Bean Dwarf Mosaic Virus Research Articles

Bean dwarf mosaic virus BV1 protein is a determinant of the hypersensitive response and avirulence in Phaseolus vulgaris.

The capacities of the begomoviruses Bean dwarf mosaic virus (BDMV) and Bean golden yellow mosaic virus (BGYMV) to differeBean dwarf mosaic viru certain common bean (Phaseolus vulgaris) cultivars were used to identify viral determinants of the hypersensitive response (HR) and avirulence (avr) in BDMV. A series of hybrid DNA-B components, containing BDMV and BGYMV sequences, was constructed and coinoculated with BDMV DNA-A (BDMV-A) or BDMVA-green florescent protein into seedlings of cv. Topcrop (susceptible to BDMV and BGYMV) and the BDMV-resistant cvs. Othello and Black Turtle Soup T-39 (BTS). The BDMV avr determinant, in bean hypocotyl tissue, was mapped to the BDMV BV1 open reading frame and, most likely, to the BV1 protein. The BV1 also was identified as the determinant of the HR in Othello. However, the HR was not required for resistance in Othello nor was it associated with BDMV resistance in BTS. BDMV BV1, a nuclear shuttle protein that mediates viral DNA export from the nucleus, represents a new class of viral avr determinant. These results are discussed in terms of the relationship between the HR and resistance.

First Report and Characterization of Rhynchosia golden mosaic virus in Honduras.

Rhynchosia minima was suspected to be a weed host of Bean golden yellow mosaic virus (BGYMV, previously designated Bean golden mosaic virus type II). Leaf tissue that exhibited yellow mosaic foliar symptoms characteristic of a geminivirus infection was collected in the Comayagua Valley in Honduras in July 1999. Extraction of viral DNA from the symptomatic leaves was accomplished with the DNeasy Plant Mini Kit (Qiagen Inc., Valencia, CA). Subsequent viral DNA amplification was accomplished with degenerative primers for the cp gene (AV494/AC1048) (4). The 570-bp fragment was cloned into the pGEM T-Easy vector (Promega Corp., Madison, WI) producing the recombinant plasmid pRhyb5. The viral insert was sequenced, and from this sequence, specific primers (RHc549 and RHv29) were designed to amplify the remaining part of DNA-A. The 2.1-kb-amplified polymerase chain reaction (PCR) fragment was cloned into the pGEM T-Easy vector producing the recombinant plasmid (pRhya-sp), and the viral insert was sequenced. Nucleotide sequence comparison (GAP program, Wisconsin Package Version 10.0, Genetics Computer Group, Madison, WI) of the complete 2,624-bp DNA-A (GenBank accession no. AF239671) to geminiviruses representing the major phylogenetic clusters (1) showed nucleotide identities ranging from 63 to 82%. Sequence comparisons for the common region and rep, trap, ren, and cp genes with the most closely related geminivirus, Pepper hausteco virus (PHV, X70418), gave 76, 82, 79, 81, and 82% nucleotide identities, respectively. There is a direct repeat (TATCGGT) of 7 nt 5' (viral sense polarity) of the conserved TATA box, and this repeat is most analogous to that in PHV (1). Specific primers were designed in the complementary sense (RGBc2414, BGBc2553) from the common region DNA-A sequence and used with a degenerative viral sense primer for the DNA-B (PBC1v2039) (3) to amplify a 647-bp fragment. Sequence comparison for the common region (134 nt from the rep gene start codon toward the 3' end) from the DNA-B sequence had 88% nt identity to the DNA-A sequence, thus indicating that this geminivirus is bipartite. These sequence analyses indicated that this geminivirus isolated from R. minima is distinct from previously described geminiviruses, and we propose the name Rhynchosia golden mosaic virus (RGMV). From rep gene sequence alignments, RGMV has an apparent genome recombination between Old and New World geminiviruses (Tomato yellow leaf curl virus and Bean dwarf mosaic virus) as previously noted for PHV (2). Our results indicate that RGMV is a distinct geminivirus from BGYMV, and, thus, additional studies are needed to establish the importance of R. minima as a reservoir for vegetable-infecting geminiviruses. This study is the first report of another virus in the PHV phylogenetic cluster and is thus of importance in the understanding of recombinant viruses and their phylogenetic relationship to other characterized geminiviruses.

Transgenic plants expressing geminivirus movement proteins: abnormal phenotypes and delayed infection by Tomato mottle virus in transgenic tomatoes expressing the Bean dwarf mosaic virus BV1 or BC1 proteins.

