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. 2016 Jul 27;3:16034. doi: 10.1038/hortres.2016.34

cDNA-AFLP analysis reveals differential gene expression in incompatible interaction between infected non-heading Chinese cabbage and Hyaloperonospora parasitica

Dong Xiao 1,2, Shi-Tuo Liu 1,2, Yan-Ping Wei 1,2, Dao-Yun Zhou 1,2, Xi-Lin Hou 1,2, Ying Li 1,2, Chun-Mei Hu 1,2,*
PMCID: PMC4962739  PMID: 27602230

Abstract

Non-heading Chinese cabbage (Brassica rapa ssp. chinensis) is one of the main green leafy vegetables in the world, especially in China, with significant economic value. Hyaloperonospora parasitica is a fungal pathogen responsible for causing downy mildew disease in Chinese cabbage, which greatly affects its production. The objective of this study was to identify transcriptionally regulated genes during incompatible interactions between non-heading Chinese cabbage and H. parasitica using complementary DNA-amplified fragment length polymorphism (cDNA-AFLP). We obtained 129 reliable differential transcript-derived fragments (TDFs) in a resistant line ‘Suzhou Qing’. Among them, 121 upregulated TDFs displayed an expression peak at 24–48 h post inoculation (h.p.i.). Fifteen genes were further selected for validation of cDNA-AFLP expression patterns using quantitative reverse transcription PCR. Results confirmed the altered expression patterns of 13 genes (86.7%) revealed by the cDNA-AFLP. We identified four TDFs related to fungal resistance among the 15 TDFs. Furthermore, comparative analysis of four TDFs between resistant line ‘Suzhou Qing’ and susceptible line ‘Aijiao Huang’ showed that transcript levels of TDF14 (BcLIK1_A01) peaked at 48 h.p.i. and 25.1-fold increased in the resistant line compared with the susceptible line. Similarly, transcript levels of the other three genes, TDF42 (BcCAT3_A07), TDF75 (BcAAE3_A06) and TDF88 (BcAMT2_A05) peaked at 24, 48 and 24 h.p.i. with 25.1-, 100- and 15.8-fold increases, respectively. The results suggested that the resistance genes tended to transcribe at higher levels in the resistance line than in the susceptible line, which may provide resistance against pathogen infections. The present study might facilitate elucidating the molecular basis of the infection process and identifying candidate genes for resistance improvement of susceptible cultivars.

Introduction

It is well known that plant–pathogen interactions activate a subset of pathogen genes so-called systemic acquired resistance to protect themselves.1–3 This interaction process is diverse and complicated because plant pathogens have evolved by developing various strategies to infect their hosts. Specific pathogen may trigger defense systems that are essential for pathogenicity. Next, molecular responses are up- or downregulations by numerous specific resistant genes. During the development of interaction, the recognition of specific host genes determines whether the interaction will be successful.

Downy mildew is an important fungal disease of Brassica specie that is caused by the obligatory biotrophic oomycete Hyaloperonospora parasitica (formerly Peronospora parasitica (Pers. Ex Fr.)), and infects most members of the Brassica family.4 It can be fatal to seedling growth in the nurseries and reduce the productivity and quality of adult plants in the field.4 Leaves become yellow after infection and then scorch. When downy mildew became epidemic, it can cause damage to >90% of the crop. The disease is more severe in spring and autumn seasons than in other seasons. Currently, the downy mildew disease is controlled by application of fungicides.5 However, chemical control is often difficult and ineffective. It has been proved that the most efficient way to manage plant diseases is to develop a host resistance in new cultivars.5 Therefore, to identify the host resistant genes is a crucial need for obtaining reliable resistant genotypes to assist plant breeding. Previous studies have shown some differentially expressed genes during infection process using various methods.6,7 The differential display-based strategy has been used to reveal genes related to downy mildew infection in B. oleracea seedlings.6 Suppression subtractive hybridization technology has been employed and revealed 37 high-quality Expressed Sequence Tags (ESTs), of which functions are known in energy metabolism, transcriptional regulation, signal transduction and defense reaction.7 However, most molecular components of the signal transduction pathway involved in gene regulation remain to be identified.

Furthermore, there is no report on pathogen virulence genes matching the resistance genes of non-heading Chinese cabbage (Brassica rapa ssp. chinensis), and their inheritance remains uncertain. In this prospect, it is important to elucidate the molecular mechanisms or gene expression profile and to identify an inventory of candidate genes during the non-heading Chinese cabbage–H. parasitica interaction.

Screening for differentially expressed genes is a direct approach to reveal the molecular basis of a biological system. The complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) method has been successfully used for the identification of genes involved in various plant–pathogen systems.8,9 In comparison with microarray technique and RNA sequencing, cDNA-AFLP costs less and does not require sequence information. When compared with subtractive hybridization, cDNA-AFLP is highly reproducible.8

The objectives of this study were to apply the cDNA-AFLP technique to the pathogenic interaction between non-heading Chinese cabbage and downy mildew. We identified a set of genes that were regulated during the incompatible interaction between the host and pathogen, and validated the expression patterns for the regulated genes.

Materials and methods

Plant material, inoculums and pathogen infection

Two non-heading Chinese cabbage inbred lines, ‘Suzhou Qing’ (resistant to H. parasitica) and ‘Aijiao Huang’ (susceptible to H. parasitica) from our lab, were used in this study. The transcript-derived fragments (TDFs) were obtained from interaction between ‘Suzhou Qing’ and downy mildew (H. parasitica). ‘Aijiao Huang’ was used for the comparison of expression patterns of the four genes related with fungal resistance between resistant and susceptible line.

