Differential gene expression in incompatible

Eur J Plant Pathol (2012) 132:393–406 DOI 10.1007/s10658-011-9885-0

Differential gene expression in incompatible interaction between turnip mosaic virus and non-heading Chinese cabbage Hai-Tao Peng & Li Wang & Ying Li & Yan-Xiao Li & Wei Guan & Yang Yang & Xiao-Hai Xu & Xi-Lin Hou

Accepted: 20 October 2011 / Published online: 12 December 2011 # KNPV 2011

Abstract Viral disease, caused by turnip mosaic virus (TuMV), is considered the most destructive disease of non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino). Although several TuMV resistance loci/genes have been mapped or characterized in Brassica vegetables, the mechanism of molecular interaction between TuMV and non-heading Chinese cabbage is poorly understood. Additionally, TuMV response genes need to be identified. The objectives of this study were to identify differentially expressed genes during the incompatible interaction between TuMV and non-heading Chinese cabbage, and validate their expressions. A total of 200 transcript-derived fragments (TDFs) obtained by complementary DNAamplified fragment length polymorphism were recovered and sequenced. The results revealed that 176 (88.0%) TDFs produced specific sequences, among which 48 (27.3%) sequences were predicted with putative functions using NCBI BLAST. Among the 48 available

Electronic supplementary material The online version of this article (doi:10.1007/s10658-011-9885-0) contains supplementary material, which is available to authorized users. H.-T. Peng : L. Wang : Y. Li : Y.-X. Li : W. Guan : Y. Yang : X.-H. Xu : X.-L. Hou (*) State Key Laboratory of Crop Genetics and Germplasm Enhancement/Key Laboratory of Southern Vegetable Crop Genetic Improvement, Ministry of Agriculture, Nanjing 210095, People’s Republic of China e-mail: [email protected]

TDFs, 22 (45.8%) sequences belonging to different functional groups were selected to monitor the changes in their expression in incompatible and compatible interactions by quantitative real-time polymerase chain reaction. To the best of our knowledge, this study provides the first global transcriptomic analysis of nonheading Chinese cabbage genes during an incompatible interaction. The results are expected to aid in characterizing TuMV response genes and clarifying the molecular mechanism of TuMV–host interaction. Keywords cDNA-AFLP . Expression profile . Pathogen–plant interaction . TuMV infection

Introduction Turnip mosaic virus (TuMV) is a member of the genus Potyvirus (Brunt 1992), which is the largest group of plant virus. In an investigation on virus diseases in 28 countries and regions, TuMV ranked second only to Cucumber mosaic virus among the most damaging viruses that infect field vegetables (Tomlinson 1987). TuMV can infect at least 318 plants in over 43 dicotyledonous families under artificial inoculation. Plants display symptoms such as mosaic patterns, stunting, and necrosis after TuMV infection (Kim et al. 2008). Moreover, black necrotic spots and streaks generally occur in cabbages (Tomlinson and Ward 1981; Tomlinson 1987), and in severe cases, death

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(Walsh and Tomlinson 1985). TuMV can cause substantial losses in the yield and marketability of non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino). Some TuMV resistance loci/genes have been mapped or cloned in Brassica crops. TuRB01, a high TuMV resistance locus (pathotype UK1), was mapped on the linkage group N6 of B. napus (cv. N-o-72-8) A-genome using doubled-haploid lines (Walsh et al. 1999). TuRB02, which controls the degree of susceptibility to TuMV isolate CHN1 in a quantitative manner, was identified on the C-genome linkage group N14 (Walsh et al. 1999). TuRB03 was mapped on chromosome N6 in B. napus (cv. Westar), which was verified to be dominant and monogenetic by segregation analysis of a backcross generation (Hughes et al. 2003). TuRB04 and TuRB05 are single dominant TuMV resistance loci, as validated by the examination of plant phenotypes induced by TuMV inoculation in B. napus line 165. TuRB04 and TuRB05 conferred extreme resistance or necrotic response to TuMV isolates UK 1 and CHN 12 in the presence of wild-type viral P3 or CI sequence (Jenner et al. 2002). Retr01, which is resistant to eight diverse TuMV isolates (UK 1, CHN 5, CZE 1, CDN 1, JPN 1, DEU 7, GK 1, and UK 4), has a recessive allele and was located on the upper position of chromosome R4 in B. rapa line RLR22 (Rusholme et al. 2007). Another conditional TuMV resistance locus, ConTR01, possesses a dominant resistance allele and was mapped on chromosome R8 (Rusholme et al. 2007). In our previous work, a TuMV resistance gene, BcTuR3, was isolated from non-heading Chinese cabbage. The translation product of BcTuR3 was homologous to other plant resistance proteins. Southern blotting showed that it belongs to a small multi-gene family. Northern hybridization confirmed its elevated expression upon TuMV infection in resistant and susceptible plants (Ma et al. 2010). To the best of our knowledge, BcTuR3 is the only TuMV resistance gene isolated and characterized in non-heading Chinese cabbage. Collectively, a number of studies on TuMV in Brassica crops are mainly in the form of molecular markers. Thus, an incompatible TuMV–cabbage pathosystem is necessary for identifying TuMV response genes and understanding their mechanism of interaction. Differential expressions of pathogenesis-related host genes have been studied in virus–host systems

