CHAPTER57

Double-Stranded RNAs andtheir Use for Characterization

of Recalcitrant VirusesR. R. Martin, W. Jelkmann, and I. E. Tzanetakis

IntroductionThe presence of high molecular weight, virus-specific,

double-stranded RNAs (dsRNAs) in virus infected plants isweIl established (Dodds et al., 1984; Morris and Dodds, 1979;Valverde et al., 1986, 1990; Yoshikawa and Converse, 1990).DsRNAs are probably formed during virus replication and con­sist of full-length genomic, subgenomic, and defective RNAsof single-stranded RNA viruses or genomes of dsRNA viruses(Dodds, 1993; Voinnet, 2005). The sizes and patterns of thesevirus-specific dsRNAs after separation on gels can be useful invirus characterization and provide useful information on thetype of virus (es) that may be infecting a plant (Dodds and Bar­Joseph, 1983; Kurppa and Martin, 1986; Morris et al., 1983;Valverde et al., 1990). The usefulness of dsRNAs from virus­infected plants is based on the premise that healthy plants notinfected with a virus do not contain dsRNAs. The presenceof multiple dsRNA bands on agarose or polyacrylamide gelsmay indicate the presence of a monopartite virus that producessubgenomic RNAs during its replication cycle (e.g., closterovi­ruses; Dodds and Bar-Joseph, 1983; Valverde et al., 1986; or vi­ruses in the family Flexiviridae; Adams et al., 2004; Jelkmann,1994; Nemchinov et al., 2000), or a multipartite virus that rep­licates via a polyprotein strategy (e.g., nepoviruses), or it maybe an indication that the plant is infected with more than onevirus (Tzanetakis et al., 2005b; Jelkmann, 1995). While thepresence of dsRNAs in plant extracts is a good indication ofvirus infection, it must be taken into consideration that virusesof fungal or bacterial pathogens, endophytes, or saprophytes,as weIl as viruses of mites or insects or plants infected withviroids or containing endornaviruses could be the source of thedsRNA.

DsRNA has been purified from infected plant tissues usinga number of methods, with that of Morris and Dodds (1979)or some variations on it being the most common. This methodinvolves total nucleic acid extraction in the presence of phenol,followed by binding of the dsRNA to cellulose in the presenceof 16% ethanol, repeated washing of the cellulose with buf­fer/ethanol, followed by elution of the dsRNA in buffer with­out ethanol. There is often some contamination of the purifieddsRNA with DNA or single-stranded RNA which can be hy­drolyzed using DNAse and RNAse under high ionic strengthconditions, respectively. The resulting sampie can then be

323

bound to cellulose again in the presence of ethanol, washed,and eluted. The eluted dsRNA is precipitated and an aliquotused for analysis on an agarose or polyacrylamide gel. Sincethere are a number of reports of dsRNA from apparently virus­free plants that may or may not be endornaviruses (Coutts,2005; Valverde and Gutierrez, 2007), whenever possible it isnecessary to use controls of the same species and cultivar as theinfected material (Stace-Smith and Martin, 1988; Wakarchukand Hamilton, 1985).

DsRNA as a Virus Detection Methodin Woody Hosts

In the case of many recalcitrant viruses of pome and stonefruit crops as well as many other woody hosts, dsRNA has longbeen the only laboratory-based means of detecting viruses ininfected plants (Jelkmann, 1998). These viruses often are nottransmitted mechanically to herbaceous hosts and in most casesit has not been possible to purify these viruses directly from theinfected woody plants, though this has been possible for severalviruses of blueberry (Lesney et al., 1978; Martin and Bristow,1988). In several cases, it has been possible to associate specificbanding patterns in gels to a viral disease (Bar-Joseph et al.,1983; Jones et al., 1986; Kurppa and Martin, 1986; Jelkmannet al., 1992). The method as described by Morris and Dodds(1979) can be used to extract dsRNA from many plant spe­cies. However, the quality of the dsRNA obtained is influencedgreatly by the host plant and tissues from which they are ex­tracted. Many plants contain compounds such as glycosides,polyphenols, and polysaccharides that co-purify with nucleicacids and can interfere with their electrophoretic mobility andare inhibitory toward enzymatic reactions. This has led to thedevelopment of modified purification techniques to improve thequality of the dsRNA for use in subsequent procedures (Bakeret al., 1990; Choi and Randles, 1997; Do and Adams, 1991;Tesniere and Vayda, 1991).

