A novel RT-PCR approach for detection and characterization of citrus viroids

Molecular and Cellular Probes 20 (2006) 105–113 www.elsevier.com/locate/ymcpr

A novel RT-PCR approach for detection and characterization of citrus viroids L. Bernad, N. Duran-Vila * Departamento de Proteccio´n Vegetal y Biotecnologı´a, Instituto Valenciano de Investigaciones Agrarias (IVIA), Apartado Oficial, 46113 Moncada (Valencia), Spain Received 6 September 2005; accepted for publication 2 November 2005 Available online 7 February 2006

Abstract Citrus plants are natural hosts of five viroid species and a large number of sequence variants. Because of their small size, viroids lend themselves to various RT-PCR approaches for their detection and further characterization. The one-step RT-PCR approach proposed here is based on the synthesis of viroid-cDNA by reverse transcription at 60 8C using a viroid specific 27-mer primer followed by standard second strand synthesis plus PCR amplification with various primer pairs. According to the primers used, full or partial length viroid-DNA is obtained. The technique avoids amplicon contamination in routine diagnosis. The suitability of the technique has been demonstrated using several nucleic acid extraction procedures and different viroid infected host species. The homogenization of tissue inside sealed plastic bags followed by nucleic acid extraction using a SDS/potassium acetate method is recommended because of its efficiency, simplicity and low cost. This extraction procedure, when coupled to the one-step RT-PCR approach, can be useful to avoid cross-contamination during routine diagnosis. A PCR strategy capable of discriminating between mild and severe strains of CEVd and identifying cachexia-inducing isolates of HSVd, is also described. q 2005 Elsevier Ltd. All rights reserved. Keywords: Exocortis; Cachexia; Citrus exocortis viroid (CEVd); Hop stunt viroid (HSVd); Citrus bent leaf viroid (CBLVd); Citrus viroid III (CVd-III); Citrus viroid IV (CVd-IV).

1. Introduction Polymerase chain reaction (PCR) offers the possibility to increase in vitro specific DNA or cDNA sequences from only trace amounts of target DNA. The technique has had an impact on basic molecular research as well as more practical aspects such as detection and characterization of pathogens. The introduction of a reverse transcription step before the PCR amplification process (RT-PCR) makes it possible to study pathogens with RNA genomes, like viruses and viroids. Viroids are small (246–401 nucleotides) covalently closed, single stranded RNAs that replicate in their host plants in which they may elicit diseases. Viroid molecules display extensive base pairing to give very stable rod-like or quasi-rodlike conformations. As viroids have small sizes, their whole genome can be amplified in a single RT-PCR reaction. This approach has been extremely useful for viroid cloning and sequencing, and today a vast number of viroid sequences are available in databases. Viroids have been classified into two families, Pospiviroidae, composed of species with a Central * Corresponding author. Tel.: C34 96 342 4066; fax: C34 96 342 4001. E-mail address: [email protected] (N. Duran-Vila).

0890-8508/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.mcp.2005.11.001

Conserved Region (CCR) and without hammerhead ribozymes, and Avsunviroidae, composed of three members lacking CCR but able to self-cleave in both polarity strands through hammerhead ribozymes [1]. Citrus plants are natural hosts of five viroid species, all of which belong to the Pospiviroidae family: Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), Citrus viroid III (CVd-III) and Citrus viroid IV (CVd-IV) [2,3]. CEVd and HSVd are the causal agents of two diseases, exocortis and cachexia, respectively [4,5]. Within these two viroid species, variants with specific sequence motifs responsible for differences in pathogenicity have been documented [6,7]. Although not inducing specific symptoms, CBLVd and CVd-III may cause a reduction of tree size and fruit harvest [8–10]. Within CBLVd, two types of variants have been identified (CVd-Ia and CVd-Ib) with similar biological properties but with discernible nucleotide changes and differences in size (327nt for CVd-Ia versus 318nt for CVdIb) [8,11]. In CVd-III, two types of variants (CVd-IIIa and CVd-IIIb) have also been identified [12]. CVd-IV, which appears to be less widespread than the other four viroids, contains a segment of 80–90 nucleotides identical to CEVd [13] and causes severe bark cracking on susceptible species [10]. Additionally, two viroid sequences, distinct from those of the above five species, have been reported to occur in Japan: (i) CVd-I-LSS (LSS, Low sequence similarity) [14], which in

