related

subtilisin-like protease promoters in transgenic Arabidopsis plants. Luciferin

induction of PR gene expression. Plant Physiology 124: 1049–1057.Karczmarek A, Overmars H, Helder J, Goverse A. 2004. Feeding cell development

by cyst and root-knot nematodes involves a similar early, local and transient

activation of a specific auxin-inducible promoter element.Molecular PlantPathology 5: 343–346.

KimSY, VolskyDJ. 2005. PAGE: parametric analysis of gene set enrichment.BMCBioinformatics 6: 144.

Kim TH, Kim MC, Park JH, Han SS, Kim BR, Moon BY, Suh MC, Cho SH.

2006. Differential expression of rice lipid transfer protein gene (LTP) classes in

response to abscisic acid, salt, salicylic acid, and the fungal pathogenMagnaporthegrisea. Journal of Plant Biology 49: 371–375.

Klink VP, Overall CC, Alkharouf NW, MacDonald MH, Matthews BF. 2007. A

time-course comparative microarray analysis of an incompatible and compatible

response byGlycine max (soybean) toHeterodera glycines (soybean cyst nematode)

infection. Planta 226: 1423–1447.Kyndt T, Denil S, Haegeman A, Trooskens G, De Meyer T, Van Criekinge W,

GheysenG. 2012a.Transcriptome analysis ofmature rice root tissue and root tips

in early development by massive parallel sequencing. Journal of ExperimentalBotany 63: 2141–2157.

Kyndt T, Nahar K, Haegeman A, De Vleesschauwer D, Hofte M, Gheysen G.

2012b. Comparing the defence-related gene expression changes upon migratory

and sedentary nematode attack in rice. Plant Biology 14: 73–82.Li Y, Fester T, Taylor CG 2008. Transcriptomic analysis of nematode infestation.

In: Berg RH, Taylor C, eds. Cell biology of plant nematode parasitism. Dordrecht,

the Netherlands: Springer, 188–220.LoharDP, Schaff JE, Laskey JG,Kieber JJ, BilyeuKD,BirdDM.2004.Cytokinins

play opposite roles in lateral root formation, and nematode and Rhizobial

symbioses. Plant Journal 38: 203–214.NaharK,KyndtT,HauseB,HofteM,GheysenG. 2012.Brassinosteroids suppress

rice defense against root knot nematodes through antagonismwith the jasmonate

pathway.Molecular Plant–Microbe interactions, in press.

Nahar K, Kyndt T, De Vleesschauwer D, Hofte M, Gheysen G. 2011. The

jasmonate pathway is a key player in systemically induced defense against root

knot nematodes in rice. Plant Physiology 157: 305–316.Peng Y, Bartley LE, Chen XW, Dardick C, Chern MS, Ruan R, Canlas PE,

Ronald PC. 2008. OsWRKY62 is a negative regulator of basal and Xa21-

mediated defense against Xanthomonas oryzae pv. oryzae in rice. Molecular Plant1: 446–458.

PieterseCMJ,Leon-ReyesA,VanderEnt S,VanWees SCM.2009.Networkingby

small-molecule hormones in plant immunity. Nature Chemical Biology 5: 308–316.

Puthoff DP, Nettleton D, Rodermel SR, Baum TJ. 2003. Arabidopsis gene

expression changes during cyst nematodeparasitism revealedby statistical analyses

of microarray expression profiles. Plant Journal 33: 911–921.Reversat G, Boyer J, Sannier C, Pando-Bahuon A. 1999.Use of a mixture of sand

and water-absorbent synthetic polymer as substrate for the xenic culturing of

plant-parasitic nematodes in the laboratory. Nematology 1: 209–212.Richards DE, King KE, Ait-ali T, Harberd NP. 2001.How gibberellin regulates

plant growth and development: a molecular genetic analysis of gibberellin -

signaling. Annual Review of Plant Physiology and Plant Molecular Biology 52:67–88.