Plants were grown in plastic nurseries (inner size: 45×45 mm; height: 57 mm) and transferred to a growth chamber under 25 °C day/ 20 °C night temperature with 85±5% relative humidity and a 12-h light/12-h dark after germination for 36 h under dark. H. parasitica was isolated from leaves of susceptible line ‘Aijiao Huang’ in the Jiangpu Farm of Nanjing Agricultural University, China.10 Conidial suspensions were adjusted to 1×105 spores per mL and Tween-20 was added as a surfactant to a final concentration of 0.1%. One hundred of 3-week-old seedlings (with four true-leaves) were sprayed with 50 mL pathogen suspension and distilled water (as control), respectively. After inoculation, the seedlings were covered with plastic film separately and transferred to a growth chamber under 20 °C, 100% relative humidity in the dark for the first 24 h to promote sporulation, then moved back to the initiatory conditions. Both control and treated third leaf of five plants were harvested and pooled at 0, 24, 48 and 72 h post inoculation (h.p.i.), immediately frozen in liquid nitrogen and stored at −70 °C until use.

RNA isolation and cDNA-AFLP analysis

Total RNAs were extracted using the RNAeasy Plant Mini kit (Qiagen; https://www.qiagen.com/cn/shop/sample-technologies/rna/rna-preparation/rneasy-mini-kit#orderinginformation) and synthesis of the first strand of cDNA is made using the M-MLV reverse transcriptase (Takara Shuzo Co., Ltd, Japan) according to the manufacturer’s protocol. To synthesize the second strand, the following components were added to the first-strand solution. A volume of 30 μL, 5×2nd strand synthesis buffer, 3 μL dNTP mixture, 89 μL RNase-free H2O, 2 μL Escherichia coli DNA polymerase I, 2 μL E. coli RNase H/E. coli DNA ligase mixture and 4 μL T4 DNA polymerase in a final volume of 150 μL. The components were gently mixed and incubated at 16 °C for 2 h. Double-stranded cDNA was purified using the DNA Fragment Purification Kit Ver.2.0 (Takara).

Second-strand cDNA was digested by two restriction enzymes Tag І (restriction site TCGA) and Ase І (restriction site ATTAAT; Takara), and then ligated to Tag І and Ase І double-strand adaptors. The AFLP adaptor primers 5′-GACGATGAGTCCTGAC-3′, 5′-CGGTCAGGACTCAT-3′ (Taq I-adaptor primers) and 5′-GCGTAGACTGCGTACC-3′, 5′-TAGGTACGCAGTC-3′ (Ase І-adaptor primers) were ligated onto the restriction fragments: Taq I pre-amplification primer, 5′-GACGATGAGTCCTGACCGA-3′; Ase I pre-amplification primer, 5-CTCGTAGACTGCGTACCTAAT-3′; Taq I selective amplification primer, 5′-GATGAGTCCAGACCGA+NN-3′; Ase I selective amplification primer, 5′-GACTGCGTACCTAAT+NN-3′ (indicated by N, representing an A, C, G or T). The initial small-scale screen using 96 AFLP primer combinations were done using six Taq I forward selective amplification primers (extension CG, CA, CT, CC, GA or GT) in combination with 16 Ase I reverse selective amplification primers (extension NN), respectively. Pre-amplification PCR was carried out with one-tenth volume of the restriction/ligation mix, the pre-amplification PCR was carried out as follows: 94 °C, 3 min; 94 °C, 30 s, 55 °C, 30 s, 72 °C, 60 s, 25 cycles; and 72 °C, 5 min. The products of pre-amplification was diluted 10-fold, and the selective amplification PCR was carried out as follows: 94 °C, 30 s; 94 °C, 30 s, 65 °C, 30 s (−0.7 °C per cycle), 72 °C, 60 s, 12 cycles; 94 °C, 30 s, 56 °C, 30 s, 72 °C, 60 s, 24 cycles; and 72 °C, 5 min.

Selective amplification products were separated on a 6% polyacrylamide gels running at 60 W for 2 h and visualized by silver staining. Differential bands were excised from the polyacrylamide gel electrophoresis gels based on the alignment between films and markers on the gels, and incubated in 30 μL of water and then at 95 °C for 30 min. The TDFs were then re-amplified by PCR using same primers under the similar conditions. The amplified fragments were retrieved from a 1% agarose gel with the Sephaglas BandPrep kit (Amersham Pharmacia Biotech.), cloned into pGEM-T Easy vector (Takara) according to the manufacturer's protocol and sequenced by Invitrogen Company (Shanghai BioWisdom Technology Co, Ltd; Shanghai, China. http://en.cellfood.com.cn/culture.aspx), and sequence information was BLASTed in the Brassica database (http://brassicadb.org/brad/).

Quantitative real-time PCR

The single-strand cDNA of resistant line ‘Suzhou Qing’ and susceptible line ‘Aijiao Huang’ were diluted to 30 ng μL−1, and were used for quantitative reverse transcription PCR (qRT-PCR) analyses. Primers were designed by the Primers 3 (http://frodo.wi.mit.edu/primer3/) based on the interested cDNA sequence. The qRT-PCR reaction mixtures contained 12.5 μL, 2× SYBR Green PCR MasterMix (Applied Biosystems; http://www.bio-rad.com/), 10 pm of each primer, 2 μL template and sterile distilled water to total volume of 25 μL, as well as also performed on CFX96 Real-Time System (C1000 Thermal Cycler, Bio-Rad, CA, USA). Thermal conditions were 2 min of denaturation at 95 °C, followed by 45 cycles of 95 °C for 10 s, annealing at 55 °C for 20 s, and extension at 72 °C for 20 s and 72 °C for 5 min. Three technical replicates were analysed for each biological replicate. All the cycle threshold (Ct) values from one gene were determined at the same threshold fluorescence value of 0.2 using the ΔΔCt method.11 The primers of gene-specific and housekeeping gene were listed in Table 1.

Table 1. Primers and reference sequence used in qRT-PCR analysis.