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spite of presenting a sequence identity of only 82–85% with CBLVd variants, has been considered as a new variant of CBLVd; (ii) CVd-OS (OS, Original sample) [15] which has a 330 nucleotide genome showing only 68% identity with CVdIII, the closed related species, and which may be considered a candidate for a new viroid species. Diagnosis of citrus viroid diseases was initially performed by biological indexing using as indicators Etrog citron (Citrus medica L.) [16] and Parson’s Special mandarin [17]. This is a sensitive and reliable method but unfortunately, it requires the incubation of the inoculated indicator species for several months at 28–30 8C to perform a correct diagnosis. Attempts to use electrophoresis or molecular hybridization analysis resulted in unreliable results because in many species and cultivars, citrus viroids do not accumulate at high enough titers to be detected [18]. However, since viroids do accumulate at detectable titers in Etrog citron, this species has been used as a secondary bio-amplification host, which, when coupled with molecular analysis, represents the most sensitive and specific procedure for indexing purposes [18,19]. Because of its great sensitivity, RT-PCR provides a desirable alternative to other diagnostic methods. Following the first report on RT-PCR to detect CEVd and HSVd from citrus [20], several protocols have been proposed to detect these and other citrus viroids [21–23]. Furthermore, when the relationship between sequence and pathogenicity is understood, strain specific primers aimed at discriminating between pathogenic and non-pathogenic strains of HSVd were designed [24]. Unfortunately, the attempts to use these strategies for routine indexing showed that in many instances, samples known to contain viroids yielded negative results, and therefore, its implementation for routine indexing has been rather limited [25]. Conversely, RT-PCR has been shown to be an extremely useful tool for cloning and sequencing purposes. In most instances, failure to amplify the target molecule was overcome by purifying the viroid from the polyacrilamide gels after electrophoresis. Several reasons may explain the difficulties encountered for the amplification of viroid sequences by RT-PCR: (i) presence of inhibitors that may interfere with the reactions, a situation that in some instances was overcome by using highly purified viroid preparations, even though this is impractical for diagnosis; (ii) inefficient synthesis of a cDNA of a suitable size during the PCR reaction. Given the tight secondary structure of the viroid molecule, it is likely that after denaturation, the molecule may reassume some of its hydrogen bonding therefore, preventing the synthesis of cDNAs suitable for amplification. In fact, at 42 8C, the temperature normally used for reverse transcription, viroids can easily re-gain their secondary structure and this is probably responsible for some of the unexpected negative results. Today, the availability of reverse transcriptases that are active at temperatures as high as 65 8C, may provide a suitable solution, provided that at these temperatures, the primers designed to that purpose anneal with the target viroid. The purpose of the present work was to define RT-PCR strategies for efficient and consistent amplification of citrus viroids for detection and characterization purposes.