DsRNA Extraction Improvementsfor Woody Plants

With some hosts such as pome and stone fruits and straw­berry, the leaf sap is quite viscous, and one or more batch

324 Chapter 57

washes of the dsRNA/cellulose matrix with ethanollbuffer isnecessary before loading onto a column for washing, but thebasic method with minor modifications can be used success­fully for a very broad range of plant species (Tzanetakis et al.,2005a). In the case of blueberry and cranberry, there appear tobe inhibitors in the leaf tissue that interfere with binding of thenucleic acid to the cellulose (Martin et al., 2006). Blueberryleaf homogenates are quite acidic (pH of about 3.0) but con­trolling the pH with higher molarity buffers, the addition ofnicotine, or high pH buffers did not result in binding of thedsRNA to cellulose in the presence of ethanol, nor did increas­ing the ethanol concentration. These inhibitors in blueberryalso blocked the binding of nucleic acids in blueberry leaf sapto glass milk, which is used routinely for binding nucleic acidsand is the basis of most nucleic acid purification kits. The na­ture of these inhibitors has not been determined.

Recently, we developed a dsRNA extraction method that waseffective in purifying dsRNA from blueberry as weil as otherhosts plants that were tested (Tzanetakis and Martin, 2008).The protocol is a scaled up version of a procedure used for ex­traction of RNA from a wide range of plant tissue for use inRT-PCR detection assays (Tzanetakis et al., 2007). This methodhas the added advantage in that it does not use any organic sol­vents other than ethanol. The method is a combination of severalRNA extraction protoco!s followed by purification of dsRNAfrom total nucleic acid preparations using glass milk. TotalRNA was extracted from plant sampies using a scaled up modi­fied Hughes and Galau (1988) method as described by Halgrenet al. (2007) through the isopropanol precipitation, enzymaticdigestion to remove DNA and ssRNA followed by the glass milkextraction method as described by ROll and Jelkmann (2001).Ten grams of leaf tissue was powdered in liquid N2, then addedto the buffer and shaken for 15 min or the leaftissue was homog­enized directly in five volumes of buffer (200 mM Tris base,pH 8.5, 300 mM lithium chloride, 1.5% lithium dodecy! sulfate,10 mM EDTA, 1% deoxycholic acid, 2% polyvinyl pyrrolidone(PVP), I% NP 40 "Tergitol" and 1% b-mercaptoethanol [b­mercaptoethanol was added just prior to use]). Fifty millilitersof 5.8 M potassium acetate (3.8 M K, 5.8 MAcetate) were addedand the solution inverted several times to mix. The sampie wascentrifuged at 15,000 x g for 10 min and the supernatant col­lected and filtered through miracloth. An equal volume of iso­propanol was added to the supernatant and mixed via inversionseveraJ times and centrifuged for 30 min at 20,000 x g. Thenucleic acid pellet was then resuspended in 10 mL of STE (100mM sodium chloride, 50 mM Tris-HCI, I mM EDTA, pH 7.1)and shaken for 5 min. Magnesium chloride was added to a finalconcentration of 0.1 M and the sampie digested with DNAse Iand RNAse Tl for I hat 37°C. EDTA was then added to chelatethe magnesium chloride and 10 mL of95% ethanol and 25 )lL ofglass milk prepared as described by ROll and Jelkmann (2001)were added and incubated at room temperature for 10 min. Thesampie was then centrifuged briefty in a c1inical centrifuge topellet the glass milk, and the pellet was washed in wash buffer3-5 times until the pellet appeared white. The final glass milkpellet was air dried and the dsRNA eluted in 100)lL ofTE, pH8.0. The dsR A can be used for gel analysis and cDNA cloningfor further characterization and identification of the virus(es)that gave rise to the dsRNAs.

