A Tomato-Free Period Delays and Reduces Begomovirus Disease in Processing Tomato Fields in a Complex Agroecosystem in Central Brazil.

A mandatory tomato-free period (TFP) was implemented in the state of Goiás, Brazil, in 2007 to help manage diseases caused by whitefly-transmitted begomoviruses. The impact of the TFP was examined in five locations across three states in Central Brazil from 2013 to 2016. Surveys revealed significant differences in begomovirus disease incidence among locations, i.e., low in Guaíra-TFP and Patos de Minas-TFP; moderate-high in Itaberaí-TFP and Morrinhos-TFP; and high in the non-TFP (NTFP) control, Cristalina-NTFP. PCR tests and DNA sequencing were used to validate the symptoms and showed that all collected symptomatic plant samples were infected with tomato severe rugose virus (ToSRV), a common indigenous bipartite begomovirus. Early season surveys (20 to 40 days after transplants [DAT]) in Itaberaí-TFP and Morrinhos-TFP revealed significantly less begomovirus disease in fields established sooner after the TFP (0 to 2 months) compared with incidences in (i) equivalent early planted fields in the Cristalina-NTFP control and (ii) fields established longer after the end of the TFP (>2 to 5 months). Whitefly infestation of crops was detected year-round in all locations and years, and all tested adults were classified in the Bemisia tabaci MEAM1 cryptic species. Infestation levels were significantly higher during the summer but did not vary significantly among locations. Results of monthly monitoring of adult whiteflies for general begomovirus and ToSRV were positively correlated and were indicators of disease incidence in the field. Notably, ToSRV was not detected in whiteflies collected from nontomato plants during the TFP, and there was a longer lag period before detection in whiteflies collected from processing tomatoes for Itaberaí-TFP and Morrinhos-TFP compared with Cristalina-NTFP. Taken together with the low levels of ToSRV infection detected in potential nontomato reservoir hosts at all locations, our results revealed low levels of primary inoculum during the TFP. Thus, even in a complex agroecosystem with year-round whitefly infestation of crops, the TFP was beneficial due to delayed and reduced begomovirus disease pressure during a critical stage of plant development (first month) and for favoring low levels of primary inoculum. Thus, we concluded that the TFP should be part of a regional integrated pest management (IPM) program targeting ToSRV in Brazil.

Tomato golden net virus and tomato yellow net virus: two novel New World begomoviruses with monopartite genomes.

Two novel tomato-infecting begomoviruses were discovered via high-throughput sequencing in Brazil. Both viruses were also Sanger-sequenced and displayed DNA-A components phylogenetically related to New World bipartite begomoviruses. The names tomato golden net virus (ToGNV) and tomato yellow net virus (ToYNV) were proposed. The majority of the New World begomoviruses has bipartite genomes. However, extensive analyses revealed that ToGNV and ToYNV have monopartite genomes, because no cognate DNA-B components were detected. Hence, they may comprise a unique group of monopartite New World begomoviruses, which have enormous biological, molecular, and plant breeding interest.

How To Be a Successful Monopartite Begomovirus in a Bipartite-Dominated World: Emergence and Spread of Tomato Mottle Leaf Curl Virus in Brazil.