The approach will take into consideration: (i) the design of complementary primers and optimization of RT protocols; (ii) the simplification of extraction procedures to avoid the release of putative inhibitors and to prevent cross-contamination among samples; (iii) the design of PCR primer pairs suitable for detection and characterization of viroid strains; (iv) the assessment of protocols for viroid detection in different citrus species and cultivars. 2. Materials and methods 2.1. Plant materials and viroid sources Unless otherwise stated, viroid infected citron plants (Citrus medica L.) were used as a source of tissue. The viroid sources used were CEVd, HSVd, CBLVd, CVd-III and CVd-IV. Whenever possible, and accordingly to the availability of viroid infected plants, assays were conducted with different hosts and different field isolates of the same viroid to verify the suitability of the technique. In all instances, the plants used had been viroid tested by sPAGE analysis [19] or imprint hybridization [18]. Assays for strain discrimination were conducted with CEVd and HSVd infected citron plants. The CEVd strains used were CEVd-117 [26] and CEVd-129 [27] previously characterized as containing in the P and V domains a set of changes characteristic of severe (Class A) and mild (Class B) isolates, respectively [28]. The HSVd strains used were HSVd (X-707) and HSVd (CVd-IIa-117) [29] previously characterized as containing in the V domain a set of changes characteristic of variants that induce and do not induce cachexia, respectively [7]. 2.2. RNA extraction methods (i) Standard viroid extraction method [30] designed to yield high viroid titers. Tissue samples (5 g) were homogenized in phenol saturated extraction medium (0.4 M Tris–HCl, pH 8.9; 1% (w/v) SDS; 5 mM EDTA, pH 7.0; 4% (v/v) mercaptoethanol). Total nucleic acids were partitioned in 2 M LiCl, and the soluble fraction was concentrated by ethanol precipitation and resuspended in 300 ml of TKM buffer (10 mM Tris–HCl, pH 7.4; 10 mM KCl; 0.1 mM MgCl2). (ii) Phenol/guanidine isothiocyanate method [31]. Briefly, tissue samples (100 mg) were homogenized in TRIZOLw reagent (Invitrogene) and the aqueous phase obtained after chloroform separation was precipitated with isopropyl alcohol and resuspended in 50 ml of water. (iii) Qiagenw method. Briefly, tissue samples (100 mg) were homogenized in extraction buffer (4 M guanidine thiocyanate, 100 mM Tris–HCl, 25 mM MgCl2, 25 mM EDTA, pH 7.5) and the soluble fraction was concentrated by isopropyl alcohol precipitation and resuspeded in TE (20 mM Tirs–HCl, 1 mM EDTA, pH 8.0) buffer. Small RNAs were recovered using the QIAGEN-tips, following the manufacturer’s instructions and resuspended in 50 ml of water.

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(iv) SDS/potassium acetate method [32,33]. Briefly, tissue samples (500 mg) were homogenized inside sealed plastic bags in extraction buffer (0.1 M Tris–HCl, pH 8.0; 50 mM EDTA; 0.5 M NaCl; 10 mM mercaptoethanol). The homogenates were treated with SDS (65 8C for 20 min) and potassium acetate (for 20 min in ice). The soluble fraction was concentrated by ethanol precipitation and resuspended in 40 ml of water.

2.3. Oligonucleotide primers For reverse transcription using the enzyme ThermoSripte RNase HK which is active at temperatures as high as 65 8C, 27nucleotide primers complementary to the upper Central Conserve Region (CCR) of each viroid sequence were used (Table 1). Several sets of forward and reverse primers were used for second strand synthesis and PCR amplification, which

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were designed as described by Olmos et al. [34] with slight modifications. Briefly, sequenced regions of each viroid were recovered using the Nucleotide Sequence Search program located in the Entrez Browser provided by the National Center for Biotechnology Information (NCBI) (http://www3.ncbi. nlm.nih.gov/Entrez) (Bethesda, MD, USA). Conserved regions for each viroid were identified using the similarity search tool Advanced BLAST 2.0, with the blast program designed to support analysis of nucleotides (http://www3.ncbi.nlm.nih. gov/blast/blast.cgi?JformZ1). The alignment view was carried out as master–slave with identities, to analyse significant nucleotide homologies in the molecular data retrieved from NCBI’s integrated databases, GenBank, EMBL and DDBJ. The reverse and forward primers designed are listed in Table 1. Fig. 1 shows the location of the primers in the secondary structure of each of the five viroids. To discriminate mild and severe strains of CEVd, two sets of primers (Table 1) designed by Chaffai [27] according to the

Table 1 Specific primers for detection and characterization of citrus viroids Viroid CEVd

