First Report of Pepper mottle virus Infecting Tomato in Hawaii.

In August 2011, tomato (Solanum lycopersicum L.) fruit from a University of Hawaii field trial displayed mottling symptoms similar to that caused by Tomato spotted wilt virus (TSWV) or other tospoviruses. The foliage from affected plants, however, appeared symptomless. Fruit and leaf tissue from affected plants were negative for TSWV analyzed by double antibody sandwich (DAS)-ELISA and/or TSWV ImmunoStrips (Agdia, Elkhart, IN) when performed following the manufacturer's instructions. Total RNA from a symptomatic and an asymptomatic plant was isolated using an RNeasy Plant Mini Kit (Qiagen, Valencia, CA) and reverse transcribed using Invitrogen SuperScript III reverse transcriptase (Life Technologies, Grand Island, NY) and primer 900 (5'- CACTCCCTATTATCCAGG(T)16-3') following the enzyme manufacturer's instructions. The cDNA was then used as template in a universal potyvirus PCR assay using primers 900 and Sprimer, which amplify sequences encoding the partial inclusion body protein (NIb), coat protein, and 3' untranslated region of potyviruses (1). A ~1,700-bp product was amplified from the cDNA of the symptomatic plant but not the asymptomatic plant. This product was cloned using pGEM-T Easy (Promega, Madison, WI) and three clones were sequenced at the University of Hawaii's Advanced Studies in Genomics, Proteomics, and Bioinformatics laboratory. The 1,747-bp consensus sequence of the three clones was deposited in GenBank (Accession No. JQ429788) and, following primer sequence trimming, found to be 97% identical to positions 7,934 through 9,640 of Pepper mottle virus (PepMoV; family Potyviridae, genus Potyvirus) accessions from Korea (isolate '217' from tomato; EU586126) and California (isolate 'C' from pepper; M96425). To determine the incidence of PepMoV in the field trial, all 292 plants representing 14 tomato cultivars were assayed for the virus 17 weeks after planting using a PepMoV-specific DAS-ELISA (Agdia) following the manufacturer's directions. Plants were considered positive if their mean absorbance at 405 nm was greater than the mean absorbance + 3 standard deviations + 10% of the negative control samples. The virus incidence ranged from 4.8 to 47.6% for the different varieties, with an overall incidence of 19.9%. Although plant growth was not noticeably impaired by PepMoV infection, the majority of fruit from infected plants was unsaleable, making PepMoV a considerable threat to tomato production in Hawaii. PepMoV has been reported to naturally infect tomato in Guatemala (3) and South Korea (2). To our knowledge, this is the first report of this virus in Hawaii and the first report of this virus naturally infecting tomato in the United States. References: (1) J. Chen et al. Arch. Virol. 146:757, 2001. (2) M.-K. Kim et al. Plant Pathol. J. 24:152, 2008. (3) J. Th. J. Verhoeven et al. Plant Dis. 86:186, 2002.

Varying genetic diversity of Papaya ringspot virus isolates from two time-separated outbreaks in Jamaica and Venezuela.

Coat protein sequences of 22 Papaya ringspot virus isolates collected from different locations in Jamaica and Venezuela in 1999 and 2004, respectively, were determined and compared with sequences of isolates from earlier epidemics in 1990 and 1993. Jamaican isolates collected in 1999 exhibited nucleotide sequence identities between 98 and 100% but shared lower identities of 92.2% with an isolate collected in 1990. Isolates from the 2004 epidemic in Venezuela exhibited more heterogeneity, with identities between 88.7 and 98.8%. However, isolates collected in 1993 were more closely related (97.7%). The viral populations of the two countries are genetically different and appear to be changing at different rates; presumably driven by introductions, movement of plant materials, geographical isolation, and disease management practices.

Field Resistance of Coat Protein Transgenic Papaya to Papaya ringspot virus in Jamaica.

Transgenic papayas (Carica papaya) containing translatable coat protein (CPT) or nontranslatable coat protein (CPNT) gene constructs were evaluated over two generations for field resistance to Papaya ringspot virus in a commercial papaya growing area in Jamaica. Reactions of R0 CPT transgenic lines included no symptoms and mild or severe leaf and fruit symptoms. All three reactions were observed in one line and among different lines. Trees of most CPNT lines exhibited severe symptoms of infection, and some also showed mild symptoms. R1 offspring showed reactions previously observed with parental R0 trees; however, reactions not previously observed or a lower incidence of the reaction were also obtained. The transgenic lines appear to possess virus disease resistance that can be manipulated in subsequent generations for the development of a product with acceptable commercial performance.

