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A novel method for constructing pathogen-regulated small RNA cDNA library
Biochemical and Biophysical Research Communications 397 (2010) 532–536
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
A novel method for constructing pathogen-regulated small RNA cDNA library Chuan Bao Sun a,*, Xian Ming Du b, Yu Ke He a a
National Key Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China b Faculty of Medicine, The University of New South Wales, Sydney, NSW 2052, Australia
a r t i c l e
i n f o
Article history: Received 18 May 2010 Available online 31 May 2010 Keywords: Small non-coding RNA cDNA/RNA subtractive hybridization Stem–loop primer
a b s t r a c t Pathogen-responsive endogenous small non-coding RNAs regulate gene expression in relation to plant immune responses by serving as RNA silencing machinery. Decay caused by the bacterium, Erwinia carotovora subsp. carotovora (Ecc), often leads to soft rot disease in the plant Brassica campestris L. ssp. pekinensis (Bcp). To discover endogenous small RNA species in Bcp in response to Ecc infection, we developed a highly efficient approach for cloning pathogen-regulated small RNAs. A group of degenerate stem–loop reverse primers was designed to synthesize first single-stranded cDNA (sscDNA) and the sscDNA was then tailed with a poly(C) at its 30 end to create a forward priming site. A novel cDNA/RNA subtractive hybridization was performed to capture Ecc-regulated small RNAs and this subsequently allowed construction of small RNA cDNA libraries for sequencing. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Small RNAs regulate gene expression in many cellular processes such as development, genome maintenance, integrity, and adaptive responses to biotic and abiotic stresses in most eukaryotes. Similar to virus-derived small RNAs that may induce plant antiviral immunity, bacterium-regulated small RNAs have recently been demonstrated to enhance plant defence systems against pathogen invasion [1]. Small non-coding RNAs, usually 20–25 nucleotides (nt) in length, are typically characterized as two classes: microRNAs (miRNAs) and small interfering RNAs (siRNAs). In the model plant, Arabidopsis thaliana, there are at least three types of endogenous siRNAs—chromatin-associated siRNAs, trans-acting siRNAs, and natural antisense transcript (NAT)-associated siRNAs (nat-siRNAs) [2–4]. To date, 191 miRNAs have been found in Arabidopsis and demonstrated active in plant development, abiotic stress tolerance, and plant defense [1,5,6]. For example, Arabidopsis miRNA393 was induced by a bacterial 22-amino acid peptide (flg22), and contributed to a basal defense against the bacterial pathogen Pseudomonas syringae (Ps) by down-regulating auxin signaling pathway [7]. NatsiRNAs are derived from the overlapping region of a NAT pair, and up to date, 1340 unique NAT pairs are known in Arabidopsis [8]. Nat-siRNAATGB2 was induced by Ps carrying effector avrRpt2 and contributed to RPS2-mediated race-specific disease resistance by repressing PPRL [9]. Another group of small RNAs consisting of
* Corresponding author. E-mail address: [email protected] (C.B. Sun). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.05.151
30–40 nt, collectively known as long siRNAs (lsiRNAs), was recently discovered in Arabidopsis. For example, AtlsiRNA-1 downregulated AtRAP mRNA and the AtRAP-knockout mutant showed enhanced resistance to infection by virulent P. syringae pv. tomato (Pst) [10]. Bacterial soft rot disease caused by Erwinia carotovora subsp. carotovora (Ecc) results in serious damage to most vegetable crops including carrot, radish, potato, and all members of cabbage family and thus a huge economic loss. It is one of the most destructive diseases for Chinese cabbage (Brassica campestris L. ssp. pekinensis) in East Asian countries where the vegetable is widely cultivated [11]. We were interested in identifying novel Ecc-regulated small RNAs of Chinese cabbage. The conventional adaptor ligation/RT-PCR method to clone a small RNA requires the presence of 50 phosphate and free 30 hydroxyl group on the small RNA. Small RNAs are usually generated by RNase III to have 50 phosphate and 30 hydroxyl termini [12]. During this procedure, it is likely to lose small RNAs, particularly low abundant species. Ligation between adaptors (selfligation) may also lower the ligation efficacy between small RNAs and adaptors. In addition, sequencing low abundant small RNA species may be hampered by the coexistence of abundant species. Therefore, to identify pathogen-associated small non-coding RNAs, it was necessary to have a better method than the conventional one. In this study, we developed a new, more efficient and reliable method for cloning small RNA than the adaptor ligation/RT-PCR, firstly by using a set of degenerate stem–loop reverse primers to synthesize first single-stranded cDNA. Subsequently sscDNA was modified to generate a poly(C) tail at its 30 end and cDNA/RNA subtractive hybridization was performed to capture specifically Ecc-regulated (either induced or suppressed) small RNAs.
C.B. Sun et al. / Biochemical and Biophysical Research Communications 397 (2010) 532–536
2. Materials and methods 2.1. Plant material and bacterial strain Seeds of 99Bre, a Chinese cabbage (Bcp) strain, were obtained from XIN-QIAO farm in Shanghai. The cabbages were grown in a controlled environment at 22 ± 1 °C with a 16 h light/8 h dark photoperiod. All experiments were performed using 7-day-old Chinese cabbage plants. Ecc strain cgmcc 1.1000, originally isolated from a Bcp in Beijing of China in 1977, was used as inoculum for Chinese cabbage.