HSVd

CBLVd

CVd-III

CVd-IV

a b

Type Reverse (CEVd-RT) Reverse (CEVd-R1) Reverse (CEVd-R2) Forward (CEVd-F1) Forward (CEVd-F2) Forward (CEVd-F3) Forward (CEVd-F4) Reverse (CEVd-SR)a Forward (CEVd-SF)a Reverse (CEVd-MR)a Forward (CEVd-MF)a Reverse (HSVd-RT) Reverse (HSVd-R1) Forward (HSVd-F1) Forward (HSVd-F2) Forward (HSVd-F3) Forward (HSVd-F4) Forward (HSVD-A) Forward (HSVD-Ca) Reverse (CBLVd-RT) Reverse (CBLVd-R1) Forward (CBLVd-F1) Forward (CBLVd-F2) Forward (CBLVd-F3) Reverse (CVd-III-RT) Reverse (CVd-III-R1) Forward (CVd-III-F1) Forward (CVd-III-F2) Forward (CVd-III-F3) Reverse (CVd-IV-RT) Reverse (CVd-IV-R1) Reverse (CVd-IV-R2)b Forward (CVd-IV-F1) Forward (CVd-IV-F2)b Forward (CVd-IV-F3)

Primers defined by Chaffai [27]. Primers defined by Ito et al. [22].

Sequence 0

Purpose 0

5 -CTTCCTCCAGGTTTCCCCGGGGATCCC-3 5 0 -CCGGGGATCCCTGAAGGA-3 0 5 0 -GGGTAGTCTCCAGAGAGAAG-3 0 5 0 -GGAAACCTGGAGGAAGTCG-3 0 5 0 -GGTGGAAACAACTGAAGCTT-3 0 5 0 -GCTCGTCTCCTTCCTTTCGCTGCTGGC-3 0 5 0 -TCGGAACCCTAGATTGGGTCCCTCGGG-3 0 5 0 -TTCTTCCCCCGCCGCCTC-3 0 5 0 -CGTCGCTGAAGCGCCTCGC-3 0 5 0 -TTCTTCCCCACCCGCCG/ATC-3 0 5 0 -CGTCGCTGAGGCGTTGCCA-3 0 5 0 -GTGTTGCCCCGGGGCTCCTTTCTCTGG-3 0 5 0 -GGGGCTCCTTTCTCAGGTAAGTC-3 0 5 0 -GGGGCAACTCTTCTCAGAATCC-3 0 5 0 -GTGGCATCACCTCTCGGTT-3 0 5 0 -GGCCGCGGTGCTCTGGAGTAG-3 0 5 0 -CTGGGGAATTCTCGAGTTGCCG-3 0 5 0 -TTTTACCTTCTCCTGGCTCTTCGAGTG-3 0 5 0 -GAATCCAGCGGGGGCGTG-3 0 5 0 -GCTGACGAGCCTTCGTCGACGACGACC-3 0 5 0 -TTCGTCGACGACGACCAGTC-3 0 5 0 -GGCTCGTCAGCTGCGGAGGT-3 0 5 0 -AGCGCTGCTTGCCGCTAGTCG-3 0 5 0 -CCCTTCACCCGAGCGCTGCTT-3 0 5 0 -CCAACTTAGCTGCCTTCGTCGACGACG-3 0 5 0 -TTCGTCGACGACGACAGGTA-3 0 5 0 -GGCAGCTAAGTTGGTGACGC-3 0 5 0 -GGAAAGACTCCGCATCCTCCG-3 0 5 0 -CCGTGTGGTTCCTGTGGGGCA-3 0 5 0 -GTCTGAAGAGATTTCCCCGGGGATCCC-3 0 5 0 -GGGGATCCCTCTTCAGGT-3 0 5 0 -GGATCCCTCTTCAGGTATGT-3 0 5 0 -GGGGAAATCTCTTCAGAC-3 0 5 0 -CCGGGGAAATCTCTTCAGACTC-3 0 5 0 -GGTGGATACAACTCTTGGG-3 0

RT PCR PCR PCR PCR PCR PCR Strain discrimination Strain discrimination Strain discrimination Strain discrimination RT PCR PCR PCR PCR PCR Strain discrimination Strain discrimination RT PCR PCR PCR PCR RT PCR PCR PCR PCR RT PCR PCR PCR PCR PCR