Update on the development of virus-resistant papaya: virus-resistant transgenic papaya for people in rural communities of Thailand.

Papaya (Carica papaya L.) is one of the most important and preferred crops in rural communities in Thailand. Papaya ringspot virus (PRSV) is a serious disease of papaya throughout Thailand. Efforts to control the virus by various methods either have not been successful or have not resulted in sustainable control. In 1995, collaborative research by the Department of Agriculture of Thailand and Cornell University to develop transgenic papaya resistant to PRSV was initiated. Two local Thai cultivars were transformed by microprojectile bombardment with the use of a nontranslatable coat protein gene of PRSV from Khon Kaen. Numerous kanamycin-resistantplants were regenerated and were inoculated with the PRSV Khon Kaen isolate for selection of resistant lines. Since 1997, promising RO transgenic lines have been transferred to the research station at Thapra for subsequent screenhouse tests and selection of the most PRSV-resistant lines. In selection set 1, three R3 lines initially derived from Khaknuan papaya showed excellent resistance to PRSV (97% to 100%) and had a yield of fruit 70 times higher than nontransgenic Khaknuan papaya. In selection set 2, one R3 line initially derived from Khakdam papaya showed 100% resistance. Safety assessments of these transgenic papayas have so far found no impact on the surrounding ecology. No natural crossing between transgenic and nonmodified papaya was observed beyond a distance of 10 m from the test plots. Analysis of the nutritional composition found no differences in nutrient levels in comparison with the nonmodified counterparts. Molecular characterization by Southern blotting revealed three copies of the transgene presented; however, no coat protein product was expressed. Data on additional topics, such as the effects offeeding the transgenic papaya to rats and the stability of the gene inserts, are currently being gathered.

Virus Coat Protein Transgenic Papaya Provides Practical Control of Papaya ringspot virus in Hawaii.

Since 1992, Papaya ringspot virus (PRSV) destroyed nearly all of the papaya hectarage in the Puna district of Hawaii, where 95% of Hawaii's papayas are grown. Two field trials to evaluate transgenic resistance (TR) were established in Puna in October 1995. One trial included the following: SunUp, a newly named homozygous transformant of Sunset; Rainbow, a hybrid of SunUp, the nontransgenic Kapoho cultivar widely grown in Puna, and 63-1, another segregating transgenic line of Sunset. The second trial was a 0.4-ha block of Rainbow, simulating a near-commercial planting. Both trials were installed within a matrix of Sunrise, a PRSV-susceptible sibling line of Sunset. The matrix served to contain and trace pollen flow from TR plants, and as a secondary inoculum source. Virus infection was first observed 3.5 months after planting. At a year, 100% of the non-TR control and 91% of the matrix plants were infected, while PRSV infection was not observed on any of the TR plants. Fruit production data of SunUp and Rainbow show that yields were at least three times higher than the industry average, while maintaining percent soluble solids above the minimum of 11% required for commercial fruit. These data suggest that transgenic SunUp and Rainbow, homozygous and hemizygous for the coat protein transgene, respectively, offer a good solution to the PRSV problem in Hawaii.

A minimum length of N gene sequence in transgenic plants is required for RNA-mediated tospovirus resistance.

We showed previously that transgenic plants with the green fluorescent protein (GFP) gene fused to segments of the nucleocapsid (N) gene of tomato spotted wilt virus (TSWV) displayed post-transcriptional gene silencing of the GFP and N gene segments and resistance to TSWV. These results suggested that a chimeric transgene composed of viral gene segments might confer multiple virus resistance in transgenic plants. To test this hypothesis and to determine the minimum length of the N gene that could trans-inactivate the challenging TSWV, transgenic plants were developed that contained GFP fused with N gene segments of 24-453 bp. Progeny from these plants were challenged with: (i) a chimeric tobacco mosaic virus containing the GFP gene, (ii) a chimeric tobacco mosaic virus with GFP plus the N gene of TSWV and (iii) TSWV. A number of transgenic plants expressing the transgene with GFP fused to N gene segments from 110 to 453 bp in size were resistant to these viruses. Resistant plants exhibited post-transcriptional gene silencing. In contrast, all transgenic lines with transgenes consisting of GFP fused to N gene segments of 24 or 59 bp were susceptible to TSWV, even though the transgene was post-transcriptionally silenced. Thus, virus resistance and post-transcriptional gene silencing were uncoupled when the N gene segment was 59 bp or less. These results provide evidence that multiple virus resistance is possible through the simple strategy of linking viral gene segments to a silencer DNA such as GFP.

