Identification of Three Distinct Classes of Satellite RNAs Associated With Two Cucumber mosaic virus Serotypes from the Ornamental Groundcover Vinca minor

Cucumber mosaic virus (CMV) is a cosmopolitan virus which may also have small satellite RNAs (satRNA) associated with it affecting symptom development. Vinca minor (periwinkle) plants exhibiting subtle mosaic symptoms tested positive for CMV by enzyme linked immunosorbent assay (ELISA). Double-stranded ribonucleic acid (dsRNA) analysis of CMV-Vinca field isolates in Nicotiana tabacum ‘Glurk’ suggested two sizes of putative satRNA associated with CMV. Immunocapture RT-PCR, cloning, and sequencing of the movement protein, coat protein, and satRNAs demonstrated serogroup 1A and serogroup 2 CMV helper strains and three distinct classes of satRNAs of four sizes. Further, two classes of satRNAs could be distinguished by their necrosis domains. Previously CMV was reported in V. minor in New Jersey. This is the first report of CMV in V. minor in Ohio and the first report of satRNA associated with CMV in V. minor in the United States. Introduction Cucumber mosaic virus (CMV) is a cosmopolitan virus with a worldwide distribution and very broad host range, infecting more than 1000 weed, crop, and ornamental hosts in over 85 families (11). The virus is the type member of the genus Cucumovirus of the family Bromoviridae with spherical particles, tripartite positive sense, single-stranded RNA genome encoding one structural (capsid) and four non-structural proteins (1,8,11). Occasionally CMV isolates have small satellite (sat) RNAs [~330 to 390 nucleotides (nt)] associated with them whose nucleotide sequences are unrelated to the CMV genome and nonessential to CMV replication but are dependent upon CMV for replication and encapsidation (2,4,12). These satRNAs often have profound effects on CMV symptoms ranging from lethal necrosis to disease attenuation (6,18), and can even alter the disease phenotype on a particular host (5,9,14,18) and among various hosts (9). Vinca minor L. (common periwinkle) is an evergreen ground cover in temperate climates (3). Previously, virus-like symptoms have been attributed to CMV isolated from V. minor in New Jersey (16). In early 2010, V. minor samples exhibiting subtle mosaic symptoms (Fig. 1) were submitted to the Ohio Plant Diagnostic Network for analysis. The samples tested positive for CMV by triple antibody sandwich (TAS) enzyme-linked immunosorbent assay (ELISA) using commercial antibodies (Agdia Inc., Elkhart, IN). Additionally, satellite RNA was detected in several of the samples by immunocapture (IC) reverse transcription (RT) polymerase chain reaction (PCR). Here we report the identification and characterization of three distinct classes of satellite RNAs supported by CMV serogroup 1A and 2 helper strains from V. minor. To our knowledge, this is the first report of CMV in V. minor in Ohio and satellite RNA associated with CMV in V. minor in the United States. These findings have 12 April 2012 Plant Health Progress important implications for commercial ornamental and vegetable growers in proximity to V. minor plantings which may serve as an overwintering reservoir for CMV and the satRNAs associated with it. The CMV helper strains and associated satRNAs described here currently appear to be benign on tobacco and tomato, but very little is known about the disease impact they may have on other ornamental and vegetable crops. Fig. 1. Subtle mosaic symptom observed on Vinca minor leaves. Characterization of CMV-Vinca Isolates and Satellite RNAs Five CMV-Vinca field isolates (designated N1-05, N1-04, N1-03, N1-02, and 0100) were used for this project. Two isolates (N1-05 and 0100) were used for IC-RT-PCR directly from