Functional categories TDFs number Gene Sequence 5′–3′ (forward ) Sequence 5′–3′ (reverse)
D TDF1 BcASN1_A06 TTCCTTCTACGCCTTATG GAATCAAGACCACCAGAT
D TDF7 BcCHS_A10 TGTGTTCTCTTCATATTGGA CACTGTCTCTACGGTAAG
D TDF11 BcTPI_A04 CTCAAGTTCCTTCACAAGA AGTTCACAAGCATCTCAG
D TDF14 BcLIK1_A01 CCTCCTCGTCTCTATCAT ATTCCAGTTAGTCTTCTTCAA
ST TDF42 BcCAT3_A07 GTCCACACCTACACTCTA CAACTACCTTAGCCTCTTC
ST TDF49 BcCCS_A08 TTCCTCATCTTCCTCTACTA CACACTTCATATCCACCAT
ST TDF58 BcNIT2_A02 GCTTCCACTGTCTATAATGA CTATGCCGAACCTATATCC
ST TDF59 BcRLK5_A01 TCATTCACATTGGTCTTCT CACATAGTAAGGCGAGAG
ST TDF60 BcKEG_A03 GCCTTACACCGTTACATA TTATAGCAGCAGCCATAC
ST TDF63 BcSAMDC_A03 GCCTTACACCGTTACATA TTATAGCAGCAGCCATAC
EM TDF75 BcAAE3_A06 CCTCCGTCAACAACATTA GGCGTCATACTTCTTCAT
EM TDF76 BcLHCB1.1_A07 GTTGAAGGTGAAGGAGAT AATGGTCAGCAAGATTCT
EM TDF88 BcAMT2_A05 ACATTAGCGGTATTCTACA GACACTACATTCCAGACA
R TDF91 BcABCG36_A07 TTGATGCTGATGAAGAGA GGTGGCTGGATTATACTT
R TDF101 BcSIG1_A08 GTCTTCTTCCTCAGTTCA TAATCTTCGCCACATCAA
Reference β-actin   GTTGCTATCCAGGCTGTTCT AGCGTGAGGAAGAGCATAAC
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Abbreviations: D, defense; ST, signal transduction; EM, energy metabolism; R, regulation; qRT-PCR, quantitative reverse transcription PCR.

Statistical analysis was performed using Student’s t-test.

Results

Isolation of differentially expressed genes

To determine the early events involved during the non-heading Chinese cabbage–H. parasitica interactions, four gene pools were constructed from resistant inbred line ‘Suzhou Qing’ at 0, 24, 48 and 72 h.p.i., respectively. TDFs displayed by cDNA-AFLP analysis ranged in size from 100 to 800 bp, depending on 96 selective primer combinations and time points. Figure 1 showed an example of the expression patterns of the genes revealed using cDNA-AFLP. A total of 180 fragments were obtained with the 96 primer pairs. After excluding repeat and error sequences, 129 TDFs were obtained. Of the 129 TDFs, 121 were upregulated and 8 downregulated. Of the 121 TDFs upregulated, 35 (28.9%), 31 (25.6%) and 4 (3.3%) TDFs were induced strongly at 24, 48 and 72 h.p.i., respectively; 12 (9.9%) and 2 (1.7%) TDFs were induced at 24 and 48 h.p.i., and 48 and 72 h.p.i., respectively. These results showed that non-heading Chinese cabbage has mainly accumulated expression at 24–48 h.p.i. and that gene expression patterns were different and complex after H. parasitica infection.

Figure 1.

Figure 1

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Expression of non-heading Chinese cabbage ‘Suzhou Qing’ genes in leaves inoculated with H. parasitica transcripts was displayed by cDNA-AFLP. An example showing that land 1, 2, 3 and 4 represents the induction time 0, 24, 48 and 72 h.p.i., respectively. The size of the differential TDFs was determined by direct sequencing. Arrow: differential bands.

Gene sequence analysis

By BLASTn search on Brassica database, 129 TDFs were successfully annotated (Table 2). One hundred and sixteen TDFs (90%) of the 129 TDFs can be divided into six functional categories, including defense (D), signal transduction (ST), energy metabolism (EM), regulation (R), protein–protein interaction (PI), others (O) and unknown (Un; Figure 2). Forty-one TDFs (31.8%) of the annotated sequences were associated with defense. Among them, four TDFs were associated with the pathogenesis-related protein, include β-1,3-glucanase (TDF3), hapless 8 (TDF8), pathogenesis-related protein (TDF28) and thaumatin-like protein (TDF16), and others had hypersensitive-induced response protein (TDF22), mannose-binding lectin superfamily protein (TDF23), respiratory burst oxidase protein (TDF13) and so on. Thirty-two TDFs (24.8%) were involved in signal transduction, for example, a member of the BEL family of homeodomain proteins (TDF49), catalase (TDF42), calcium ion binding (TDF48) and so on. Followed that, 19 TDFs (14.7%) mainly involved in regulation, including ATP-binding cassette G36 (TDF91), heat-shock cognate protein (TDF102) and so on. Fifteen TDFs (11.7%) were mainly involved in energy metabolism, for example, TDF81 was predicted to involve in Arabidopsis thaliana photosynthetic electron transfer chain. Four TDFs (3.1%) and five TDFs (3.9%) were involved in protein–protein interaction and others metabolic pathways, respectively. These genes might function to protect cells from the fungal pathogen in non-heading Chinese cabbage. No function was assigned to 13 (10.0%) of the TDFs as they showed no or low sequence similarities in the Brassica database search. In conclusion, gene expression patterns are more complex after infection and involved in many different metabolic pathways. It indicates that it is the common effect of these different metabolic pathways that improved the plant resistance to fungus, thereby reducing the hypersensitive response (HR) in resistance line against fungus pathogen.

Table 2. Homology of obtained TDFs in the non-heading Chinese cabbage–H. parasitica interaction.