Rupestris stem pitting associated virus-1 consists of a family of sequence variants.

Rupestris stem pitting (RSP) seems to be one of the most widespread virus diseases of grapevines. A virus, designated as rupestris stem pitting associated virus-1 (RSPaV-1), is consistently associated with, and likely to be the causative agent of RSP. Sequence analyses of cDNA clones derived from several RSP-affected grapevines suggested that a family of sequence variants of RSPaV-1 was associated with RSP. The genome structure of the sequence variants is identical to that of RSPaV-1 in that they had five open reading frames (ORF) and sequence identities ranging from 75 to 93% in nucleotide sequence and from 80 to 99% in amino acid sequence. ORF5 (coat protein) and the carboxyl-terminal portion of ORF1 (replicase) appeared to be the most conserved regions. The coat proteins of the sequence variants exhibited highly similar antigenic indices, suggesting serological relatedness among them. The cDNA clones obtained through reverse transcription-polymerase chain reaction from RSP-infected grapevines were heterogeneous in nt sequence with identities of 77-99% relative to RSPaV-1. Furthermore, a number of sequence variants were identified in several grapevines infected with RSP. Baselines for defining RSPaV-1 and possible mechanisms accounting for infection of grapevines with multiple sequence variants of RSPaV-1 are proposed. Findings from this study should have practical applications toward understanding the etiology of RSP and developing reliable assays to rapidly detect the disease.

Turnip mosaic potyvirus resistance in Nicotiana benthamiana derived by post-transcriptional gene silencing.

The coat protein (CP) gene of turnip mosaic potyvirus isolate ESC8 (TuMV-ESC8) was cloned and sequenced. Comparisons of the 867-nucleotide (nt) CP region with those of 11 TuMV isolates showed 86.7-89.3% nucleotide identity and 92.4-95.5% amino acid identity. The CP gene was cloned into a plant expression vector and transformed into Nicotiana benthamiana plants via Agrobacterium tumefaciens-mediated leaf disk transformation. Progeny from R0 lines was screened for resistance to TuMV-ESC8. Five of 29 tested lines showed TuMV protection in more than 50% of their progeny. Interestingly, some of the resistant plants transformed with the CP gene of TuMV displayed mild mosaicism in the new growing leaves at the later stages of evaluation; but these mosaic symptoms disappeared when the leaves were fully expanded. Collective data from steady-state RNA analysis and nuclear run-on assay of a line showed that the resistance was RNA-mediated through the post-transcriptional gene silencing mechanism.

Nucleotide sequence and genome structure of grapevine rupestris stem pitting associated virus-1 reveal similarities to apple stem pitting virus.

Rupestris stem pitting (RSP), a component of the rugose wood complex, is one of the most widespread graft-transmissible diseases of grapevines. Here we report on the consistent association of a high molecular mass dsRNA (ca. 8.7 kbp) with RSP. The dsRNA was reverse-transcribed and cDNAs generated were cloned into Lambda ZAP II. Sequence analysis of the cDNA clones showed that the dsRNA was of viral origin and the putative virus was designated rupestris stem pitting associated virus-1 (RSPaV-1). The genome of RSPaV-1 consists of 8726 nt excluding a poly(A) tail at the 3' terminus. It has five potential open reading frames which have the capacity to code for the replicase (ORF1), the triple gene block (ORF2-4) and the coat protein (ORF5). Comparison of the genome structure and nucleotide and amino acid sequences indicated similarities of RSPaV-1 to apple stem pitting virus, and to a lesser extent, to potato virus M carlavirus. The possibility that different strains of RSPaV-1 or other viruses are associated with RSP is discussed.

