The nuclear shuttle protein of Tomato leaf curl New Delhi virus is a pathogenicity determinant

The role of the movement protein (MP) and nuclear shuttle protein (NSP) in the pathogenicity of Tomato leaf curl New Delhi virus (ToLCNDV), a bipartite begomovirus, was studied. Both genes were expressed in Nicotiana benthamiana, Nicotiana tabacum, and Lycopersicon esculentum plants with the Potato virus X (PVX) expression vector or by stable transformation of gene constructs under the control of the 35S promoter in N. tabacum. No phenotypic changes were observed in any of the three species when the MP was expressed from the PVX vector or constitutively expressed in transgenic plants. Expression of the ToLCNDV NSP from the PVX vector in N. benthamiana resulted in leaf curling that is typical of the disease symptoms caused by ToLCNDV in this species. Expression of NSP from PVX in N. tabacum and L. esculentum resulted in a hypersensitive response (HR), demonstrating that the ToLCVDV NSP is a target of host defense responses in these hosts. The NSP, when expressed as a transgene under the control of the 35S promoter, resulted in necrotic lesions in expanded leaves that initiated from a point and then spread across the leaf. The necrotic response was systemic in all the transgenic plants. Deletion of 100 amino acids from the C terminus did not compromise the HR response, suggesting that this region has no role in HR. Deletion of 60 or 100 amino acids from the N terminus of NSP abolished the HR response, suggesting that these sequences are required for the HR response. These findings demonstrate that the ToLCNDV NSP is a pathogenicity determinant as well as a target of host defense responses.     

Gene C2 of the monopartite geminivirus tomato yellow leaf curl virus-China encodes a pathogenicity determinant that is localized in the nucleus

Expression of the Tomato yellow leaf curl virus-China (TYLCV-C) C2 protein and green fluorescent protein (GFP) fused to the C2 protein (C2-GFP) in Nicotiana benthamiana from a Potato virus X (PVX) vector induced necrotic ringspots on inoculated leaves as well as necrotic vein banding and severe necrosis on systemically infected leaves. The localization of GFP fluorescence in plant cells infected with PVX/C2-GFP and in insect cells transfected with Baculovirus expressing C2-GFP indicates that the TYLCV-C C2 protein is capable of shuttling GFP into plant and insect cell nuclei. Our data demonstrate that the TYLCV-C C2 protein may contribute to viral pathogenicity in planta and is nuclear localized.     

A novel shaggy-like kinase interacts with the Tomato leaf curl virus pathogenicity determinant C4 protein

Tomato leaf curl virus-Australia (ToLCV) C4 protein has been shown to be associated with virus pathogenesis. Here, we demonstrate that C4 acts as a suppressor of gene silencing. To understand the multifunctional role of C4, a novel shaggy-like kinase (SlSK) from tomato, which interacts with ToLCV C4 in a yeast two-hybrid assay, was isolated and interaction between these proteins was confirmed in vitro and in planta. Using deletion analysis of C4, a 12 amino acid region in the C-terminal part of C4 was identified which was shown to be essential for its binding to SlSK. We further demonstrate that this region is not only important for the interaction of C4 with SlSK, but is also required for C4 function to suppress gene silencing activity and to induce virus symptoms in a PVX system. The potential significance of ToLCV C4 and SlSK interaction is discussed.     

Regulation of tomato leaf curl viral gene expression in host tissues

The regulation of expression of the two virion-sense (V1 and V2) and four complementary-sense (C1, C2, C3, and C4) open reading frames (ORFs) of Tomato leaf curl virus (TLCV) was studied in both stably and transiently transformed Nicotiana tabacum tissues with fusions with the beta-glucuronidase (GUS) reporter gene. GUS-expressing transgenic lines were obtained with each of the four complementary-sense gene-GUS fusion constructs and with truncated versions of the virion-sense gene-GUS fusion constructs (V1GUSdeltaC and V2GUSdeltaC) lacking complementary-sense sequences encoding the C1, C2, and C3 ORFs. However, little or no GUS expression was observed in kanamycin-resistant plants transformed with full-length, virion-sense gene constructs (V1GUS and V2GUS) constituting the complete viral genome. In contrast, V1GUS and V2GUS were found to direct high-level GUS expression in transient assays with tobacco protoplasts, suggesting that integration of viral constructs containing functional, complementary-sense genes may lead to repression or deletion of the introduced constructs in transgenic tissues. V2GUS expression in the transient protoplast assay was found to be severely curtailed by specific mutation of the C2 ORF, supporting a role for the C2 protein in transactivation of TLCV virion-sense gene expression. TLCV ORF-GUS constructs displayed distinctive tissue expression patterns in transgenic tobacco plants that could be divided into constitutive (C1, C4, and V2GUSdeltaC), predominantly vascular (C2, C3), or reduced expression in cells associated with the vascular bundles (V1GUSdeltaC). The significance of these results is discussed in terms of current models of gene function and regulation in geminiviruses.     