Eur J Plant Pathol (2012) 132:393–406

for decades. In general, five techniques or approaches are utilized (Whitham et al. 2006): (1) profiling host responses to wild-type or engineered viruses (Golem and Culver 2003; Fregene et al. 2004; Ishihara et al. 2004; Klausmeyer et al. 2008); (2) comparative analysis of host responses to different viruses, pathogens, and abiotic stresses (Dietrich et al. 2003; Whitham et al. 2003; Senthil et al. 2005); (3) differential analysis of host responses to viruses in host mutants and genotypes (Ventelon-Debout et al. 2004; Huang et al. 2005); (4) expression of individual virus proteins and nucleic acids (Geri et al. 1999; Trinks et al. 2005); and (5) determination of spatial and temporal relationships between viral infection and host responses (Aranda et al. 1996; Havelda and Maule 2000). Large-scale transcriptional profiling has shown novel aspects in compatible and/or incompatible interactions between plants and their pathogens (Tao et al. 2003; Caldo et al. 2004; Chisholm et al. 2006; Fung et al. 2008; Wang et al. 2009; Baldo et al. 2010; Wang et al. 2010). The complementary DNAamplified fragment length polymorphism (cDNAAFLP) technique is a valid method for genomewide expression analysis. It is independent of genetic information and has been successfully employed in various pathogen–host systems for the identification of differential gene expression, and helps clarify the molecular mechanism of interaction (Qin et al. 2000; Eckey et al. 2004; Gabriels et al. 2006; Polesani et al. 2008). Considering the availability of studies on the altered gene expressions of hosts and the necessity of exploring the molecular interaction of TuMV–cabbage, the cDNA-AFLP technique was applied in this study to analyze the global gene expression changes in the incompatible interaction between TuMV and nonheading Chinese cabbage. A total of 176 genes were identified as possibly involved in TuMV–cabbage interaction and 22 transcript-derived fragments (TDF) were selected to monitor their expression changes in the resistant cultivar “Taisankang” and the susceptible cultivar “Xiangqingcai” by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). The aims of this study were to identify the genes regulated during the incompatible interaction of non-heading Chinese cabbage and TuMV, validate the expression patterns for some of the regulated genes by qRT-PCR, and break down the molecular mechanism of the interaction.