108 L. Bernad, N. Duran-Vila / Molecular and Cellular Probes 20 (2006) 105–113 Fig. 1. Location of viroid specific primers (Table 1) in the secondary structure of CEVd (CEVd-A) [6], HSVd (cachexia variant X-704) [29], CBLVd (variant CVd-Ia) [8], CVd-III (variant CVd-IIIb) [12] and CVdIV [13]. Grey boxes show the nucleotide changes discriminating severe and mild strains of CEVd [28], and cachexia and non-cachexia variants of HSVd [24,29].

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sequences of the V and P domains (Fig. 1) in which the pathogenicity determinants appear to be located [28] were tested. Similarly, to discriminate variants of HSVd that induce cachexia from those that replicate as latent, symptomless infections in cachexia sensitive hosts, several primer sets were designed according to the sequences of the upper and lower strands of the V domain where the pathogenicity domain is located [7,24]. These primers were tested. Only the primers that yielded the expected results are shown in Table 1 and Fig. 1.

109

A L

C

1 –

B

2 +

3 –

4 +

L

C

1 –

2 +

3 –

4 +

2.4. RT-PCR protocols Samples containing the viroid template were denatured at 95 8C for 5 min. First-strand viroid cDNA was synthesized with 12.5 U of avian myeloblastosis virus reverse transcriptase (AMV-RT) (Promega Corp., Madison, WI) or 15 U ThermoScripte RNase HK reverse transcriptase (Thermoscript-RT) (Invitrogenw) using the reverse specific primer (0.75 mM), and dNTPs (1 mM each). The AMV-RT reaction buffer contained 50 mM Tris–HCl (pH 8.3), 10 mM MgCl2, 50 mM KCl, 0.5 mM spermidine, 10 mM DTT and 40 U of RNase Out (Invitrogenw). The Thermoscript-RT reaction buffer contained 50 mM Tris–acetate (pH 8.4), 75 mM potassium acetate, 8 mM magnesium acetate and 40 U of RNase Out. The reaction mixture (20 ml final volume) was incubated at 42 8C (AMV-RT) or 60 8C (Thermoscript-RT) for 1 h. Second-strand cDNA synthesis plus PCR amplification (final 50 ml volume) were performed using 4 ml of the first-strand cDNA reaction mixture, 1 U Taq DNA polymerase (Roche), the selected forward and reverse primer pair (Table 1) (0.5 mM each) and dNTPs (0.12 mM each) in a buffer containing 10 mM Tris–HCl (pH 9.0), 50 mM KCl and 1 mM MgCl2. PCR parameters consisted of a denaturation step at 94 8C for 5 min, followed by 35 cycles (94 8C for 30 s, 60 8C for 30 s and 72 8C for 1 min) to finish with an extension step at 72 8C for 5 min. Electrophoretic analysis in 2% agarose gels was used to confirm the synthesis of a DNA product of the expected size. For one-step-RT-PCR, samples containing the viroid template were denatured at 95 8C for 5 min. The reaction (25 ml final volume) was performed using 3 U Thermoscript-RT, 1 U Taq DNA polymerase, the selected forward and reverse primer pair (Table 1) (0.16 mM each) and dNTPs (0.4 mM each) in a buffer containing 10 mM Tris– HCl, pH 9.0, 50 mM KCl and 3 mM MgCl2 and 4 U RNase Out.

Fig. 2. Agarose gel electrophoresis of RT-PCR products from healthy (K) and CEVd infected (C) Gynura aurantiaca (lanes 1, 2) and citron (lanes 3, 4). Retrotranscription was performed at 42 8C using AMV-RT (A) and at 60 8C using Thermoscript-RT (B). C, Water control subjected to RT-PCR; L, 100 bp DNA ladder.