The contribution of tomato and alternative host plants to tomato leaf curl virus inoculum pressure in different areas of South India

Indian tomato leaf curl virus (ToLCV) (Geminiviridae: Sub-group III) was detected both in field-collected and laboratory-reared B. tabaci using a triple-antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA). ToLCV was detected in six of the 10 group samples of field collected B. tabaci. ToLCV was also identified in 13 weed species commonly found in Karnataka, both by symptom expression and TAS-ELISA. ToLCV from c. 61% of infected plants was transmitted successfully to tomato by B. tabaci. Tomato plots were planted at three locations on the University of Agricultural Sciences Campus, Bangalore. Indian tomato leaf curl virus disease (ToLCVD) incidence increased most rapidly when the tomato plot was situated adjacent to an older ToLCVD-infected tomato field. When the plots were positioned in a dryland or a wetland area, at least 500 m away from any infected tomato fields, the ToLCVD incidence increased less rapidly, although in all sites it was 100% by 11 wk after transplanting. The numbers of B. tabaci caught on yellow traps in all sites increased during weeks 1–3 after transplanting and thereafter remained at between 10–15 adults trap^-1 24 h^_1. Adult numbers recorded on tomato plants by direct counts remained approximately constant at 2–4 adults plant“”¹. Tomato fields were planted in three taluks (administrative areas) of Karnataka, that had different current and previous histories of tomato production. ToLCVD incidence increased most and least rapidly, respectively, in Kolar taluk where tomato is grown continuously and Doddaballapur tuluk where tomato was grown in the area for the first time. In Malur tuluk, where tomato was grown discontinuously (once a year), the incidence of ToLCVD increased at an intermediate rate. Weed host-plant species growing near the experimental sites had averages of between 1.5–10.0 B. tabaci nymphs per plant, whereas the tomato plants had only 0.3 nymphs per plant. The percentage parasitism of B. tabaci nymphs on tomato and weed species, respectively, was 0.7% and 2–6%. Nymphs and pupae were parasitised by an Encarsia sp. and Eretmocerus mundus Mercet. The relevance and implications of these findings for the epidemiology and management of ToLCVD in Karnataka State, South India is discussed.     

Newcastle disease virus V protein is a determinant of host range restriction

It has been demonstrated that the V protein of Newcastle disease virus (NDV) functions as an alpha/beta interferon (IFN-alpha/beta) antagonist (M. S. Park, M. L. Shaw, J. Mu?oz-Jordan, J. F. Cros, T. Nakaya, N. Bouvier, P. Palese, A. García-Sastre, and C. F. Basler, J. Virol. 77:1501-1511, 2003). We now show that the NDV V protein plays an important role in host range restriction. In order to study V functions in vivo, recombinant NDV (rNDV) mutants, defective in the expression of the V protein, were generated. These rNDV mutants grow poorly in both embryonated chicken eggs and chicken embryo fibroblasts (CEFs) compared to the wild-type (wt) rNDV. However, insertion of the NS1 gene of influenza virus A/PR8/34 into the NDV V(-) genome [rNDV V(-)/NS1] restores impaired growth to wt levels in embryonated chicken eggs and CEFs. These data indicate that for viruses infecting avian cells, the NDV V protein and the influenza NS1 protein are functionally interchangeable, even though there are no sequence similarities between the two proteins. Interestingly, in human cells, the titer of wt rNDV is 10 times lower than that of rNDV V(-)/NS1. Correspondingly, the level of IFN secreted by human cells infected with wt rNDV is much higher than that secreted by cells infected with the NS1-expressing rNDV. This suggests that the IFN antagonist activity of the NDV V protein is species specific. Finally, the NDV V protein plays an important role in preventing apoptosis in a species-specific manner. The rNDV defective in V induces apoptotic cell death more rapidly in CEFs than does wt rNDV. Taken together, these data suggest that the host range of NDV is limited by the ability of its V protein to efficiently prevent innate host defenses, such as the IFN response and apoptosis.     

The Tomato yellow leaf curl virus (TYLCV) V2 protein interacts with the host papain-like cysteine protease CYP1

The V2 protein of Tomato yellow leaf curl geminivirus (TYLCV) is an RNA-silencing suppressor that counteracts the innate immune response of the host plant. However, this anti-host defense function of V2 may include targeting of other defensive mechanisms of the plant. Specifically, we show that V2 recognizes and directly binds the tomato CYP1 protein, a member of the family of papain-like cysteine proteases which are involved in plant defense against diverse pathogens. This binding occurred both in vitro and in vivo, within living plant cells. The V2 binding site within mCYP1 was identified in the direct proximity to the papain-like cysteine protease active site.     