Nucleotide sequence and genome organization of grapevine leafroll-associated virus-2 are similar to beet yellows virus, the closterovirus type member.

The entire genome of grapevine leafroll-associated closterovirus-2 (GLRaV-2), except the exact 5' terminus, was cloned and sequenced. The sequence encompasses nine open reading frames (ORFs) which include, in the 5' to 3' direction, an incomplete ORF1a encoding a putative viral polyprotein and eight ORFs that encode proteins of 52 kDa (ORF1b), 6 kDa (ORF2), 65 kDa (ORF3), 63 kDa (ORF4), 25 kDa (ORF5), 22 kDa (ORF6), 19 kDa (ORF7) and 24 kDa (ORF8) respectively, and 216 nucleotides of the 3' untranslated region. An incomplete ORF1a potentially encoded a large polyprotein containing the conserved domains characteristic of a papain-like protease, methyltransferase and helicase. ORF1b potentially encoded a putative RNA-dependent RNA polymerase. The expression of ORF1b may be via a +1 ribosomal frameshift mechanism, similar to other closteroviruses. A unique gene array, which is conserved in other closteroviruses, was also identified in GLRaV-2; it includes genes encoding a 6 kDa small hydrophobic protein, 65 kDa heat shock protein 70, 63 kDa protein of function unknown, 25 kDa coat protein duplicate and 22 kDa coat protein. Identification of ORF6 (22 kDa) as the coat protein gene was further confirmed by in vivo expression in E. coli and immunoblotting. Phylogenetic analysis comparing different genes of GLRaV-2 with those of other closteroviruses demonstrated a close relationship with beet yellows virus (BYV), beet yellow stunt virus and citrus tristeza virus. GLRaV-2 is the only closterovirus, so far, that matches the genome organization of the type member of the group, BYV, and thus can be unambiguously classified as a definitive member of the genus Closterovirus.

Nucleotide sequence of the 3'-terminal two-thirds of the grapevine leafroll-associated virus-3 genome reveals a typical monopartite closterovirus.

The RNA genome of grapevine leafroll-associated closterovirus-3 (GLRaV-3) was cloned as a cDNA generated from GLRaV-3-specific dsRNA, and a partial genome sequence of 13154 nucleotides (nt) including the 3' terminus was determined. The sequenced portion contained 13 open reading frames (ORFs) potentially encoding, in the 5'-3' direction, proteins of > 77 kDa (ORF1a; helicase, HEL), 61 kDa (ORF1b; RNA-dependent RNA polymerase, RdRp), 6 kDa (ORF2), 5 kDa (ORF3, small transmembrane protein), 59 kDa (ORF4; heat shock protein 70, HSP70), 55 kDa (ORF5), 35 kDa (ORF6; coat protein, CP), 53 kDa (ORF7; diverged coat protein, CPd), 21 kDa (ORF8), 20 kDa (ORF9), 20 kDa (ORF10), 4 kDa (ORF11), 7 kDa (ORF12), and an untranslated region of 277 nt. ORF1b is probably expressed via a +1 ribosomal frameshift mechanism, most similar to that of lettuce infectious yellows virus (LIYV). Phylogenetic analysis using various gene sequences (HEL, RdRp, HSP70 and CP) clearly demonstrated that GLRaV-3, a mealybug-transmissible closterovirus, is positioned independently from aphid-transmissible monopartite closteroviruses (beet yellows, citrus tristeza and beet yellows stunt) and whitefly-transmissible bipartite closterovirus (lettuce infectious yellows, LIYV). However, another alleged mealybug-transmissible closterovirus, little cherry virus, was shown to be more closely related to the whitefly-transmissible LIYV than to GLRaV-3.

Transgenic peanut plants containing a nucleocapsid protein gene of tomato spotted wilt virus show divergent levels of gene expression.

The nucleocapsid protein (N) gene of the lettuce isolate of tomato spotted wilt virus (TSWV) was inserted into peanut (Arachis hypogaea L.) via microprojectile bombardment. Constructs containing the hph gene for resistance to the antibiotic hygromycin and the TSWV N gene were used for bombardment of peanut somatic embryos. High frequencies of transformation and regeneration of plants containing the N gene were obtained. Southern blot analysis of independent transgenic lines revealed that one to several copies of the N gene were integrated into the peanut genome. Northern blot, RT-PCR and ELISA analyses indicated that a gene silencing mechanism may be operating in primary transgenic lines containing multiple copy insertions of the N transgene. One transgenic plant which contained a single copy of the transgene expressed the N protein in the primary transformant, and the progeny segregated in a 3 :1 ratio based upon ELISA determination.