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PCR products were subjected to 2% agarose gel electrophoresis. The bands spanning 70–105 bps were purified from gel using QIAquick gel extraction kit (Qiagen) and cloned into pMD18-T Vector (Takara) for sequencing. The sequencing results were processed for BLAST against the Brassica sequences in the Brassica Information Resource database (http://brassica.bbsrc.ac.uk/BrassicaDB) and other plant sequences in the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov/BLAST/). 3. Results 3.1. First single-stranded cDNA were synthesized
2.2. Plant inoculation method A single colony of Ecc was proliferated on a nutrient agar plate (10.0 g peptone, 3.0 g beef extract, 5.0 g NaCl and 15.0 g agar per liter) at 28 °C for two days. The cells were harvested, washed, and suspended in sterile water to a density of 109 cells/ml. Seven-dayold Chinese cabbage seedlings were roots-inoculated with the bacterial suspension, while the seedlings inoculated with sterile water served as mock controls. The seedlings were incubated on MS plate at 22 °C under 95% humidity. Symptoms were monitored from the first day post inoculation (dpi) till the Ecc-infected plants decayed (two weeks after inoculation). The seedlings were harvested and flash-frozen in liquid nitrogen at 7 dpi for total RNA extraction. 2.3. First single-stranded cDNA synthesis Total RNA was isolated from plant samples using TrizolÒ Reagent (Invitrogen). Low molecular weight (LMW) RNA were separated from high molecular weight (HMW) RNA following a lithium chloride (LiCl) method as described elsewhere [13]. AMV reverse transcriptase, RNasinÒ ribonuclease inhibitor, terminal transferase, and Magnotex-SA beads were purchased from Takara Bio.Inc. To the mixture of annealed small RNA templates and 60 nM degenerate stem–loop reverse primers (primer A–D, Supplementary Table 1), 1 mM dNTP mix, 20U RNasinÒ ribonuclease inhibitor, 2U AMV reverse transcriptase and 1 reaction buffer were added and topped up with nuclease-free water to a final volume of 30 ll. The reaction was incubated at 16 °C for 30 min, 42 °C for 30 min and 85 °C for 5 min. 2.4. sscDNA tailing and subtractive hybridization of cDNA/RNA Tailing at the 30 terminus of sscDNA was performed by incubation at 37 °C for 60 min with 2 pmol sscDNA, 2 mM dCTP, and 2 U terminal transferase, and was terminated by incubation at 70 °C for 10 min. For subtractive hybridization, 2.5 lg each of small RNAs and sscDNA were mixed in a total volume of 60 ll. The reaction was overlayered with mineral oil and incubated in a thermal cycler at 98 °C for 1.5 min and then at 68 °C for 12 h. The cDNA/RNA hybrid and cDNA labeled with biotin were incubated with streptavidin magnetic beads to achieve biotin–avidin binding. The mixture stood undisturbed at room temperature for 1 h and then on a magnetite separation stand for 1 min. The supernatant was gently transferred to a new microtube and the pathogen-regulated small RNAs were precipitated by ethanol for further amplification. 2.5. Amplification and cloning of small RNAs The tailed cDNA was amplified with a pair of forward (f) and reverse (r) primers. PCR conditions were as follows: 2 min at 94 °C followed by 15 s at 94 °C, 15 s at 55 °C, and 10 s at 72 °C for 20 cycles. The primers for amplifying Ecc-induced or -suppressed small RNA populations were designated primer1f and primer1r or primer2f and primer2r, respectively (Supplementary Table 1). The
To investigate whether endogenous small RNAs of Bcp were generated specifically in response to Ecc infection, 7-day-old seedlings 99Bre were divided into two groups. One group was inoculated with Ecc, and the other with mock. The former was further divided into two subgroups in terms of their soft rot severity at 7 dpi, one exhibiting typical root decay and the other mild or no symptoms. Total RNA was extracted from all these groups of seedlings by Trizol reagent and 2 ll of total RNA in each group were checked for its purity and degradation by capillary electrophoresis (CE) and Agilent 2100 Bioanalyzer RNA chip (Agilent Technologies). For the Ecc-infected RNA sample, the ratios of 260–280 nm and 28S–18S ribosome were 1.83 or 1.4, respectively, showing a slight degradation. For the mock RNA sample, the ratios were 1.85 for 260/280 nm and 1.7 for 28S/18S, demonstrating that the total RNA was intact (Supplementary Fig. 1). This is normal since the degradation occurs as a result of Ecc infection on seedlings. As LMW RNA can be dissolved in 4 M lithium chloride (LiCl) but not HMW RNA, total RNA was dissolved in 4 M LiCl and centrifuged to precipitate HMW RNA. LMW RNA in supernatant was resolved on a 17%, 8 M urea PAGE gel. Small RNAs with a size range of 15–50 nt were extracted from the gel by 0.4 M NaCl at 4 °C overnight. They were recovered by ethanol precipitation and used for first single-stranded cDNA synthesis. A group of four degenerate stem–loop reverse primers (Supplementary Table 1) was used to generate cDNA. A typical stem–loop primer comprises an universal sequence of 35 nt at the 50 end, 5 nt at the 30 end complementary to a specific small RNA, and a stretch of nucleotides in the middle of the primer to form a stem–loop structure (Fig. 1). The sequence of 35 nt at the 50 end shares no homology with any sequences registered in public databases but contains a 454 sequencing adaptor B (454 Life Sciences, http:// www.454.com) [14] for high-throughput DNA sequencing. Being unique to a small RNA, the 30 end 5 nt of a primer allows itself to specifically bind to the small RNA, which ensures the initiation of reverse transcription. The internal nucleotides form a stem–loop structure within the primer that may limit nonspecific annealing to primary small RNA (genomic DNA). Therefore, the stem–loop primers have multiple functions. They can initiate synthesis of cDNA from a small RNA template, introduce a specific site at the 30 end of the cDNA for further amplification and provide a site for annealing high-throughput sequencing primer. To capture as many small RNA species as possible, four primers, A–D were designed. The first nucleotide at the 30 end of each primer begins with either A, T, G, or C and the 50 end was labeled with biotin. These stem–loop reverse primers can be used to synthesize both highand low-abundant small RNAs. 3.2. Subtractive hybridization removed the same background small RNAs Plant endogenous small RNAs may be induced or suppressed by pathogenic invasion. To isolate these endogenous small RNAs, each total RNA extracted from Bcp treated with either Ecc or mock was
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Fig. 1. Synthesis of first single-stranded cDNA of small RNA, miR166 as an example using a degenerate stem–loop reverse primer.
divided into two halves. One was used as template for first sscDNA synthesis, and the other for hybridization with the first sscDNA that was transcribed from the counterpart small RNAs. To capture Ecc-induced small RNAs, the first sscDNA of mocked seedlings were hybridized with small RNAs of Ecc-infected. If a small RNA matches with nucleotide sequence of certain sscDNA, it will anneal to form a double-stranded cDNA/RNA hybrid with a labeled biotin at the 50 end. Incubation with magnotex-labeled streptavidin beads followed by centrifugation may remove both cDNA/RNA hybrids and all unhybridized sscDNA via biotin–streptavidin binding. The Ecc-induced small RNAs exclusively remained in the hybridization solution. Similarly, to capture Ecc-suppressed small RNAs, the first sscDNA of Ecc-infected seedlings were hybridized with the small RNAs of mock, the cDNA/RNA hybrids, and unhybridized sscDNA were removed by magnotex-labeled streptavidin beads. The Eccsuppressed small RNAs remained in the solution. In this way, the
pathogen-regulated endogenous small RNAs, either down- or upregulated, were captured by the cDNA/RNA subtractive hybridization. The procedure is shown in Fig. 2A. 3.3. Ecc-regulated endogenous small RNAs were cloned Terminal Deoxynucleotidyl Transferase (TdT) catalyzes addition of mononucleotides (dNTPs) to the terminal 30 -OH of a DNA initiator. It added a poly (C) tail to the 30 termini of first sscDNA and this generated a priming site for the second-stranded cDNA synthesis. A primer that comprises a 454 sequencing adaptor A sequence with an oligo (dG) was used to generate the second-stranded cDNA. Two different bar codes were inserted between the 454 sequencing adaptor A and oligo (dG) to mark origins of endogenous small RNA. For PCR amplification, a pair of primers (Supplementary Table 1) consisting of 454 sequencing adaptor A (or B) sequence were used to perform a standard PCR as shown in Fig. 2B. The
Fig. 2. Schematic flowchart of construction of pathogen-regulated small RNA cDNA library. (A) cDNA/RNA hybridization for capturing Ecc-regulated endogenous small RNAs. (B) cloning of Ecc-regulated endogenous small RNAs.