Materials and methods Plant materials and inoculation Non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino), cv. Taisankang (introduced from Thailand, highly resistant to TuMV), is an advanced inbred line. It showed no clear disease symptoms of TuMV infection. Seeds were disinfected in hot water (50–60°C, 15 min) and pre-germinated in a culture dish for 36 h, 25°C in the dark. They were then transferred into 20 cm×25 cm plastic pots filled with sterilized soil at 24°C and 80% relative humidity (RH) under a 14 h photoperiod in the greenhouse. TuMV (pathotype C4) was extracted from fresh leaves with severe mosaic patterns and tested by electron microscopy. The susceptible leaves were collected and ground in phosphate buffer (pH 7.2, 0.05 mol l−1) for inoculation. Mechanical inoculation was applied onto the second and third seedling leaves. Parallel mock inoculation was performed with the same buffer and exactly in the same manner, except the extraction of the virus. Subsequently, all the plants were kept under a 14 h photoperiod at 28°C in the daytime and 22°C at night, with constant 85% RH in the greenhouse. The inoculated and mock-inoculated leaves were sampled at 1, 2, 3, and 4 d post inoculation (dpi), as well as the mock-inoculated plants near 0 h post inoculation (hpi). Sampling was conducted on the inoculated leaves on each plant. Three replicates were performed for three independent plants. The materials sampled at each time point were pooled for cDNAAFLP analysis. A mock inoculation served as the control for each time point. RNA extraction and cDNA synthesis RNA was extracted using an RNA kit (Tiangen, China) following the manufacturer’s instructions. RNA was treated with DNase I (Sigma, USA) for purification. The amount and quality of RNA were verified on 1% agarose gel and by BioPhotometer Plus (Eppendorf, Germany). Double-stranded cDNA (dscDNA) was synthesized using an M-MLV RTase cDNA Synthesis Kit (TaKaRa, Japan) according to the manufacturer’s protocols. cDNA-AFLP analysis About 80 ng dscDNA was digested using EcoRI and MseI (NEB, USA) for 4 h at 37°C. The digested

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products were incubated for 20 min at 80°C to inactivate the restriction enzymes. The products were then ligated to EcoRI and MseI adaptors with T4 DNA ligase (TaKaRa, Japan) at 16°C for 8 h or overnight. The sequences of the adaptors were as follows: EcoRIadaptor top strand, 5′-CTC GTA GAC TGC GTA CC3′, EcoRI-adaptor bottom strand, 5′-AAT TGG TAC GCA GTC-3′, MseI-adaptor top strand, 5′-GAC GAT GAG TCC TGA GC-3′, MseI-adaptor bottom strand, 5′TAC TCA GGA CTC AT-3′. The core sequences of the primers for pre-amplification and selective amplification were assigned E00 and M00, respectively, E00: 5′-GAC TGC GTA CCA ATT C-3′, M00: 5′-GAT GAG TCC TGA GTA A-3′. 3′-ends of E00 and M00 added to zero nucleotides were regarded as pre-amplification primers and two or three nucleotides (A/T/G/C) were regarded as selective primers. Pre-amplification was carried out in a 20 μl volume containing 10 ng adaptor-linked dscDNA, 10 pM of EcoRI and MseI pre-amplification primer, 0.25 mM dNTPs, 1.25 U rTaq polymerase (TaKaRa, Japan), and 2.0 μl PCR buffer (10×, Mg2+). PCR was performed on a thermal cycler (Eppendorf, Germany). The procedure for pre-amplification involved initial denaturation for 2 min at 94°C, 25 cycles (94°C denaturation for 30 s, 56°C annealing for 30 s, and 72°C extension for 1 min), and a final extension of 10 min at 72°C. The mixture was diluted 30× for selective amplification. Selective amplification was also conducted in a 20 μl volume. A “touchdown” program (12 cycles, at a scale down of 0.7°C per cycle) was added to the preamplification program as selective amplification. The PCR mixture of the selective amplification is as follows: 2.0 μl diluted template, 10 pM primer, 2.0 μl PCR buffer (10×), 0.2 mM dNTPs, 1.5 mM Mg2+, and 1.0 U rTaq polymerase (TaKaRa, Japan). The products mixed with 5 μl loading buffer were heat denatured at 95°C for 5 min. The 3-μl mixtures were then loaded in the 6.5% denaturing polyacrylamide gel and ran for 3 h at 40 W. Electrophoresis images were displayed by silver staining. Isolation of TDFs and sequence analysis Three independent replications were performed to every primer combination in the cDNA-AFLP analysis to ensure the reproducibility of the TDFs. The bands of interest were excised from gels and placed in sterilized tubes with 50 μl TE buffer. The tubes were then placed

