Seediscussions,stats,andauthorprofilesforthispublicationat:https://www.researchgate.net/publication/5508208

Associationofthetranscriptionalresponseofsoybeanplantswithsoybeanmosaicvirussystemicinfection.JGenVirol

ArticleinJournalofGeneralVirology·April2008

ImpactFactor:3.18·DOI:10.1099/vir.0.83531-0·Source:PubMed

CITATIONS

39

READS

52

4authors:

MohanBabu

UniversityofToronto

149PUBLICATIONS2,107CITATIONS

SEEPROFILE

AllaGagarinova

UniversityofToronto

15PUBLICATIONS386CITATIONS

SEEPROFILE

JimBrandle

VinelandResearchandInnovationCentre

83PUBLICATIONS2,117CITATIONS

SEEPROFILE

AimingWang

AgricultureandAgri-FoodCanada

138PUBLICATIONS1,479CITATIONS

SEEPROFILE

Allin-textreferencesunderlinedinbluearelinkedtopublicationsonResearchGate,

lettingyouaccessandreadthemimmediately.

Availablefrom:MohanBabu

Retrievedon:12May2016

Association of the transcriptional response ofsoybean plants with soybean mosaic virus systemicinfection

Mohan Babu,13 Alla G. Gagarinova,1,23 James E. Brandle1

and Aiming Wang1,2

Correspondence

Aiming Wang

wanga@agr.gc.ca

1Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada (AAFC),1391 Sandford St, London, ON N5V 4T3, Canada

2Department of Biology, The University of Western Ontario, Biological and Geological Building,1151 Richmond St, London, ON N6A 5B7, Canada

Received 18 October 2007

Accepted 24 December 2007

Compatible virus infection induces and suppresses host gene expression at the global level.

These gene-expression changes are the molecular basis of symptom development and general

stress and defence-like responses of the host. To assess transcriptional changes in soybean

plants infected with soybean mosaic virus (SMV), the first soybean trifoliate leaf, immediately

above the SMV-inoculated unifoliate leaf, was sampled at 7, 14 and 21 days post-inoculation

(p.i.) and subjected to microarray analysis. The identified changes in gene expression in soybean

leaves with SMV infection at different time points were associated with the observed symptom

development. By using stringent selection criteria (¢2- or ¡”2-fold change and a Q value of

¡0.05), 273 (1.5 %) and 173 (0.9 %) transcripts were identified to be up- and downregulated,

respectively, from 18 613 soybean cDNAs on the array. The expression levels of many transcripts

encoding proteins for hormone metabolism, cell-wall biogenesis, chloroplast functions and

photosynthesis were repressed at 14 days p.i. and were associated with the highest levels of viral

RNA in the host cells. A number of transcripts corresponding to genes involved in defence were

either downregulated or not affected at the early stages of infection, but upregulated at the

late stages, indicating that the plant immune response is not activated until the late time points of

infection. Such a delayed defence response may be critical for SMV to establish its systemic

infection.

INTRODUCTION

Systemic infections of plant viruses result from thecomplex molecular interplay between the host plant andthe invading virus (Maule et al., 2000; Golem & Culver,2003; Whitham et al., 2003; Pompe-Novak et al., 2005).The magnitude of physiological and phenotypic changes inthe host during viral infection suggests the involvement ofa large number of host genes (Golem & Culver, 2003;Whitham et al., 2006). Thus, the intimate interactionbetween a plant virus and its host is complicated by thesystemic nature of infection and global alterations in host

gene expression (Maule et al., 2002; Whitham & Wang,2004). Dissecting the plant gene-expression network thatoccurs in response to virus infection should assist in abetter understanding of the infection process and ulti-mately in the control of plant viruses. To achieve this,microarray technology has been adopted to profile globalgene expression of the host (mainly model plant species)infected with several positive-sense RNA viruses (Golem &Culver, 2003; Whitham et al., 2003; Ishihara et al., 2004;Marathe et al., 2004; Pompe-Novak et al., 2005; Senthilet al., 2005; Dardick, 2007; Shimizu et al., 2007; Yang et al.,2007).

Soybean mosaic virus (SMV) is a member of the genusPotyvirus in the family Potyviridae, which is the largestplant virus family. SMV is the most prevalent viralpathogen of soybean [Glycine max (L.) Merr.] in the world.Infection by SMV usually causes yield losses of between 35and 50 % under natural field conditions and of up to 50–100 % in severe outbreaks (Arif & Hassan, 2002; Liao et al.,2002). SMV has a single-stranded, positive-sense [ss(+)]RNA genome approximately 9600 nt in length (Jayaram

3Present address: Department of Molecular Genetics, University ofToronto, Toronto M5S 1A8, Canada.

The NCBI Gene Expression Omnibus (GEO) accession numbers for themicroarray data from this work are GSE9824, GPL6258, GSM247941,GSM247942 and GSM247943.

A supplementary figure showing confirmation of microarray data byNorthern hybridizations, as well as five supplementary tables andreferences cited therein, are available with the online version of thispaper.

Journal of General Virology (2008), 89, 1069–1080 DOI 10.1099/vir.0.83531-0

0008-3531 G 2008 Agriculture and Agri-Food Canada Printed in Great Britain 1069

et al., 1992). Whilst the virus itself has been relatively wellstudied, the molecular mechanisms underlying soybeanresponses to SMV infection remain poorly understood.

In this work, we report analysis of gene expression in SMV-infected soybean plants during the course of infection. Themost pronounced changes in gene expression occurredwhen viral RNA accumulation reached the highest leveland the leaf underwent moderate symptom development.The SMV-induced and -repressed transcripts were clas-sified based on their putative functions and clusteredaccording to their expression patterns. A cross-sequencecomparison of the transcriptional response to SMV and toother ss(+) RNA viruses identified transcripts withsimilar changes in gene expression. Cumulatively, thegene-expression analysis presented in this study revealscorrelations between host gene-expression changes anddisease-symptom development in soybean.

METHODS

Plant materials, virus inoculation and RNA extraction. Soybean[G. max (L.) Merr.] ‘Williams 82’ plants were maintained in a

greenhouse at 80 % relative humidity with a day/night regime of 16 h

light at 24 uC followed by 8 h dark at 16 uC. A Canadian SMV isolate

(strain G2) was used as the inoculum. The unifoliate leaves of 10-day-

old soybean plants were rub-inoculated with SMV and control plants

were mock-inoculated. The first trifoliate leaf immediately above the

SMV-inoculated unifoliate leaf was harvested at 7, 14 and 21 days

post-inoculation (p.i.) for RNA extraction. Leaf-to-leaf and plant-to-

plant variation was minimized by pooling the total RNA isolated from

the first trifoliate leaf of at least 25 plants for each time point. Three

independent biological replicates for each time point were performed.

All of the mock- and SMV-inoculated soybean plants used in the

microarray experiment were randomized and grown in different

locations within a greenhouse under the same growth conditions.

Total RNA isolation from soybean leaves was carried out by using

TRIzol reagent (Invitrogen). The isolated total RNA was purified by

using RNeasy Mini columns (Qiagen). SMV was detected by RT-PCR

and quantified by Northern blot analysis.