C.B. Sun et al. / Biochemical and Biophysical Research Communications 397 (2010) 532–536
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Fig. 3. Gel electrophoresis for cDNA libraries constructed by both novel and traditional methods. (A) PCR products of Ecc-regulated endogenous small RNAs resolved on 2% agarose gel. Ecc: Ecc-suppressed small RNAs. Ecc+: Ecc-induced small RNAs. (B) PCR products of microRNAs for testing the cDNA libraries separated on 2% agarose gel. Top: cDNA library A constructed with adaptor ligations. MiR162, miR173, and miR393 were unable to be detected. Bottom: cDNA library B constructed with cDNA/RNA subtractive hybridization and stem–loop reverse primers.
PCR products were purified from agarose gel for 454 sequencing amplification (Fig. 3A). 4. Discussion Plant genes can be induced or suppressed by pathogen infection [15–17]. RNA silencing has been known to function in antiviral defense systems by post-transcriptional gene silence (PTGS) [18–20], and recent evidence suggests that PTGS also functions in basal defense and race-specific resistance to microbes in plants [7,9,10]. To identify small RNA species induced or suppressed specifically under soft rot pathogenic invasion, we developed a simple, rapid, and highly efficient method to clone Ecc-regulated endogenous small RNAs. Traditional protocols to amplify small RNAs employ total RNA preparation, separation of LMW and HMW RNA, purification of small RNAs, 30 adaptor ligation, 50 adaptor ligation, reverse transcription, and PCR amplification (Supplementary Fig. 2). To prevent either small RNA or 30 adaptor from self-ligation in the 30 adaptor ligation, the small RNA needs to be dephosphorylated at the 50 -terminus and excess amount of the adaptor over the small RNA is required. In contrast, the 50 -terminus of 30 ligation product is required to be phosphorylated prior to 50 adaptor ligation. Thus, the ligation efficiency is remarkably reduced as a result of the dephosphorylation and phosphorylation, especially for low abundant endogenous small RNAs. Furthermore, it could lead to loss of some RNAs in PCR amplification (Fig. 3B top). There have been a few reports about tailing small RNAs with a 30 -poly(A) tail to provide reverse priming sites for cDNA synthesis, but only those RNAs such as mammalian miRNAs with a 30 hydroxyl end were efficiently polyadenylated [21,22]. Plant miRNAs and endogenous siRNAs often contain a 20 -O-methy group at the 30 end, and cannot be tailed by Poly(A) polymerase [23,24]. To discover novel small RNAs, we introduced a group of biotinlabeled degenerate stem–loop reverse primers which bound to the 30 end of small RNAs. The small RNAs were efficiently reverse-transcribed to their first sscDNA (Fig. 1). These primers have the following advantages: the ability of a short reverse priming sequence annealed to the 30 end of small RNA discriminates small RNA by only one base. Second, a double-stranded stem structure inhibits hybridization of the reverse primer to small RNA precursors and other long RNAs, improving the reverse transcription efficacy [23,24]. Here we have randomly chosen three microRNAs, miR166, miR168, and miR172 to check the first single-stranded cDNA population I (Ecc) and II (Ecc+) (Supplementary Table 2). All PCR reactions were positive, which demonstrated the effectiveness of degenerate stem–loop reverse primers for small RNA
reverse transcription (Supplementary Fig. 3). For the secondstranded cDNA synthesis, a poly(C) tail was added to the 30 end of the first single-stranded cDNA by TdT, hence providing a forward priming site for further PCR. Therefore, our method offers a highly specific and quantitative mean to clone small RNAs using a very small amount of starting material (1–10 ng of total RNA) [25,26]. Although high-throughput sequencing was applied to sequence small RNAs, it was difficult to isolate signals of low abundant small RNAs from high-throughput sequencing reads. To address this problem, we performed a subtractive cDNA/RNA hybridization to remove any small RNA of the same abundance in both infected and mock samples and thus, only those species with different abundances remained. The biotin-labeled first sscDNA allowed removal of double-stranded cDNA/RNA hybrids by magnotex-labeled streptavidin beads. To examine the sensitivity of our method, we randomly selected 10 microRNAs, miR156, miR159, miR160, miR162, miR166, miR172, miR173, miR393, miR396, and miR398 to test their cDNA libraries A (Ecc+) and B (Ecc+). The primers for the two libraries were listed in Supplementary Table 1 (our method) or 2 (traditional method) respectively. The PCR products analyzed on 2% agarose gel are shown in Fig. 3B. The construction of cDNA library A (Ecc+) was performed by traditional method using Takara small RNA cloning kit. The results clearly showed that our approach successfully amplified small RNAs such as miR162, miR173, and miR393 (Fig. 3B bottom) whereas the commercial kit did not (Fig. 3B top). In this study, we constructed the cDNA 454 sequencing libraries for small RNA species spanning 15–50 nt (Fig. 3A). The libraries can be directly processed with high-throughput sequencing. Endogenous small RNA-mediated gene silencing underlies the mechanism for gene regulation in plant defense responses [1,9,10], thus, the bacterial pathogen, e.g. Ecc, regulated small RNAs may play an important role in host defense systems. The bioinformatics analysis of small RNA cDNA libraries would help elucidate the molecular events related to plant disease resistance. Modifying pathogen-regulated small RNAs or their target genes could potentially be useful in molecular breeding procedure to increase disease resistance in plants. Identification and characterization of small RNAs by novel amplification approaches may contribute to the understanding of plant immune systems in bacterial pathogenic diseases. Acknowledgments We especially thank Dr. Mi-Jurng Kim for her critical reading of the paper, and Dr. Naweed I.N. for helpful discussion and comments on the paper. Chuan Bao Sun was supported by a scholarship from SA-SIBS Scholarship Program. This work was supported by a