Leafroll Virus Is Common in Cultivated American Grapevines in Western New York.

American grapevines (Vitis labrusca L. 'Niagara'; Vitis × labruscana L. H. Bailey 'Concord' and 'Catawba'; V. labrusca × V. riparia Michx. 'Elvira') from 24 vineyards in the New York portion of the Lake Erie production region (>13,000 ha cultivated) were tested to explore a possible relationship between virus infection and an unexplained fruit set malady in the district. One-year-old cane segments were collected 4 to 6 weeks before budbreak from 65 individual vines, which previously had been identified as malady positive or negative. Preparations from bark scrapings were tested for the presence of double-stranded (ds) RNA and for fan leaf degeneration virus, tobacco streak virus, and grapevine leafroll associated closterovirus-3 (GLRaV-3) by enzyme-linked immunosorbent assay (ELISA). Mechanical transmission of other potential viruses to Chenopodium quinoa was attempted with sap extracted from young shoots forced from intact segments of sampled canes. GLRaV-3 was detected in 17 (26%) of the sampled vines from eight (33%) of the vineyards, but there was no apparent relationship between infected vines and the fruit set malady. Vines of all four cultivars were infected. dsRNA was detected in all 17 samples positive for GLRaV-3 plus four additional samples. No other viruses were detected. Near harvest, nine vines (from two vineyards) previously testing positive for GLRaV-3 were examined and retested; all nine tested positive again, although none showed any overt symptoms of viral infection. This is believed to be the first report of GLRaV-3 from American grape vineyards in New York. The source of these infections is unknown: all vines were self rooted, the individual vineyards had been planted independently at different times, and V. vinifera and its hybrids are rare in the district. Wild grapevines (primarily V. riparia) are abundant in the region, although it has been reported that leafroll disease does not occur naturally in wild North American grapes (1). Nevertheless, our results indicate that cultivated American grapevines can be common reservoirs of GLRaV-3, and furthermore suggest the need to reassess the possibility that wild grapes also may serve as reservoirs of the virus. Trials are currently underway to determine possible effects of GLRaV-3 on cv. Concord, the most widely planted variety in the region. Reference: (1) A. C. Goheen. 1988. Leafroll. Page 52 in: Compendium of Grape Diseases. R. C. Pearson and A. C. Goheen, eds. American Phytopathological Society, St. Paul, MN.

Control of papaya ringspot virus in papaya: a case study.

The papaya crop is severely affected by papaya ringspot virus (PSRV) worldwide. This review focuses on efforts to control the destructiveness of the disease caused by PSRV in Hawaii, starting from the use of cross protection to parasite-derived resistance with transgenic papaya expressing the PSRV coat protein gene. A chronology of the research effort is given and related to the development of technologies and the pressing need to control PSRV in Hawaii. The development of commercial virus-resistant transgenic papaya provides a tangible approach to control PSRV in Hawaii. Moreover, the development of transgenic papaya by other laboratories and employment of a mechanism of effective technology transfer to different countries hold promise for control of PSRV worldwide.

Nontarget DNA sequences reduce the transgene length necessary for RNA-mediated tospovirus resistance in transgenic plants.

RNA-mediated virus resistance has recently been shown to be the result of post-transcriptional transgene silencing in transgenic plants. This study was undertaken to characterize the effect of transgene length and nontarget DNA sequences on RNA-mediated tospovirus resistance in transgenic plants. Transgenic Nicotiana benthamiana plants were generated to express different regions of the nucleocapsid (N) protein of tomato spotted wilt (TSWV) tospovirus. Transgenic plants expressing half-gene segments (387-453 bp) of the N gene displayed resistance through post-transcriptional gene silencing. Although smaller N gene segments (92-235 bp) were ineffective in conferring resistance when expressed alone in transgenic plants, these segments conferred resistance when fused to the nontarget green fluorescent protein gene DNA. These results demonstrate that (i) a critical length of N transgene (236-387 bp) is required for a high level of transgene expression and consequent gene silencing, and (ii) the post-transcriptional gene silencing mechanism can trans-inactivate the incoming tospovirus genome with homologous transgene segments that are as short as 110 bp. Therefore, the activation of post-transcriptional transgene silencing requires a significantly larger transgene than is required for the trans-inactivation of the incoming viral genome. These results raise the possibility of developing a simple new strategy for engineering multiple virus resistance in transgenic plants.