Begomoviruses are members of the family Geminiviridae, a large and diverse group of plant viruses characterized by a small circular single-stranded DNA genome encapsidated in twinned quasi-icosahedral virions. Cultivated tomato (Solanum lycopersicum L.) is particularly susceptible and is infected by >100 bipartite and monopartite begomoviruses worldwide. In Brazil, 25 tomato-infecting begomoviruses have been described, most of which are bipartite. Tomato mottle leaf curl virus (ToMoLCV) is one of the most important of these and was first described in the late 1990s but has not been fully characterized. Here, we show that ToMoLCV is a monopartite begomovirus with a genomic DNA similar in size and genome organization to those of DNA-A components of New World (NW) begomoviruses. Tomato plants agroinoculated with the cloned ToMoLCV genomic DNA developed typical tomato mottle leaf curl disease symptoms, thereby fulfilling Koch's postulates and confirming the monopartite nature of the ToMoLCV genome. We further show that ToMoLCV is transmitted by whiteflies, but not mechanically. Phylogenetic analyses placed ToMoLCV in a distinct and strongly supported clade with other begomoviruses from northeastern Brazil, designated the ToMoLCV lineage. Genetic analyses of the complete sequences of 87 ToMoLCV isolates revealed substantial genetic diversity, including five strain groups and seven subpopulations, consistent with a long evolutionary history. Phylogeographic models generated with partial or complete sequences predicted that the ToMoLCV emerged in northeastern Brazil >700 years ago, diversifying locally and then spreading widely in the country. Thus, ToMoLCV emerged well before the introduction of MEAM1 whiteflies, suggesting that the evolution of NW monopartite begomoviruses was facilitated by local whitefly populations and the highly susceptible tomato host. IMPORTANCE Worldwide, diseases of tomato caused by whitefly-transmitted geminiviruses (begomoviruses) cause substantial economic losses and a reliance on insecticides for management. Here, we describe the molecular and biological properties of tomato mottle leaf curl virus (ToMoLCV) from Brazil and establish that it is a NW monopartite begomovirus indigenous to northeastern Brazil. This answered a long-standing question regarding the genome of this virus, and it is part of an emerging group of these viruses in Latin America. This appears to be driven by widespread planting of the highly susceptible tomato and by local and exotic whiteflies. Our extensive phylogenetic studies placed ToMoLCV in a distinct strongly supported clade with other begomoviruses from northeastern Brazil and revealed new insights into the origin of Brazilian begomoviruses. The novel phylogeographic analysis indicated that ToMoLCV has had a long evolutionary history, emerging in northeastern Brazil >700 years ago. Finally, the tools used here (agroinoculation system and ToMoLCV-specific PCR test) and information on the biology of the virus (host range and whitefly transmission) will be useful in developing and implementing integrated pest management (IPM) programs targeting ToMoLCV.

Curly Top Disease of Hemp (Cannabis sativa) in California Is Caused by Mild-Type Strains of Beet curly top virus Often in Mixed Infection.

Interest in industrial hemp (Cannabis sativa) as a potential crop led to the establishment of commercial fields in a number of counties in California in 2019 and 2020. Plants in these fields developed different types of virus-like symptoms. The most prevalent type was stunted and bushy plants with distorted, upcurled, and yellowed leaves, which were similar to those associated with curly top disease (CTD) caused by the beet curly top virus (BCTV). This beet leafhopper-vectored virus is endemic in California and can cause economic losses to processing tomato production. Using a multiplex PCR test, BCTV infection was detected in 89% of hemp samples with CTD-like symptoms from Fresno, San Bernardino, and Ventura counties. Other symptom types had low incidence of BCTV infection and were associated with other factors. Hemp plants in California were infected only with the mild-type strains, BCTV-CO and BCTV-Wor, and often in mixed infection (43% of samples). Finally, using an infectious clone of a BCTV-CO isolate from hemp, we demonstrated that agroinoculated hemp plants developed these CTD-like symptoms, thereby fulfilling Koch's postulates for the disease.

The invasion biology of tomato begomoviruses in Costa Rica reveals neutral synergism that may lead to increased disease pressure and economic loss.