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Grant from the National Natural Science Foundation of China (No. 30871617) and a Grant from the Science and Technology Commission of Shanghai Municipality (No. 09JC1416000) and the National High-tech R&D Program of China (863 Program No. 2008AA 02Z111). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2010.05.151. References [1] H. Jin, Endogenous small RNAs and antibacterial immunity in plants, FEBS Lett. 582 (2008) 2679–2684. [2] X. Chen, MicroRNA biogenesis and function in plants, FEBS Lett. 579 (2005) 5923–5931. [3] A.M. Gustafson, E. Allen, S. Givan, D. Smith, J.C. Carrington, K.D. Kasschau, ASRP: the Arabidopsis small RNA project database, Nucleic Acids Res. 33 (2005) D637–D640. [4] B. Zhang, X. Pan, G.P. Cobb, T.A. Anderson, Plant microRNA: a small regulatory molecule with big impact, Dev. Biol. 289 (2006) 3–16. [5] U. Ellendorff, E.F. Fradin, R. de Jonge, B.P. Thomma, RNA silencing is required for Arabidopsis defence against Verticillium wilt disease, J. Exp. Bot. 60 (2009) 591–602. [6] R. Sunkar, J.K. Zhu, Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis, Plant Cell 16 (2004) 2001–2019. [7] L. Navarro, P. Dunoyer, F. Jay, B. Arnold, N. Dharmasiri, M. Estelle, O. Voinnet, J.D. Jones, A plant miRNA contributes to antibacterial resistance by repressing auxin signaling, Science 312 (2006) 436–439. [8] X.J. Wang, T. Gaasterland, N.H. Chua, Genome-wide prediction and identification of cis-natural antisense transcripts in Arabidopsis thaliana, Genome Biol. 6 (2005) R30. [9] S. Katiyar-Agarwal, R. Morgan, D. Dahlbeck, O. Borsani, A. Villegas Jr., J.K. Zhu, B.J. Staskawicz, H. Jin, A pathogen-inducible endogenous siRNA in plant immunity, Proc. Natl Acad. Sci. USA 103 (2006) 18002–18007. [10] S. Katiyar-Agarwal, S. Gao, A. Vivian-Smith, H. Jin, A novel class of bacteriainduced small RNAs in Arabidopsis, Genes Dev. 21 (2007) 3123–3134. [11] J. Ren, R. Petzoldt, M.H. Dickson, Screening and identification of resistance to bacterial soft rot in Brassica rapa, Euphytica 118 (2001) 271–280. [12] S. Boutet, F. Vazquez, J. Liu, C. Beclin, M. Fagard, A. Gratias, J.B. Morel, P. Crete, X. Chen, H. Vaucheret, Arabidopsis HEN1: a genetic link between endogenous miRNA controlling development and siRNA controlling transgene silencing and virus resistance, Curr. Biol. 13 (2003) 843–848.
[13] S. Katiyar-Agarwal, H. Jin, Discovery of pathogen-regulated small RNAs in plants, Methods Enzymol. 427 (2007) 215–227. [14] M. Margulies, M. Egholm, W.E. Altman, S. Attiya, J.S. Bader, L.A. Bemben, J. Berka, M.S. Braverman, Y.J. Chen, Z. Chen, S.B. Dewell, L. Du, J.M. Fierro, X.V. Gomes, B.C. Godwin, W. He, S. Helgesen, C.H. Ho, G.P. Irzyk, S.C. Jando, M.L. Alenquer, T.P. Jarvie, K.B. Jirage, J.B. Kim, J.R. Knight, J.R. Lanza, J.H. Leamon, S.M. Lefkowitz, M. Lei, J. Li, K.L. Lohman, H. Lu, V.B. Makhijani, K.E. McDade, M.P. McKenna, E.W. Myers, E. Nickerson, J.R. Nobile, R. Plant, B.P. Puc, M.T. Ronan, G.T. Roth, G.J. Sarkis, J.F. Simons, J.W. Simpson, M. Srinivasan, K.R. Tartaro, A. Tomasz, K.A. Vogt, G.A. Volkmer, S.H. Wang, Y. Wang, M.P. Weiner, P. Yu, R.F. Begley, J.M. Rothberg, Genome sequencing in microfabricated highdensity picolitre reactors, Nature 437 (2005) 376–380. [15] S.T. Chisholm, G. Coaker, B. Day, B.J. Staskawicz, Host–microbe interactions: shaping the evolution of the plant immune response, Cell 124 (2006) 803–814. [16] J.L. Dangl, J.D. Jones, Plant pathogens and integrated defence responses to infection, Nature 411 (2001) 826–833. [17] R.P. Wise, M.J. Moscou, A.J. Bogdanove, S.A. Whitham, Transcript profiling in host–pathogen interactions, Annu. Rev. Phytopathol. 45 (2007) 329– 369. [18] Q.W. Niu, S.S. Lin, J.L. Reyes, K.C. Chen, H.W. Wu, S.D. Yeh, N.H. Chua, Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance, Nat. Biotechnol. 24 (2006) 1420–1428. [19] J. Qu, J. Ye, R. Fang, Artificial microRNA-mediated virus resistance in plants, J. Virol. 81 (2007) 6690–6699. [20] X. Zhang, Y.R. Yuan, Y. Pei, S.S. Lin, T. Tuschl, D.J. Patel, N.H. Chua, Cucumber mosaic virus-encoded 2b suppressor inhibits Arabidopsis Argonaute1 cleavage activity to counter plant defense, Genes Dev. 20 (2006) 3255–3268. [21] S. Ro, C. Park, J. Jin, K.M. Sanders, W. Yan, A. PCR-based, A PCR-based method for detection and quantification of small RNAs, Biochem. Biophys. Res. Commun. 351 (2006) 756–763. [22] S. Ro, C. Park, R. Song, D. Nguyen, J. Jin, K.M. Sanders, J.R. McCarrey, W. Yan, Cloning and expression profiling of testis-expressed piRNA-like RNAs, RNA 13 (2007) 1693–1702. [23] P. Mestdagh, T. Feys, N. Bernard, S. Guenther, C. Chen, F. Speleman, J. Vandesompele, High-throughput stem–loop RT-qPCR miRNA expression profiling using minute amounts of input RNA, Nucleic Acids Res. 36 (2008) e143. [24] T.D. Schmittgen, E.J. Lee, J. Jiang, A. Sarkar, L. Yang, T.S. Elton, C. Chen, Realtime PCR quantification of precursor and mature microRNA, Methods 44 (2008) 31–38. [25] C. Chen, D.A. Ridzon, A.J. Broomer, Z. Zhou, D.H. Lee, J.T. Nguyen, M. Barbisin, N.L. Xu, V.R. Mahuvakar, M.R. Andersen, K.Q. Lao, K.J. Livak, K.J. Guegler, Realtime quantification of microRNAs by stem–loop RT-PCR, Nucleic Acids Res. 33 (2005) e179. [26] H. Yang, J.J. Schmuke, L.M. Flagg, J.K. Roberts, E.M. Allen, S. Ivashuta, L.A. Gilbertson, T.A. Armstrong, A.T. Christian, A novel real-time polymerase chain reaction method for high throughput quantification of small regulatory RNAs, Plant Biotechnol. J. 7 (2009) 621–630.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