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in a boiling-water bath for 20 min to separate the DNA from the gels. After centrifugation, the extracts were amplified using the corresponding selective amplification primers under the same reaction conditions as the selective amplification program, but without the touchdown. The re-amplified DNA fragments were detected by agarose gel electrophoresis and cloned into pMD18-T vector (TaKaRa, Japan), following the manufacturer’s instructions. Positive recombinant plasmids were chosen from Amp LB medium (solid) and propagated in LB medium (liquid) for 12 h at 37°C. At least three clones of each re-amplified TDF were sent for sequencing. Sequences were identified by BLASTN search against the nr database of NCBI. The E-value of 1e×10−05 was regarded as the cutoff point of similar sequence and>1e×10−05 was considered to be no hits found. Expression analysis of the selected TDFs The plants were kept in the same growing conditions as the materials used in the cDNA-AFLP analysis. The seedling leaves at the three-leaf stage were sampled for both TuMV- and mock-inoculated plants at 2, 6, 12, 24, 48, 72, 96, 120, and 144 hpi, as well as the mock-inoculated plants near 0 hpi. All the plants were sampled in resistant cultivar Taisankang and susceptible cultivar Xiangqingcai. RNA was extracted using an RNA kit (Tiangen, China) following the manufacturer’s protocols. Single-strand cDNA was synthesized on 2 μg RNA by AMV reverse transcriptase (TaKaRa, Japan) according to the manufacturer’s instructions. The quantitative analysis of genes was performed using the Rotor Gene system (Corbett, Australia). The qRT-PCR reaction mixtures had a volume of 25 μl and contained 12.5 μl SYBR Green PCR mix (TaKaRa, Japan), 2.0 μl template (5× diluted cDNA), 10 pM of each primer, and 8.5 μl sterile water. The thermal conditions were 2 min at 95°C for denaturation, followed by 40 cycles of 90°C for 20 s, 56°C for 15 s, and 72°C for 20 s. Subsequently, a melting curve from 65 to 95°C was performed to detect primer dimerization or other artifacts of amplification. The results were standardized by comparing the data with one non-heading Chinese cabbage glyceradehyde3-phosphate dehydrogenase gene (GAPDH, DDBJ accession number AB303568), which retained constant expression under different treatment conditions in our evaluation (data not shown). Transcript abundance


was assessed with three independent biological replicates. Relative gene quantification was calculated by comparative ΔCT method (Livak and Schmittgen 2001). Statistical analysis was carried out by one-way ANOVA using SAS software. The data were presented as Mean±SD.

Results Isolation of differentially expressed genes The differentially expressed genes after TuMV infection in non-heading Chinese cabbage were identified by cDNA-AFLP. Each primer combination produced 35– 40 TDF. About 4000 TDF were obtained with 100 primer combinations. The fragments were highly reproducible in three biological replications for each time-point treatment. In general, approximately four differential TDF were observed for each primer combination. The bands displayed lengths ranging from 50 to 1000 bp. Among the 400 TDF, 314 (78.5%) genes displayed upregulation and 86 exhibited (21.5%) downregulation, illustrating that a large number of genes are induced in the incompatible interaction. In total, 200 differentially expressed TDF were selected on the basis of their differential expression intensity on gels, among which 176 (88.0%) produced specific bands by reamplification. These TDF were then sequenced. The sequences were submitted to GenBank (Supplementary file 1). Figure 1 shows the partial results of the TDF gel image of five primer combinations at five sampling time points. Sequence analysis The 176 TDF produced reproducible and reliable sequences. At least three clones were sequenced for each TDF to verify the sequences for the respective bands; the clones produced identical sequencing results. The obtained sequences were identified by BLASTN search against the nr database of NCBI. The complete information on the isolated TDF is provided in Supplementary File 1, and the 48 genes of interest are shown in Table 1. The functions of the genes were classified into 12 groups on the basis of their homology to known proteins or unknown proteins; no hits were found as determined by Bevan’s method


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qRT-PCR analysis

Fig. 1 Expression of non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) transcripts determined by cDNA-AFLP. The example shows selective amplification with six different primer combinations. Lines 1 to 5 represent 0, 1, 2, 3, and 4 d post inoculation. M: Marker

(Bevan et al. 1998). Figure 2 shows the percentages of genes assigned to different functional categories. The largest group that accounted for 71 (40.34%) sequences was assigned as an unclear category. A group of 16 (9.09%) sequences belongs to the unknown function category. Forty-one (23.3%) sequences had no hits in the gene database. Approximately 5.68% of the annotated sequences have metabolic roles, including protein and carbohydrate metabolism. The genes involved in disease/defence, such as virus-resistance protein and RPP5 disease resistance locus, accounted for 4.55%. Approximately 5.11% of the sequences participate in signal transduction, 3.98% in cell structure, 2.84% in transporting, 2.27% in transcription, 1.70% in energy, 0.57% in protein synthesis, and 0.57% in cell growth/division.