Since the late 1980s, tomato production in Costa Rica has been affected by diseases caused by whitefly-transmitted begomoviruses. The first was tomato yellow mottle virus (ToYMoV), a locally evolved New World (NW) bipartite begomovirus associated with the tomato yellow mottle disease (ToYMoD). In the late 1990s, the invasive NW bipartite tomato leaf curl Sinaloa virus (ToLCSiV) was detected in Costa Rica and has become established and associated with ToYMoD. Finally, the invasive Old World (OW) monopartite tomato yellow leaf curl virus (TYLCV) was detected in Costa Rica in 2012 and has also become established and is causing tomato yellow leaf curl disease (TYLCD). In the present study, we investigated the invasion biology of these tomato-infecting begomoviruses in Costa Rica in terms of (i) their biological and genetic properties and (ii) disease symptoms and viral DNA accumulation in tomato plants having single and mixed infections. We first generated infectious DNA-A and DNA-B clones and agroinoculation systems for ToYMoV and ToLCSiV isolates recovered from archival ToYMoD samples collected in Costa Rica in 1990 and 2002, respectively. Tomato plants agroinoculated with the infectious clones of both viruses developed ToYMoD symptoms, completing Koch's postulates for ToYMoV, and showing that ToLCSiV also causes this disease. However, pseudorecombinants formed between the DNA components of these viruses were not infectious, which is consistent with independent evolution in different lineages and limits genetic interactions. Furthermore, ToYMoV is well-adapted to tomato, has a narrow host range and is mechanically transmissible. The DNA-A component has a recombination event in the hot spot area and induced a symptomless infection in agroinoculated Nicotiana benthamiana and tomato plants. Tomato plants co-infected with two or all three viruses developed more severe symptoms compared with plants infected with each virus alone. Symptoms induced by the NW bipartite ToYMoV and ToLCSiV appeared earlier (∼7 d post-inoculation [dpi]) than those induced by TYLCV (∼10 dpi), but TYLCD symptoms became predominant in single and mixed infections by 14 dpi. Viral DNA accumulation was quantified by qPCR and generally revealed a neutral synergistic interaction in which the viruses co-existed in mixed infections. A transient reduction in accumulation of ToYMoV and ToLCSiV was detected in mixed infections at 7 dpi, whereas TYLCV accumulation was not affected in mixed infections and was uniform among treatments and time points. Together our results suggest that this neutral synergistic interaction will lead to increased begomovirus disease severity in Costa Rica. We discuss this in terms of begomovirus invasion biology and disease management.

Weed-infecting viruses in a tropical agroecosystem present different threats to crops and evolutionary histories.

In the Caribbean Basin, malvaceous weeds commonly show striking golden/yellow mosaic symptoms. Leaf samples from Malachra sp. and Abutilon sp. plants with these symptoms were collected in Hispaniola from 2014 to 2020. PCR tests with degenerate primers revealed that all samples were infected with a bipartite begomovirus, and sequence analyses showed that Malachra sp. plants were infected with tobacco leaf curl Cuba virus (TbLCuCV), whereas the Abutilon sp. plants were infected with a new bipartite begomovirus, tentatively named Abutilon golden yellow mosaic virus (AbGYMV). Phylogenetic analyses showed that TbLCuCV and AbGYMV are distinct but closely related species, which are most closely related to bipartite begomoviruses infecting weeds in the Caribbean Basin. Infectious cloned DNA-A and DNA-B components were used to fulfilled Koch's postulates for these diseases of Malachra sp. and Abutilon sp. In host range studies, TbLCuCV also induced severe symptoms in Nicotiana benthamiana, tobacco and common bean plants; whereas AbGYMV induced few or no symptoms in plants of these species. Pseudorecombinants generated with the infectious clones of these viruses were highly infectious and induced severe symptoms in N. benthamiana and Malachra sp., and both viruses coinfected Malachra sp., and possibly facilitating virus evolution via recombination and pseudorecombination. Together, our results suggest that TbLCuCV primarily infects Malachra sp. in the Caribbean Basin, and occasionally spills over to infect and cause disease in crops; whereas AbGYMV is well-adapted to an Abutilon sp. in the Dominican Republic and has not been reported infecting crops.

The Begomovirus Species Melon Chlorotic Leaf Curl Virus is Composed of Two Highly Divergent Strains that Differ in Their Genetic and Biological Properties.