A novel method for constructing pathogen-regulated small RNA cDNA library Chuan Bao Sun a,*, Xian Ming Du b, Yu Ke He a a
National Key Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China b Faculty of Medicine, The University of New South Wales, Sydney, NSW 2052, Australia
a r t i c l e
i n f o
Article history: Received 18 May 2010 Available online 31 May 2010 Keywords: Small non-coding RNA cDNA/RNA subtractive hybridization Stem–loop primer
a b s t r a c t Pathogen-responsive endogenous small non-coding RNAs regulate gene expression in relation to plant immune responses by serving as RNA silencing machinery. Decay caused by the bacterium, Erwinia carotovora subsp. carotovora (Ecc), often leads to soft rot disease in the plant Brassica campestris L. ssp. pekinensis (Bcp). To discover endogenous small RNA species in Bcp in response to Ecc infection, we developed a highly efficient approach for cloning pathogen-regulated small RNAs. A group of degenerate stem–loop reverse primers was designed to synthesize first single-stranded cDNA (sscDNA) and the sscDNA was then tailed with a poly(C) at its 30 end to create a forward priming site. A novel cDNA/RNA subtractive hybridization was performed to capture Ecc-regulated small RNAs and this subsequently allowed construction of small RNA cDNA libraries for sequencing. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Small RNAs regulate gene expression in many cellular processes such as development, genome maintenance, integrity, and adaptive responses to biotic and abiotic stresses in most eukaryotes. Similar to virus-derived small RNAs that may induce plant antiviral immunity, bacterium-regulated small RNAs have recently been demonstrated to enhance plant defence systems against pathogen invasion [1]. Small non-coding RNAs, usually 20–25 nucleotides (nt) in length, are typically characterized as two classes: microRNAs (miRNAs) and small interfering RNAs (siRNAs). In the model plant, Arabidopsis thaliana, there are at least three types of endogenous siRNAs—chromatin-associated siRNAs, trans-acting siRNAs, and natural antisense transcript (NAT)-associated siRNAs (nat-siRNAs) [2–4]. To date, 191 miRNAs have been found in Arabidopsis and demonstrated active in plant development, abiotic stress tolerance, and plant defense [1,5,6]. For example, Arabidopsis miRNA393 was induced by a bacterial 22-amino acid peptide (flg22), and contributed to a basal defense against the bacterial pathogen Pseudomonas syringae (Ps) by down-regulating auxin signaling pathway [7]. NatsiRNAs are derived from the overlapping region of a NAT pair, and up to date, 1340 unique NAT pairs are known in Arabidopsis [8]. Nat-siRNAATGB2 was induced by Ps carrying effector avrRpt2 and contributed to RPS2-mediated race-specific disease resistance by repressing PPRL [9]. Another group of small RNAs consisting of
* Corresponding author. E-mail address: [email protected] (C.B. Sun). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.05.151
30–40 nt, collectively known as long siRNAs (lsiRNAs), was recently discovered in Arabidopsis. For example, AtlsiRNA-1 downregulated AtRAP mRNA and the AtRAP-knockout mutant showed enhanced resistance to infection by virulent P. syringae pv. tomato (Pst) [10]. Bacterial soft rot disease caused by Erwinia carotovora subsp. carotovora (Ecc) results in serious damage to most vegetable crops including carrot, radish, potato, and all members of cabbage family and thus a huge economic loss. It is one of the most destructive diseases for Chinese cabbage (Brassica campestris L. ssp. pekinensis) in East Asian countries where the vegetable is widely cultivated [11]. We were interested in identifying novel Ecc-regulated small RNAs of Chinese cabbage. The conventional adaptor ligation/RT-PCR method to clone a small RNA requires the presence of 50 phosphate and free 30 hydroxyl group on the small RNA. Small RNAs are usually generated by RNase III to have 50 phosphate and 30 hydroxyl termini [12]. During this procedure, it is likely to lose small RNAs, particularly low abundant species. Ligation between adaptors (selfligation) may also lower the ligation efficacy between small RNAs and adaptors. In addition, sequencing low abundant small RNA species may be hampered by the coexistence of abundant species. Therefore, to identify pathogen-associated small non-coding RNAs, it was necessary to have a better method than the conventional one. In this study, we developed a new, more efficient and reliable method for cloning small RNA than the adaptor ligation/RT-PCR, firstly by using a set of degenerate stem–loop reverse primers to synthesize first single-stranded cDNA. Subsequently sscDNA was modified to generate a poly(C) tail at its 30 end and cDNA/RNA subtractive hybridization was performed to capture specifically Ecc-regulated (either induced or suppressed) small RNAs.
C.B. Sun et al. / Biochemical and Biophysical Research Communications 397 (2010) 532–536
2. Materials and methods 2.1. Plant material and bacterial strain Seeds of 99Bre, a Chinese cabbage (Bcp) strain, were obtained from XIN-QIAO farm in Shanghai. The cabbages were grown in a controlled environment at 22 ± 1 °C with a 16 h light/8 h dark photoperiod. All experiments were performed using 7-day-old Chinese cabbage plants. Ecc strain cgmcc 1.1000, originally isolated from a Bcp in Beijing of China in 1977, was used as inoculum for Chinese cabbage.
