Indigenous double-stranded RNA from pepper (Capsicum annuum)

Plant Science, 67 (1990) 1 9 5 - 2 0 1

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Elsevier Scientific Publishers Ireland Ltd.

I N D I G E N O U S DOUBLE-STRANDED RNA FROM P E P P E R (CAPSICUMANNUUM)

RODRIGO A. VALVERDE a, STEPHEN NAMETH b, OMER ABDALLHA ¢, 0. AL-MUSA ~, PAUL DESJARDINS ¢ and ALLAN DODDS c

*Department of Plant Pathology and CropPhysiology, Louisiana Agricultural Experiment Station, Louisiana State University A gricultural Center, Baton Rouge, LA 70805, bDepartment of Plant Pathology, The Ohio State University, Columbus, OH 43210 and ~Department @Plant Pathology, University of Californi~ Riverside CA 9~521 (U.S.A./ (Received July 24th, 1989} (Revision received November 7th, 1989) (Accepted November 20th, 1989}

A unique 12 kb linear double-stranded RNA (dsRNA) was detected in the chloroplast fraction of tissue extracts from pepper (Capsicum annuum). This dsRNA was detected in 10 out of 14 pepper cultivars tested. Molecular hybridization using eDNA to this dsRNA indicated t h a t the 12 kb dsRNAs found in these pepper cultivars had nucleotide sequence similarity. This dsRNA did not hybridize to dsRNAs of similar size detected in bean and melon, nor to other dsRNAs found in non-virus inoculated plants. No evidence was obtained for a virus-like particle associated with this dsRNA.

Key words: dsRNA; pepper; chloroplast

Introduction Nucleic acid extracts from plants infected with RNA viruses are expected to contain viral-specific double-stranded RNA (dsRNA) [1]. The detection of these dsRNAs has been used successfully as a tool for identification of plant viruses [1,2]. DsRNAs similar in size and number to those normally associated with virus infection of plants have been found in several non-virus inoculated plant species [2-6]. Those found in avocado and french bean have been studied in detail [3,6]. In these 2 examples, the dsRNAs were usually restricted to particular cultivars, were not closely associated with any virus-like disease, and virus particles could not be detected in tissues which contained the dsRNAs. During a study of the dsRNAs that accumulate in plants infected with viruses representing five plant viral groups, we noticed that some non-virus-inoculated plants had relatively large quantities of dsRNAs [2]. These dsRNAs were similar in size to viral specific dsRNAs detected in virus inoculated plants but

apparently were not the product of an RNA virus infection. Among those dsRNAs a particular one was consistently obtained in relatively large amounts from pepper (Capsicum annuum) cv. California Wonder. In this study we report properties of this unique dsRNA. Materials and Methods

Plant species DsRNAs of the following plant species were analyzed: pepper (Capsicum annuum L.), bean (Phaseolus vulgaris L.), melon (Cucumis melo L.), sugarbeet {Beta vulgaris L.) and spinach (Spinacia oleraceae L.). All plants were grown in 4 inch pots in steam sterilized soil mix. Plants were placed in a greenhouse with an average temperature of 22°C and a 12-h per day photoperiod. Generally, leaf tissue was harvested and analyzed for dsRNA 2 months after planting.

Nucleic acid extraction and electrophoresis DsRNAs were extracted from 7.0 g of plant tissue and purified by 2 cycles of fractionation

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196 on columns of cellulose powder (Whatman CF11) [3l. DsRNAs retained by the column in STE (containing 0.1 M NaC1, 0.05 M Tris--HC1 and 0.001 M ethylenediamine tetraacetic acid (EDTA}, pH 7.0) buffered ethanol {16% v/v) and eluted in ethanol-free STE were precipitated with 3 volumes of 95% ethanol and resuspended in 300 ~l of electrophoresis buffer (TAE) (containing 0.04 M T r i s - H C 1 , 0.02 M sodium acetate and 0.001 M EDTA, pH 7.8). Aliquots of 30 ~l (approximately 0.5 ~g of dsRNA) were loaded on 6% polyacrylamide gels (40:1 acrylamide, bisacrylamide) in a verticle slab gel apparatus. Electrophoresis was at a constant voltage of 100 V for 3 h. Gels were stained in ethidium bromide (20 ng/ml) and viewed under an ultraviolet light transilluminator (300 nm). Different tissues from pepper cv. California Wonder (leaves, petals, and roots) were analyzed for dsRNA. Pepper (cv. California Wonder} callus from in vitro cell culture (provided by Dr. H. Murakishi, Department of Botany and Plant Pathology, Michigan State University) was also examined for the prescence or absence of dsRNA. For analysis of fractions, 10 g of pepper leaf tissue were processed for isolation of chloroplasts and mitochondria according to Hrubec et al. [7]. For nuclear and cytoplasmic fractions, another 10 g of tissue was processed as described by Jackson et al. [8]. DsRNA was extracted from all four fractions and analyzed by electrophoresis as described.