3. Results and discussion 3.1. RT-PCR protocol for detection of CEVd When nucleic acid preparations from CEVd-infected Gynura aurantiaca and citron plants obtained using the standard viroid extraction protocol were subjected to retrotranscription at 42 8C with AMV-RT or ThermoscriptRT and the oligonucleotide CEVd specific primer (CEVdRT), followed by second strand synthesis and PCR amplification using contiguous CEVd primers of opposite polarity (CEVd-F1 and CEVd-R1), no specific amplification products were generally obtained (Fig. 2A). However, when A L

–

B +

–

C +

–

D +

–

E +

–

+

2.5. Amplicon analysis Full-length viroid amplicons were sequenced with an ABI PRISM DNA sequencer 377 (Perkin-Elmer). Partial length amplicons were subjected to molecular hybridization using viroid-specific probes [18].

Fig. 3. Agarose gel electrophoresis of RT-PCR products from healthy (K) and CEVd infected (C) citron obtained using different nucleic acid extraction procedures: Standard viroid extraction (A); phenol/guanidine isothiocyanate method (B); Qiagenw (C), SDS/potassium acetate using fresh tissue (D) and dried tissue (E). L, 100 bp DNA ladder.

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retrotranscription was performed at 60 8C using Thermoscript-RT and the 27-mer CEVd-specific reverse primer (CEVd-RT), followed by second strand synthesis and PCR amplification using primers CEVd-F1 and CEVd-R1, products of the expected size of CEVd were consistently obtained (Fig. 2B). Sequencing confirmed that the amplicon corresponded to the full-length CEVd sequence. The protocol was also suitable using RNA preparations obtained with three additional methods (phenol/guanidine isothiocyanate, Qiagenw, SDS/potassium acetate) (Fig. 3A– D). The SDS/potassium acetate method was chosen for further assays because it has several advantages: (i) it does not require the use of organic solvents; (ii) it is cheap and easy to handle; (iii) the homogenization in sealed plastic bags minimizes cross

contamination risks. Additionally, this method can be successfully used to obtain RNA preparations from dried leaf samples that had been stored in silica gel for several weeks (Fig. 3E). The use of contiguous viroid-specific primers of opposite polarities for second strand synthesis plus PCR amplification yielded full length viroid-DNAs, a strategy that has been widely adopted for cloning and sequencing purposes. However, since the amplification of smaller fragments is equally suitable for detection purposes, additional primer pairs (CEVdRT/CEVd-F3, CEVd-RT/CEVd-F4, CEVd-R1/CEVd-F2, CEVd-R2/CEVd-F1,) yielding 305, 137, 174 and 196 bp DNA products, respectively, were also tested (Fig. 4). Molecular hybridization using a CEVd-specific probe

Fig. 4. PCR-specific primer pair combinations and expected amplicon size to be obtained by RT-PCR. In all instances retrotranscription was performed at 60 8C using Thermoscript-RT and primers CEVd-RT, HSVd-RT, CBLVd-RT, CVd-III-RT and CVd-IV-RT. Agarose gel electrophoresis shows that the reactions yielded amplicons of the expected sizes (numbers in the gel refer to the each primer set used in the PCR amplification). The specificity of the RT-PCR reaction was further tested by molecular hybridization with viroid specific probes (numbers refer to the amplicons shown in the gel). C, Water control subjected to RT-PCR; –, healthy control subjected to RT-PCR; L, 100 bp DNA ladder.

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confirmed that in all instances the amplicons contained CEVd sequence fragments. The availability of different primer sets is critical to avoid amplicon cross contamination when performing routine tests in detection laboratories. The consistency of these RT-PCR protocols was assessed and confirmed using nucleic acid preparations from different CEVd infected plant species: Etrog citron (84 tests), sour orange (14 tests), trifoliate orange (19 tests), G. aurantiaca (9 tests), Washington navel sweet orange, Fino lemon, Mexican lime, calamondin, C. depressa, C. karna, C. bergamia, C. pyriformis and eggplant (three tests each).