Another non-phenol-chloroform extraction method ofdsRNA from plant and fungal tissues has been described by(Balijja et al., 2008). The extraction takes no more than 2 handrequires about 200 mg of fresh plant material. The versatilityand reliability of the method was demonstrated by extractionof dsRNA and RI-dsRNA from virus-infected fungal and plantspecies. However, since Cueumber mosaie virus-infected to­bacco was used as the model for plant viruses, it remains to be

shown if this protocol is suitable for recalcitrant viruses fromhosts, such as pome and stone fruits, which contain more in­hibitors, and generally have viruses in low titers.

DsRNA as Templates for Virus CharacterizationPurified dsRNA has been the starting material for the clon­

ing and sequencing of viruses with (Potgieter et al., 2002) orwithout (Jelkmann et al., 1989; Jelkmann, 1994; Nemchinovand Hadidi, 1998; Nemchinov et al., 2000; Tzanetakis et al.,2005a; Zhang and Rowhani, 2000) PCR amplification prior tothe initial cloning or can be used as templates in RT-PCR assays(Davis and Boyle, 1990). Over the past 15 years, there has beensuccess in the sequencing, characterization, and identificationof a number of viruses of grapevines (Routh et al., 1998; Zhangand Rowhani, 2000), tree fruit (James et al., 2000; Jelkmann,1995; KeimKonrad and Jelkmann, 1996; Zhang et al. , 1998;Nemchinov and Hadidi, 1998; Nemchinov et al., 2000; RaUand Jelkmann, 2001; Marini et al., 2002) and small fruitcrops (Jelkmann et a1., 1990; Schoen et a1., 1998; Thompsonet al., 2002; Martin and Tzanetakis, 2006; Tzanetakis et al.,2006, 2007). The sequence information has been used to de­velop diagnostic tests for these viruses. In some instances thecoat protein genes have been expressed in E. eoli and used tomake antibodies that were used successfully to develop ELISAbased detection methods (Jelkmann, 1998). More commonly,the nucleotide sequence information is used to develop prim­ers for PCR based detection tests or for developing primersto sequence the gaps between the sequence of the fragmentsobtained from the initial cloning. This, combined with 5' and3' RACE (Rapid Amplification of cDNA Ends) to obtain thesequence of the ends of the RNA, has been a useful strategyto sequence viral genomes (ROll and Jelkmann, 2001, 2005;Tzanetakis and Martin, 2004; Menzel et al., 2008). The use ofdsRNA as templates for cloning and sequencing has increasedgreatly over the past several years. One of the major difficul­ties that need to be overcome in using dsRNA as templates forcloning is the extremely stable nature of the molecule. Thoughthe stability of dsRNA is very beneficial during its purification,this stability presents a hurdle that must be overcome before itcan be used as a template for reverse transcription. The bondsof double-stranded RNA are stronger than those of double­stranded DNA, thus the challenge is in complete success ofdenaturing dsRNA.