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PCR products were subjected to 2% agarose gel electrophoresis. The bands spanning 70–105 bps were purified from gel using QIAquick gel extraction kit (Qiagen) and cloned into pMD18-T Vector (Takara) for sequencing. The sequencing results were processed for BLAST against the Brassica sequences in the Brassica Information Resource database (http://brassica.bbsrc.ac.uk/BrassicaDB) and other plant sequences in the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov/BLAST/). 3. Results 3.1. First single-stranded cDNA were synthesized
2.2. Plant inoculation method A single colony of Ecc was proliferated on a nutrient agar plate (10.0 g peptone, 3.0 g beef extract, 5.0 g NaCl and 15.0 g agar per liter) at 28 °C for two days. The cells were harvested, washed, and suspended in sterile water to a density of 109 cells/ml. Seven-dayold Chinese cabbage seedlings were roots-inoculated with the bacterial suspension, while the seedlings inoculated with sterile water served as mock controls. The seedlings were incubated on MS plate at 22 °C under 95% humidity. Symptoms were monitored from the first day post inoculation (dpi) till the Ecc-infected plants decayed (two weeks after inoculation). The seedlings were harvested and flash-frozen in liquid nitrogen at 7 dpi for total RNA extraction. 2.3. First single-stranded cDNA synthesis Total RNA was isolated from plant samples using TrizolÒ Reagent (Invitrogen). Low molecular weight (LMW) RNA were separated from high molecular weight (HMW) RNA following a lithium chloride (LiCl) method as described elsewhere [13]. AMV reverse transcriptase, RNasinÒ ribonuclease inhibitor, terminal transferase, and Magnotex-SA beads were purchased from Takara Bio.Inc. To the mixture of annealed small RNA templates and 60 nM degenerate stem–loop reverse primers (primer A–D, Supplementary Table 1), 1 mM dNTP mix, 20U RNasinÒ ribonuclease inhibitor, 2U AMV reverse transcriptase and 1 reaction buffer were added and topped up with nuclease-free water to a final volume of 30 ll. The reaction was incubated at 16 °C for 30 min, 42 °C for 30 min and 85 °C for 5 min. 2.4. sscDNA tailing and subtractive hybridization of cDNA/RNA Tailing at the 30 terminus of sscDNA was performed by incubation at 37 °C for 60 min with 2 pmol sscDNA, 2 mM dCTP, and 2 U terminal transferase, and was terminated by incubation at 70 °C for 10 min. For subtractive hybridization, 2.5 lg each of small RNAs and sscDNA were mixed in a total volume of 60 ll. The reaction was overlayered with mineral oil and incubated in a thermal cycler at 98 °C for 1.5 min and then at 68 °C for 12 h. The cDNA/RNA hybrid and cDNA labeled with biotin were incubated with streptavidin magnetic beads to achieve biotin–avidin binding. The mixture stood undisturbed at room temperature for 1 h and then on a magnetite separation stand for 1 min. The supernatant was gently transferred to a new microtube and the pathogen-regulated small RNAs were precipitated by ethanol for further amplification. 2.5. Amplification and cloning of small RNAs The tailed cDNA was amplified with a pair of forward (f) and reverse (r) primers. PCR conditions were as follows: 2 min at 94 °C followed by 15 s at 94 °C, 15 s at 55 °C, and 10 s at 72 °C for 20 cycles. The primers for amplifying Ecc-induced or -suppressed small RNA populations were designated primer1f and primer1r or primer2f and primer2r, respectively (Supplementary Table 1). The
To investigate whether endogenous small RNAs of Bcp were generated specifically in response to Ecc infection, 7-day-old seedlings 99Bre were divided into two groups. One group was inoculated with Ecc, and the other with mock. The former was further divided into two subgroups in terms of their soft rot severity at 7 dpi, one exhibiting typical root decay and the other mild or no symptoms. Total RNA was extracted from all these groups of seedlings by Trizol reagent and 2 ll of total RNA in each group were checked for its purity and degradation by capillary electrophoresis (CE) and Agilent 2100 Bioanalyzer RNA chip (Agilent Technologies). For the Ecc-infected RNA sample, the ratios of 260–280 nm and 28S–18S ribosome were 1.83 or 1.4, respectively, showing a slight degradation. For the mock RNA sample, the ratios were 1.85 for 260/280 nm and 1.7 for 28S/18S, demonstrating that the total RNA was intact (Supplementary Fig. 1). This is normal since the degradation occurs as a result of Ecc infection on seedlings. As LMW RNA can be dissolved in 4 M lithium chloride (LiCl) but not HMW RNA, total RNA was dissolved in 4 M LiCl and centrifuged to precipitate HMW RNA. LMW RNA in supernatant was resolved on a 17%, 8 M urea PAGE gel. Small RNAs with a size range of 15–50 nt were extracted from the gel by 0.4 M NaCl at 4 °C overnight. They were recovered by ethanol precipitation and used for first single-stranded cDNA synthesis. A group of four degenerate stem–loop reverse primers (Supplementary Table 1) was used to generate cDNA. A typical stem–loop primer comprises an universal sequence of 35 nt at the 50 end, 5 nt at the 30 end complementary to a specific small RNA, and a stretch of nucleotides in the middle of the primer to form a stem–loop structure (Fig. 1). The sequence of 35 nt at the 50 end shares no homology with any sequences registered in public databases but contains a 454 sequencing adaptor B (454 Life Sciences, http:// www.454.com) [14] for high-throughput DNA sequencing. Being unique to a small RNA, the 30 end 5 nt of a primer allows itself to specifically bind to the small RNA, which ensures the initiation of reverse transcription. The internal nucleotides form a stem–loop structure within the primer that may limit nonspecific annealing to primary small RNA (genomic DNA). Therefore, the stem–loop primers have multiple functions. They can initiate synthesis of cDNA from a small RNA template, introduce a specific site at the 30 end of the cDNA for further amplification and provide a site for annealing high-throughput sequencing primer. To capture as many small RNA species as possible, four primers, A–D were designed. The first nucleotide at the 30 end of each primer begins with either A, T, G, or C and the 50 end was labeled with biotin. These stem–loop reverse primers can be used to synthesize both highand low-abundant small RNAs. 3.2. Subtractive hybridization removed the same background small RNAs Plant endogenous small RNAs may be induced or suppressed by pathogenic invasion. To isolate these endogenous small RNAs, each total RNA extracted from Bcp treated with either Ecc or mock was
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Fig. 1. Synthesis of first single-stranded cDNA of small RNA, miR166 as an example using a degenerate stem–loop reverse primer.
divided into two halves. One was used as template for first sscDNA synthesis, and the other for hybridization with the first sscDNA that was transcribed from the counterpart small RNAs. To capture Ecc-induced small RNAs, the first sscDNA of mocked seedlings were hybridized with small RNAs of Ecc-infected. If a small RNA matches with nucleotide sequence of certain sscDNA, it will anneal to form a double-stranded cDNA/RNA hybrid with a labeled biotin at the 50 end. Incubation with magnotex-labeled streptavidin beads followed by centrifugation may remove both cDNA/RNA hybrids and all unhybridized sscDNA via biotin–streptavidin binding. The Ecc-induced small RNAs exclusively remained in the hybridization solution. Similarly, to capture Ecc-suppressed small RNAs, the first sscDNA of Ecc-infected seedlings were hybridized with the small RNAs of mock, the cDNA/RNA hybrids, and unhybridized sscDNA were removed by magnotex-labeled streptavidin beads. The Eccsuppressed small RNAs remained in the solution. In this way, the
pathogen-regulated endogenous small RNAs, either down- or upregulated, were captured by the cDNA/RNA subtractive hybridization. The procedure is shown in Fig. 2A. 3.3. Ecc-regulated endogenous small RNAs were cloned Terminal Deoxynucleotidyl Transferase (TdT) catalyzes addition of mononucleotides (dNTPs) to the terminal 30 -OH of a DNA initiator. It added a poly (C) tail to the 30 termini of first sscDNA and this generated a priming site for the second-stranded cDNA synthesis. A primer that comprises a 454 sequencing adaptor A sequence with an oligo (dG) was used to generate the second-stranded cDNA. Two different bar codes were inserted between the 454 sequencing adaptor A and oligo (dG) to mark origins of endogenous small RNA. For PCR amplification, a pair of primers (Supplementary Table 1) consisting of 454 sequencing adaptor A (or B) sequence were used to perform a standard PCR as shown in Fig. 2B. The
Fig. 2. Schematic flowchart of construction of pathogen-regulated small RNA cDNA library. (A) cDNA/RNA hybridization for capturing Ecc-regulated endogenous small RNAs. (B) cloning of Ecc-regulated endogenous small RNAs.