Since the early 1990s, squash production in Costa Rica has been affected by a whitefly-transmitted disease characterized by stunting and yellow mottling of leaves. The squash yellow mottle disease (SYMoD) was shown to be associated with a bipartite begomovirus, originally named squash yellow mild mottle virus (SYMMoV). It was subsequently established that SYMMoV is a strain of melon chlorotic leaf curl virus (MCLCuV), a bipartite begomovirus that causes a chlorotic leaf curl disease of melons in Guatemala. In the present study, the complete sequences of the DNA-A and DNA-B components of a new isolate of the strain MCLCuV-Costa Rica (MCLCuV-CR) were determined. Comparisons of full-length DNA-A sequences revealed 97% identity with a previously characterized isolate of MCLCuV-CR and identities of 90 to 91% with those of isolates of the strain MCLCuV-Guatemala (MCLCuV-GT), which is below or at the current begomovirus species demarcation threshold of 91%. A more extensive analysis of the MCLCuV-CR and -GT sequences revealed substantial divergence in both components and different histories of recombination for the DNA-A components. The cloned full-length DNA-A and DNA-B components of this new MCLCuV-CR isolate were infectious and induced SYMoD in a range of squashes and in pumpkin, thereby fulfilling Koch's postulates for this disease. However, in contrast to MCLCuV-GT, MCLCuV-CR induced mild symptoms in watermelon and no symptoms in melon and cucumber. Taken together, our results indicate that MCLCuV-CR and -GT have substantially diverged, genetically and biologically, and have evolved to cause distinct diseases of different cucurbit crops. Taxonomically, these viruses are at the strain/species boundary, but retain the designation as strains of Melon chlorotic leaf curl virus under current International Committee on Taxonomy guidelines.

Rasta Disease of Tomato in Ghana is Caused by the Pospiviroids Potato spindle tuber viroid and Tomato apical stunt viroid.

Rasta is a virus-like disease of unknown etiology affecting tomato (Solanum lycopersicum) plants in Ghana. Symptoms include stunting; epinasty, crumpling, and chlorosis of leaves; and necrosis of leaf veins, petioles, and stems. Leaf samples with rasta symptoms were collected from commercial tomato fields in Ghana in October 2012 and applied to FTA cards, and RNA extracts were prepared. Reverse-transcription polymerase chain reaction (RT-PCR) tests with primers for Columnea latent viroid, which causes rasta-like symptoms in tomato plants in Mali, were negative, whereas tests with degenerate viroid primer pairs were inconclusive. However, tomato seedlings (Early Pak 7) mechanically inoculated with RNA extracts of 10 of 13 samples developed rasta-like symptoms. In RT-PCR tests with RNA from leaves of the 10 symptomatic seedlings and primers for Potato spindle tuber viroid (PSTVd) or Tomato apical stunt viroid (TASVd), the expected size (approximately 360 bp) of DNA fragment was amplified from eight and two seedlings, respectively. Sequence analyses confirmed that these fragments were from PSTVd and TASVd isolates, and revealed a single PSTVd haplotype and two TASVd haplotypes. The PSTVd and TASVd isolates from Ghana had high nucleotide identities (>94%) with isolates from other geographic regions. In a host range study, PSTVd and TASVd isolates from Ghana induced rasta symptoms in the highly susceptible tomato cultivar Early Pak 7 and mild or no symptoms in Glamour, and symptomless infections in a number of other solanaceous species. PSTVd and TASVd isolates were seed associated and possibly seed transmitted.

A Bipartite Begomovirus Infecting Boerhavia erecta (Family Nyctaginaceae) in the Dominican Republic Represents a Distinct Phylogenetic Lineage and has a High Degree of Host Specificity.

Boerhavia erecta plants in and around agricultural fields in the Azua Valley of the southeastern Dominican Republic often show striking golden mosaic symptoms. Leaf samples from B. erecta plants showing these symptoms were collected in 2012 and 2013, and PCR tests with degenerate primers revealed begomovirus DNA-A and DNA-B components. The complete sequences of the DNA-A and DNA-B components of four isolates show a high degree of sequence identity (>96%) and a genome organization typical of New World (NW) bipartite begomoviruses. Sequence comparisons and phylogenetic analyses revealed that these isolates composed a new phylogenetic lineage of NW bipartite begomoviruses. The most closely related begomovirus is Merremia mosaic virus, a weed-infecting species from Puerto Rico. Because DNA-A sequence identities are well below the 91% threshold, these isolates represent a new begomovirus species, for which the name Boerhavia golden mosaic virus (BoGMV) is proposed. Infectious cloned BoGMV DNA-A and DNA-B components induced golden mosaic symptoms in agroinoculated B. erecta plants, thereby fulfilling Koch's postulates for this disease. Agroinoculation and mechanical transmission experiments revealed that BoGMV has an unusually narrow host range, limited to members of the family Nyctaginaceae and not including the permissive host Nicotiana benthamiana. The inability of BoGMV to infect N. benthamiana was due to a deficiency in cell-to-cell movement but not to a unique amino acid residue in the movement protein.