To validate the results of the cDNA-AFLP and monitor the expression patterns of the differentially expressed genes, qRT-PCR was conducted for 22 representative TDF in compatible and incompatible interactions using 10 time points (0, 2, 6, 12, 24, 48, 72, 96, 120, and 144 hpi) (Fig. 3). These genes were chosen because they represented different functional categories, including 5 genes for disease/defence response, 5 for signal transduction, 3 for transporting, 4 for cell structure, 2 for metabolism, 1 for energy, and 1 for transcription. In addition, to demonstrate whether function-unknown genes were also involved in the interactions, a transcript with high homology (e=1e×10−61) to an Arabidopsisunknown gene was chosen for quantitative analysis. The following were differentially upregulated in both of the interactions, as determined by quantitative analysis: 18 genes (77.3%), encoding LRR-RLK (HR505263), PRR5 disease resistance locus (HR505266), glutathione reductase (HR505270), virus-resistant protein (HR505358), heat shock protein 70 (HR505409), GTPbinding protein (HR505267), ankyrin repeat family protein (HR505261), calmodulin binding protein (HR505357), Ca-dependent solute carrier protein (HR505264), Transducin family protein, Brassica rapa chloroplast (HR505276, HR505408, HR505346), structural constituent of cytoskeleton (HR505382), enoyl-CoA hydratase (HR505293), diaminopimelate decarboxylase (HR505359), glycolate oxidase (HR505322), ring zinc finger protein (HR505269), and an unknown protein (HR505424). Four genes showed depressed expression, among of which three were related to signal transduction. The three transcripts were homologous to GTP-binding protein (HR505267), phytochrome kinase substrate (HR505268), and protein tyrosine phosphatase (HR505357). Another downregulated transcript was potassium ion transmembrane transporter (HR505271) with a putative transporting function. The accumulation of all the induced transcripts peaked after 24 hpi, except that encoding chloroplast (Fig. 3n), indicating that most of the identified genes were involved in systemic infection. For each TDF, the same expression pattern observed in the cDNAAFLP analysis was found in the qRT-PCR analyses as, except for only one TDF (E18M17-4, Ankyrin repeat family protein), which may be a wrong fragment. The cDNA-AFLP technique efficiently

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Table 1 Forty-eight TDFs with putative functions from TuMV-infected non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) leaves Clone no.