The coat protein gene of grapevine leafroll associated closterovirus-3: cloning, nucleotide sequencing and expression in transgenic plants.

A lambda ZAP II cDNA library was constructed by cloning cDNA prepared from a high molecular weight double-stranded RNA (dsRNA, ca. 18 kb) isolated from grapevine leafroll associated closterovirus-3 (GLRaV-3) infected tissues. This cDNA library was immuno-screened with GLRaV-3 coat protein specific polyclonal and monoclonal antibodies and three immuno-positive clones were identified. Analysis of nucleotide sequences from these clones revealed an open reading frame (ORF) which was truncated at the 3' end; the remainder of this ORF was obtained by sequencing a fourth clone that overlapped with one of the immunopositive clones. A total of 2028 bp was sequenced. The putative GLRaV-3 coat protein ORF, 939 bp, encodes a protein (referred to as p35) with a calculated M(r) of 34866. Multiple alignment of the p35 amino acid sequence with coat protein sequences from other closteroviruses revealed that the consensus amino acid residues (R and D) of filamentous plant viruses are preserved in the expected locations. The GLRaV-3 coat protein gene was then engineered for sense and antisense expression in transgenic plants. Transgenic Nicotiana benthamiana plants that contain the sense GLRaV-3 coat protein gene produced a 35 kDa protein that reacted with GLRaV-3 antibody in Western blot.

Biolistic transformation of chrysanthemum with the nucleocapsid gene of tomato spotted wilt virus.

In vitro regeneration and biolistic transformation procedures were developed for several commercial chrysanthemum Dendranthema grandiflora Tzvelev, syn. Chrysanthemum morifolium Ramat. cultivars using leaf and stem explants. Studies on the effect of several growth regulators and kanamycin on chrysanthemum regeneration were conducted, and a step-wise procedure to optimize kanamycin selection and recovery of transgenic plants was developed. A population of putative transformed chrysanthemum plants cvs. Blush, Dark Bronze Charm, Iridon, and Tara, was obtained after bombardment with tungsten microprojectiles coated with the binary plasmid pBIN19 containing the nucleocapsid (N) gene of tomato spotted wilt virus (TSWV) and the marker gene neomycin phosphotransferase (NPT II). PCR analysis of 82 putative transgenic plants selected on kanamycin indicated that the majority of the lines (89%) were transformed and contained both genes (71%). However, some transgenic lines contained only one of the genes: either the NPT II (15%) or the TSWV (N) gene (14%). Southern blot analysis on selected transgenic lines confirmed the integration of the TSWV (N) gene into the chrysanthemum genome. These results demonstrate the development of an efficient procedure to transfer genetic material into the chrysanthemum genome and selectively regenerate transgenic chrysanthemum plants at frequencies higher than previously reported.

Transgenic plums (Prunus domestica L.) express the plum pox virus coat protein gene.

Plum hypocotyl slices were transformed with the coat protein (CP) gene of plum pox virus (PPV-CP) following cocultivation with Agrobacterium tumefaciens containing the plasmid pGA482GG/PPVCP-33. This binary vector carries the PPV-CP gene construct, as well as the chimeric neomycin phosphotransferase and ß-glucuronidase genes. Integration and expression of the transferred genes into regenerated plum plants was verified through kan resistance, GUS assays, and PCR amplification of the PPV-CP gene. Twenty-two transgenic clones were identified from approximately 1800 hypocotyl slices. DNA, mRNA, and protein analyses of five transgenic plants confirmed the integration of the engineered CP gene, the accumulation of CP mRNA and of PPV-CP-immunoreactive protein. CP mRNA levels ranged from high to undetectable levels, apparently correlated with gene structure, as indicated by DNA blot analysis. Western analysis showed that transgenic plants produced amounts of CP which generally correlated with amounts of detected mRNA.