Microarray probe labelling, hybridization, scanning and data

analysis. Soybean cDNA microarrays (18K A series) containing

18 613 soybean cDNAs of low redundancy were obtained from Dr Lila

Vodkin (University of Illinois, Urbana–Champaign, IL, USA). The

GEO accession number of these expressed sequence tags (ESTs) is

GPL3015. Two separate cDNA-labelling reactions (Cy3 and Cy5), one

for the SMV-infected leaf and the other for the mock-inoculated leaf,

were carried out. In total, 18 microarray slides were used for this

study: six array hybridizations, including three reciprocal labelling

experiments from three independent biological replications of either

SMV-infected or mock-inoculated leaf, at each of the three time

points. Dye swaps were performed to ensure that the results were not

biased by dye effects. Total RNA (10 mg), isolated from each of the

virus-infected and mock-inoculated leaf tissues at different time

points, was labelled with either Cy3 or Cy5 fluorescent dye by using a

CyScribe post-labelling kit (Amersham Biosciences). Synthesized

probes were purified by CyScribe GFX (Amersham Biosciences) and

hybridized to soybean chips following the manufacturer’s protocol.

Subsequent processing of the slides was essentially as described by

Moy et al. (2004). The quantified data extracted from the 16-bit TIFF

images from ArrayVision v. 6.0 (Imaging Research) were background-

subtracted and then analysed with GeneSpring microarray-analysis

software version 7.3 (Silicon Genetics), where LOWESS normaliza-tion was used to correct for any spatial or intensity-dependent biaseswithin each array. Data analysis was essentially as described by Senthilet al. (2005). In brief, the mean normalized signal-intensity values foreach transcript were calculated from six replicate hybridizations(three biological replicates and a dye-swap hybridization for eachbiological replicate) for each time point. log2 ratios were thencalculated as described in the GeneSpring microarray-analysisinstruction manual, where the mean of normalized signal-intensityvalues from the virus-infected samples was divided by the mean ofrespective values from control samples (Cy5-infected/Cy3-control orCy3-infected/Cy5-control in a dye-swap experiment). The foldchanges of differentially regulated transcripts in virus-infectedsamples compared with the control samples were calculated basedon these ratios. To select transcripts with significant changes duringvirus infection, P values (P¡0.05) derived from ANOVA wereadjusted by using the multiple testing correction of Benjamini &Hochberg (1995) with a 5 % false-discovery rate (FDR), correspond-ing to a Q value of ¡0.05 (Storey & Tibshirani, 2003). This multipletesting correction procedure is used to correct for the occurrence offalse positives and to maximize the likelihood of finding significantgene sets. In addition to these statistical criteria, we searched fortranscripts that showed significant up- (¢2.0-fold) or down-(¡22.0-fold) regulation at at least one time point. Expressionprofiles from each time point were clustered based on their similarityin expression pattern by using a hierarchical average linkageclustering algorithm and Pearson correlation distance metric,implemented in the GeneSpring v. 7.3 software. A gene tree heatmap was also built based on this analysis.

Functional categorization of transcripts. The 59 and 39 sequencesof each differentially expressed soybean unique sequence of expressedsequence tag (uniEST) were queried against the non-redundant (nr)protein database by using the BLASTX algorithm (Altschul et al., 1997).In addition, we identified the protein in the Dana Farber CancerInstitute (DFCI) Soybean Gene Index database (http://compbio.df-ci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=soybean), using thehighest BLASTX score to identify the function of each nr uniEST.Significantly induced and repressed soybean transcripts were groupedinto functional groups by performing searches using annotatedsoybean transcripts against the Arabidopsis MIPS (MunichInformation Center for Protein Sequences) functional classificationscheme to create a custom set of functional gene categories andsubcategories. Transcripts with no matches in the database werelabelled as ‘unknown’. Both x2 and Fisher’s exact tests were carriedout to confirm the significance on the representation of the numbersof genes in each functional category at each time point (P¡0.05).Statistical measurement to calculate the significant category wasperformed as described by Draghici et al. (2003).

Cross-sequence comparison of transcripts regulated differ-

entially in soybean infected with SMV and in plants infected

with other ss(+) RNA viruses. The EST sequences of soybeantranscripts regulated differentially in SMV-infected leaf tissues werecross-compared by BLAST-searching against the sequences of thetranscripts that were significantly differentially expressed in responseto infection by other ss(+) RNA viruses (Golem & Culver, 2003;Whitham et al., 2003; Ishihara et al., 2004; Marathe et al., 2004;Dardick, 2007; Yang et al., 2007). Sequence searches were performedby using the TBLASTX algorithm with default settings. The resultingBLAST output for each transcript was then parsed for high-scoring pair(HSP) and E value. Any hits with the existence of HSP¢100 andE¡10220 were indicative of significant similarity (Rubin et al., 2000).

Northern hybridization. Three independent Northern blots wereconducted to validate the microarray data. Total RNA (10 mg) wasseparated and transferred to a nylon membrane. RT-PCR products

M. Babu and others

1070 Journal of General Virology 89

derived from primers (given in Supplementary Table S1, available in

JGV Online) were used as a probe. The probes were labelled with[a-32P]dCTP by using a Ready-To-Go DNA-labelling kit (Amersham

Biosciences). A phosphorimager and QualityOne quantification v. 4.2

software (Bio-Rad) were used for visualization and quantification of

radioactive signals.

RESULTS AND DISCUSSION

Symptoms of SMV infection and viral RNAaccumulation in soybean

The unifoliate leaf (the first true leaf) of 10-day-oldsoybean plants was inoculated mechanically with SMV. At7 days p.i., the earliest symptoms appeared, analogous tomild mosaic in the first trifoliate leaf (Fig. 1a). Moderatemosaic and vein-clearing symptoms were evident in thetrifoliate leaf at approximately 14 days p.i. Symptoms ofthe trifoliate leaf turned to severe mosaic and mildmottling at about 21 days p.i. (Fig. 1a). Accumulation ofviral RNA was detected by Northern hybridizations in thefirst trifoliate leaf at 7 days p.i., reached its peak level at14 days p.i. and then decreased by 1.7-fold (58 % of themaximum viral RNA level) at 21 days p.i. (Fig. 1b).

Experimental design and data normalization forconsistency of the microarray data

Reliability of the microarray data was assessed bynormalizing the mean signal intensities of mock-inoculatedsamples against all replicates for each time point on ascatter plot (data not shown). A high correlation coefficient(0.90–0.93) among mock-inoculated samples for each timepoint was observed, indicating low technical and biologicalvariability. In addition, variation differences between dye-swap experiments from mock-inoculated samples resultedin a correlation coefficient of 0.90–0.91 for each time point,indicating that dye effects were insignificant.