World Management of Geminiviruses.

Management of geminiviruses is a worldwide challenge because of the widespread distribution of economically important diseases caused by these viruses. Regardless of the type of agriculture, management is most effective with an integrated pest management (IPM) approach that involves measures before, during, and after the growing season. This includes starting with resistant cultivars and virus- and vector-free transplants and propagative plants. For high value vegetables, protected culture (e.g., greenhouses and screenhouses) allows for effective management but is limited owing to high cost. Protection of young plants in open fields is provided by row covers, but other measures are typically required. Measures that are used for crops in open fields include roguing infected plants and insect vector management. Application of insecticide to manage vectors (whiteflies and leafhoppers) is the most widely used measure but can cause undesirable environmental and human health issues. For annual crops, these measures can be more effective when combined with host-free periods of two to three months. Finally, given the great diversity of the viruses, their insect vectors, and the crops affected, IPM approaches need to be based on the biology and ecology of the virus and vector and the crop production system. Here, we present the general measures that can be used in an IPM program for geminivirus diseases, specific case studies, and future challenges.

Characterization of a new world monopartite begomovirus causing leaf curl disease of tomato in Ecuador and Peru reveals a new direction in geminivirus evolution.

All characterized whitefly-transmitted geminiviruses (begomoviruses) with origins in the New World (NW) have bipartite genomes composed of a DNA-A and DNA-B component. Recently, an NW begomovirus lacking a DNA-B component was associated with tomato leaf curl disease (ToLCD) in Peru, and it was named Tomato leaf deformation virus (ToLDeV). Here, we show that isolates of ToLDeV associated with ToLCD in Ecuador and Peru have a single, genetically diverse genomic DNA that is most closely related to DNA-A components of NW bipartite begomoviruses. Agroinoculation of multimeric clones of the genomic DNA of three ToLDeV genotypes (two variants and a strain) resulted in the development of tomato leaf curl symptoms indistinguishable from those of ToLCD in Ecuador and Peru. Biological properties of these ToLDeV genotypes were similar to those of Old World (OW) monopartite tomato-infecting begomoviruses, including lack of sap transmissibility, phloem limitation, a resistance phenotype in tomato germplasm with the Ty-1 gene, and functional properties of the V1 (capsid protein) and C4 genes. Differences in symptom phenotypes induced by the ToLDeV genotypes in tomato and Nicotiana benthamiana plants were associated with a highly divergent left intergenic region and C4 gene. Together, these results establish that ToLDeV is an emergent NW monopartite begomovirus that is causing ToLCD in Ecuador and Peru. This is the first report of an indigenous NW monopartite begomovirus, and evidence is presented that it emerged from the DNA-A component of a NW bipartite progenitor via convergent evolution and recombination.

Occurrence of Squash yellow mild mottle virus and Pepper golden mosaic virus in Potential New Hosts in Costa Rica.

Leaf samples of Solanum lycopersicum, Capsicum annuum, Cucurbita moschata, Cucurbita pepo, Sechium edule and Erythrina spp. were collected. All samples were positive for begomoviruses using polymerase chain reaction and degenerate primers. A sequence of ∼1,100 bp was obtained from the genomic component DNA-A of 14 samples. In addition, one sequence of ∼580 bp corresponding to the coat protein (AV1) was obtained from a chayote (S. edule) leaf sample. The presence of Squash yellow mild mottle virus (SYMMoV) and Pepper golden mosaic virus (PepGMV) were confirmed. The host range reported for SYMMoV includes species of the Cucurbitaceae, Caricaceae and Fabaceae families. This report extends the host range of SYMMoV to include the Solanaceae family, and extends the host range of PepGMV to include C. moschata, C. pepo and the Fabaceae Erythrina spp. This is the first report of a begomovirus (PepGMV) infecting chayote in the Western Hemisphere.

