The complete nucleotide sequence of the RNA genome of papaya ringspot virus (PRSV) was determined from four overlapping cDNA clones and by direct sequencing of viral RNA. The genomic RNA is 10326 nucleotides in length, excluding the poly(A) tract, and contains one large open reading frame that starts at nucleotide positions 86 to 88 and ends at positions 10118 to 10120, encoding a polyprotein of 3344 amino acids. The highly conserved sequence AAAUAAAANANCUCAACACAACAUA at the 5' end of the RNA of PRSV and those of the other five reported potyviruses shows 80% similarity, suggesting that this region may play a common important role for potyvirus replication. Two cleavage sites of the polyprotein were determined by amino acid sequencing of the N termini of helper component (HC-Pro, amorphous inclusion) and cylindrical inclusion (CI) proteins. Other cleavage sites were predicted by analogy with the other potyviruses. The genetic organization of PRSV is similar to that of the other potyviruses except that the first protein processed from the N terminus of the polyprotein (NT protein) has an Mr of 63K, 18K to 34K larger than those of the other potyviruses. The cleavage site for liberating the N terminus of the HCPro protein was found at the same location downstream from the consensus sequence FI(V)VRG as that reported for tobacco vein mottling virus. The NT protein of potyviruses is the most variable and may be considered important for identification of individual potyviruses. The most conserved protein ofpotyviruses appears to be the Nlb protein, the putative polymerase for the replication of the potyviral RNA. The genetic organization of PRSV RNA is tentatively proposed to be VPg-5' leader-63K NT-52K HC-Pro-46K-72K CI-6K-48K NIa-59K NIb-35K coat protein-3' noncoding region-poly(A) tract.

Most strains of Papaya ringspot virus (PRSV) belong to type W, causing severe loss on cucurbits worldwide, or type P, devastating papaya in tropical areas. While the host range of PRSV W is limited to plants of the families Chenopodiaceae and Cucuribitaceae, PRSV P, in addition, infects plants of the family Caricaceae (papaya family). To investigate one or more viral genetic determinants for papaya infection, recombinant viruses were constructed between PRSV P-YK and PRSV W-CI. Host reactions to recombinant viruses indicated that the viral genomic region covering the C-terminal region (142 residues) of NIaVPg, full NIaPro, and N-terminal region (18 residues) of NIb, is critical for papaya infection. Sequence analysis of this region revealed residue variations at position 176 of NIaVPg and positions 27 and 205 of NIaPro between type P and W viruses. Host reactions to the constructed mutants indicated that the amino acid Lys27 of NIaPro determines the host-specificity of PRSV for papaya infection. Predicted three-dimensional structures of NIaPros of parental viruses suggested that Lys27 does not affect the protease activity of NIaPro. Recovery of the infected plants from certain papaya-infecting mutants implied involvement of other viral factors for enhancing virulence and adaptation of PRSV on papaya.

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Zucchini yellow mosaic virus (ZYMV) and Papaya ringspot virus type W (PRSV W) are major limiting factors for production of watermelon worldwide. For the effective control of these two viruses by transgenic resistance, an untranslatable chimeric construct containing truncated ZYMV coat protein (CP) and PRSV W CP genes was transferred to commercial watermelon cultivars by Agrobacterium-mediated transformation. Using our protocol, a total of 27 putative transgenic lines were obtained from three cultivars of 'Feeling' (23 lines), 'China baby' (3 lines), and 'Quality' (1 line). PCR and Southern blot analyses confirmed that the chimeric construct was incorporated into the genomic DNA of the transformants. Greenhouse evaluation of the selected ten transgenic lines of 'Feeling' cultivar revealed that two immune lines conferred complete resistance to ZYMV and PRSV W, from which virus accumulation were not detected by Western blotting 4 weeks after inoculation. The transgenic transcript was not detected, but small interfering RNA (siRNA) was readily detected from the two immune lines and T(1) progeny of line ZW 10 before inoculation, indicating that RNA-mediated post-transcriptional gene silencing (PTGS) is the underlying mechanism for the double-virus resistance. The segregation ratio of T(1) progeny of the immune line ZW10 indicated that the single inserted transgene is nuclearly inherited and associated with the phenotype of double-virus resistance as a dominant trait. The transgenic lines derived from the commercial watermelon cultivars have great potential for control of the two important viruses and can be implemented directly without further breeding.

Papaya ringspot virus (PRSV) HA 5-1, a nitrous acid-induced mild mutant of severe strain HA, widely applied for control of PRSV by cross-protection, was used to study the genetic basis of attenuation. Using infectious clones, a series of recombinants was generated between HA 5-1 and HA and their infectivity was analyzed on the systemic host papaya and the local lesion host Chenopodium quinoa. The recombinants that contained mutations in PI and HC-Pro genes caused attenuated infection on papaya without conspicuous symptoms, similar to HA 5-1. The recombination and sequence analyses strongly implicated two amino acid changes in the C-terminal region of PI and two in HC-Pro of HA 5-1 involved in the attenuated infection on papaya. The recombinants that infected C. quinoa plants without local lesions contained the same mutations in the C-terminal region of HC-Pro for attenuated infection on papaya. We conclude that both PI and HC-Pro bear important pathogenicity determinants for the infection on the systemic host papaya and that the mutations in HC-Pro affecting pathogenicity on papaya are also responsible for the inability to induce hypersensitive reaction on C. quinoa