DNA helicase activity is associated with the replication initiator protein rep of tomato yellow leaf curl geminivirus

The Rep protein of tomato yellow leaf curl Sardinia virus (TYLCSV), a single-stranded DNA virus of plants, is the replication initiator essential for virus replication. TYLCSV Rep has been classified among ATPases associated with various cellular activities (AAA+ ATPases), in superfamily 3 of small DNA and RNA virus replication initiators whose paradigmatic member is simian virus 40 large T antigen. Members of this family are DNA- or RNA-dependent ATPases with helicase activity necessary for viral replication. Another distinctive feature of AAA+ ATPases is their quaternary structure, often composed of hexameric rings. TYLCSV Rep has ATPase activity, but the helicase activity, which is instrumental in further characterization of the mechanism of rolling-circle replication used by geminiviruses, has been a longstanding question. We present results showing that TYLCSV Rep lacking the 121 N-terminal amino acids has helicase activity comparable to that of the other helicases: requirements for a 3' overhang and 3'-to-5' polarity of unwinding, with some distinct features and with a minimal AAA+ ATPase domain. We also show that the helicase activity is dependent on the oligomeric state of the protein.     

Resistance to Tomato yellow leaf curl virus accumulation in the tomato wild relative Solanum habrochaites associated with the C4 viral protein

Tomato yellow leaf curl disease (TYLCD) is a severe threat to tomato crops worldwide and is caused by Tomato yellow leaf curl virus (TYLCV) and several other begomoviruses (genus Begomovirus, family Geminiviridae). Host plant resistance is the best TYLCD control method but limited sources of resistance are available. In this study, two Solanum habrochaites TYLCD-resistance sources, EELM-388 and EELM-889, were found after a wide germplasm screening and were further characterized. A consistent resistance to the widely distributed strain TYLCV-IL was observed when plants were inoculated by Bemisia tabaci or by agroinoculation using an infectious clone, with no symptoms or virus accumulation observed in inoculated plants. Moreover, the resistance was effective under field conditions with high TYLCD pressure. Two independent loci, one dominant and one recessive, were associated with EELM-889 resistance. The study shows these loci to be distinct from that of the resistance gene (Ty-1 gene) commonly deployed in commercial tomato cultivars. Therefore, both kinds of resistance could be combined to provide improved resistance to TYLCD. Four additional TYLCD-associated viruses were challenged, showing that the resistance always prevented symptom expression, although systemic infection could occur in some cases. By using chimeric and mutant expression constructs, the C4 protein was shown to be associated with the ability to result in effective systemic infection.     

Fine mapping of the tomato yellow leaf curl virus resistance gene Ty-2 on chromosome 11 of tomato

Resistances to begomoviruses, including bipartite tomato mottle virus and monopartite tomato yellow leaf curl virus (TYLCV), have been introgressed to cultivated tomato (Solanum lycopersicum) from wild tomato accessions. A major gene, Ty-2 from S. habrochaites f. glabratum accession “B6013,” that confers resistance to TYLCV was previously mapped to a 19-cM region on the long arm of chromosome 11. In the present study, approximately 11,000 plants were screened and nearly 157 recombination events were identified between the flanking markers C2_At1g07960 (82.5 cM, physical distance 51.387 Mb) and T0302 (89 cM, 51.878 Mb). Molecular marker analysis of recombinants and TYLCV evaluation of progeny from these recombinants localized Ty-2 to an approximately 300,000-bp interval between markers UP8 (51.344 Mb) and M1 (51.645 Mb). No recombinants were identified between TG36 and C2_At3g52090, a region of at least 115 kb, indicating severe recombination suppression in this region. Due to the small interval, fluorescence in situ hybridization analysis failed to clarify whether recombination suppression is caused by chromosomal rearrangements. Candidate genes predicted based on tomato genome annotation were analyzed by RT-PCR and virus-induced gene silencing. Results indicate that the NBS gene family present in the Ty-2 region is likely not responsible for the Ty-2-conferred resistance and that two candidate genes might play a role in the Ty-2-conferred resistance. Several markers very tightly linked to the Ty-2 locus are presented and useful for marker-assisted selection in breeding programs to introgress Ty-2 for begomovirus resistance.     