Histone H3 interacts and colocalizes with the nuclear shuttle protein and the movement protein of a geminivirus.

Geminiviruses are plant-infecting viruses with small circular single-stranded DNA genomes. These viruses utilize nuclear shuttle proteins (NSPs) and movement proteins (MPs) for trafficking of infectious DNA through the nuclear pore complex and plasmodesmata, respectively. Here, a biochemical approach was used to identify host factors interacting with the NSP and MP of the geminivirus Bean dwarf mosaic virus (BDMV). Based on these studies, we identified and characterized a host nucleoprotein, histone H3, which interacts with both the NSP and MP. The specific nature of the interaction of histone H3 with these viral proteins was established by gel overlay and in vitro and in vivo coimmunoprecipitation (co-IP) assays. The NSP and MP interaction domains were mapped to the N-terminal region of histone H3. These experiments also revealed a direct interaction between the BDMV NSP and MP, as well as interactions between histone H3 and the capsid proteins of various geminiviruses. Transient-expression assays revealed the colocalization of histone H3 and NSP in the nucleus and nucleolus and of histone H3 and MP in the cell periphery and plasmodesmata. Finally, using in vivo co-IP assays with a Myc-tagged histone H3, a complex composed of histone H3, NSP, MP, and viral DNA was recovered. Taken together, these findings implicate the host factor histone H3 in the process by which an infectious geminiviral DNA complex forms within the nucleus for export to the cell periphery and cell-to-cell movement through plasmodesmata.

Roles and interactions of begomoviruses and satellite DNAs associated with okra leaf curl disease in Mali, West Africa.

Okra leaf curl disease (OLCD) is a major constraint on okra (Abelmoschus esculentus) production in West Africa. Two monopartite begomoviruses (okra virus-1 and okra virus-2), a betasatellite and a DNA1 satellite are associated with OLCD in Mali. Okra virus-1 is an isolate of okra yellow crinkle virus (OYCrV), okra virus-2 is a recombinant isolate of cotton leaf curl Gezira virus (CLCuGV) and the betasatellite is a variant of cotton leaf curl Gezira betasatellite (CLCuGB). Cloned DNA of OYCrV and CLCuGV were infectious and induced leaf curl symptoms in Nicotiana benthamiana plants, but did not induce OLCD in okra. However, when these clones were individually co-inoculated with the cloned CLCuGB DNA, symptom severity and viral DNA levels were increased in N. benthamiana plants and typical OLCD symptoms were induced in okra. The CLCuGB was also replicated by, and increased symptom severity of, three monopartite tomato-infecting begomoviruses, including two from West Africa. The sequence of the DNA1 satellite was highly divergent, indicating that it represents a distinct West African lineage. DNA1 replicated autonomously, and replication required the DNA1-encoded Rep protein. Although DNA1 reduced helper begomovirus DNA levels, symptoms were not attenuated. In the presence of CLCuGB, DNA levels of the helper begomoviruses and DNA1 were substantially increased. Together, these findings establish that OLCD in Mali is caused by a complex of monopartite begomoviruses and a promiscuous betasatellite with an associated parasitic DNA1 satellite. These findings are discussed in terms of the aetiology of OLCD and the evolution of new begomovirus/satellite DNA complexes.

Reciprocal phosphorylation and glycosylation recognition motifs control NCAPP1 interaction with pumpkin phloem proteins and their cell-to-cell movement.