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Fig. 3. Gel electrophoresis for cDNA libraries constructed by both novel and traditional methods. (A) PCR products of Ecc-regulated endogenous small RNAs resolved on 2% agarose gel. Ecc: Ecc-suppressed small RNAs. Ecc+: Ecc-induced small RNAs. (B) PCR products of microRNAs for testing the cDNA libraries separated on 2% agarose gel. Top: cDNA library A constructed with adaptor ligations. MiR162, miR173, and miR393 were unable to be detected. Bottom: cDNA library B constructed with cDNA/RNA subtractive hybridization and stem–loop reverse primers.
PCR products were purified from agarose gel for 454 sequencing amplification (Fig. 3A). 4. Discussion Plant genes can be induced or suppressed by pathogen infection [15–17]. RNA silencing has been known to function in antiviral defense systems by post-transcriptional gene silence (PTGS) [18–20], and recent evidence suggests that PTGS also functions in basal defense and race-specific resistance to microbes in plants [7,9,10]. To identify small RNA species induced or suppressed specifically under soft rot pathogenic invasion, we developed a simple, rapid, and highly efficient method to clone Ecc-regulated endogenous small RNAs. Traditional protocols to amplify small RNAs employ total RNA preparation, separation of LMW and HMW RNA, purification of small RNAs, 30 adaptor ligation, 50 adaptor ligation, reverse transcription, and PCR amplification (Supplementary Fig. 2). To prevent either small RNA or 30 adaptor from self-ligation in the 30 adaptor ligation, the small RNA needs to be dephosphorylated at the 50 -terminus and excess amount of the adaptor over the small RNA is required. In contrast, the 50 -terminus of 30 ligation product is required to be phosphorylated prior to 50 adaptor ligation. Thus, the ligation efficiency is remarkably reduced as a result of the dephosphorylation and phosphorylation, especially for low abundant endogenous small RNAs. Furthermore, it could lead to loss of some RNAs in PCR amplification (Fig. 3B top). There have been a few reports about tailing small RNAs with a 30 -poly(A) tail to provide reverse priming sites for cDNA synthesis, but only those RNAs such as mammalian miRNAs with a 30 hydroxyl end were efficiently polyadenylated [21,22]. Plant miRNAs and endogenous siRNAs often contain a 20 -O-methy group at the 30 end, and cannot be tailed by Poly(A) polymerase [23,24]. To discover novel small RNAs, we introduced a group of biotinlabeled degenerate stem–loop reverse primers which bound to the 30 end of small RNAs. The small RNAs were efficiently reverse-transcribed to their first sscDNA (Fig. 1). These primers have the following advantages: the ability of a short reverse priming sequence annealed to the 30 end of small RNA discriminates small RNA by only one base. Second, a double-stranded stem structure inhibits hybridization of the reverse primer to small RNA precursors and other long RNAs, improving the reverse transcription efficacy [23,24]. Here we have randomly chosen three microRNAs, miR166, miR168, and miR172 to check the first single-stranded cDNA population I (Ecc) and II (Ecc+) (Supplementary Table 2). All PCR reactions were positive, which demonstrated the effectiveness of degenerate stem–loop reverse primers for small RNA
reverse transcription (Supplementary Fig. 3). For the secondstranded cDNA synthesis, a poly(C) tail was added to the 30 end of the first single-stranded cDNA by TdT, hence providing a forward priming site for further PCR. Therefore, our method offers a highly specific and quantitative mean to clone small RNAs using a very small amount of starting material (1–10 ng of total RNA) [25,26]. Although high-throughput sequencing was applied to sequence small RNAs, it was difficult to isolate signals of low abundant small RNAs from high-throughput sequencing reads. To address this problem, we performed a subtractive cDNA/RNA hybridization to remove any small RNA of the same abundance in both infected and mock samples and thus, only those species with different abundances remained. The biotin-labeled first sscDNA allowed removal of double-stranded cDNA/RNA hybrids by magnotex-labeled streptavidin beads. To examine the sensitivity of our method, we randomly selected 10 microRNAs, miR156, miR159, miR160, miR162, miR166, miR172, miR173, miR393, miR396, and miR398 to test their cDNA libraries A (Ecc+) and B (Ecc+). The primers for the two libraries were listed in Supplementary Table 1 (our method) or 2 (traditional method) respectively. The PCR products analyzed on 2% agarose gel are shown in Fig. 3B. The construction of cDNA library A (Ecc+) was performed by traditional method using Takara small RNA cloning kit. The results clearly showed that our approach successfully amplified small RNAs such as miR162, miR173, and miR393 (Fig. 3B bottom) whereas the commercial kit did not (Fig. 3B top). In this study, we constructed the cDNA 454 sequencing libraries for small RNA species spanning 15–50 nt (Fig. 3A). The libraries can be directly processed with high-throughput sequencing. Endogenous small RNA-mediated gene silencing underlies the mechanism for gene regulation in plant defense responses [1,9,10], thus, the bacterial pathogen, e.g. Ecc, regulated small RNAs may play an important role in host defense systems. The bioinformatics analysis of small RNA cDNA libraries would help elucidate the molecular events related to plant disease resistance. Modifying pathogen-regulated small RNAs or their target genes could potentially be useful in molecular breeding procedure to increase disease resistance in plants. Identification and characterization of small RNAs by novel amplification approaches may contribute to the understanding of plant immune systems in bacterial pathogenic diseases. Acknowledgments We especially thank Dr. Mi-Jurng Kim for her critical reading of the paper, and Dr. Naweed I.N. for helpful discussion and comments on the paper. Chuan Bao Sun was supported by a scholarship from SA-SIBS Scholarship Program. This work was supported by a
