Joseph A. J. Raja

A Single Amino Acid of NIaPro of Papaya ringspot virus Determines Host Specificity for Infection of Papaya

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.

Generation of transgenic oriental melon resistant to Zucchini yellow mosaic virus by an improved cotyledon-cutting method

Production of melon (Cucumis melo L.) worldwide is often limited by the potyvirus, Zucchini yellow mosaic virus (ZYMV). In order to engineer melon lines resistant to ZYMV, a construct containing the translatable coat protein (CP) sequence coupled with the 3′ non-translatable region of the virus was generated and used to transform an elite cultivar of oriental melon (Silver light) mediated by Agrobacterium using an improved cotyledon-cutting method. Removal of 1-mm portion from the proximal end of cotyledons greatly increased the frequency of transgenic regenerants by significantly decreasing the incidence of false positive and aberrant transformants. Results of greenhouse evaluation of transgenic lines by mechanical challenge with ZYMV identified transgenic lines exhibiting different levels of resistance or complete immunity to ZYMV. Southern hybridization of transgenic lines revealed random insertion of the transgene in host genome, with insert numbers differing among transformants. Northern hybridization revealed great variations in the levels of accumulation of the transgene transcripts among transgenic lines, and evidenced an inverse correlation of the levels of accumulation of transgene transcript to the degrees of virus resistance, indicating post-transcriptional gene silencing (PTGS)-mediated transgenic resistance. These transgenic melon lines with high degrees of resistance to ZYMV have great potential for the control of ZYMV in East Asia.

Complete genomic sequence of watermelon bud necrosis virus

Watermelon bud necrosis virus (WBNV) is one of the major limiting factors in production of cucurbits in India, causing losses up to 100% in watermelon in some growing areas [8, 15]. Based on the previously determined nucleocapsid (N) protein coding sequence of WBNV S RNA, this virus is considered to belong to a tentative new tospovirus species [9]. Although the complete S RNA and M RNA sequences of WBNV were determined recently [11], these sequences were obtained from different virus isolates, and knowledge of the detailed characteristics of the complete genome of a WBNV isolate is therefore still lacking. In this investigation, the full-length genomic sequence, including the L, M and S RNA segments, of a watermelon isolate of WBNV originating from southern India was completely determined and analyzed. Phylogenetic analysis of the sequences of all of the viral proteins, N, NSs, NSm, glycoprotein (GP) and RNA-dependent RNA polymerase (RdRp) indicates that WBNV is closely related to watermelon silver mottle virus (WSMoV) [16], peanut bud necrosis virus (PBNV) [14] and capsicum chlorosis virus (CaCV) [10].

Genetic analysis of an attenuated Papaya ringspot virus strain applied for cross-protection

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 P1 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 P1 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 P1 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.

Broad-Spectrum Transgenic Resistance against Distinct Tospovirus Species at the Genus Level

Thrips-borne tospoviruses cause severe damage to crops worldwide. In this investigation, tobacco lines transgenic for individual WLm constructs containing the conserved motifs of the L RNA-encoded RNA-dependent RNA polymerase (L) gene of Watermelon silver mottle virus (WSMoV) were generated by Agrobacterium-mediated transformation. The WLm constructs included: (i) translatable WLm in a sense orientation; (ii) untranslatable WLmt with two stop codons; (iii) untranslatable WLmts with stop codons and a frame-shift; (iv) untranslatable antisense WLmA; and (v) WLmhp with an untranslatable inverted repeat of WLm containing the tospoviral S RNA 3′-terminal consensus sequence (5′-ATTGCTCT-3′) and an NcoI site as a linker to generate a double-stranded hairpin transcript. A total of 46.7–70.0% transgenic tobacco lines derived from individual constructs showed resistance to the homologous WSMoV; 35.7–100% plants of these different WSMoV-resistant lines exhibited broad-spectrum resistance against four other serologically unrelated tospoviruses Tomato spotted wilt virus, Groundnut yellow spot virus, Impatiens necrotic spot virus and Groundnut chlorotic fan-spot virus. The selected transgenic tobacco lines also exhibited broad-spectrum resistance against five additional tospoviruses from WSMoV and Iris yellow spot virus clades, but not against RNA viruses from other genera. Northern analyses indicated that the broad-spectrum resistance is mediated by RNA silencing. To validate the L conserved region resistance in vegetable crops, the constructs were also used to generate transgenic tomato lines, which also showed effective resistance against WSMoV and other tospoviruses. Thus, our approach of using the conserved motifs of tospoviral L gene as a transgene generates broad-spectrum resistance against tospoviruses at the genus level.

Double-virus resistance of transgenic oriental melon conferred by untranslatable chimeric construct carrying partial coat protein genes of two viruses.