高抗番茄黄化曲叶病毒病番茄新品种浙粉702的选育及抗病性鉴定

高抗黄化曲叶病毒病番茄新品种浙粉702是以自育株系材料T7969F2-19-1-1-3为母本,T4078F2-3-3-3为父本,结合分子标记辅助技术选育的杂交一代粉红大果型番茄品种。母本‘T7969F2-19-1-1-3’系从以色列引进的耐贮运番茄品种‘NEMO-TAMMI’（F1）与抗叶霉病粉红株系材料‘T9179’杂交分离后代中经连续9代单株选择而成。父本‘T4078F2-3-3-3’系从荷兰引进的抗TYLCVD番茄品种‘奇诺亚’（F1）与粉红株系材料‘T9178’杂交分离后代中经连续8代单株选择而成。该品种2011年通过浙江省非主要农作物认定委员会认定。通过对浙粉702园艺性状、产量性状、品质性状和抗病性等进行研究,结果表明：浙粉702 品质优良,早熟,丰产,高抗番茄黄化曲叶病毒病,枯萎病,抗叶霉病和番茄花叶病毒病,适合我国喜食粉果地区种植,平均可达73.83 t.hm-2。     

Rinderpest virus phosphoprotein gene is a major determinant of species-specific pathogenicity

We previously demonstrated that the rinderpest virus (RPV) hemagglutinin (H) protein plays an important role in determining host range but that other viral proteins are clearly required for full RPV pathogenicity to be manifest in different species. To examine the effects of the RPV nucleocapsid (N) protein and phosphoprotein (P) genes on RPV cross-species pathogenicity, we constructed two new recombinant viruses in which the H and P or the H, N, and P genes of the cattle-derived RPV RBOK vaccine were replaced with those from the rabbit-adapted RPV-Lv strain, which is highly pathogenic in rabbits. The viruses rescued were designated recombinant RPV-lapPH (rRPV-lapPH) and rRPV-lapNPH, respectively. Rabbits inoculated with RPV-Lv become feverish and show leukopenia and a decrease in body weight gain, while clinical signs of infection are never observed in rabbits inoculated with RPV-RBOK or with rRPV-lapH. However, rabbits inoculated with either rRPV-lapPH or rRPV-lapNPH became pyrexic and showed leukopenia. Further, histopathological lesions and high virus titers were clearly observed in the lymphoid tissues from animals infected with rRPV-lapPH or rRPV-lapNPH, although they were not observed in rabbits infected with RPV-RBOK or rRPV-lapH. The clinical, virological, and histopathological signs in rabbits infected with the two new recombinant viruses did not differ significantly; therefore, the RPV P gene was considered to be a key determinant of cross-species pathogenicity.     

Genomic dissection of ROS detoxifying enzyme encoding genes for their role in antioxidative defense mechanism against Tomato leaf curl New Delhi virus infection in tomato

In the present study, genes encoding for six major classes of enzymatic antioxidants, namely superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), Peroxidase (Prx) and glutathione S-transferase (GST) are identified in tomato. Their expression was studied in tomato cultivars contrastingly tolerant to ToLCNDV during virus infection and different hormone treatments. Significant upregulation of SlGR3, SlPrx25, SlPrx75, SlPrx95, SlGST44, and SlGST96 was observed in the tolerant cultivar during disease infection. Virus-induced gene silencing of SlGR3 in the tolerant cultivar conferred disease susceptibility to the knock-down line, and higher accumulation (~80%) of viral DNA was observed in the tolerant cultivar. Further, subcellular localization of SlGR3 showed its presence in cytoplasm, and its enzymatic activity was found to be increased (~65%) during ToLCNDV infection. Knock-down lines showed ~3- and 3.5-fold reduction in GR activity, which altogether underlines that SlGR3 is vital component of the defense mechanism against ToLCNDV infection.     

Identification of a distinct strain of Cotton leaf curl Gezira virus infecting tomato in Oman

Tomato (Solanum lycopersicum) plants exhibiting yellowing, curling and stunting symptoms were identified in fields of the Tawoos Agricultural Systems, in Al‐Batinah in Oman. Cloning and sequencing of restriction endonuclease digested rolling circle amplified viral DNA identified a cotton begomovirus (family Geminiviridae) associated with the symptomatic tomato plants. Detailed analysis of complete sequences showed the virus to be a previously unknown strain of Cotton leaf curl Gezira virus (CLCuGeV) in association with the betasatellite Tomato leaf curl betasatellite (ToLCB). The new CLCuGeV strain, for which the name “Al Batinah” strain is suggested, has the greatest levels of sequence identity (91.9%) to an isolate of CLCuGeV recently reported from the neighbouring United Arab Emirates. Additionally, CLCuGeV‐Al Batinah was shown to have a recombinant origin with sequences donated by an African cassava mosaic virus‐like parent. This is the first identification of this Malvaceae‐adapted begomovirus in tomato. Although ToLCB is common in Oman, being one of only two betasatellites identified there so far, this is the first identification of this betasatellite with CLCuGeV. The significance of these findings is discussed.