Identification of soybean transcripts regulateddifferentially over time

Gene-expression data were analysed by one-way ANOVAto identify differentially expressed transcripts. By usingstringent criteria with a 5 % FDR that corresponds to a Qvalue of ¡0.05, we identified approximately 4.8 % nrtranscripts (894 of the 18 613 soybean transcripts printedon the chip) that were significantly differentially expressed(induced or repressed) in response to SMV infection atthree different time points. These significantly differentiallyregulated transcripts were filtered further by using a 2.0-fold increase or a 22.0-fold decrease in signal intensity. At7 days p.i., 62 nr transcripts were induced and 46 wererepressed by SMV; at 14 days p.i., 154 were induced and 90repressed; at 21 days p.i., 100 were induced and 45repressed. This final filtering resulted in 316 transcripts(representing 273 nr transcripts) that were induced by¢2.0-fold, and 181 transcripts (representing 173 nr

transcripts) that were repressed by ¡22.0-fold, as illustratedby Venn diagrams (Fig. 2a, b). Comparison analysis betweenthe 273 induced and the 173 repressed nr transcriptsidentified 168 transcripts that were significantly inducedonly at one time point but not significantly repressed at othertime points, 68 transcripts significantly repressed only at onetime point but not significantly induced at other time points

Fig. 1. Symptom development and SMV detection in systematic-ally infected soybean trifoliate leaves at different times post-inoculation (days p.i.). (a) The left panel shows symptomdevelopment in the first trifoliate leaf of soybean plants in whichthe unifoliate leaf was inoculated mechanically with SMV and theright panel shows the corresponding leaf from mock-inoculatedcontrol plants. (b) Northern blot hybridizations showing viral RNAaccumulation in the infected trifoliate leaf. The actin gene(GenBank accession no. AI494739) was used as an internalcontrol to normalize the differences in total RNA samples loaded.The relative amount of SMV genomic RNA after normalization isgiven as a percentage.

Transcriptional response of SMV-infected soybean

http://vir.sgmjournals.org 1071

and 105 transcripts significantly induced and repressed atdifferent time points (Fig. 2c).

Functional classification of soybean transcriptsinduced and repressed in SMV-infected leaftissues

Soybean transcripts induced or repressed during SMVinfection were classified according to the predictedfunctions by using BLASTX searches against the NCBI nrdatabase. These transcripts were grouped further, essen-tially following the method of the Arabidopsis MIPSfunctional classification scheme. With the information

gathered from the MIPS Arabidopsis database, we wereable to assign putative functions to 63.2 % of the 18 613transcripts on the slide, whilst the remaining 36.8 % (6854transcripts) with no matches in the database were classifiedinto the ‘unknown’ category (Table 1). uniESTs wereassigned with a putative function only if the best matchhad an E value of ¡10220. Based on the putative functions,the differentially expressed transcripts were classified into 11functional categories (Fig. 3; see Supplementary Tables S2,S3 and S4, available in JGV Online). In four instances,subcategories were created to interpret the data better. x2 andFisher’s exact tests suggested that the majority of trans-cripts are associated with defence, chromatin regula-tion and cytoskeleton reorganization, protein synthesisand translation, metabolism (including cell wall-relatedand sulphur-assimilation transcripts) and development/storage proteins (including hormone-, chloroplast- andphotosynthesis-related transcripts), comprising approxi-mately 60 % of all differentially regulated transcripts changedsignificantly at a P value of ¡0.05 (Table 2; Fig. 3).

To visualize expression profiling of the transcripts at allthree time points, hierarchical clustering was employed,using the 273 induced and 173 repressed transcripts (Fig. 4).A gene tree heat map was built by using the Pearsoncorrelation distance metric with an average linkage cluster-ing algorithm. Horizontal time points, representing differ-ences in expression of transcripts, were plotted against thevertical grouping, corresponding to expression similarity.Six distinct groups of expression pattern were identifiedfrom the clustering analysis. A complete list of genes inducedor repressed in different cluster groups, along with theirexpression levels and putative functions, is provided inSupplementary Table S2 (available in JGV Online).

Clustering analysis also revealed that the greatest changesin gene expression took place in soybean leaves withmoderate mosaic symptoms (14 days p.i.), whilst fewerdifferentially expressed transcripts were identified at theearlier or later stages when infected leaves displayed mild orsevere mosaic symptom development. Thus, the magnitudeof host gene-expression changes correlated with theamount of viral RNA detected in the leaves. Theseobservations may reflect specific host responses to activevirus replication and spread, apparently peaking at 14 daysp.i. and declining thereafter. Another intriguing aspect isthe large shift in host gene-expression patterns observedbetween 14 and 21 days p.i., coinciding with the onset ofmore severe disease symptoms in the infected leaves.

Confirmation of microarray data by Northernhybridization

As most gene changes occurred at 14 and 21 days p.i., ninetranscripts either up- or downregulated significantly at 14 or21 days p.i. were selected for Northern hybridizations tovalidate the microarray data. Overall expression patterns ofall nine transcripts analysed by Northern blotting wereconsistent with those obtained by microarray hybridizations

Fig. 2. Venn diagrams showing the differential distribution ofinduced (¢2.0-fold) and repressed (¡”2.0-fold) transcripts inSMV-infected leaf tissues at three different time points. Astatistical cut-off with a 5 % FDR corresponding to a Q value of¡0.05 was used to determine whether transcripts were differ-entially expressed. Number of transcripts shown in the non-overlapping sectors represents unique significant transcripts ateach time point, whereas transcripts within the intersectionrepresent the number of shared significant transcripts. (a) nrtranscripts induced significantly by ¢2.0-fold at at least one timepoint (n5273); (b) nr transcripts repressed significantly by¡”2.0-fold at one time point at least (n5173); (c) overlappingof 273 and 173 nr transcripts that were induced and repressedsignificantly by SMV infection, respectively.

M. Babu and others

1072 Journal of General Virology 89

(Fig. 5; Supplementary Fig. S1, available in JGV Online). Therelative expression ratios of the transcripts analysed byNorthern hybridizations were lower than those frommicroarray hybridizations. This could be attributed to

methodological variations, for example, normalization toactin expression in Northern blotting compared with a moreglobal normalization method used in microarray hybridiza-tions (Taniguchi et al., 2001; Yao et al., 2004).

Table 1. Assignment of up- and downregulated transcripts that change in expression by ¢2- or ¡”2-fold in each functional class

Functional category* No. transcripts on

the arrayD

No. (%) transcripts up- or downregulated

Upregulated (¢2.0-fold) Downregulated (¡”2.0-fold)

A Defence and virulence 797 34 (3.01) 16 (2.01)

B Cellular communication or signal-transduc-

tion mechanism

917 19 (2.07) 11 (1.20)

C Chromatin regulation and cytoskeleton

reorganization

192 10 (5.21) 2 (1.04)

D Transcription 627 13 (2.07) 9 (1.44)

E Protein synthesis and translation 287 13 (4.53) 5 (1.74)

F Transporters 295 8 (2.71) 5 (1.69)

G Metabolism 3607 76 (1.95) 32 (0.82)

G1 Cell wall-related 389 10 (2.57) 10 (2.57)

G2 Sulphur assimilation 286 13 (4.54) 4 (1.39)

H Protein fate 395 11 (2.78) 4 (1.01)

I Proteins with binding function or cofactor

requirement

1385 19 (1.37) 9 (0.65)

J Development and storage proteins 1662 12 (0.72) 7 (0.42)

J1 Hormone-related 764 4 (0.52) 6 (0.79)

J2 Chloroplast and photosynthesis 156 11 (7.05) 22 (14.10)

K Unknown 6854 43 (0.63) 35 (0.51)

*Soybean transcripts were categorized following the method of the Arabidopsis MIPS (http://mips.gsf.de/proj/thal/db/) functional classification

scheme.