TDF no. Accession number Length (bp) A. thaliana Gene name in Arabidopsis Gene name in B. rapa Gene name in B. campestris ssp. chinensis Functional categories 0 h 24 h 48 h 72 h
1 AB474661 302 AT3G47340 ASN1 Bra018160 BcASN1_A06 D — X x x
2 AB474690 202 AT1G33970 AT1G33970 Bra028016 AT1G33970_A09 D — x X x
3 AB474678 149 AT3G57260 PR2 Bra014636 BcBG3_A04 D — x X x
4 AB474674 313 AT1G52400 ATBG1 Bra018969 BcATBG1_A06 D x X x x
5 AB474696 127 AT1G55490 CPN60B Bra011919 BcCPN60B_A07 D — x x x
6 AB474682 297 AT1G02305 AT1G02305 Bra030498 AT1G02305_A08 D — x x x
7 AB474660 221 AT5G13930 CHS Bra008792 BcCHS_A10 D — x X x
8 AB474679 122 AT5G56250 HAP8 Bra003169 BcHAP8_A07 D — x x x
9 AB474688 80 AT1G08400 AT1G08400 Bra018627 AT1G08400_A06 D — x x x
10 AB474667 228 AT2G16600 ROC3 Bra037296 BcROC3_A09 D — x x x
11 AB474666 441 AT3G55440 TPI Bra014743 BcTPI_A04 D — x X x
12 AB474665 132 AT1G01040 ASU1 Bra033293 BcASU1_A10 D x x X x
13 AB474676 326 AT2G03820 NMD3 Bra040033 BcNMD3_A01 D — x x x
14 AB474643 304 AT3G14840 LIK1 Bra021579 BcLIK1_A01 D — x X x
15 AB474693 293 AT2G47070 SPL1 Bra041037 BcSPL1_Scaffold000403 D — x x x
16 FJ605478 954 AT1G18250 ATLP-1 Bra025923 BcATLP-1_A06 D — x x x
17 AB474697 223 AT1G60950 FD2 Bra031471 BcFD2_A01 D — x x x
18 AB474681 204 AT1G08540 SIG1 Bra030732 BcSIG1_A08 D — x x x
19 AB474684 160 AT3G60120 BGLU27 Bra004840 BcBGLU27_A05 D — x x x
20 AB474664 383 AT2G41480 PRX25 Bra000228 BcPRX25_A03 D — x x x
21 AB474689 265 AT1G18360 AT1G18360 Bra016558 AT1G18360_A08 D — x x x
22 AB474694 119 AT3G01290 HIR2 Bra039130 BcHIR2_A05 D — x x x
23 AB474671 204 AT1G52120 AT1G52120 Bra018940 AT1G52120_A06 D — x x x
24 AB474685 258 AT5G42870 PAH2 Bra027461 BcPAH2_A09 D — x x x
25 AB474659 100 AT2G23760 BLH4 Bra032147 BcBLH4_A04 D — x x x
26 AB474677 100 AT1G25540 MED25 Bra038087 BcMED25_A08 D — — x x
27 AB474686 67 AT3G14210 ESM1 Bra027359 BcESM1_A05 D x X x x
28 AB474683 126 AT2G14580 PRB1 Bra013123 BcPR1_A03 D — x x x
29 AB474662 138 AT3G27310 PUX1 Bra025265 BcPUX1_A06 D — — x —
30 AB474669 112 AT2G35100 ARAD1 Bra023001 BcARAD1_A03 D — x x x
31 AB474663 255 AT5G63110 HDA6 Bra035858 BcHDA6_A09 D — — x —
32 AB474695 150 AT3G04790 EMB3119 Bra040120 BcEMB3119_A01 D — X X x
33 AB474658 80 AT4G34850 LAP5 Bra011566 BcLAP5_A01 D — x x x
34 AB474687 363 AT5G67360 ARA12 Bra037113 BcARA12_A09 D — x x x
35 AB474668 183 AT5G58070 TIL Bra006784 BcTIL_A03 D — x x x
36 AB474691 263 AT3G16560 AT3G16560 Bra022166 AT3G16560_A05 D X x x x
37 AB474675 336 AT5G38430 RBCS1B Bra028181 BcRBCS1B_A04 D — x x X
38 AB439291 556 AT1G54410 ATHIRD11 Bra013206 BcATHIRD11_A03 D x X x x
39 AB439836 528 AT3G09390 ATMT-1 Bra029765 BcATMT-1_A05 D — x X x
40 AB474649 315 AT5G44790 RAN1 Bra025102 BcRAN1_A06 D x X x x
41 AB474717 316 AT5G53300 UBC10 Bra022645 BcUBC10_A02 D — x x x
42 AB474628 266 AT1G20620 CAT3 Bra012238 BcCAT3_A07 ST — X X x
43 AB495004 917 AT5G06290 2CPB Bra009181 Bc2CPB_A10 ST x x X x
44 AB474632 843 AT1G12520 CCS Bra016768 BcCCS_A08 ST x x x x
45 AB474627 239 AT5G56500 CPN60BETA3 Bra028922 BcCPN60BETA3_A03 ST — x X x
46 AB474645 121 AT5G18020 SAUR20 Bra026598 BcSAUR20_A02 ST — x x x
47 AB474656 264 AT4G28080 AT4G28080 Bra024230 AT4G28080_A03 ST — X x x
48 AB474629 140 AT3G56800 CAM3 Bra014671 BcCAM3_A04 ST — X x x
49 AB474632 843 AT1G12520 CCS Bra016768 BcCCS_A08 ST — — X x
50 AB474637 266 AT4G39220 ATRER1A Bra010695 BcATRER1A_A08 ST — — x x
51 AB474646 136 AT2G33150 PKT3 Bra005522 BcPKT3_A05 ST — x X x
52 AB474648 128 AT5G13650 SVR3 Bra008811 BcSVR3_A10 ST — x X x
53 AB474647 252 AT2G31060 EMB2785 Bra021694 BcEMB2785_A04 ST — X X x
54 AB474635 638 AT4G00700 AT4G00700 Bra000963 AT4G00700_A03 ST — X X x
55 AB474640 95 AT2G01400 AT2G01400 Bra026670 BcAT2G01400_A02 ST — — x —
56 AB474652 160 AT4G26240 AT4G26240 Bra019116 AT4G26240_A03 ST — x x x
57 AB474657 224 AT3G63070 HULK3 Bra040426 BcHULK3_A04 ST — — x X
58 AB474655 186 AT3G44300 NIT2 Bra023598 BcNIT2_A02 ST X x x x
59 AB474634 267 AT4G28490 RLK5 Bra011033 BcRLK5_A01 ST — X x x
60 AB474641 226 AT5G13530 KEG Bra006205 BcKEG_A03 ST X x x x
61 AB474633 231 AT2G22260 ALKBH2 Bra038536 BcALKBH2_A09 ST — x x —
62 AB474639 166 AT4G13940 MEE58 Bra032750 BcMEE58_A04 ST x x X x
63 AB474638 306 AT3G02470 SAMDC Bra001046 BcSAMDC_A03 ST X x x x
64 AB474651 405 AT3G55800 SBPASE Bra007192 BcSBPASE_A09 ST — — x x
65 AB474636 736 AT4G32200 ASY2 Bra039766 BcASY2_A01 ST — — x x
66 AB474630 369 AT5G16070 AT5G16070 Bra006338 AT5G16070_A03 ST — X X x
67 AB474654 236 AT4G21450 AT4G21450 Bra013528 AT4G21450_A01 ST — X x x