In plants, cell-to-cell trafficking of non-cell-autonomous proteins (NCAPs) involves protein-protein interactions, and a role for posttranslational modification has been implicated. In this study, proteins contained in pumpkin (Cucurbita maxima cv Big Max) phloem sap were used as a source of NCAPs to further explore the molecular basis for selective NCAP trafficking. Protein overlay assays and coimmunoprecipitation experiments established that phosphorylation and glycosylation, on both Nicotiana tabacum NON-CELL-AUTONOMOUS PATHWAY PROTEIN1 (Nt-NCAPP1) and the phloem NCAPs, are essential for their interaction. Detailed molecular analysis of a representative phloem NCAP, Cm-PP16-1, identified the specific residues on which glycosylation and phosphorylation must occur for effective binding to NCAPP1. Microinjection studies confirmed that posttranslational modification on these residues is essential for cell-to-cell movement of Cm-PP16-1. Lastly, a glutathione S-transferase (GST)-Cm-PP16-1 fusion protein system was employed to test whether the peptide region spanning these residues was required for cell-to-cell movement. These studies established that a 36-amino acid peptide was sufficient to impart cell-to-cell movement capacity to GST, a normally cell-autonomous protein. These findings are consistent with the hypothesis that a phosphorylation-glycosylation recognition motif functions to control the binding of a specific subset of phloem NCAPs to NCAPP1 and their subsequent transport through plasmodesmata.

Characterization of a novel Toll/interleukin-1 receptor (TIR)-TIR gene differentially expressed in common bean (Phaseolus vulgaris cv. Othello) undergoing a defence response to the geminivirus Bean dwarf mosaic virus.

SUMMARY Common bean (Phaseolus vulgaris L.) cultivar (cv.) Othello develops a hypersensitive response-associated vascular resistance to infection by Bean dwarf mosaic virus (BDMV), a single-stranded DNA virus (genus Begomovirus, family Geminiviridae). A PCR-based cDNA subtraction approach was used to identify genes involved in this resistance response. Eighteen clones, potentially involved with BDMV resistance, were identified based upon being up-regulated in BDMV-infected tissues and/or having sequence similarity with known resistance-associated genes. Analysis of these clones revealed potential genes involved in pathogen defence, including pathogenesis-related protein genes and resistance gene analogues (RGAs). Further characterization of one RGA, F1-10, revealed that it encodes a predicted protein with a double Toll/interleukin-1 receptor (TIR) motif. Full-length (F1-10) and spliced (F1-10sp) forms of the RGA were strongly up-regulated in BDMV-infected cv. Othello hypocotyl tissues by 4 days post-inoculation, but not in equivalent mock-inoculated tissues. In agroinfiltration experiments, F1-10, but not F1-10sp, mediated resistance to BDMV in the susceptible common bean cv. Topcrop. By contrast, transgenic Nicotiana benthamiana lines expressing F1-10 or F1-10sp were not resistant to BDMV. Interestingly, when these transgenic lines were inoculated with the potyvirus Bean yellow mosaic virus, some F1-10 lines showed a more severe symptom phenotype compared with non-transgenic control plants. Based on these findings, F1-10 was named: Phaseolus vulgaris VIRUS response TIR-TIR GENE 1 (PvVTT1).

Permeabilized mammalian cells as an experimental system for nuclear import of geminiviral karyophilic proteins and of synthetic peptides derived from their nuclear localization signal regions.

The plant-infecting geminiviruses deliver their genome and viral proteins into the host cell nucleus. Members of the family Geminiviridae possess either a bipartite genome composed of two approximately 2.6 kb DNAs or a monopartite genome of approximately 3.0 kb DNA. The bipartite genome of Bean dwarf mosaic virus (BDMV) encodes several karyophilic proteins, among them the capsid protein (CP) and BV1 (nuclear shuttle protein). A CP is also encoded by the monopartite genome of Tomato yellow leaf curl virus (TYLCV). Here, an in vitro assay system was used for direct demonstration of nuclear import of BDMV BV1 and TYLCV CP, as well as synthetic peptides containing their putative nuclear localization signals (NLSs). Full-length recombinant BDMV BV1 and TYLCV CP mediated import of conjugated fluorescently labelled BSA molecules into nuclei of permeabilized mammalian cells. Fluorescently labelled and biotinylated BSA conjugates bearing the synthetic peptides containing aa 3-20 of TYLCV CP (CP-NLS) or aa 84-106 of BDMV BV1 (BV1-NLS) were also imported into the nuclei of permeabilized cells. This import was blocked by the addition of unlabelled BSA-NLS peptide conjugates or excess unlabelled free NLS peptides. The CP- and BV1-NLS peptides also mediated nuclear import of fluorescently labelled BSA molecules into the nuclei of microinjected mesophyll cells of Nicotiana benthamiana leaves, demonstrating their biological function in intact plant tissue. BV1-NLS and CP-NLS were shown to mediate specific binding to importin alpha, both in vitro and in vivo. These results are consistent with a common nuclear-import pathway for CP and BV1, probably via importin alpha.