Primer comb. Length(bp) Accession no. Closest to database match

Similarity E-Value percentage

Predicted functions

E18M17–11

CTC-TCC

237

HR505270

glutathione reductase

97%

2.00E-91

Disease/defense

E17M15-7

TCC-ATC

204

HR505263

90%

2.00E-58

Disease/defense

E18M15-5

CTC-ATC

202

HR505266

leucine-rich repeat receptor-like protein kinase RPP5 disease resistance locus

77%

7.00E-25

Disease/defense

E22M17-2

AGC-TCC

132

HR505354

drought induced protein

91%

4.00E-31

Disease/defense

E18M16-7

CTC-TAC

237

HR505318

glutathione reductase

96%

7.00E-90

Disease/defense

E21M15-2

GAC-ATC

305

HR505415

multi-drug resistance protein

75%

7.00E-09

Disease/defense

E21M26-1

GAC-GCA

277

HR505358

virus-resistant protein

93%

1.00E-100 Disease/defense

E22M20-1

AGC-GTC

351

HR505409

heat shock protein 70-1

86%

1.00E-101 Disease/defense

E18M15-7

CTC-ATC

178

HR505267

GTP-binding protein

95%

3.00E-42

Signaling

E21M23-6

GAC-TCA

133

HR505357

90%

6.00E-29

Signaling

E18M17-4

CTC-TCC

219

HR505261

protein tyrosine phosphatase (PTP1) ankyrin repeat family protein

74%

1.00E-22

Signaling

3-1

TCC-ATC

204

HR505259

92%

6.00E-64

Signaling

E18M15-4

CTC-ATC

504

HR505268

84%

4.00E-142 Signaling

E20M23-3

GTC-TCA

203

HR505340

88%

3.00E-28

Signaling

E21M23-2

GAC-TCA

133

HR505405

90%

6.00E-29

Signaling

E23M21-5

TCA-GAC

224

HR505423

85%

2.00E-58

Signaling

E16M16-4

TAC-TAC

137

HR505264

90%

1.00E-24

Transporter

E23M16-5

TCA-TAC

185

E23M21-4

TCA-GAC

224

E23M16-1-1 TCA-TAC E23M16-4

TCA-TAC

E16M19-6

HR505386

leucine-rich repeat transmembrane protein PHYTOCHROME KINASE SUBSTRATE protein tyrosine phosphatase(PTP1) protein tyrosine phosphatase (PTP1) kinesin calmodulin-binding protein Ca-dependent solute carrier protein transducin family protein

75%

2.00E-32

Transporter

HR505375

calmodulin binding protein

87%

3.00E-62

Transporter

212

HR505398

transducin family protein

86%

1.00E-53

Transporter

185

HR505381

transducin family protein

75%

7.00E-31

Transporter

TAC-TGC

283

HR505271

93%

2.00E-90

Transporter

E18M13-6

CTC-CAC

201

HR505269

80%

3.00E-35

Transcription

E20M25-1

GTC-GCT

391

HR505321

92%

2.00E-143 Transcription

E19M26-3-1 TGC-GCA

159

HR505339

86%

3.00E-34

Transcription Transcription

E19M26-4

TGC-GCA

159

HR505306

potassium ion transmembrane transporter RING zinc finger protein-like protein RNA polymerase II transcription factor processing protein-related mRNA rRNA processing protein-related

86%

3.00E-34

E20M25-4

GTC-GCT

233

HR505322

glycolate oxidase

95%

1.00E-85

Energy

E20M18-1-2 GTC-CTC

137

HR505341

85%

4.00E-19

Energy

91%

8.00E-33

Energy

90%

4.00E-81

Metabolism

E21M26-11

GAC-GCA

269

HR505352

NADPH-dependent thioredoxin reductase isocitrate lyase (ILA)

E20M25-3

GTC-GCT

255

HR505323

insulinase family protein

E23M20-1

TCA-GTC

154

HR505429

cycloartenol synthase

92%

7.00E-42

Metabolism

E19M25-2b

TGC-GCT

209

HR505309

proline dehydrogenase

97%

4.00E-35

Metabolism

E19M17-4

TGC-TCC

162

HR505293

enoyl-CoA hydratase (AIM1)

89%

9.00E-35

Metabolism

E20M21-5

GTC-GAC

128

HR505372

88%

5.00E-16

Metabolism

E21M25-1

GAC-GCT

222

HR505359

NONPHOTOCHEMICAL QUENCHING diaminopimelate decarboxylase

89%

2.00E-64

Metabolism

E20M21-4b

GTC-GAC

105

HR505348

94%

3.00E-23

Metabolism

NONPHOTOCHEMICAL QUENCHING


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Table 1 (continued) Clone no.

Primer comb. Length(bp) Accession no. Closest to database match

Similarity E-Value percentage

Predicted functions

E20M22-4

GTC-AGC

135

HR505406

93%

3.00E-33

Metabolism

E23M20-3

TCA-GTC

154

HR505428

92%

7.00E-42

Metabolism

E17M14-2

TCC-GCC

136

HR505345

95%

1.00E-37

Metabolism

E16M19-3

TAC-TGC

285

HR505276

99%

6.00E-132 Cell structure

E20M18-1-1 GTC-CTC

296

HR505308

99%


E22M20-2

311

HR505408

99%


AGC-GTC

E21M21-2

GAC-GAC

238

HR505404

E23M17-3

TCA-TCC

196

HR505382

E17M14-4

TCC-GCC

160

HR505288

E16M19-4

TAC-TGC

285

HR505346

E18M13-6

CTC-CAC

216

E23M21-3-1 TCA-GAC

251

AUDP-glucose:sterol glucosyltransferase cycloartenol synthase phosphoinositide binding mRNA Brassica rapa subsp. pekinensis chloroplast Camelina sativa18S rRNA gene Brassica napus strain ZY036 chloroplast Arabidopsis thaliana mRNA