68 AB474650 180 AT4G31800 WRKY18 Bra023983 BcWRKY18_A03 ST x X x x
69 AB474714 291 AT3G22650 AT3G22650 Bra005858 AT3G22650_A03 ST — x x x
70 AB474715 199 AT1G47210 CYCA3;2 Bra040753 BcCYCA3;2_Scaffold000249 ST — X X x
71 AB474720 188 AT2G33800 EMB3113 Bra021879 BcEMB3113_A04 ST — x x x
72 AB474716 126 AT5G42220 AT5G42220 Bra027974 AT5G42220_A09 ST — — x x
73 AB474644 121 AT2G33620 AHL10 Bra021865 BcAHL10_A04 ST — x x x
74 AB474713 129 AT3G15290 AT3G15290 Bra027263 AT3G15290_A05 EM — X X x
75 AB474703 127 AT3G48990 AAE3 Bra018019 BcAAE3_A06 EM — x X x
76 AB474710 384 AT1G29920 LHCB1.1 Bra010807 BcLHCB1.1_A07 EM X x x x
77 AB474711 405 AT1G29920 CAB2 Bra010807 BcCAB2_A08 EM x x x x
78 AB474704 150 AT3G45190 AT3G45190 Bra038298 AT3G45190_A10 EM X x x x
79 AB474705 427 AT4G18760 RLP51 Bra040730 BcRLP51_A06 EM — x x —
80 AB474702 150 AT5G18800 AT5G18800 Bra002186 AT5G18800_A10 EM — x x x
81 AB474709 266 AT4G03280 PGR1 Bra000837 BcPGR1_A03 EM — x — x
82 AB474708 75 AT3G16560 AT3G16560 Bra022166 AT3G16560_A05 EM X x x x
83 AB474706 222 AT5G47910 RBOHD Bra020724 BcRBOHD_A02 EM — x x x
84 AB474712 239 AT5G38430 RBCS1B Bra028181 BcRBCS1B_A04 EM — X x x
85 AB439290 733 AT5G38430 RBCS1B Bra028174 BcRBCS1B_A04 EM — X x x
86 AB474707 53 AT3G60750 TKL1 Bra007555 BcTKL1_A09 EM — — x x
87 AB474626 190 AT5G36880 ACS Bra030286 BcACS_A04 EM — x X x
88 AB474613 210 AT2G38290 AMT2 Bra005125 BcAMT2_A05 EM — X x x
89 AB474614 169 AT2G33150 PKT3 Bra022927 BcPKT3_A03 R — — x x
90 AB474609 106 AT4G00810 AT4G00810 Bra037409 AT4G00810_A09 R — x — —
91 AB474616 349 AT1G59870 ABCG36 Bra003527 BcABCG36_A07 R X x x x
92 AB474621 144 AT4G34970 ADF9 Bra017683 BcADF9_A03 R — x x x
93 AB474610 140 AT3G23240 ERF1 Bra023744 BcERF1_A01 R — x x x
94 AB474624 310 AT3G55360 GLH6 Bra007154 BcGLH6_A09 R — x x x
95 AB474619 211 AT4G27640 AT4G27640 Bra026329 AT4G27640_A01 R — x x x
96 AB474612 119 AT1G35160 14-3-3PHI Bra028068 Bc14-3-3PHI_A09 R — x X X
97 AB439837 1062 AT1G01050 PPA1 Bra033292 BcPPA1_A10 R x — x x
98 AB474615 230 AT5G14400 CYP724A1 Bra023464 BcCYP724A1_A02 R — x x x
99 AB474718 200 AT3G62310 AT3G62310 Bra007671 AT3G62310_A09 R — X X x
100 AB495003 1418 AT4G11260 EDM1 Bra035239 BcEDM1_A09 R x X x x
101 AB474611 393 AT1G08540 SIG1 Bra030732 BcSIG1_A08 R — X X x
102 AB474719 173 AT5G02500 AT-HSC70-1 Bra009584 BcAT-HSC70-1_A10 R — x x x
103 AB474622 429 AT5G64940 ATH13 Bra024339 BcATH13_A06 R — X x x
104 AB474623 218 AT5G54770 THI4 Bra022742 BcTHI4_A02 R — X x x
105 AB474625 150 AT3G55360 GLH6 Bra007154 BcGLH6_A09 R x X X X
106 AB474618 220 AT1G65550 AT1G65550 Bra036544 AT1G65550_A09 R x x x x
107 AB474620 183 AT1G55620 CLCF Bra038007 BcRRN23S.2_A06 R — x x x
108 AB474699 478 AT3G32195 AT3G32195 Bra007659 AT3G32195_A09 PPI — x x x
109 AB474701 448 AT4G00700 AT4G00700 Bra000963 AT4G00700_A03 PPI — x x x
110 AB474698 180 AT1G78120 TPR12 Bra015628 BcTPR12_A07 PPI — x x x
111 AB474700 423 AT4G28270 ZF Bra026271 BcZF_A01 PPI — x x x
112 AB474727 308 AT5G53370 PMEPCRF Bra003062 BcPMEPCRF_A10 O x — x x
113 AB474721 113 AT3G62770 ATG18A Bra003509 ATG18A_A07 O — X X x
114 AB474726 583 AT1G19480 AT1G19480 Bra025741 AT1G19480_A06 O x X x x
115 AB474723 175 AT1G62970 AT1G62970 Bra027007 AT1G62970_A09 O — x X x
116 AB474730 340 AT4G34640 SQS1 Bra011548 BcSQS1_A01 O x x x —
117 AB474741 209 AT3G13720 PRA1.F3 Bra027406 BcPRA1.F3_A05 Un — X x x
118 AB474742 149 AT1G44191 AT1G44191 Bra010149 AT1G44191_A06 Un — — x x
119 AB474745 149 AT3G13080 MRP3 Bra039368 BcMRP3_Scaffold000164 Un — — — x
120 AB474733 126 AT1G14740 TTA1 Bra026192 BcTTA1_A06 Un — X x x
121 AB474737 205 AT3G29075 AT3G29075 Bra025663 AT3G29075_A04 Un x X x x
122 AB474740 149 AT3G13080 MRP3 Bra039368 BcMRP3_A06 Un x X x x
123 AB474736 121 AT5G42050 AT5G42050 Bra025450 AT5G42050_A04 Un — X x x
124 AB474731 130 AT5G09805 IDL3 Bra009082 BcIDL3_A10 Un x — X —
125 AB474738 191 AT1G70900 AT1G70900 Bra016176 AT1G70900_A07 Un x X x x
126 AB474735 239 AT3G29075 AT3G29075 Bra025663 AT3G29075_A04 Un x — — x
127 AB474739 77 AT3G49601 AT3G49601 Bra017971 AT3G49601_A06 Un — X X —
128 AB474732 398 AT4G19430 AT4G19430 Bra013396 AT4G19430_A01 Un — — X —
129 AB474746 301 AT5G44790 RAN1 Bra025102 BcRAN1_A06 Un — x x —
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Abbreviations: D, defense; EM, energy metabolism; O, others; PPI, protein-protein interaction; R, regulation; ST, signal transduction; Un, unknown. Legend: (X,X) Different signal intensity in cDNA-AFLP analysis (6% polyacrylamide gel). The bigger legend "X", the more signal intensity.