source plants. Three isolates (N1-02, N1-03, and N104) were passaged to Nicotiana tabacum ‘Glurk’ by triturating Vinca tissue (1:5 ratio) in 100 mM sodium phosphate buffer (pH 7.0) containing 1% (w/v) celite and rubbing on plants at the four leaf stage. CMV in symptomatic inoculated tobacco was confirmed by TAS-ELISA. Additionally, CMV-Vinca N1-03 in Vinca and tobacco ‘Glurk’ was arbitrarily screened by ELISA for impatiens necrotic spot, tomato spotted wilt, tobacco ring spot, tobacco mosaic, alfalfa mosaic, arabis mosaic, and tomato ring spot viruses using commercial antibodies (Agdia Inc.). Test samples and controls were loaded in duplicate wells and evaluated at 405 nm (MRX Revelation plate reader, Dynex Technologies, Chantilly, VA). A sample was considered positive if the OD was twice the mean of the negative controls and a plate was valid only if the positive controls reacted. Healthy tissue and extraction buffer alone were used as negative controls. None of these other viruses were detected. Asymptomatic V. minor was pre-screened for CMV prior to back-inoculation from tobacco infected with the N1-03 isolate. Four healthy Vinca plants were inoculated with CMV-Vinca N1-03 and one plant mock inoculated with extraction buffer. Three of the four experimental plants developed the subtle mosaic symptom observed on the original samples, and the mock inoculated plant remained symptomless. All plants were tested for CMV by ELISA 28 days post inoculation. The three symptomatic plants tested positive. The asymptomatic experimental plant and mock inoculated plant tested negative. These results satisfy Koch’s postulates for the CMV-Vinca N1-03 isolate and demonstrate with some certainty that CMV is the causal agent of the mosaic symptom observed on V. minor and not due to a mixed infection with another common virus. 12 April 2012 Plant Health Progress Double-stranded ribonucleic acid (dsRNA) was purified from CMV-Vinca infected tobacco (17), analyzed on 1% or 1.5% ethidium bromide-stained agarose gels, and results recorded (Gel Logic 112, Eastman Kodak, Rochester, NY). DsRNA analysis of CMV-Vinca N1-02, N1-03, and N1-04 infected tobacco ‘Glurk’ produced banding profiles typical for CMV, with major genomic bands corresponding to approximately 3.3 Kb, 3.0 Kb, 2.2 Kb, and a sub-genomic band at approximately 1.0 Kb (1,11), but with apparent size variation among the genomic RNAs as well as several additional lower MW RNAs in the 0.5 to 0.75 Kb range (Fig. 2). N1-03 and N1-04 also had an additional low molecular weight (~0.3 to 0.4 Kb) molecule that N1-02 apparently lacked, and the low MW species present in the N1-03 and N1-04 isolates did not co-migrate (Fig. 2). This result suggested the N1-03 and N1-04 isolates possessed a putative satellite RNA absent in N1-02, and that the N1-04 satRNA was of greater MW than the N1-03 satRNA. Fig. 2. DsRNA analysis of CMV-Vinca isolates N1-02 (lane 1), N1-03 (lane 2), and N1-04 (lane 3) in N. tabacum ‘Glurk.’ Approximate locations of dsRNAs 1, 2, 3, 4, and putative satRNAs indicated on left. 1 Kb DNA ladder (lane 4). 1% agarose gel, 100 volts, 2 hours. 12 April 2012 Plant Health Progress Tomato (Lycopersicon esculentum Mill.) cv ‘Rutgers’ was used as a lethal necrosis bioassay (19). Seed was sown in sterile potting mix (Miracle-Gro Lawn Products, Marysville, OH) and grown to the fully expanded cotyledon stage. CMV-Vinca infected N. tabacum ‘Glurk’ inoculum was prepared as above. Inoculum or buffer alone was rubbed on cotyledons using sterile gauze pads and the plants maintained in indoor growth rooms (25°C, ~12 h day/night) and monitored for symptoms over a 28-day period. All plants were screened for CMV by TAS-ELISA. A minimum of ten seedlings was inoculated with each