Gel purification of dsRNA After cellulose chromatography, the partially purified pepper dsRNA was subjected to electrophoresis from a wide sample well in a 1.2% agarose gel in TAE buffer. After staining a small portion of the gel, the region of the remaining gel containing the dsRNA was excised and placed in a dialysis bag containing a minimal volume of 1/5th strength TAE buffer. Elution of the dsRNA from the gel was carried out by electrophoresis (60 mA for 1 h). Eluted dsRNA was recovered in the solution from the bag and precipitated by addition of ethanol to a final concentration of 66% (v/v}. The dsRNA nature of the nucleic acids was confirmed by appropriate nuclease digestions. A 10 ~g/ml

(assuming Ae80 = 20 for 1 mg/ml solution of dsRNA} solution of pepper dsRNA was mixed with pancreatic RNase A {type I, Sigma) and DNase I (Sigma). RNase and DNase were used at a final concentration of 25 ~g/ml. Preparations were incubated at room temperature for 1 h. DNase digestion was carried out in a solution (SSC) containing 0.15 M NaC1, 0.005 M MgCl 2 and 0.015 M sodium citrate (pH 7.0). RNase digestion was carried out in either 1 × or 0.1 x SSC.

Denaturation and blotting Prior to electroblotting, dsRNAs in polyacrylamide gels were denatured with 50% (v/v) dimethyl sulfoxide (DMSO} and 1 M glyoxal in 0.025 M sodium phosphate buffer (pH 6.5) at 50°C for 1 h (all reagents are given at final concentrations). Gels were electroblotted to nylon membranes in the presence of 0.01 M Tris - H C I , 0.005 M sodium acetate, and 0.005 M EDTA (pH 7.8) at 60 mA for 5 h. Purified preparations of dsRNA were denatured in 0.01 M methylmercury hydroxide and spotted on nitrocellulose membranes. Membranes were baked under vacuum at 80°C for 2 h and stored at room temperature until used for hybridization.

DNA synthesis and hybridization Randomly primed cDNA was transcribed from denatured ssRNA with reverse transcriptase from avian myeloblastosis virus (Amersham) in the presence of [32P]dCTP (3000 Ci/mmol; Amersham) essentially as described by Maniatis et al. [9]. Prior to transcription, the dsRNA was partially hydrolyzed and denatured by boiling for 3 min in 100% formamide. Prehybridization was carried out overnight in 20 ml of prehybridization solution [11 ml of 100% deionized formamide {deionized with Dowex XG8 mixed bed resin for 1 h), 5 ml of 15 x SSC containing 0.05 M sodium phsophate (pH 7.0}; 1 ml of'2% bovine serum albumin; 1 ml of 2% ficoll; 1 ml of 2% polyvinylpyrrolidone; 0.2 ml of 10% SDS and 0.8 ml of sonicated calf thymus DNA (10 mg/ml), s2P-labeled cDNA (approx. 250 000 cpm/ml) was added and hybridized for 24 h at 42°C. After hybridization, membranes were washed in: 2 × SSC

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containing 0.1% (w/v) SDS for 2 h at room temperature, 0.1 × SSC containing 0.1°/0 SDS for 30 rain at room termperature and 0.1 × SSC containing 0.1°/0 SDS for 30 rain at 55°C. Membranes were then dried and exposed to X-ray film.

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Transmission Attempts to transmit dsRNA from pepper to plants of the same species that lacked it were carried out using several approaches. These included: (a) mechanical inoculations by grinding foliar samples of pepper cv. California Wonder in 0.02 M phosphate buffer (pH 7.0) and rubbing, the suspension on leaves of pepper cv. Mexican Chili; (b) grafting scions of Mexican Chili on California Wonder rootstocks; (c) establishing dodder (Cuscuta campestris) first on California Wonder and then transferring the same dodder plants to Mexican Chili; (d) planting 24 seeds obtained from California Wonder in order to determine seed transmissibility. In all cases, all the experimental plants were tested for presence or absence of dsRNA by gel electrophoresis and molecular hybridization.