Figure 2.

Figure 2

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Classification of differentially accumulated TDFs after inoculation of H. parasitica. A total of 129 TDFs were classified based on the Chinese cabbage database.

Validation of expression patterns using qRT-PCR analysis

To investigate the reliability of cDNA-AFLP for detecting differentially expressed genes, qRT-PCR analysis was carried out for 15 TDFs. These TDFs were selected based on significantly different expression patterns in the time course of the cDNA-AFLP experiment and homology to genes known to have a role in defense, signal transduction, regulation and energy metabolism. Expression patterns of the 15 TDFs in non-heading Chinese cabbage leaves after infection are shown in Figure 3. The same expression pattern was found for each TDF with qRT-PCR analysis as observed in the cDNA-AFLP tests, except for TDF60 (BcKEG_A03) and TDF91 (BcABCG36_A07). As shown in Figure 3, TDFs that involved in defense (TDF1 (BcASN1_A06), TDF7 (BcCHS_A10), TDF11 (BcTPI_A04) and TDF14 (BcLIK1_A01)) had maximum expression at 48 h.p.i., except for TDF1 that had maximum expression peaked at 24 h.p.i. TDFs involved in signal transduction included TDF42 (BcCAT3_A07), TDF49 (BcCCS_A08), TDF58 (BcNIT2_A02), TDF59 (BcRLK5_A01), TDF60 (BcKEG_A03) and TDF63 (BcSAMDC_A03)). Among of them, TDF42, TDF49 and TDF59 had similar expression patterns and peaked at 24 or 48 h.p.i. TDF58 and TDF63 had minimum expression at 72 h.p.i., suggesting that these two genes may be repressed after infection. TDFs involved in energy metabolism included TDF75 (BcAAE3_A06), TDF76 (BcLHCB1.1_A07) and TDF88 (BcAMT2_A05). Among them, TDF75 and TDF88 were expressed highly at 0 h.p.i. compared with other TDFs, and showed maximum expression at 48 and 24 h.p.i., respectively. The results suggested that they may have been involved in the earlier stageinteraction between non-heading Chinese cabbage and H. Parasitica. TDF76 expressed very low at the 0 h.p.i. TDF91 (BcABCG36_A07) and TDF101 (BcSIG1_A08)) were related to regulation. TDF91 was slowly increased after 0 h.p.i. and maximum expression peaked at 48 h.p.i. TDF101 was strongly upregulated at 24 h.p.i. and decreased after 24 h.p.i. These results suggested that the selected TDFs with putative four categories of functions might be triggered rapidly and have an active role during the early incompatible interaction between non-heading Chinese cabbage and H. parasitica.