DNumber of transcripts in a given category represented on the chip.

Fig. 3. Functional distribution of soybeantranscripts significantly induced and repressedin SMV-infected soybean leaf tissues at eachtime point. Bars represent the number oftranscripts downregulated (¡”2.0-fold) orupregulated (¢2.0-fold) in each functionalcategory at each time point. Text in boldindicates subcategories created under a majorfunctional category.

Transcriptional response of SMV-infected soybean

http://vir.sgmjournals.org 1073

Table 2. Statistical analysis to determine the enriched functional categories at each time point post-infection using x2 and Fisher’sexact tests

Functional category* Time p.i.

(days)

No. differentially regulated

transcriptsD

x2d x2 P value§ Fisher’s exact test§

Observed Expected

A Defence and virulence 7 8 5 1.8460 1.74E201 1.44E201

14 21 11 9.8373 1.70E”03 3.66E”03

21 14 6 8.7852 3.00E”03 5.74E”03

B Cellular communication or signal

transduction mechanism

7 6 5 0.0087 9.26E201 6.55E201

14 17 12 1.7894 1.81E201 1.36E201

21 9 7 0.2868 5.92E201 4.37E201

C Chromatin regulation and

cytoskeleton reorganization

7 8 1 33.9443 1.00E”04 2.63E”05

14 5 3 1.3553 2.44E201 1.95E201

21 1 2 0.0011 9.73E201 1.00E+00

D Transcription 7 7 4 2.2263 1.36E201 1.00E201

14 9 8 0.0048 9.45E201 7.22E201

21 9 5 2.6341 1.05E201 1.00E201

E Protein synthesis and translation 7 3 2 0.3618 5.48E201 2.47E201

14 13 4 18.7031 1.00E”04 2.00E”04

21 5 2 2.0977 1.48E201 8.37E202

F Transporters 7 2 2 0.0394 8.43E201 6.96E201

14 8 4 3.0842 7.91E202 6.69E202

21 3 2 0.0077 9.30E201 5.14E201

G Metabolism 7 28 19 5.6949 1.70E”02 1.94E”02

14 63 41 13.2049 3.00E”04 4.04E”04

21 35 24 4.9621 2.59E”02 2.54E”02

G1 Cell wall-related 7 8 2 11.6873 6.00E”04 2.43E”03

14 11 5 5.3144 2.11E”02 2.24E”02

21 5 3 0.6362 4.25E201 2.44E201

G2 Sulphur assimilation 7 2 2 0.0259 8.72E201 6.89E201

14 10 4 8.0983 4.40E”03 6.45E”03

21 5 2 2.1198 1.45E201 8.28E202

H Protein fate 7 3 2 0.0103 9.19E201 5.10E201

14 8 5 0.9171 3.38E201 2.62E201

21 6 3 1.7697 1.83E201 1.39E201

I Proteins with binding function or

cofactor requirement

7 6 8 0.2283 6.33E201 7.07E201

14 15 18 0.2655 6.06E201 6.18E201

21 13 10 0.4237 5.15E201 4.18E201

J Development and storage proteins 7 5 10 2.4173 1.20E201 1.68E201

14 7 23 11.6195 7.00E”04 6.79E”04

21 9 14 1.4436 2.30E201 3.72E201

J1 Hormone-related 7 1 4 2.0240 1.55E201 1.36E201

14 3 10 4.4086 3.58E”02 2.07E”02

21 6 6 0.0377 8.46E201 1.00E+00

J2 Chloroplast and photosynthesis 7 6 1 21.5481 1.00E”04 4.25E”04

14 22 2 156.8722 1.00E”04 2.80E”15

21 7 1 21.0469 1.00E”04 3.43E”04

K Unknown 7 20 30 4.1533 4.16E”02 3.12E”02

14 43 68 11.9551 5.00E”04 3.04E”04

21 27 40 5.5897 1.81E”02 1.52E”02

M. Babu and others

1074 Journal of General Virology 89

Associations of differentially expressedtranscripts in general metabolism with virusinfection and symptom development

To gain insight into the spatial association of significantlydifferentially regulated transcripts with SMV infection andsymptom development, we examined transcripts related togeneral metabolism. Lipoxygenase (LOX) came to ourattention immediately, as seven of eight transcripts encodingLOX enzymes were repressed at 7 days p.i., with a mean of21.7-fold hybridization intensity (see Supplementary TableS3, available in JGV Online). Expression levels of these LOXtranscripts increased by a mean of 1.2-fold at 14 days p.i.and 2.6-fold at 21 days p.i. (Supplementary Table S3). LOXis a dioxygenase that catalyses the hydroperoxidation of

polyunsaturated fatty acids. Induction of LOX activity hasbeen consistently considered as a pathogen-induced defenceresponse, such as in potato infected by a fungal pathogen(Kolomiets et al., 2000). In virus–plant interactions, Arandaet al. (1996) showed that the level of LOX1 transcript wasdepleted significantly in pea embryo cells undergoing peaseed-borne mosaic virus replication, and recovered at ashorter distance behind the infection front when virusreplication declined. Based on the above study, LOX isprobably associated with virus infection. However, pastmicroarray studies on soybean have shown that LOXtranscripts change dramatically under diverse treatments(Thibaud-Nissen et al., 2003; Moy et al., 2004). Therefore, itis also possible that the expression changes of this genefamily may simply result from biotic stress.

*Soybean transcripts were categorized following the method of Arabidopsis MIPS (http://mips.gsf.de/proj/thal/db/) functional classification scheme.

DTranscripts that are significantly (Q¡0.05) upregulated (¢2.0-fold) or downregulated (¡22.0-fold) at each time point were used for this

analysis. Observed values are the total number of transcripts that were significantly differentially regulated at each time point; expected values are

derived based on the formula described by Draghici et al. (2003).

dx2 value is calculated based on the formula described by Draghici et al. (2003).

§Values highlighted in bold are the statistically significant functional group at a specfic time point with a P value of ¡0.05.

Table 2. cont.

Fig. 4. Hierarchical clustering of significantlydifferentially expressed soybean transcripts.Gene tree heat map consists of 273 up- and173 downregulated transcripts. Expressionvalues are colour-coded, with red indicatingupregulation (.1.0) by SMV infection, greenindicating downregulation (,1.0) and blackindicating no change (#1.0) in expression.The intensity of colour represents the degreeof gene-expression level. A complete list ofgenes in each cluster is given in SupplementaryTable S2 (available in JGV Online).

Transcriptional response of SMV-infected soybean

http://vir.sgmjournals.org 1075

Another interesting metabolic transcript repressed at7 days p.i., but induced at 14 days p.i., is a transcriptencoding callose synthase (GenBank accession no.AW278243). This enzyme is deposited as a layer betweenthe plasma membrane and the cell wall (Jacobs et al., 2003;Nishimura et al., 2003; Ueki & Citovsky, 2005). Beffa et al.(1996) showed that the increased deposition of callose inand around tobacco mosaic virus (TMV)-induced lesionsin b-1,3-glucanase-deficient tobacco plants resulted indecreased susceptibility to TMV. In the current study,repression of callose synthase at 7 days p.i. and upregula-tion of this gene at 14 days p.i. may be associated with adelayed resistance response in soybean (see SupplementaryTable S3, available in JGV Online).