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Electron microscopy A method commonly used to purify cryptic viruses was followed using pepper cv. California Wonder [10]. Final preparations were negatively stained with 20/0 (w/v) uranyl acetate and examined with the electron microscope. Sap extracts were also stained and examined. For nucleic acid spreading, an electroeluted preparation of pepper dsRNA was used. Purified dsRNA was prepared for electron microscopy by the droplet technique of Arcidiacono et al. [11]. After rotary shadowcasting with platinum/palladium alloy (80:20), the preparations were examined and photographed in a Hitachi 600 electron microscope using a 30-nm objective aperature. Results

dsRNAs and hybridization This nucleic acid isolated from pepper was dsRNA since it resisted digestion by DNase

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Fig. 1. Polyacrylamide gel electrophoresis of dsRNAs obtained from various pepper cultivars and dot spot hybridization. (A) One tenth of the dsRNA obtained from 7 g of pepper tissue (0.5 ~g of dsRNA) was analysed in lanes 1-14. Lane 1, Gypsy Hybrid; lane 2, Hungarian Wax; lane 3, Bell Boy Hybrid; lane 4, Zippy Hybrid; lane 5, California Wonder; lane 6, Golden California Wonder; lane 7, Avelar; lane 8, New Ace Hybrid; lane 9, Yolo Wonder; lane 10, PI 342947; lane 11, Long Red Cayenne; lane 12, Jalapeno M; lane 13, Yolo Wonder B; and lane 14, Mexican Chili. Migration of dsRNA in this gel and those of figures 2 and 3 is from top to bottom [cathode ( - ) to anode (+)]. (B) Results of hybridization using cDNA to pepper cv California Wonder dsRNA as probe. Denatured dsRNAs from Fig. 1A were spotted (numbers correspond to those of Fig. 1A).

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and by RNase at high salt concentrations. It also behaved like dsRNA in cellulose chromatography. Five distinct patterns of dsRNAs were found associated with 13 pepper cultivars out of 14 analyzed (Fig. 1A). A dsRNA of about 12 kb was most commonly found and was used in most of the studies. Other dsRNAs detected were smaller and ranged from I to 2 kb. cDNA transcribed from pepper cv. California Wonder dsRNA hybridized to the same dsRNA and to those of similar size found in 9

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other pepper cultivars (Fig. 1B). It did not hybridize to the small dsRNAs found in two pepper cultivars and an experimental line. No relationship between the pepper high molecular weight dsRNA and dsRNAs from sugarbeet, spinach, cantaloupe, bean, and small dsRNAs from two other pepper cultivars was found (Fig. 2). Tissue and cell localization The 12-kb dsRNA was detected in leaf, flower, and root tissue of pepper. It was also obtained from pepper callus culture. Fractionation of tissue extracts yielded a significant amount of dsRNA in the chloroplast fraction (Fig. 3). Little or none was detected in the nuclear, cytoplasmic, and mitochondrial fractions. Transmission All attempts to transmit the dsRNA other than through seed failed, including graft transmission. All 24 plants obtained from 24 seeds of pepper cv. California Wonder were dsRNA positive.

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Fig. 2. Polyacrylamide gel (A) of dsRNAs stained with ethidium bromide and equivalent northern blot (B) hybridized with cDNA to pepper dsRNA, dsRNAs (0.5/~g/lane) were extracted from non-virus inoculated dsRNA positive plants. Lane 1, sugarbeet; lane 2, spinach; lane 3, melon; lane 4, bean; lane 5, pepper cv. California Wonder; lane 6, pepper cv. Jalapeno M; and lane 7, pepper cv. Long Red Cayenne.

Fig. 3. Polyacrylamide gel electrophoresis of dsRNAs from various cell fractions of pepper cv. California Wonder. Each lane contains the total dsRNA extracted from fractions obtained from 10 g of pepper leaf tissue. Gel was stained with ethidium bromide. Lane 1, cytoplasmic; lane 2, nuclear; lane 3, mitochondrial; and lane 4, chloroplast fraction.

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Discussion

Fig. 4. Electron m i c r o g r a p h of a linear d s R N A molecule from p e p p e r cv. California W o n d e r . Bar = 100 nm.