Figure 3.

Figure 3

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Quantitative real-time PCR analysis of 15 selected genes. (a–o) Both control and treated third leaf of five plants ‘Suzhou Qing’ were harvested and pooled at 0, 24, 48 and 72 h.p.i. The relative expression level for H. parasitica-inoculated plants at each time point was calculated as fold of the control plants at 0 using the LOG method. All data were normalized to the β-actin gene expression level. Error bars indicate s.d. of the three technical repeats.

Through BLAST searching in the Arabidopsis database (http://www.arabidopsis.org/), we found that four of the 15 TDFs were related with fungal resistance. To verify these expectations were related to fungal resistance, we performed a qRT-PCR experiment with a resistant and a susceptible line. Results are shown in Figure 4.

Figure 4.

Figure 4

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The comparison of partial TDFs (a–d) expression patterns between resistant line and susceptible line. The results show that these four genes has almost the same expression trend between two lines, but the expression of gene in the resistant line ‘Suzhou Qing’ is higher than that of in susceptible line ‘Aijiao Huang’. Both control and treated third leaf of five plants were harvested and pooled at 0, 24, 48 and 72 h.p.i. The gene expression level was calculated using the LOG method. The gene expression level in both lines was compared using LOG value directly. All data were normalized to the β-actin gene expression level. Error bars indicate s.d. of the three technical repeats. Asterisks indicate statistically significant differences compared ‘Aijiao Huang’ and ‘Suzhou Qing’ at each TDFs (Student’s t-test: *P<0.05; **P<0.01).

TDF14 (BcLIK1_A01) encodes LRR-RLK protein, is involved in regulation of innate immune response, and have a role against pathogens according to the homologous alignment in the Brassica database. As shown in Figure 4, gene expression of TDF14 was increased slowly after inoculation and expression peaked at 48 h.p.i. Although both have the same expression trends, the expression of TDF14 in resistant line ‘Suzhou Qing’ is higher than that in susceptible line ‘Aijiao Huang’, especially in 48 h.p.i.

TDF42 (BcCAT3_A07) encodes catalase and is involved in the regulation of defense. Catalase is one of the key enzymes in vivo anti-oxidative defense systems, which has a special role in removing the hydrogen peroxide to avoid the body to produce oxidative stress in the process. The expression patterns of TDF42 in two lines were similar to that of TDF14. TDF75 (BcAAE3_A06) encodes an oxalyl-CoA synthetase and involved in defense response to fungus. The gene expression in resistant line ‘Suzhou Qing’ was much higher than that in susceptible line ‘Aijiao Huang’, with fold change reaching to 100 times at 48 h.p.i.

TDF88 (BcAMT2_A05) encodes a high-affinity ammonium transporter and involved in ammonium transmembrane transport and defense response to fungus. The expression of TDF88 was almost the same in 0 h.p.i. in two lines, strongly induced subsequently and maximum expression both peaked at 24 h.p.i. But gene expression of TDF88 in resistant line ‘Suzhou Qing’ was always higher than that in susceptible line ‘Aijiao Huang’. The expression of the genes related with fungal resistance in resistance line were higher than that in susceptible line.

Discussion

We identified 129 TDFs, of which 121 TDFs were upregulated and eight were down-regulated using cDNA-AFLP.2,3 By BLAST searching in the Brassica database, these TDFs were classified according to their different functions. The functional categorization showed a complex linkage between proteins encoding by the TDFs. Information obtained from this study may provide a foundation for better understanding defense mechanisms of the non-heading Chinese cabbage with H. parasitica incompatible interaction.

Defense

Our data showed that several transcripts encoding the group of PR proteins were differentially expressed in the interaction (Table 2). For example, TDF3 (β-1, 3-glucanase) was induced within 24 h.p.i. and its expression peaked at 48 h.p.i. The expression levels of TDF8 (Hapless 8), TDF16 (a thaumatin-like protein) and TDF28 (PR 1-like protein) were induced within 24–72 h.p.i. Previously, we cloned the full length of β-1, 3-glucanase, hapless 8 and PR 1 genes, and analysed their expression patterns in response to H. parasitica infection in ‘Suzhou Qing’ cultivar of non-heading Chinese cabbage.10 The accumulations of these two transcripts were upregulated during the infection period, suggesting that these proteins may participate in the defence reaction for non-heading Chinese cabbage against H. parasitica. We also found that expression of TDF39 (ATMT-1) peaked at 48 h.p.i. In rice and barley, MT2A genes were induced by stresses such as drought, cold treatment and wounding or in response to pathogen attacks.12–14 Further research revealed that products of homologous MT scavenged the reactive oxygen species (ROS), such as OH to H2O.15 Evidences suggest that the generation of ROS occurs at early stage in the plant–pathogen interaction. Rapid accumulation of ROS causes oxidative burst that results in hypersensitive cell death and cell wall cross-link.16 Our data may indicate that the upregulation of MT2A in non-heading Chinese cabbage leaves may contribute to ROS accumulation for inducing the hypersensitive response of the plant.

Signal transduction

Studies suggest that several signal transduction-related proteins are involved in the plant–fungus interactions.1–3,17 We also identified many TDFs related to the signal transduction, such as TDF42 (Catalase 3), TDF43 (2-Cys PrxB), TDF45 (ATP binding), TDF50 (ATRER1A), TDF58 (Nitrilase), TDF48 (BcCAM3_A04, calcium ion binding) and TDF68 (WRKY DNA-binding protein).

Calcium binding-like proteins may have a role in signalling pathways against pathogens and wounding.18 A number of downstream targets of calmodulin (CaM), including nitric oxide synthase,19 barley MLO protein,20 maize Ca2+-CaM21 and transcriptional regulators,22 are involved in plant responses to pathogens. Given that calcium ion-binding proteins are important modulators of defence response in pathways for pathogen sensing in plants, the CAM 3 gene could have a special role as Ca2+ sensors during the plant immune response to the fungus H. Parasitica.