An intriguing subset of transcripts that were inducedsubstantially at 14 days p.i. includes enzymes critical in theKrebs cycle, e.g. NADP-dependent malate dehydrogenase,cytosolic malate dehydrogenase and acetyl-CoA carboxylase;the oxidative pentose phosphate pathway, e.g. phospho-enolpyruvate/phosphate translocator; amino acid synthesis,such as amine oxidase, adenosine 5-phosphosulphatereductase, adenosylhomocysteinase and S-adenosylmethio-nine decarboxylase; sugar metabolism, e.g. sucrose synthase;and carbohydrate synthesis, e.g. b-amylase 1, mannosyloli-gosaccharide 1,2-a-mannosidase, GDP-mannose 3,5-epi-merase and glutamate carboxypeptidase. Previous studieshave suggested that the appearance of disease symptomssuch as chlorosis on cucumber mosaic virus (CMV)-infectedleaf tissues in cucumber is correlated with increasedglycolysis, phosphoenolpyuruvate phosphate, respiration

and starch accumulation, decreased photosynthesis andreduction in total protein synthesis (Tecsi et al., 1996). Theaccumulation of sugar and starch in virus-infected plants iscorrelated with symptom development, such as alteredcoloration in leaf, leaf distortion, chlorosis, mosaic andstunted phenotype (von Schaewen et al., 1990; Herbers et al.,1997). Based on the above findings, it is possible that hostgene-expression changes related to sugar, starch and aminoacid metabolism constitute part of the host response toinfection and that these changes may contribute to symptomdevelopment in soybean.

Repression of transcripts related to hormone,anti-oxidative metabolism, cell wall, sulphurassimilation, chloroplast and photosynthesis withSMV infection and symptom development

A wide range of symptoms in virus-infected plants can belinked to plant hormones, such as auxin or gibberellin(Padmanabhan et al., 2005, 2006; Culver & Padmanabhan,2007). Interaction between the TMV replicase and auxin/indole acetic acid proteins has been shown to affect auxin-mediated pathways and contribute to disease symptoms inArabidopsis (Padmanabhan et al., 2005, 2006). Transcriptsencoding auxin-repressed protein were downregulatedsignificantly in potato plants infected with potato virus Yat 7 days p.i. (Pompe-Novak et al., 2005). In rice plantsinfected with rice dwarf virus, the expression of agibberellin-biosynthetic enzyme, ent-kaurene oxidase, wasrepressed, leading to the appearance of dwarf phenotype

Fig. 5. Confirmation of relative expressionlevels of the transcripts selected from themicroarray analysis with Northern blot hybridi-zations. Nine transcripts were chosen tovalidate the microarray data. The GenBankaccession number of each transcript is pro-vided. The y-axis indicates the fold change ofthe transcripts from microarray (indicated byblack bars) and Northern (indicated by greybars) hybridizations. The x-axis represents timepost-inoculation (days). Signals for Northernhybridizations are from three biological repli-cates carried out in this study.

M. Babu and others

1076 Journal of General Virology 89

(Itoh et al., 2004; Zhu et al., 2005). In this study, fiveauxin-associated transcripts and one gibberellin-associatedtranscript were repressed in SMV-infected soybean leaftissues at 14 or 21 days p.i. (see Supplementary Table S4,available in JGV Online). It is not clear whether thesehormone-related gene changes also contribute to SMVsymptom development in soybean.

Reactive or activated oxygen species have been suggested tobe key mediators of local and systemic resistance responsesin incompatible plant–pathogen reactions and to beinvolved in symptom development and pathogenesis incompatible plant–virus interactions (Sandermann, 2000;Hernandez et al., 2004). In this study, the expression of twoanti-oxidative metabolism-related ESTs encoding catalase(CAT; GenBank accession no. AW349008) and glutathioneS-transferase (GST; GenBank no. AW472161) was sup-pressed significantly in the SMV-infected leaf at 14 daysp.i. Another key detoxification gene, superoxide dismutase(SOD), was downregulated significantly at 7 days p.i. Incontrast, two ESTs (GenBank accession nos AW471843 andAI938378) encoding peroxidase (PO) were inducedsignificantly at 7 days p.i. (by about 1.7-fold) and 14 daysp.i. (by .4-fold). Consistent with these transcriptionalprofiles, Zhuang et al. (1993) reported that SOD activitydecreased with an increase in PO activity in the SMV-infected soybean leaf. A decrease of CAT and SOD activityand an increase of PO activity were also found in manyother compatible host–virus interactions, such as inPhaseolus vulgaris infected with white clover mosaic virus(Clarke et al., 2002). Upregulation of PO transcriptsand downregulation of CAT, SOD and GST transcriptsmay induce an oxidative stress in the early infectionprocess. Such an anti-oxidative metabolism imbalancemay be associated with the progression of SMV infectionand symptom development, as suggested for the plumpox virus (PPV)–peach interaction (Hernandez et al.,2004).

The other interesting changes observed in this study werethe significant repression of cell wall-related genes. Thisgene group, containing several hundred different structuralproteins and cell wall-related enzymes, is known to be amajor determinant of cell morphogenesis in plants(Milioni et al., 2001; Huckelhoven, 2007). In SMV-infectedleaves, 10 cell wall-related genes encoding proteins forreassembly (six genes), matrix polymers (two genes) andexpansins (two genes) (see Supplementary Table S4,available in JGV Online) were downregulated significantlyat 14 days p.i. Downregulation of cell wall-related tran-scripts during SMV infection may be associated with areduction in cell wall cross-linking that contributes todisease-symptom development. This is consistent with therecent finding that cell wall-related transcripts weredownregulated significantly in Arabidopsis infected withturnip mosaic virus (TuMV) (Yang et al., 2007) and in riceinfected with rice dwarf virus (Shimizu et al., 2007). Inthese two studies, repression of cell wall-related transcriptswas correlated with symptom development.

Chlorosis is usually associated with susceptible interactionsin which virus replicates and moves throughout the plant.Chlorotic symptoms appear as yellowed areas in expandedleaves. In fully developed leaves, virus is largely confined tolight-green areas, where it seems to interfere withchloroplast structure, function and/or development(Culver et al., 1991). In this study, 18 chloroplast andfour photosynthesis transcripts belonging to the ‘chloro-plast and photosynthesis’ functional category were down-regulated in the SMV-infected leaf at 14 days p.i. (seeSupplementary Table S4, available in JGV Online).Recently, Yang et al. (2007) showed that a large fractionof the metabolic genes encoding chloroplast- and pho-tosynthesis-related proteins were repressed significantly inArabidopsis plants infected with TuMV. Downregulation ofphotosynthesis-related genes is also correlated with thedevelopment of infection symptoms, such as chlorosis,stunting or mosaic, in plants (Tecsi et al., 1994, 1996;Maule et al., 2002; Pompe-Novak et al., 2005; Espinozaet al., 2007).