Electron microscopy No virus-like particles were observed in negative stained sap extracts. Attempts to purify a virus-like particle failed. Purified dsRNA was resolved by electron microscopy as a linear molecule (Fig. 4). This molecule corresponds in length and size to the 12-kb pepper dsRNA detected by gel electrophoresis.

In plants and fungi, dsRNAs of uncertain origin have been found to be associated with some phenotypic characters. Male sterile Vicia faba [12], corn [13] and sunflower [14] are all sources of dsRNA. Male sterile V. faba contains a 10-15-kb dsRNA which can be transmitted by dodder, thereby transmitting the male sterile factor. Loss of dsRNA results in the recovery of fertility in this case. In corn and sunflower, the correlation between dsRNA and male sterility is not clear and is still controversial [15]. The presence of high molecular weight dsRNAs in phenotypically normal non-virus inoculated plants seem to be a feature of some cultivars of some plant species [6,12-14,16]. Other cultivars lack such dsRNAs, indicating that they are not constitutively essential. In some cases, it would not be surprising if some of the dsRNAs constitute the genomes of cryptic viruses. Cryptic viruses contain divided dsRNA genomes [10]. Other properties of these viruses are: (a) lack of transmissibility by graft or mechanical inoculation, (b) lack of symptoms in the carrying host, (c) high rates of seed transmission and (d) low concentration in plant tissues. Some dsRNA patterns obtained from two pepper cultivars (Long Red Cayenne and Jalapeno M) and an experimental line (PI 342947) resemble the genome of cryptic viruses based on the size and number of dsRNA bands. The 12-kb dsRNA found in pepper is unlikely to be the genome of either a plant reovirus or a cryptic virus. Reasons for this include the failure to detect any virus-like particle in tissue extracts, the large size of the molecule, and lack of multiple dsRNAs. The dsRNAs of known seed-transmitted viruses in pepper such as cucumber mosaic virus and tobacco mosaic virus differ from the 12-kb dsRNA in size and also in frequency of detection in non-inoculated seedlings. These viruses also cause symptoms and are mechanically transmitted. The 12-kb dsRNA detected in pepper was similar in size to those obtained from bean and melon. Despite this similarity, there was no

200 d e t e c t a b l e nucleotide sequence similarity b e t w e e n t h e m . All 12-kb d s R N A s o b t a i n e d f r o m nine p e p p e r c u l t i v a r s had nucleotide sequence similarity with t h e d s R N A isolated f r o m California W o n d e r p e p p e r . This m a y be an indication of a c o m m o n g e n e t i c origin a m o n g all t h e s e p e p p e r cultivars. T h e lack of r e l a t i o n s h i p with o t h e r c o m p a r a b l e d s R N A s e s t a b l i s h e s its uniqueness. The chloroplast fraction f r o m p e p p e r leaf tissue contained m o s t of the 12-kb d s R N A . N e v e r t h e l e s s , t h e d i s t r i b u t i o n in g r e e n and non-green t i s s u e s indicates t h a t it is not r e s t r i c t e d to fully d e v e l o p e d chloroplasts. L a c k of g r a f t t r a n s m i s s i o n and failure to d e t e c t this d s R N A in d o d d e r a t t a c h e d to p e p p e r plants (data not shown) p r o v i d e f u r t h e r evidence for organelle localization or association. T h e s e cell fractionation studies do not a d d r e s s t h e n a t u r e of the association of t h e d s R N A with t h e comp o n e n t s p r e s e n t in t h e chloroplast fraction. A low molecular w e i g h t d s R N A has b e e n r e p o r t e d in h e a l t h y tobacco p l a n t s [17]. L i k e t h e p e p p e r d s R N A it w a s found to be a s s o c i a t e d m o s t l y with t h e chloroplast fraction. T h e a u t h o r s p r o p o s e d it to be a p r o d u c t of an R N A dependent RNA polymerase previously shown to occur in h e a l t h y plants. W a k a r c h u k and H a m i l t o n [5] r e p o r t e d a d s R N A similar to t h e one r e p o r t e d h e r e in t h e b e a n cultivar Black Turtle. P r e s e n c e of this d s R N A was found to be a s s o c i a t e d with t h e color of t h e s e e d coat, and it was r e p o r t e d to h a v e s o m e s e q u e n c e similarity to host p l a n t DNA. W e h a v e used the Black T u r t l e d s R N A in our c o m p a r i s o n s and found no e v i d e n c e of nucleic acid s i m i l a r i t y with p e p p e r d s R N A , which is also slightly smaller. This s u g g e s t s that the pepper dsRNA may have a different origin t h a n the one found in bean. T h e origin and function of this d s R N A in p e p p e r is unclear. E x p e r i m e n t s d e s i g n e d to t r a n s f e r this molecule to hosts which do not h a v e it will be e s s e n t i a l in a n s w e r i n g questions a b o u t its functions.