A L

C 1 2 – +

B C

3 4 – +

111 C

C

5 6 – +

D C

7 8 – +

E C 9 10 – +

L

3.2. RT-PCR protocol for detection of other citrus viroids The RT-PCR protocol based on retrotranscription at 60 8C using Thermoscript-RT and 27-mer viroid specific primers (HSVd-RT1, CBLVd-RT1, CVd-III-RT1 and CVd-IV-RT1) followed by second strand synthesis plus PCR amplification using contiguous primers of opposite polarity specific for each viroid (HSVd-F1/HSVd-R1, CBLVd-F1/CBLVd-R1, CVd-IIIF1/CVd-III-R1 and CVd-IV-F1/CVd-IV-R1), was tested using RNA preparations obtained with the SDS/potassium acetate method. In all instances the samples consistently yielded DNAs of the expected full-length size of each viroid (Fig. 4). Sequencing confirmed that the amplicons of 299, 327, 294, and 284 bp corresponded respectively to the full-length HSVd, CBLVd, CVd-III, and CVd-IV sequences. Smaller fragments suitable for detection purposes were consistently obtained using additional primer pairs for second strand synthesis plus PCR amplification of HSVd (HSVdR1/HSVd-F2, HSVd-R1/HSVd-F4, HSVd-R1/HSVd-F3), CBLVd (CBLVd-R1/CBLVd-F2, CBLVd-R1/CBLVd-F3), CVd-III (CVd-III-R1/CVd-III-F2, CVd-III-R1/CVd-III-F3) and CVd-IV (CVd-IV-R1/CVd-IV-F2, CVd-IV-R1/CVd-IVF3) (Fig. 1, Table 1). In all instances the RT-PCR reaction yielded amplicons of the expected size (Fig. 4). Molecular hybridization using a specific probes for each viroid confirmed that the amplicons consisted of HSVd, CBLVd, CVd-III and CVd-IV sequence fragments (Fig. 4). The consistency of the protocol was confirmed using nucleic acid preparations from different viroid infected plant species: Etrog citron (39 HSVd tests, 21 CBLVd tests, 30 CVd-III tests and 17 CVd-IV 17 tests), sour orange (24 HSVd tests), trifoliate orange (26 HSVd tests), Orlando tangelo (18 HSVd tests), Washington navel sweet orange (2 HSVd tests, 4 CVdIII tests) and Verna lemon (2 HSVd tests) (results not shown). Further assays were conducted to test the suitability of the One-Step RT-PCR. As shown in Fig. 5, amplicons of the expected sizes for CEVd (lane 2), HSVd (lane 4), CBLVd (lane 6), CVd-III (lane 8) and CVd-IV (lane 10) were obtained using contiguous primer sets of opposite polarities specific for each viroid. The protocol has been tested using preparations from fresh and dried citron and trifoliate orange tissues. Since both reactions are conducted in the same mixture, without opening the tubes in between the two reactions, this protocol minimizes the contamination risks inherent to conventional RT-PCR

Fig. 5. Agarose gel electrophoresis of RT-PCR products from healthy (K) and viroid infected (C) citrons obtained by One-Step RT-PCR. Results were obtained with samples of citron infected CEVd (A), HSVd (B), CBLVd (C), CVd-III (D) and CVd-IV (E) using the primer sets CEVd-R1/CEVd-F1, HSVdR1/HSVd-F1, CBLVd-R1/CBLVd-F1, CVd-III-R1/CVd-III-F1 and CVd-IVR1/CVd-IV-F1. C, Water control subjected to RT-PCR; L, 100 bp DNA ladder.