In this study, 10 transcripts involved in sulphur assimila-tion and utilization were upregulated significantly at14 days p.i., followed by downregulation at 21 days p.i.These transcripts encode enzymes such as methioninesynthase, adenosine 5-phosphosulphate reductase, adeno-sylhomocysteinase and adenosylmethionine decarboxylase,which play a major role in sulphur uptake. Recently, Yanget al. (2007) also reported that TuMV infection suppressedexpression of genes involved in the sulphur-uptakepathway, affecting plant growth and development. Takentogether, these results suggest that gene-expression changesin hormone, cell-wall biogenesis, chloroplast, photosyn-thesis and sulphur-assimilation pathways may contributeto symptom development in SMV-infected soybean plants.

Association of upregulated transcripts in proteinsynthesis and disease resistance with SMVinfection

Translational regulation is a critical component of thecellular response to a variety of types of stress, such as viralinfection, nutrient deprivation and heat shock. In thecurrent study, 11 differentially regulated transcripts relatedto protein synthesis and translation were identified. Thesetranscripts, including two translation-elongation factorsand nine ribosomal transcripts, were affected slightly at7 days p.i., but upregulated substantially at 14 days p.i. (seeSupplementary Table S3, available in JGV Online). Similarinduction of ribosomal genes was also observed inNicotiana benthamiana infected with PPV (Dardick,2007) and in Arabidopsis infected with TuMV (Yang et al.,2007). It is not known whether the increased expression ofthese ribosomal proteins is a simple stress response tocompensate for the host cell that may lack sufficienttranslation components to maintain its viability, becausemany such components are hijacked by the virus for itsgenome translation and replication.

Transcriptional response of SMV-infected soybean

http://vir.sgmjournals.org 1077

Transcripts encoding proteins related to defence andvirulence were significantly over-represented at 14 and21 days p.i., but not at 7 days p.i. (Table 2), suggesting ageneral delayed defence in the SMV-infected leaf. Of the 24upregulated defence-related transcripts, a subset of 17defence-related transcripts, such as pathogenesis-related(PR) protein 3 (chitinase), GST10, HSP, SOD and PO,were either downregulated or slightly affected at 7 days p.i.,but substantially upregulated at 14 or 21 days p.i. (seeSupplementary Table S3, available in JGV Online). Thesetranscripts have been found to be vital in disease signalling,plant defence and stress responses (Cardinale et al., 2002;Marathe et al., 2004; Garcia-Brugger et al., 2006; Whithamet al., 2006). It is possible that, at late infection stages, thesoybean plant responds to SMV infection by expressingdefence-related genes, as in the case of other virus-infectedplants (Whitham et al., 2003; Senthil et al., 2005; Shimizuet al., 2007). Collectively, these data suggest that there is adelayed host defence response; the immune reponse insoybean plants is not activated until the relatively late stageof infection.

Association of a common set of inducedtranscripts in general stress- and defence-relatedcategories with infection by SMV and other ss(+)RNA viruses

To examine whether the diverse ss(+) RNA viruses havethe ability to elicit common gene-expression changes, across-sequence comparison analysis was performed. TheuniEST sequences of 894 significantly differentiallyexpressed (either up- or downregulated, regardless of foldchange) soybean transcripts identified in this study wereBLAST-searched against all of the differentially regulatedtranscripts identified in potato in response to infection bythree distinct fruit tree viruses, namely PPV, tomatoringspot virus (ToRSV) and Prunus necrotic ringspot virus(Dardick, 2007), and in Arabidopsis in response to infectionby CMV (Ishihara et al., 2004; Marathe et al., 2004), TuMV(Yang et al., 2007), TMV (Golem & Culver, 2003) and fivepositive-sense plant viruses including TuMV, oilseed rapemosaic virus, turnip vein clearing virus, potato virus X andCMV (Whitham et al., 2003). As a result, 107 uniquetranscripts were identified (see Supplementary Table S5,available in JGV Online).

The 107 unique transcripts were classified into four majorgroups. Group A contains 43 soybean transcripts inducedby SMV infection whose sequences match those oftranscripts induced by infection with other ss(+) RNAviruses. Six of them encode ribosomal proteins (three 40Sand three 60S ribosomal proteins). Group B includes 34soybean transcripts induced during SMV infection, butthese transcripts are repressed in plants infected by otherss(+) RNA viruses. For example, a transcript encodingplasma membrane ATPase (GenBank accession no.BE020672) was induced 1.3-fold by SMV infection at21 days p.i., but was downregulated 1.5-fold by CMV

infection at 6 h p.i. Three histone proteins related tochromatin regulation were induced approximately 1.2-foldby SMV infection at 7 days p.i. and were downregulatedapproximately 1.2-fold by ToRSV infection at 14 days p.i.Group C contains 19 SMV-induced soybean transcripts,each of which shares sequence similarity with more thanone transcript induced or repressed in plants infected withother ss(+) RNA viruses. One example is a transcriptinduced by SMV infection at 14 days p.i. encoding heat-shock protein 70 kDa (Hsp70) that shares sequencesimilarity with four Arabidopsis transcripts (AT5g02500,AT3g09440, AT3g12580 and AT5g02490) induced byTuMV infection at 10 days p.i. (Yang et al., 2007).Group D consists of 11 SMV-induced soybean transcripts.In this group, more than one SMV-induced soybeantranscript matches the sequence of a transcript induced orrepressed by infection with other ss(+) RNA viruses. Forexample, the sequences of two photosystem I transcripts(GenBank accession nos AI461105 and AI495711) inducedby SMV infection at 21 days p.i. match the sequence of anArabidopsis transcript (At4g12800) induced by CMVinfection at 12 h p.i. (Ishihara et al., 2004).

Among the 107 unique soybean transcripts, 62 (approx.58 %), 14 (13 %), 12 (approx. 11 %) and 35 (33 %)transcripts share sequence similarity with transcriptsregulated differentially by infection with other virusesidentified by Ishihara et al. (2004), Marathe et al. (2004),Yang et al. (2007) and Dardick (2007), respectively (seeSupplementary Table S5, available in JGV Online). Asdiverse plant RNA viruses may elicit a general stress anddefence response to plant virus infection (Whitham et al.,2003), we directed our searches specifically to a commonsubset of such transcripts. Indeed, soybean transcriptsinvolved in general stress and defence, such as LRR proteinkinase (GenBank accession no. AW186515), pyruvate kinase(AW830175), protein phosphatase 2C (AW830157), proteinkinase (AW831515), calmodulin (AI441176), peroxidase(AI496108), 2-Cys peroxiredoxin (AI443769), hypersens-itive-induced response protein (AT3G01290) and universalstress protein (USP; AI735896), were identified from thisanalysis. It seems that, despite the differences in symptoms,hosts and viruses, there is a commonly shared stress anddefence response in plants to the infections of ss(+) RNAviruses. Interestingly, all of these transcripts were upregu-lated by SMV infection at 14 or 21 days p.i., but not at7 days p.i., further suggesting a delayed defence response inSMV-infected soybean plants.