Acknowledgement This p a p e r was a p p r o v e d for publication b y

the D i r e c t o r of t h e L o u i s i a n a A g r i c u l t u r a l E x p e r i m e n t Station as m a n u s c r i p t No. 89-383310. References 1 J.A. ])odds, T.J. Morris and R.L. Jordan, Plant viral double-stranded RNA. Annu. Rev. Phytopatbol., 22 (1984) 151-168. 2 R.A. Valverde, J.A. Dodds and J.A. I-Ieick, Doublestranded ribonucleic acid from plants infected with viruses having elongated particles and undivided genomes. Phytopatbology, 76 (1986)459--465. 3 R.L. Jordan, J.A. Dodds and H.D. Ohr, Evidence for virus-like agents in avocado. Phytopathology, 73 (1983) 1130-1135. 4 J.A. Dodds, R.A. Valverde and D.M. Mathews, Detection and interpretation of dsRNA, in: Y. Koltin and M.J. Leibowitz (Eds.), Viruses of Fungi and Simple Eukaryotes, Marcel Dekker, new York, 1988, pp. 309-326. 5 S.T. Nameth, J.A. Dodds, Double-stranded RNA detected in cucurbit varieties not inoculated with viruses. Phytopathology, 75 (1985) 1293. 6 D.A. Wakarchuk and R.I. Hamilton, Cellular doublestranded RNA in Phaseolus vulgaris cultivar Black Turtle Soup. Plant Mol. Biol., 5 (1985)55--64. 7 T.C.Hrubec, J.M. Robinson and R.P. Donaldson, Isolation of mitochondria from soybean on discontinuous Percell gradients. Plant Physiol., 77 (1985) 1010-1012. 8 A.O. Jackson, Replication of tobacco mosaic virus. I. Isolation and characterization of double-stranded forms of ribonucleic acid. Virology, 45 (1971) 182-191. 9 T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular cloning: A laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982, pp. 229-- 234. 10 G. Boccardo, V. Lisa and R.G. Milne, Cryptic viruses in plants, in: R.W. Compans and D.H.L. Bishop (Eds.), Double Stranded RNA viruses, Elsevier, New York, 1983, pp. 425- 430. 11 A. Arcidiacono, A. Stasiak, and T. Koller, Protein-free specimen preparation of nucleic acids and nucleic acidprotein complexes. Proc. Eur. Congr. Elect. Micr., 2 (1980) 516- 523. 12 L.K. Grill and S.J. Garger, Identification and characterization of double-stranded RNA associated with cytoplasmic male sterility in Vicia fabo. Proc. Natl. Acad. Sci. U.S.A., 78 (1981)7043-- 7046. 13 P.H. Sisce, F. Garcia-Arenal, M. Zaitlin, V.E. Gracen and E.D. Earle, LBN, a male sterile cytoplasm of maize contains 2 double stranded RNAs. Plant Sci. Lett., 34 (1984) 127-- 134. 14 G.G. Brown, H. Bussey and L.J. Desrosiers, Analysis of mitocbondrial DNA, chloroplast DNA and doublestranded RNA in fertile and cytoplasmic male-sterile sunflower, Helianthus annuus. Can. J. Genet. Cytol., 28 (1986) 121 - 129.

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M~R.Hanson and M.F. Conde, Functioning and variation of cytoplasmic genomes: lessons from cytoplasmic nuclear interactions affecting male fertility in plants. Int. Rev. Cytol., 94 (1985~213-267. W. Zong-Yang, Z. Fei-Qin, G. Xiao-Li and H. Meng-Min, Occurrence and partial characterization of an RNA

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species in rice mitochondira. Plant Sci., 61 (1989) 227234. M. Ikegami and H. Fraenkel-Conrat, Characterization of double-stranded ribonucleic acid in tobacco leaves. Proc. Natl. Acad. Sci. U.S.A., 76 (1979)3637-3640.