Production of oriental melon (Cucumis melo var. makuwa) in Asia is often limited by two potyviruses, the watermelon infecting type of Papaya ringspot virus (PRSV W) and Zucchini yellow mosaic virus (ZYMV). In order to engineer transgenic resistance to these two viruses, an untranslatable chimeric DNA comprising partial coat protein (CP) sequences of ZYMV and PRSV W was constructed and used to transform the elite cultivar of oriental melon, Silver Light, by Agrobacterium. Greenhouse evaluation by mechanical challenges with ZYMV and PRSV W, alone or together, identified transgenic lines exhibiting different levels of resistance or complete immunity to ZYMV and PRSV W. Molecular analyses of transgenic lines revealed random insertion of transgene into the host genome, with insert numbers differing among transformants. There was no correlation between transgene insert numbers and the degree of resistance expressed by transgenic lines. The levels of accumulation of transgene transcript varied among transgenic lines. However, an inverse correlation was observed between the level of accumulation of transgene transcripts and the degree of virus resistance. Moreover, small interfering (si)RNA was readily detected from the immune and highly resistant lines, but not from the weakly resistant and susceptible lines. Altogether, our results indicated that RNA-mediated post-transcriptional gene silencing (PTGS) was the underlying mechanism of double-virus resistance of the transgenic melon lines. The segregation analysis of the R1 progeny of the immune line ZW-1 indicated that the single inserted transgene is associated with the resistance phenotype and is inherited as a dominant trait. These transgenic melon lines with high degrees of resistance to ZYMV and PRSV W have great potential for the control of ZYMV and PRSV W in C. melo in Asia and elsewhere.

Generation of Marker-free Transgenic Plants Concurrently Resistant to a DNA Geminivirus and a RNA Tospovirus

Global threats of ssDNA geminivirus and ss(-)RNA tospovirus on crops necessitate the development of transgenic resistance. Here, we constructed a two-T DNA vector carrying a hairpin of the intergenic region (IGR) of Ageratum yellow vein virus (AYVV), residing in an intron inserted in an untranslatable nucleocapsid protein (NP) fragment of Melon yellow spot virus (MYSV). Transgenic tobacco lines highly resistant to AYVV and MYSV were generated. Accumulation of 24-nt siRNA, higher methylation levels on the IGR promoters of the transgene, and suppression of IGR promoter activity of invading AYVV indicate that AYVV resistance is mediated by transcriptional gene silencing. Lack of NP transcript and accumulation of corresponding siRNAs indicate that MYSV resistance is mediated through post-transcriptional gene silencing. Marker-free progenies with concurrent resistance to both AYVV and MYSV, stably inherited as dominant nuclear traits, were obtained. Hence, we provide a novel way for concurrent control of noxious DNA and RNA viruses with less biosafety concerns.

Nucleotide Sequence-Homology-Independent Breakdown of Transgenic Resistance by More Virulent Virus Strains and a Potential Solution

Controlling plant viruses by genetic engineering, including the globally important Papaya ringspot virus (PRSV), mainly involves coat protein (CP) gene mediated resistance via post-transcriptional gene silencing (PTGS). However, the breakdown of single- or double-virus resistance in CP-gene-transgenic papaya by more virulent PRSV strains has been noted in repeated field trials. Recombination analysis revealed that the gene silencing suppressor HC-Pro or CP of the virulent PRSV strain 5-19 is responsible for overcoming CP-transgenic resistance in a sequence-homology-independent manner. Transient expression assays using agro-infiltration in Nicotiana benthamiana plants indicated that 5-19 HC-Pro exhibits stronger PTGS suppression than the transgene donor strain. To disarm the suppressor from the virulent strain, transgenic papaya lines were generated carrying untranslatable 5-19 HC-Pro, which conferred complete resistance to 5-19 and other geographic PRSV strains. Our study suggested the potential risk of the emergence of more virulent virus strains, spurred by the deployment of CP-gene-transgenic crops, and provides a strategy to combat such strains.

Two Novel Motifs of Watermelon Silver Mottle Virus NSs Protein Are Responsible for RNA Silencing Suppression and Pathogenicity

The NSs protein of Watermelon silver mottle virus (WSMoV) is the RNA silencing suppressor and pathogenicity determinant. In this study, serial deletion and point-mutation mutagenesis of conserved regions (CR) of NSs protein were performed, and the silencing suppression function was analyzed through agroinfiltration in Nicotiana benthamiana plants. We found two amino acid (aa) residues, H113 and Y398, are novel functional residues for RNA silencing suppression. Our further analyses demonstrated that H113 at the common epitope (CE) (109KFTMHNQ117), which is highly conserved in Asia type tospoviruses, and the benzene ring of Y398 at the C-terminal β-sheet motif (397IYFL400) affect NSs mRNA stability and protein stability, respectively, and are thus critical for NSs RNA silencing suppression. Additionally, protein expression of other six deleted (ΔCR1-ΔCR6) and five point-mutated (Y15A, Y27A, G180A, R181A and R212A) mutants were hampered and their silencing suppression ability was abolished. The accumulation of the mutant mRNAs and proteins, except Y398A, could be rescued or enhanced by co-infiltration with potyviral suppressor HC-Pro. When assayed with the attenuated Zucchini yellow mosaic virus vector in squash plants, the recombinants carrying individual seven point-mutated NSs proteins displayed symptoms much milder than the recombinant carrying the wild type NSs protein, suggesting that these aa residues also affect viral pathogenicity by suppressing the host silencing mechanism.

Agbiotechnology: Costs and Benefits of Genetically Modified Papaya

Papaya is the fruit of angels; however, its production is limited by the destructive aphid-borne Papaya ringspot virus (PRSV) worldwide. The transgenic resistance conferred by the PRSV coat protein (CP) gene in genetically modified (GM) papaya has become the most effective way to prevent papaya from PRSV infection. In this article, the success in Hawaii, the difficulty in Taiwan, and the hurdle in Thailand for application of GM papaya are addressed in terms of costs and benefits. Moreover, advances in papaya biotechnology, including tissue culture, micropropagation, somatic-embryo transformation, and marker-assistance breeding for fixing hermaphroditic sex and pyramiding transgenes are reviewed.