In conclusion, high-throughput microarray analysis usedin this study permitted us to draw some generalassociations between SMV infection and gene-expressionchanges in the SMV-infected leaf. Even though large sets ofinformative data were generated, our study is limited incertain ways. First, the total RNA samples derived fromSMV-infected soybean leaves contained a mixture ofuninfected, infected and post-infected cells, and thereforespatial and temporal information on the patterns of geneexpression was not optimized. Second, certain zones in the

M. Babu and others

1078 Journal of General Virology 89

SMV-infected leaf tissue might contain little or no viralRNA, commonly referred to as dark-green islands(Atkinson & Matthews, 1970; Moore & MacDiarmid,2006). These zones may dilute or mask expression changesin the infected cells. Despite these limitations, this analysissuggests clearly that a number of genes are probablyassociated with SMV-compatible infection and symptomdevelopment, and activation of defence-like genes seemsnot to occur until virus accumulation reaches its highestlevel. Such a delayed defence response may be critical forSMV to establish its systemic infection. Further targetedfunctional studies of these differentially expressed tran-scripts, particularly those involved in defence, cell wall,sulphur assimilation, hormone, chloroplast and photosyn-thesis pathways, will provide new insights into SMV–soybean interactions.

ACKNOWLEDGEMENTS

The authors wish to thank Dr Vaino Poysa (AAFC) for soybean seeds,

Dr Lila Vodkin (University of Illinois, Urbana–Champaign, IL, USA)

for the microarray slides, Drs Mark Gijzen, Taiyun Wei, Dinah Qutob

(AAFC) and Martina Stromvik (McGill University, Montreal, QC,

Canada) for critical reading of the manuscript and Alex Molnar for

photography. This work was supported in part by Ontario Soybean

Growers, the AAFC Canadian Crop Genomics Initiative and the

National Science and Engineering Council of Canada.

REFERENCES

Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z.,Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new

generation of protein database search programs. Nucleic Acids Res 25,

3389–3402.

Aranda, M. A., Escaler, M., Wang, D. & Maule, A. J. (1996). Induction

of HSP70 and polyubiquitin expression associated with plant virus

replication. Proc Natl Acad Sci U S A 93, 15289–15293.

Arif, M. & Hassan, S. (2002). Evaluation of resistance in soybean

germplasm to soybean mosaic potyvirus under field conditions. J Biol

Sci 2, 601–604.

Atkinson, P. H. & Matthews, R. E. F. (1970). On the origin of dark

green tissue in tobacco leaves infected with tobacco mosaic virus.

Virology 40, 344–356.

Beffa, R. S., Hofer, R.-M., Thomas, M. & Meins, F., Jr (1996).Decreased susceptibility to virus disease of b-1,3-glucanase-deficient

plants generated by antisense transformation. Plant Cell 8, 1001–1011.

Benjamini, Y. & Hochberg, Y. (1995). Controlling the false discovery

rate: a practical and powerful approach to multiple testing. J R Stat

Soc Ser B Methodol 57, 289–300.

Cardinale, F., Meskiene, I., Ouaked, F. & Hirt, H. (2002). Convergence

and divergence of stress-induced mitogen-activated protein kinase

signaling pathways at the level of two distinct mitogen-activated

protein kinases. Plant Cell 14, 703–711.

Clarke, S. F., Guy, P. L., Burritt, D. J. & Jameson, P. E. (2002). Changes

in the activities of antioxidant enzymes in response to virus infection

and hormone treatment. Physiol Plant 114, 157–164.

Culver, J. N. & Padmanabhan, M. S. (2007). Virus-induced disease:

altering host physiology one interaction at a time. Annu Rev

Phytopathol 45, 221–243.

Culver, J. N., Lindbeck, A. G. C. & Dawson, W. O. (1991). Virus-hostinteractions: induction of chlorotic and necrotic responses in plantsby tobamoviruses. Annu Rev Phytopathol 29, 193–217.

Dardick, C. (2007). Comparative expression profiling of Nicotianabenthamiana leaves systemically infected with three fruit tree viruses.Mol Plant Microbe Interact 20, 1004–1017.

Draghici, S., Khatri, P., Martins, R. P., Ostermeier, C. & Krawetz, S. A.(2003). Global functional profiling of gene expression. Genomics 81,98–104.

Espinoza, C., Vega, A., Medina, C., Schlauch, K., Cramer, G. & Arce-Johnson, P. (2007). Gene expression associated with compatible viraldiseases in grapevine cultivars. Funct Integr Genomics 7, 95–110.

Garcia-Brugger, A. G., Lamotte, O., Vandelle, E., Bourque, S.,Lecourieux, D., Poinssot, B., Wendehenne, D. & Pugin, A. (2006).Early signaling events induced by elicitors of plant defenses. Mol PlantMicrobe Interact 19, 711–724.

Golem, S. & Culver, J. N. (2003). Tobacco mosaic virus inducedalterations in the gene expression profile of Arabidopsis thaliana. MolPlant Microbe Interact 16, 681–688.

Herbers, K., Tacke, E., Hazirezaei, M., Krause, K. P., Melzer, M.,Rohde, W. & Sonnewald, U. (1997). Expression of a luteoviralmovement protein in transgenic plants leads to carbohydrateaccumulation and reduced photosynthetic capacity in source leaves.Plant J 12, 1045–1056.

Hernandez, J. A., Rubio, M., Olmos, E., Ros-Barcelo, A. & Martınez-Gomez, P. (2004). Oxidative stress induced by long-term plum poxvirus infection in peach (Prunus persica). Physiol Plant 122, 486–495.

Huckelhoven, R. (2007). Cell wall-associated mechanisms of diseaseresistance and susceptibility. Annu Rev Phytopathol 45, 101–127.

Ishihara, T., Sakurai, N., Sekine, K. T., Hase, S., Ikegami, M., Shibata, D.& Takahashi, H. (2004). Comparative analysis of expressed sequencetags in resistance and susceptible ecotypes of Arabidopsis thalianainfected with cucumber mosaic virus. Plant Cell Physiol 45, 470–480.

Itoh, H., Tatsumi, T., Sakamoto, T., Otomo, K., Toyomasu, T.,Kitano, H., Ashikari, M., Ichihara, S. & Matsuoka, M. (2004). A ricesemi-dwarf gene, Tan-Ginbozu (D35), encodes the gibberellinbiosynthesis enzyme, ent-kaurene oxidase. Plant Mol Biol 54, 533–547.

Jacobs, A. K., Lipka, V., Burton, R. A., Panstruga, R., Strizhov, N.,Lefert, P. S. & Fincher, G. B. (2003). An Arabidopsis callose synthase,GSL5, is required for wound and papillary callose formation. PlantCell 15, 2503–2513.

Jayaram, C. H., Hill, J. H. & Miller, W. A. (1992). Complete nucleotidesequences of two soybean mosaic virus strains differentiated byresponse of soybean containing the Rsv resistance gene. J Gen Virol 73,2067–2077.

Kolomiets, M. V., Chen, H., Gladon, R. J., Braun, E. J. & Hannapel, D. J.(2000). A leaf lipoxygenase of potato induced specifically by pathogeninfection. Plant Physiol 124, 1121–1130.

Liao, L., Chen, P., Buss, G. R., Yang, Q. & Tolin, S. A. (2002).Inheritance and allelism of resistance to soybean mosaic virus inZao18 soybean from China. J Hered 93, 447–452.