HR505269

structural constituent of cytoskeleton Brassica rapa subsp. pekinensis mRNA Brassica napus strain ZY036 chloroplast 18S ribosomal RNA

HR505425

Mo25 family protein

identified gene expression. Conclusively, most of the genes isolated by the cDNA-AFLP in this work were involved in the TuMV-cabbage interaction.

3.00E-75

Cell structure

3.00E-42

Cell structure

100%

1.00E-57

Cell structure

99%


100%

4.00E-79

Protein synthesis

91%

2.00E-78

Cell growth/division

cDNA-AFLP is also widely used for the study of hostpathogen interactions because it can facilitate gene discovery (Eckey et al. 2004; Gabriels et al. 2006; Polesani et al. 2008). As shown in the present study, the transcript fragments obtained by cDNA-AFP are reproducible. They can be considered newly discovered genes responsive to TuMV infection. The 176 genes can help illustrate the molecular mechanism of the TuMVcabbage interactions and can provide broader insights into the signal pathways modulated by viral infection. Among the 48 TDF with putative functions (Table 1), the disease/defence genes are direct effectors in TuMV

Discussion cDNA-AFLP is a powerful technique for global transcriptional analysis. It is appropriate for gene expression study of various species because prior sequence data are not required for the identification of differentially expressed transcripts, and the primers used are universal. Fig. 2 Classification of differential TDFs under TuMV infection in nonheading Chinese cabbage (Brassica campestris ssp. chinensis Makino). A total of 176 TDFs were classified based on the BLASTN homology search in NCBI. E1e×10−5) as determined by similarity search in NCBI (Supplementary File 1). The pathogen– host interactions are complicated by the fact that the infected leaves comprised a heterogeneous mixture of hosts as pathogens spread from the primary inoculated cells to adjacent tissues. Plant response to pathogen challenges can evoke a large quantity of transcriptomic components that have not been published (de Torres et al. 2003). Because the genome of non-heading Chinese cabbage has not been completely sequenced, the abundance of genes provides the possibility of acquisition of unknown TDFs. The quantitative analysis revealed that 20 (90.9%) out of the 22 representative genes exhibited earlier expression changes in the incompatible interaction than in the compatible interaction, and shared similar expression patterns, demonstrating kinetic difference between the two interactions. Seventeen (77.3%) genes had high but diverse expression peaks, indicating that the response to TuMV in the compatible and incompatible interactions reflects a largely quantitative difference. This conclusion is in accordance with the research on the compatible and incompatible interactions between Arabidopsis and the bacterial pathogen Pseudomonas syringae (Tao et al. 2003). Most changes in mRNA transcript abundance occurred later in the TuMV-infected non-heading Chinese cabbage, a result consistent with the conclusions of Yang et al. (Yang et al. 2007). However, Maule et al. (Maule et al. 2002) proposed that the major changes in host genes occur at the early stage of viral infection. Some time is required before many significant changes begin to show, indicating that viral RNA and proteins require a certain threshold.

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In the current work, the efficient cDNA-AFLP technique was successfully used to determine the gene expression patterns in the incompatible interaction between TuMV and non-heading Chinese cabbage. The sequence analysis of 176 TDFs identified the genes involved in various molecular events during the incompatible interaction. These genes and their putative functions provide insight into the TuMV-cabbage incompatible interaction, and provide candidate genes for future function analysis. To clarify the interaction system further, carrying out compatible interactions with the same materials, methods, and conditions is currently underway. Moreover, further research is required, such as those involving the validation of more differentially expressed genes at large-scale sampling times or with different experimental methods (including comparative analysis between compatible and incompatible interaction systems), and the functional verification of TuMV response genes. Acknowledgements This study was supported by Innovative Scholars Project of Jiangsu Provincial Natural Science Foundation of China (No. BK2008035) and the earmarked fund for Modern Agro-industry Technology Research System.

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