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Grant from the National Natural Science Foundation of China (No. 30871617) and a Grant from the Science and Technology Commission of Shanghai Municipality (No. 09JC1416000) and the National High-tech R&D Program of China (863 Program No. 2008AA 02Z111). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2010.05.151. References [1] H. Jin, Endogenous small RNAs and antibacterial immunity in plants, FEBS Lett. 582 (2008) 2679–2684. [2] X. Chen, MicroRNA biogenesis and function in plants, FEBS Lett. 579 (2005) 5923–5931. [3] A.M. Gustafson, E. Allen, S. Givan, D. Smith, J.C. Carrington, K.D. Kasschau, ASRP: the Arabidopsis small RNA project database, Nucleic Acids Res. 33 (2005) D637–D640. [4] B. Zhang, X. Pan, G.P. Cobb, T.A. Anderson, Plant microRNA: a small regulatory molecule with big impact, Dev. Biol. 289 (2006) 3–16. [5] U. Ellendorff, E.F. Fradin, R. de Jonge, B.P. Thomma, RNA silencing is required for Arabidopsis defence against Verticillium wilt disease, J. Exp. Bot. 60 (2009) 591–602. [6] R. Sunkar, J.K. Zhu, Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis, Plant Cell 16 (2004) 2001–2019. [7] L. Navarro, P. Dunoyer, F. Jay, B. Arnold, N. Dharmasiri, M. Estelle, O. Voinnet, J.D. Jones, A plant miRNA contributes to antibacterial resistance by repressing auxin signaling, Science 312 (2006) 436–439. [8] X.J. Wang, T. Gaasterland, N.H. Chua, Genome-wide prediction and identification of cis-natural antisense transcripts in Arabidopsis thaliana, Genome Biol. 6 (2005) R30. [9] S. Katiyar-Agarwal, R. Morgan, D. Dahlbeck, O. Borsani, A. Villegas Jr., J.K. Zhu, B.J. Staskawicz, H. Jin, A pathogen-inducible endogenous siRNA in plant immunity, Proc. Natl Acad. Sci. USA 103 (2006) 18002–18007. [10] S. Katiyar-Agarwal, S. Gao, A. Vivian-Smith, H. Jin, A novel class of bacteriainduced small RNAs in Arabidopsis, Genes Dev. 21 (2007) 3123–3134. [11] J. Ren, R. Petzoldt, M.H. Dickson, Screening and identification of resistance to bacterial soft rot in Brassica rapa, Euphytica 118 (2001) 271–280. [12] S. Boutet, F. Vazquez, J. Liu, C. Beclin, M. Fagard, A. Gratias, J.B. Morel, P. Crete, X. Chen, H. Vaucheret, Arabidopsis HEN1: a genetic link between endogenous miRNA controlling development and siRNA controlling transgene silencing and virus resistance, Curr. Biol. 13 (2003) 843–848.
[13] S. Katiyar-Agarwal, H. Jin, Discovery of pathogen-regulated small RNAs in plants, Methods Enzymol. 427 (2007) 215–227. [14] M. Margulies, M. Egholm, W.E. Altman, S. Attiya, J.S. Bader, L.A. Bemben, J. Berka, M.S. Braverman, Y.J. Chen, Z. Chen, S.B. Dewell, L. Du, J.M. Fierro, X.V. Gomes, B.C. Godwin, W. He, S. Helgesen, C.H. Ho, G.P. Irzyk, S.C. Jando, M.L. Alenquer, T.P. Jarvie, K.B. Jirage, J.B. Kim, J.R. Knight, J.R. Lanza, J.H. Leamon, S.M. Lefkowitz, M. Lei, J. Li, K.L. Lohman, H. Lu, V.B. Makhijani, K.E. McDade, M.P. McKenna, E.W. Myers, E. Nickerson, J.R. Nobile, R. Plant, B.P. Puc, M.T. Ronan, G.T. Roth, G.J. Sarkis, J.F. Simons, J.W. Simpson, M. Srinivasan, K.R. Tartaro, A. Tomasz, K.A. Vogt, G.A. Volkmer, S.H. Wang, Y. Wang, M.P. Weiner, P. Yu, R.F. Begley, J.M. Rothberg, Genome sequencing in microfabricated highdensity picolitre reactors, Nature 437 (2005) 376–380. [15] S.T. Chisholm, G. Coaker, B. Day, B.J. Staskawicz, Host–microbe interactions: shaping the evolution of the plant immune response, Cell 124 (2006) 803–814. [16] J.L. Dangl, J.D. Jones, Plant pathogens and integrated defence responses to infection, Nature 411 (2001) 826–833. [17] R.P. Wise, M.J. Moscou, A.J. Bogdanove, S.A. Whitham, Transcript profiling in host–pathogen interactions, Annu. Rev. Phytopathol. 45 (2007) 329– 369. [18] Q.W. Niu, S.S. Lin, J.L. Reyes, K.C. Chen, H.W. Wu, S.D. Yeh, N.H. Chua, Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance, Nat. Biotechnol. 24 (2006) 1420–1428. [19] J. Qu, J. Ye, R. Fang, Artificial microRNA-mediated virus resistance in plants, J. Virol. 81 (2007) 6690–6699. [20] X. Zhang, Y.R. Yuan, Y. Pei, S.S. Lin, T. Tuschl, D.J. Patel, N.H. Chua, Cucumber mosaic virus-encoded 2b suppressor inhibits Arabidopsis Argonaute1 cleavage activity to counter plant defense, Genes Dev. 20 (2006) 3255–3268. [21] S. Ro, C. Park, J. Jin, K.M. Sanders, W. Yan, A. PCR-based, A PCR-based method for detection and quantification of small RNAs, Biochem. Biophys. Res. Commun. 351 (2006) 756–763. [22] S. Ro, C. Park, R. Song, D. Nguyen, J. Jin, K.M. Sanders, J.R. McCarrey, W. Yan, Cloning and expression profiling of testis-expressed piRNA-like RNAs, RNA 13 (2007) 1693–1702. [23] P. Mestdagh, T. Feys, N. Bernard, S. Guenther, C. Chen, F. Speleman, J. Vandesompele, High-throughput stem–loop RT-qPCR miRNA expression profiling using minute amounts of input RNA, Nucleic Acids Res. 36 (2008) e143. [24] T.D. Schmittgen, E.J. Lee, J. Jiang, A. Sarkar, L. Yang, T.S. Elton, C. Chen, Realtime PCR quantification of precursor and mature microRNA, Methods 44 (2008) 31–38. [25] C. Chen, D.A. Ridzon, A.J. Broomer, Z. Zhou, D.H. Lee, J.T. Nguyen, M. Barbisin, N.L. Xu, V.R. Mahuvakar, M.R. Andersen, K.Q. Lao, K.J. Livak, K.J. Guegler, Realtime quantification of microRNAs by stem–loop RT-PCR, Nucleic Acids Res. 33 (2005) e179. [26] H. Yang, J.J. Schmuke, L.M. Flagg, J.K. Roberts, E.M. Allen, S. Ivashuta, L.A. Gilbertson, T.A. Armstrong, A.T. Christian, A novel real-time polymerase chain reaction method for high throughput quantification of small regulatory RNAs, Plant Biotechnol. J. 7 (2009) 621–630.