Marathe, R., Guan, Z., Anandalakshmi, R., Zhao, H. & Dinesh-Kumar,S. P. (2004). Study of Arabidopsis thaliana resistome in response tocucumber mosaic virus infection using whole genome microarray.Plant Mol Biol 55, 501–520.

Maule, A. J., Escaler, M. & Aranda, M. A. (2000). Programmedresponses to virus replication in plants. Mol Plant Pathol 1, 9–15.

Maule, A., Leh, V. & Lederer, C. (2002). The dialogue between virusesand hosts in compatible interactions. Curr Opin Plant Biol 5, 279–284.

Milioni, D., Sado, P. E., Stacey, N. J., Domingo, C., Roberts, K. &McCann, M. C. (2001). Differential expression of cell-wall-related

Transcriptional response of SMV-infected soybean

http://vir.sgmjournals.org 1079

genes during the formation of tracheary elements in the Zinnia

mesophyll cell system. Plant Mol Biol 47, 221–238.

Moore, C. J. & MacDiarmid, R. M. (2006). Dark green islands: the

phenomenon. In Natural Resistance Mechanisms of Plants to Viruses,

pp. 187–209. Edited by G. Loebenstein & J. P. Carr. Dordrecht:

Springer.

Moy, P., Qutob, D., Chapman, B. P., Atkinson, I. & Gijzen, M. (2004).Patterns of gene expression upon infection of soybean plants by

Phytophthora sojae. Mol Plant Microbe Interact 17, 1051–1062.

Nishimura, M. T., Stein, M., Hou, B. H., Vogel, J. P., Edwards, H. &Somerville, S. C. (2003). Loss of callose synthase results in salicylic

acid dependent disease resistance. Science 301, 969–972.

Padmanabhan, M. S., Goregaoker, S. P., Golem, S., Shiferaw, H. &Culver, J. N. (2005). Interaction of the tobacco mosaic virus replicase

protein with the Aux/IAA protein PAP1/IAA26 is associated with

disease development. J Virol 79, 2549–2558.

Padmanabhan, M. S., Shiferaw, H. & Culver, J. N. (2006). The

Tobacco mosaic virus replicase protein disrupts the localization and

function of interacting Aux/IAA proteins. Mol Plant Microbe Interact

19, 864–873.

Pompe-Novak, M., Gruden, K., Baebler, S., Krecic-Stres, H., Kovac, M.,Jongsma, M. & Ravnikar, M. (2005). Potato virus Y induced changes in

the gene expression of potato (Solanum tuberosum L.). Physiol Mol Plant

Pathol 67, 237–247.

Rubin, G. M., Yandell, M. D., Wortman, J. R., Gabor Miklos, G. L.,Nelson, C. R., Hariharan, I. K., Fortini, M. E., Li, P. W., Apweiler, R. &other authors (2000). Comparative genomics of the eukaryotes.

Science 287, 2204–2215.

Sandermann, H. (2000). Active oxygen species as mediators of plant

immunity: three case studies. Biol Chem 381, 649–653.

Senthil, G., Liu, H., Puram, V. G., Clark, A., Stromberg, A. & Goodin,M. M. (2005). Specific and common changes in Nicotiana benthami-

ana gene expression in response to infection by enveloped viruses.

J Gen Virol 86, 2615–2625.

Shimizu, T., Satoh, K., Kikuchi, S. & Omura, T. (2007). The repression

of cell wall- and plastid-related genes and the induction of defense-

related genes in rice plants infected with rice dwarf virus. Mol Plant

Microbe Interact 20, 247–254.

Storey, J. D. & Tibshirani, R. (2003). Statistical significance for

genomewide studies. Proc Natl Acad Sci U S A 100, 9440–9445.

Taniguchi, M., Miura, K., Iwao, H. & Yamanaka, S. (2001).Quantitative assessment of DNA microarrays – comparison with

northern blot analyses. Genomics 71, 34–39.

Tecsi, L. I., Maule, A. J., Smith, A. M. & Leegood, R. C. (1994).Complex, localized changes in CO2 assimilation and starch contentassociated with the susceptible interaction between cucumber mosaicvirus and a cucurbit host. Plant J 5, 837–847.

Tecsi, L. I., Smith, A. M., Maule, A. J. & Leegood, R. C. (1996). Aspatial analysis of physiological changes associated with infection ofcotyledons of marrow plants with cucumber mosaic virus. PlantPhysiol 111, 975–985.

Thibaud-Nissen, F., Shealy, R. T., Khanna, A. & Voldkin, L. O. (2003).Clustering of microarray data reveals transcript patterns associatedwith somatic embryogenesis in soybean. Plant Physiol 132, 118–136.

Ueki, S. & Citovsky, V. (2005). Identification of an interactor ofcadmium ion-induced glycine-rich protein involved in regulation ofcallose levels in plant vasculature. Proc Natl Acad Sci U S A 102,12089–12094.

von Schaewen, A., Stitt, M., Schmidt, R., Sonnewald, U. & Willmitzer, L.(1990). Expression of a yeast-derived invertase in the cell wall of tobaccoand Arabidopsis plants leads to accumulation of carbohydrate andinhibition of photosynthesis and strongly influences growth andphenotype of transgenic tobacco plants. EMBO J 9, 3033–3044.

Whitham, S. A. & Wang, Y. (2004). Roles for host factors in plant viralpathogenicity. Curr Opin Plant Biol 7, 365–371.

Whitham, S. A., Quan, S., Chang, H. S., Cooper, B., Estes, B., Zhu, T.,Wang, X. & Hou, Y. M. (2003). Diverse RNA viruses elicit theexpression of common sets of genes in susceptible Arabidopsisthaliana plants. Plant J 33, 271–283.

Whitham, S. A., Yang, C. & Goodin, M. M. (2006). Global impact:elucidating plant responses to viral infection. Mol Plant MicrobeInteract 19, 1207–1215.

Yang, C., Guo, R., Jie, F., Nettleton, D., Peng, J., Carr, T., Yeakley, J. M.,Fan, J. B. & Whitham, S. A. (2007). Spatial analysis of Arabidopsisthaliana gene expression in response to turnip mosaic virus infection.Mol Plant Microbe Interact 20, 358–370.

Yao, B., Rakhade, S. N., Li, Q., Ahmed, S., Krauss, R., Draghici, S. &Loeb, J. A. (2004). Accuracy of cDNA microarray methods to detectsmall gene expression changes induced by neuregulin on breastepithelial cells. BMC Bioinformatics 5, 99.

Zhu, S., Gao, F., Cao, X., Chen, M., Ye, G., Wei, C. & Li, Y. (2005). Therice dwarf virus P2 protein interacts with ent-kaurene oxidases in vivo,leading to reduced biosynthesis of gibberellins and rice dwarfsymptoms. Plant Physiol 139, 1935–1945.

Zhuang, B. C., Xu, B. & Liao, L. (1993). Change of superoxide dismutase,peroxidase and storage protein in soybean leaves after inoculation withsoybean mosaic virus. Acta Phytopathol Sin 23, 261–265.

M. Babu and others

1080 Journal of General Virology 89