The most effective means of denaturing the dsRNA is withthe use of methylmercury hydroxide (Jelkmann et al., 1989)but some success has been achieved with the use of heat de­naturation at 99°C for 10 min (Imai et al., 1983) or heat com­bined with DMSO (Cashdollar et al., 1982). The widest useof reverse transcriptase in cloning has been its use to catalyzefirst-strand cDNA synthesis from RNA templates. Typicalprotocols then remove or inactivate the reverse transcripta e(Jelkmann et al., 1989). This is usually followed by treatmentwith RNase H before second-strand synthesis utilizing E. eoliDNA polymerase 1. Once the double-stranded cDNA has beenobtained it is usually cloned into a bacterial plasmid, followedby transformation of E. eoli, and size determination of the in­sert. Various methods have been described for manipulation ofthe double-stranded cDNA following second-strand synthesisto increase the efficiency of cloning, including adapter/linkerligation, homopolymer tailing, and generation of blunt ends forcloning into appropriate vectors (Gubler and Hoffman, 1983;Jelkmann et a1., 1989; Okayama and Berg, 1982). Some cDNAsynthesis/cloning protocols use PCR amplification to obtainsufficient template concentration to allow efficient cloning intoplasmid vectors (Ono and Nakane, 1990). Another method forrapid cDNA cloning from small amounts of purified dsR A

Double-Stranded R As and their Use for Characterization of Recalcitrant Viruses 325

of unknown origin is using a modification of degenerate oligoprimed polymerase chain reaction (DOP-PCR) (Rott andJelkmann, 2001). In DOP-PCR, a single primer consisting of a5' and 3' defined sequence flanking a random sequence of sixnucleotides is used for amplification. The first five PCR cyclesconsist of low stringency annealing followed by a slow raise intemperature to the elongation temperature to facilitate primingof the oligonucleotide. A further 35 cycles at high stringencypreferentially amplify the products generated during the firstfive cycles. When applied to viral dsRNA of unknown origin,the method generally results in a few small cDNAs. These cansubsequently be used for an initial analysis followed by useof specific primers to generate cDNA covering gaps betweenfragments or by using RACE technologies to complete genomicsequences.

Recently, Tzanetakis et al. (2005a) developed a protocol forefficient cloning of dsRNA that made use of the methylmer­cury denaturation as described by Jelkmann et al. (1989) andthen used reverse transcriptase for the first- and second-strandsynthesis of the cDNA. RNAse H was used after first-strandsynthesis to digest the RNA in the RNA:DNA hybrid as inother methods. Then an additional aliquot of reverse tran scrip­tase was added and the reaction continued. The advantage ofthis protocol is that there are relatively few steps, and the re­verse transcriptase is much more robust in terms of not beingaffected by various inhibitors than is the DNA Polymerase Idu ring second-strand synthesis. In this protocol, the double­stranded cDNA was A-tailed using Taq polymerase and thencloned into a T-tailed vector (Tzanetakis et al., 2005a). In theoriginal manuscript, the authors had used this method to obtainpartial sequence for eight different viruses. They then used thepartial sequences to design primers to fill in the gaps and used5' and 3' RACE to obtain the ends. They were able to get theentire sequence of an ilarvirus, f1exivirus, Closterovirus, anda picorna-like plant virus. This method has since been usedto obtain partial or entire sequences of over 20 viruses fromdsRNA templates (Kraus et al., 2008; Susaimuthu et al., 2008a;Tzanetakis and Martin, 2007).

SummaryDsR A has been the basis for the characterization and de­

velopment of diagnostic tests for many viruses of woody hostssince 1990. In many cases it has been shown that "severe"strains of a disease are caused by virus complexes rather thanstrains of a single virus (Karyeija et al., 2000; Susaimuthu etal., 2008b). The use of dsRNA will continue to be the basisfor development of information on viruses that cause diseasesin pome and stone fruit crops. With the development of virusgenus-specific primers the potential to identify a broad range ofviruses using RT-PCR is becoming more common (Adkins etal., 2006; Foi sac et al., 2005; Guaragna et al., 2004; Marini etal., 2002; Routh et al., 1998). Even as the number of these broadspectrum primers increases, the use of dsRNA to examine dis­eases will be necessary since there is always the possibilitythat a completely unexpected virus may be present in diseasedplants (Sabanadzovic et aJ., 2009). Due to the presence or po­tential for the unknown being present in pome and stone fruitsas weil as other woody crops, broad spectrum techniques suchas dsRNA analysis and grafting will be part of the detection,diagnostic, and characterization technology tool box for sometime into the future.

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