isolate with a further five plants mock inoculated with buffer, and the experiment was repeated three times. ELISA positive tomato ‘Rutgers’ inoculated with CMV-Vinca N1-04 never developed symptoms, and those inoculated with N1-03 and N1-02 developed a fern leaf symptom and were stunted compared to buffer mock inoculations. None of the seedlings developed a lethal necrosis symptom. These results demonstrate that none of the tested satellite RNAs (N1-04, N1-03, and N1-02) induces lethal necrosis on tomato. For IC-RT-PCR magnetic beads conjugated with sheep anti-rabbit IgG (Dynabeads M-280, Dynal Biotech/Invitrogen, Carlsbad, CA) were incubated with polyclonal rabbit anti-CMV IgG (Agdia Inc.). Tissue was ground in phosphate sucrose buffer (116.1 ml 1M Na H PO , 33.9 ml 1M NaH PO , 50g sucrose; pH 7.4; 1L) at a 1:10 ratio and 10to 20-μl antibody-coated beads incubated for two hours with shaking. Beads were pulled out of suspension on a magnetic rack (Dynal/Invitrogen), washed four times, and used for first strand synthesis by addition of 20 μl MMLV-RT cocktail [10 μl H O, 4 μl 5X MMLV buffer, 2 μl 5 mM dNTPs, 0.5 μl 0.5 μg/μl random hexamers, 1 μl 1 mg/ml BSA, 1 μl 0.1 mM dithiothreitol, 0.5 μl 40 units/μl RNAsin, 1 μl 200 units/μl MMLVRT (Promega Inc., Madison, WI)] and incubating for one hour in a 42°C water bath. 5 μl cDNA or sterile water was used as template for separate PCR reactions using three primer pairs (Integrated DNA Technologies Inc., Coralville, IA) to amplify the movement protein (MP), coat protein (CP), and satRNA (Table 1) in 25 μl reactions [1.5 mM MgCl , 0.2 mM dNTP mix, 0.2 μM primer pair, 0.625 units Taq polymerase (Promega Inc.)]. Amplification was done in an Eppendorf Mastercycler (Hamburg, Germany) with the cycling parameters: 94°C (2 min), 40 cycles of 94°C (30 sec), 59°C (30 sec), 55°C (30 sec), 52°C (30 sec), 72°C (45 sec), final extension 72°C (10 min). PCR products were analyzed on 0.8% agarose gels, DNA cut from the gel, purified (QIAquick gel extraction kit, Qiagen, Inc., Valencia, CA), ligated into pGEM-T Easy plasmid (Promega Inc.), the ligation used to transform competent E. coli JM109 cells, and colonies screened for insert by PCR using M13 primers. Selected colonies were grown in LB broth containing ampicillin and plasmid purified using the Wizard Plus SV mini-prep kit (Promega Inc.). DNA was quantified (Nanodrop 2000, Thermo Scientific, Waltham, MA) and sequenced (Plant Microbe Genomics Facility, The Ohio State University, Columbus, OH) in both directions using M13 primers. Vector was trimmed from raw sequences (Chromas v. 2.33, Technelysium Pty Ltd., Australia), contigs assembled, pairwise and multiple sequence alignments with reference sequences performed (Contig Express, ClustalW, Vector NTI 11, Invitrogen), sequences edited and open reading frames translated (Genedoc v. 2.6.001, 2000, Karl Nicholas). Known CMV strains C, Fny, K, LS, My17, Q, Rb, Sn, SO, WL, Y, Z1, and peanut stunt virus PSV-ER (GenBank Accession numbers D00462.1, D10538.1, AF127977.1, AF127976.1, AF103993.1, J02059.1, GU327365.1, U22822.1, AF103992.1, D00463.1, M57602.1, GU327368.1, and U15730.1, respectively) were used as reference sequences for movement and coat protein comparison. Known satRNAs D, E, G, B1, B2, B3, Fny1, Fny2, Ky, WL, WL2, S, T43, Y, OY2, and D27 (GenBank Accession numbers M20350.1, M20844.1, M16585.1, M16586.1, M16587.1, M16588.1, X54065.1, X54066.1, DQ975201.1, M30589.1, M16590.1, M14934.1, D10039.1, D00542.1, M20845.1, and U31661.1, respectively) were used as reference sequences for satRNA comparison. Sixteen CMV-Vinca isolate sequences were deposited in GenBank under Accession numbers JF918960-918963 (MP), JF918964-918967 (CP), and JF918968918975 (satRNA). 2 2 4 2 4