WRKY proteins are signal transcriptional factors recognizing the TTGAC (C/T) W-box elements in the promoters of a large number of plant defence-related genes.23 Many of WRKY genes are upregulated particularly in pathogen-infected, wounded or abiotic-treated plants.24 In this study, expression of WRKY DNA-binding protein peaked at 24 h.p.i., suggesting that the possible role of WRKYs is in the regulation of the genes associated with plant defence responses. However, we found that expression pattern of TDF60 (WRKY gene) determined by qRT-PCR was inconsistent with that of cDNA-AFLP. The inconsistence may be caused by different paralogues in the genome.

Regulation

An ethylene response factor (BcERF1_A01, TDF93; Table 2), a regulator of ethylene responses after pathogen attack in Arabidopsis,25 may have a key role in the non-heading Chinese cabbage–H. parasitica interaction. Previous studies have been demonstrated that ERFs are involved in regulating the expression of the defence-related genes during the disease resistance responses.26,27

We found that TDF91 (BcABCG36_A07, ATP-binding cassette g36) was inhibited after inoculation by cDNA-AFLP analysis. However, its expression was induced weakly at 24 h.p.i., peaked at 48 h.p.i. and decreased weakly at 72 h.p.i. afterwards by qRT-PCR analysis. TDF100 (BcEDM1_A09) coding for an enhanced downy mildew 1 homolog was found to be induced during the infection period. Its relationship with the fungi, bacteria and viruses has been identified to be regulators of R gene-mediated resistance in other crop species.28,29 Recent studies have revealed that EDM1 homologue gene SGT1 is required for pathogen-induced disease-associated cell death during both compatible and incompatible interactions in tobacco.30

Energy metabolism

Energy metabolism has an important role in plants–pathogen interaction. The photosynthetic carbon cycle (PCC) is part of the dark reactions of photosynthesis and can be roughly divided into three steps: carboxylation, reduction reaction and regeneration of RuBP.31 In this study, we found that some TDFs relating to energy metabolism were downregulated, such as TDF76 (chlorophyll a/b binding protein), TDF78 (SIT4 phosphatase-associated family protein) and TDF82 (PP2C-related protein), whereas some were upregulated, such as TDF79 (receptor like protein 51), TDF80 (NADH-ubiquinone oxidoreductase), TDF83 (respiratory burst oxidase protein), TDF85 (rubisco small subunit 1b; Table 2). Previous reports have identified that they are involved in PCC cycle, for example, TDF86 (reduction of transketolase) inhibited ribulose-1,5-bisphosphate regeneration and photosynthesis.31,32 These results are consistent with previous report that the expression of energy metabolism-related genes are induced and/or suppressed in photosynthesis during abiotic and biotic stresses.33,34 Our results may suggest that PCC cycle could provide protection function in energy metabolism during non-heading Chinese cabbage against H. parasitica.

Protein–protein interaction

A number of genes related to protein–protein interaction were induced after inoculation, such as TDF110 (BcTPR12_A07, tetratricopeptide repeat protein) and TDF111 (BcZF_A01, zinc-finger family protein). Of which, the gene expression of PAT is induced in the presence of ozone in Arabidopsis.35 The tryptophan biosynthetic enzymes, including anthranilate synthase (ASA) and PAT, are co-ordinately upregulated at both the messenger RNA and protein level during biotic and abiotic stress.36 We found that one of BcZF orthologous to A. thaliana was induced after inoculation (Table 2). Rizhsky et al. speculate that a zinc-finger protein is required for the expression of ascorbate peroxidase, which provides some measure of resistance for plant during oxidative stress.37 In this study, fact that pathogen-induced accumulation of these protein–protein interaction-related genes suggested that these genes may be involved in some defence mechanisms against H. parasitica indirectly.

Using the cDNA-AFLP method, we also detected several unknown functional genes. Their biological role is still unclear.

In this study, we examined gene expression patterns in an incompatible interaction between non-heading Chinese cabbage ‘Suzhou Qing’ and the downy mildew pathogen. We obtained 129 TDFs with different expression patterns and classified functional categories using cDNA-AFLP. Fifteen TDFs were randomly selected for validation of cDNA-AFLP expression patterns using qRT-PCR. Results showed that reliability of cDNA-AFLP is suitable for detecting differentially expressed genes. Among the 15 TDFs, four TDFs are related with fungal resistance, namely, TDF14 (BcLIK1_A01), TDF42 (BcCAT3_A07), TDF75 (BcAAE3_A06) and TDF88 (BcAMT2_A05). We further compared expression patterns in ‘Suzhou Qing’ and ‘Aijiao Huang’ using qRT-PCR. Results showed that the four genes displayed similar expression trend in the two lines. Importantly, the expression of genes in the resistant line is higher than that in susceptible line. These genes expression patterns and their putative functions may provide insight in understanding the non-heading Chinese cabbage–downy mildew incompatible interaction. Our study may also provide a foundation for better understanding molecular mechanisms and can be beneficial in selecting candidate resistance genes for the incompatible interaction between non-heading Chinese cabbage and H. Parasitica. Further research is needed to study the comparison between compatible and incompatible interactions to identify novel and common genes that regulate non-heading Chinese cabbage–downy mildew pathosystem.

Acknowledgments

The research was supported by the following: the Independent Innovation Fund for Agricultural Science and Technology of Jiangsu Province (CX (15)1015); the Science-technology Support Plan of Jiangsu Province (BE2012325, BE2013429); Natural Science Foundation of China (31272173); and Jiangsu Natural Science Foundation (BK20140704).

The authors declare no conflict of interest.

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