A viral resistance gene from common bean functions across plant families and is up-regulated in a non-virus-specific manner.

Genes involved in a viral resistance response in common bean (Phaseolus vulgaris cv. Othello) were identified by inoculating a geminivirus reporter (Bean dwarf mosaic virus expressing the green fluorescent protein), extracting RNA from tissue undergoing the defense response, and amplifying sequences with degenerate R gene primers. One such gene (a TIR-NBS-LRR gene, RT4-4) was selected for functional analysis in which transgenic Nicotiana benthamiana were generated and screened for resistance to a range of viruses. This analysis revealed that RT4-4 did not confer resistance to the reporter geminivirus; however, it did activate a resistance-related response (systemic necrosis) to seven strains of Cucumber mosaic virus (CMV) from pepper or tomato, but not to a CMV strain from common bean. Of these eight CMV strains, only the strain from common bean systemically infected common bean cv. Othello. Additional evidence that RT4-4 is a CMV R gene came from the detection of resistance response markers in CMV-challenged leaves of RT4-4 transgenic plants, and the identification of the CMV 2a gene product as the elicitor of the necrosis response. These findings indicate that RT4-4 functions across two plant families and is up-regulated in a non-virus-specific manner. This experimental approach holds promise for providing insights into the mechanisms by which plants activate resistance responses against pathogens.

Exploiting chinks in the plant's armor: evolution and emergence of geminiviruses.

The majority of plant-infecting viruses utilize an RNA genome, suggesting that plants have imposed strict constraints on the evolution of DNA viruses. The geminiviruses represent a family of DNA viruses that has circumvented these impediments to emerge as one of the most successful viral pathogens, causing severe economic losses to agricultural production worldwide. The genetic diversity reflected in present-day geminiviruses provides important insights into the evolution and biology of these pathogens. To maximize replication of their DNA genome, these viruses acquired and evolved mechanisms to manipulate the plant cell cycle machinery for DNA replication, and to optimize the number of cells available for infection. In addition, several strategies for cell-to-cell and long-distance movement of the infectious viral DNA were evolved and refined to be compatible with the constraints imposed by the host endogenous macromolecular trafficking machinery. Mechanisms also evolved to circumvent the host antiviral defense systems. Effectively combatting diseases caused by geminiviruses represents a major challenge and opportunity for biotechnology.

Protein interactions involved in nuclear import of the Agrobacterium VirE2 protein in vivo and in vitro.

Agrobacterium, the only known organism capable of trans-kingdom DNA transfer, genetically transforms plants by transferring a segment of its DNA, T-DNA, into the nucleus of the host cell where it integrates into the plant genome. One of the central events in this genetic transformation process is nuclear import of the T-DNA molecule, which to a large degree is mediated by the bacterial virulence protein VirE2. VirE2 is distinguished by its nuclear targeting, which occurs only in plant but not in animal cells and is facilitated by the cellular VIP1 protein. The molecular mechanism of the VIP1 function is still unclear. Here, we used in vitro assays for nuclear import and quantification of protein-protein interactions to directly demonstrate formation of ternary complexes between VirE2, VIP1, and a component of the cellular nuclear import machinery, karyopherin alpha. Our results indicate that VIP1 functions as a molecular bridge between VirE2 and karyopherin alpha, allowing VirE2 to utilize the host cell nuclear import machinery even without being directly recognized by its components.