reactions. In addition, it is quicker and uses less amounts of chemicals. 3.3. RT-PCR protocols for strain characterization of CEVd and HSVd Initial assays were conducted using templates for PCR, plasmids containing known full-length CEVd and HSVd inserts. Using the standard PCR parameters (60 8C annealing temperature), the primer pairs CEVd-SR/CEVd-SF and CEVdMR/CEVd-MF yielded amplicons of the expected size (230 bp), each primer pair being highly selective for those CEVd sequences containing the nucleotide changes characteristic of severe and mild CEVd strains, respectively. The discriminating properties of these primer pairs was further confirmed using as a template the cDNA obtained with nucleic acid preparations from citron, Washington navel sweet orange and G. aurantiaca infected with a mild (CEVd-129) and a severe strain (CEVd117) subjected to retrotranscription using Thermoscript-RT and the reverse primer CEVd-RT. PCR with primers CEVdMR/CEVd-MF yielded 230 bp amplicons only with samples infected with mild CEVd (Fig. 6A) whereas primers CEVdSR/CEVd-SF yielded amplicons on the same size only with samples infected with severe CEVd (Fig. 6B). A similar strategy was followed to discriminate among HSVd variants. Using the standard PCR parameters (60 8C annealing temperature), the primer pair HSVd-F1/HSVd-Ca yielded amplicons of the expected size (283 bp) only with sequences containing the nucleotide changes characteristic of cachexia variants (CVd-IIb) of HSVd. Several approaches were undertaken in terms of designing primers for the specific amplification of non-cachexia variants (CVd-IIa) of HSVd. Unexpectedly, in all instances, the primers tested yielded amplicons of the expected size both with CVd-IIa and CVd-IIb sequences, a difficulty also found and discussed by Reanwarakorn and Semancik [24]. Specificity was only accomplished by increasing the PCR annealing temperature up to 72 8C

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A L

1 2 3 4 + – + –

B L 1 2 3 4 + – + –

C

D

L 5 6 7 8 L 5 6 7 8 + – + – + – + –

Fig. 6. Agarose gel electrophoresis of RT-PCR products from healthy (K) and viroid infected (C) citrons obtained using primers designed for strain discrimination. Mild and severe strains of CEVd were subjected to RT-PCR using the primer sets CEVd-MR/CEVd-MF (A) and CEVd-SR/CEVd-SF (B): (1) citron infected with the mild strain CEVd-129; (3) citron infected with the severe strain CEVd-117; (2,4) uninoculated citrons. Cachexia and non-cachexia isolates of HSVd were subjected to RT-PCR using the primer sets HSVd-R1/HSVd-A (C) and HSVd-R1/HSVd-Ca (D); (5) citron infected with the non-cachexia isolate CVd-IIa-117; (7) citron infected with the cachexia isolated X-707; (6,8) uninoculated citrons. L, 100 bp DNA ladder.

and using a 27-mer primer (Table 1: HSVd-A). The discriminating properties of these primer pairs were further confirmed with nucleic acid preparations from citron, trifoliate orange and Orlando tangelo infected with a cachexia variant (X-707) and a non-cachexia variant (CVd-IIa-117) of HSVd. These preparations were subjected to retrotranscription using Thermoscript-RT and the reverse primer HSVd-RT, followed by PCR with the discriminating primer pairs. PCR with primers HSVd-R1/HSVd-A yielded 220 bp amplicons only with samples infected with non-cachexia variants of HSVd (Fig. 6C) whereas primers HSVd-R1/HSVd-Ca yielded 283 bp amplicons only with samples infected with cachexia variants of HSVd (Fig. 6D). 4. Conclusion With the protocols described here, reliable and consistent detection and characterization of all citrus viroids can be achieved by RT-PCR. Discrimination between mild and severe strains of CEVd, and between cachexia inducing variants and non-cachexia variants of HSVd was also obtained. Key points in the RT-PCR protocol developed are: (i) for viroid cDNA synthesis, it is essential to use a reverse transcriptase which is active at temperatures as high as 65 8C, thus preventing internal re-annealing of the viroid molecules; (ii) for second strand synthesis and PCR amplification, several primer sets specific for each viroid must be used; (iii) reverse transcription and PCR are carried out concomitantly in the same PCR tube (onestep RT-PCR); (iv) strain specific PCR primers can be used to determine the virulence of field isolates; (v) the simplified nucleic acid extraction procedure, followed by the one-step RT-PCR, can be applied to any infected host, does not require incubation of an indicator host for several months, and minimizes cross contaminations. The technique permits routine viroid detection, and in conjunction with other procedures such as sPAGE, it can be useful for certification purposes.

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