Papaya ringspot virus (PRSV) HA5-1, a mild mutant of type P Hawaii severe strain (PRSV P-HA), has been widely used for the control of PRSV type P strains in papaya, but did not provide practical protection against PRSV type W strains in cucurbits. In order to widen the protection effectiveness against W strains, chimeric mild strains were constructed from HA5-1 to carry the heterologous 3' genomic region of a type W strain W-CI. Virus accumulation of recombinants and their crossprotection effectiveness against W-CI and P-HA were investigated. In horn melon and squash plants, the recombinant carrying both the heterologous coat protein (CP) coding region and the 3' untranslated region (3'UTR), but not the heterologous CP coding region alone, significantly enhanced the protection against W-CI. The heterologous 3'UTR alone is critical for the enhancement of the protection against W-CI in horn melon, but not in zucchini squash. In papaya, the heterologous CP coding region or 3'UTR alone, but not both together, significantly reduced the effectiveness of cross protection against P-HA. Our recombinants provide broader protection against both type W and P strains in cucurbits; however, the protective effectiveness is also affected by virus accumulation, the organization of the 3' genomic region, and host factors.

Viral diseases are very detrimental to watermelon production. Watermelon silver mottle virus (WSMoV) is a major limiting factor for the production of watermelon and other cucurbit fruits. There are no effective natural sources of resistance to WSMoV, making transgenic resistance an appropriate solution for attenuating virus infection. Hyperhydricity is an important problem in watermelon culture in vitro, resulting from lower multiplication rates, poor quality shoots and tissue necrosis. In this study, we report an Agrobacterium-mediated genetic transfer protocol for commercial watermelon cultivars expressing the nucleocapsid (N) gene of WSMoV and a suitable approach to overcome hyperhydricity in watermelon culture in vitro. Murashige and Skoog (MS) salts containing Schenk and Hildebrandt (SH) vitamins ? 50 mg l -1 thiamine HCl could diminish the hyperhydric phenotype. The proximal halves of cotyledons from 3-dayold seedlings were cut into 1.5 9 1.5 mm segments as explants. Four days after co-cultivation, the explants were transferred to a selection medium for shoot regeneration. The putative transgenic shoots developed within 6 weeks of culture and were then transferred to stringent medium for 8 weeks to eliminate 'escape type' shoots. Fifty putative transgenic watermelon lines were obtained from three cultivars. PCR and Southern blot analysis confirmed that the foreign gene was incorporated into the genomic DNA of the transgenic lines.

Bell pepper (Capsicum annuum L.) plants exhibiting systemic mild mosaic, vein yellowing, and leaf malformation were collected from Puli City in 2006. Double-antibody sandwich (DAS)-ELISA was used to test these samples for Chilli veinal mottle virus (ChiVMV) infection using polyclonal antibodies. In addition, Chenopodium quinoa, C. amaranticolor, and Nicotiana benthamiana plants were mechanically inoculated with sap extracted from collected samples. Ten days postinoculation, chlorotic local lesions were observed on inoculated leaves of C. quinoa and C. amaranticolor plants, whereas, systemic mosaic and foliar distortion symptoms were developed on upper leaves of N. benthamiana plants. The DAS-ELISA test showed that field-collected pepper samples and inoculated leaves of C. quinoa and C. amaranticolor were infected with ChiVMV, while N. benthamiana with mosaic symptoms did not react with ChiVMV antibodies. To confirm ChiVMV, field-collected samples as well as mechanically inoculated plants were tested by reverse transcription (RT)-PCR using the potyvirus degenerate primers Hrp5/Pot1 (2). Amplified RT-PCR products were cloned and sequenced. Sequence analysis of amplified fragments (1.4 kb) revealed that field-collected pepper samples were infected with ChiVMV and Pepper mottle virus (PepMoV). The DNA fragment amplified from C. quinoa and C. amaranticolor showed high (99.2%) sequence identities with the CP gene of ChiVMV (3) (GenBank Accession No. AM909717). However, amplicons obtained from N. benthamiana plants (GenBank Accession No. HQ329082) that showed mosaic symptoms showed 83.6% to 98.7% nucleotide identities with PepMoV (GenBank Accession Nos. AB126033, AF227728, AF440801, AF501591, EU586133, and M96425). Next, a pure isolate of PepMoV was established on N. benthamiana by mechanical inoculation of diluted plant sap obtained from a PepMoV-infected N. benthamiana plant. Bell pepper plants inoculated with the Taiwan isolate of PepMoV developed mosaic and leaf distortion symptoms. Antiserum against the PepMoV Taiwan isolate was subsequently prepared by immunizing rabbits with purified virus particles. Using the prepared antiserum and specific primers (1) to detect PepMoV, ChiVMV, and Pepper veinal mottle virus (PVMV), three viruses could be readily detected and differentiated from diseased bell peppers in the field. In a survey done in 2007, 18 of 33 pepper samples from southern Taiwan were found with mixed infections of PepMoV and ChiVMV, seven samples were infected with PepMoV and PVMV, five samples were infected with PVMV, and another three samples were infected with ChiVMV. To our knowledge, this is the first report of the occurrence of PepMoV in bell peppers in Taiwan. References: (1) Y. H. Cheng et al. Plant Dis. 93:107, 2009. (2) S. S. Pappu et al. Plant Dis. 82:1121, 1998. (3) W. S. Tsai et al. Plant Pathol. 58:408, 2008.