Robinson MD, Oshlack A. 2010. A scaling normalization method for differential

expression analysis of RNA-seq data. Genome Biology 11: R25.Scheller HV, Ulvskov P. 2010.Hemicelluloses. Annual Review of Plant Biology 61:263–289.

Surget-Groba Y, Montoya-Burgos JI. 2010.Optimization of de novo

transcriptome assembly from next-generation sequencing data. Genome Research20: 1432–1440.

Szakasits D,Heinen P,Wieczorek K,Hofmann J,Wagner F, Kreil DP, Sykacek P,

Grundler FMW, BohlmannH. 2009.The transcriptome of syncytia induced by

the cyst nematode Heterodera schachtii in Arabidopsis roots. Plant Journal 57:771–784.

ThimmO, Blasing O, Gibon Y, Nagel A,Meyer S, Kruger P, Selbig J, Muller LA,

Rhee SY, Stitt M. 2004.MAPMAN: a user-driven tool to display genomics data

sets onto diagrams of metabolic pathways and other biological processes. PlantJournal 37: 914–939.

Trapnell C, Pachter L, Salzberg SL. 2009. TopHat: discovering splice junctions

with RNA-Seq. Bioinformatics 25: 1105–1111.TrapnellC,WilliamsBA, PerteaG,Mortazavi A,KwanG, vanBarenMJ, Salzberg

SL,Wold BJ, Pachter L. 2010.Transcript assembly and quantification by RNA-

Seq reveals unannotated transcripts and isoform switching during cell

differentiation. Nature Biotechnology 28: 511–515.Walters DR, McRoberts N. 2006. Plants and biotrophs: a pivotal role for

cytokinins? Trends in Plant Science 11: 581–586.Yamamoto Y, Kamiya N, Morinaka Y, Matsuoka M, Sazuka T. 2007.

Auxin biosynthesis by the YUCCA genes in rice. Plant Physiology 143: 1362–1371.

Zerbino DR, Birney E. 2008. Velvet: Algorithms for de novo short read assembly

using de Bruijn graphs. Genome Research 18: 821–829.



Supporting Information

Additional Supporting Information may be found in the onlineversion of this article.

Table S1 Chromosomal location and primer pairs of the loci thatwere independently experimentally validated by qRT-PCR

Table S2 List of 4684 putative novel transcriptionally activeregions identified in nematode-infected rice root tissues, with theirgenomic location, the match found in the rice root transcriptomedata of Kyndt et al. (2012a), BLASTn results against cDNA andgenomic DNA of Oryza sativa cv ‘Japonica’, BLASTx resultsagainst rice proteins, SwissProt/trEMBL

Table S3 Results of parametric analysis of gene set enrichment oftranscriptome data of root knot- or migratory root rot nematode-infected rice roots at 3 (early) or 7 d (late) after infection, showingthe fivemost important up-regulated andfivemost down-regulatedGO terms

Table S4 List of loci that are significantly differentially expressed inrice root tissues upon infection with M. graminicola (Mg) orH. oryzae (Ho), at 3 or 7 dai, including locus number or nTARnumber, MSU6 annotation and the log2FC between infected andhealthy tissues

Table S5 List of 382 loci that are significantly differentiallyexpressed between root galls and uninfected root tips, but notdifferentially expressed upon root rot nematode infection, includ-ing locus number or nTAR number, MSU6 annotation and thelog2FC between infected and healthy tissues

Table S6 List of 578 loci that are significantly differentiallyexpressed between migratory root rot nematode-infected rice rootsand healthy roots, but not differentially expressed upon root knotnematode infection, including locus number or nTAR-number,MSU6 annotation and the log2FC between infected and healthytissues

Notes S1 Schematic representation and more detailed explanationof the bioinformatics workflow that was followed during theanalysis of mRNA-Seq data obtained from nematode-infected anduninfected rice root tissues.

Please note: Wiley-Blackwell are not responsible for the content orfunctionality of any supporting information supplied by theauthors. Any queries (other than missing material) should bedirected to the New Phytologist Central Office.



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