Transgenic tomato plants expressing wild-type or mutated BV1 or BC1 movement proteins from Bean dwarf mosaic virus (BDMV) were generated and examined for phenotypic effects and resistance to Tomato mottle virus (ToMoV). Fewer transgenic plants were recovered with the wild-type or mutated BC1 genes, compared with the wild-type or mutated BV1 genes. Transgenic tomato plants expressing the wild-type or mutated BV1 proteins appeared normal. Interestingly, although BDMV induces only a symptomless infection in tomato (i.e., BDMV is not well adapted to tomato), transgenic tomato plants expressing the BDMV BC1 protein showed a viral disease-like phenotype (i.e., stunted growth, and leaf mottling, curling, and distortion). This suggests that the symptomless phenotype of BDMV in tomato is not due to a host-specific defect in the BC1 protein. One transgenic line expressing the BC1 gene did not show the viral disease-like phenotype. This was associated with a deletion in the 3' region of the gene, which resulted in expression of a truncated BC1 protein. Several R0 plants, expressing either wild-type or mutated BV1 or BC1 proteins, showed a significant delay in ToMoV infection, compared with non-transformed plants. R1 progeny plants also showed a significant delay in ToMoV infection, but this delay was less than that in the R0 parents. These results also demonstrate that expression of viral movement proteins, in transgenic plants, can have deleterious effects on various aspects of plant development.

A Bipartite Geminivirus Infecting Tomatoes in Cuba.

Whitefly-transmitted geminiviruses have increased in their distribution and importance during the last decade, apparently due to a wider distribution of their vector Bemisia tabaci (Gennadius). In Cuba, symptoms of geminivirus disease have been observed since the early 1990s. A survey of geminivirus-caused diseases in tomato plants has been conducted since 1994 in the province of La Habana. The most severe symptoms were observed in the commercial varieties Campbell 28, Criollo Quivican, and HC 3880. In some plants the Israeli strain of tomato yellow leaf curl geminivirus (TYLCV) was detected (1). In other cases, a viral DNA with a genome size smaller than that of TYLCV that could not be amplified with primers specific for the Israeli strain was also detected by hybridization. In these samples a polymerase chain reaction amplification with broad-spectrum, degenerate primers designed for whitefly-transmitted bipartite geminivirus (2) was carried out. Two fragments of about 1.1 and 0.6 kb, corresponding to components A and B, respectively, were amplified, indicating that a bipartite geminivirus was present. The 1.1-kb fragment amplified from a sample showing strong leaf yellowing and slight curling was cloned and sequenced. A sequence of 1,174 nucleotides was obtained and compared with geminivirus sequences deposited in the GenBank, by multiple alignment (CLUSTAL W, European Bioinformatics Institute, Hinxton Hall, UK). Genetic distances were calculated by Kimura's two parameters method. Percentages of similarities obtained with the sequence were as follows: tomato mottle virus 78%, abutilon mosaic virus 73.8%, and tomato golden mosaic 61.8%. Partial sequences of the open reading frames AL1 and AR1 and the intergenic region were present. Percentages of similarities obtained with the intergenic region were as follows: tomato mottle virus 78.1%, sida golden mosaic virus 63%, bean dwarf mosaic virus 62.6%, abutilon mosaic virus 57.1%, and tomato golden mosaic virus 32%. These results suggest that we have detected in Cuban tomatoes a new bipartite geminivirus related to tomato mottle virus, and we propose that it be named Havana tomato virus. Frequency of distribution of viruses in commercial varieties and their relationship with the presence of whiteflies are currently being studied.

Taino Tomato Mottle Virus, a New Bipartite Geminivirus from Cuba.

Geminiviruses have become the most important virus group affecting tomatoes (Lycopersicon lycopersicum (L.) Karsten) in Cuba since they have been detected in all tomato-producing areas, causing serious losses. Recently, a whitefly-transmitted, monopartite geminivirus was detected in Cuba and identified as tomato yellow leaf curl virus-Israel (TYLCV-Is) (1). Samples collected from the main tomato-producing areas during the period 1995 to1996 were further analyzed by polymerase chain reaction (PCR) with degenerate primers (PAL1v1978 and PAR1c496) (2). Whereas in samples from most areas only TYLCV was detected, in some samples from the Havana area, two DNA fragments (approximately 1.4 and 1.1 kb) were amplified by PCR. The larger fragment was identified as part of the TYLCV-Is genome, confirming the previous report (1). The 1.1-kb fragment was cloned and its nucleotide sequence suggested that a new bipartite geminivirus was also present in those tomato samples. To clone the entire genome, tomato plants were inoculated by biolistics with DNA extract from field samples. After symptom expression, a viral DNA-enriched preparation from the inoculated tomatoes was independently digested with several restriction enzymes and the products were ligated into pZero plasmid (Invitrogen, San Diego, CA). Several clones in the 2.6-kb size range were characterized by restriction mapping and hybridization against component A and B heterologous probes. Two clones were selected as containing putative A and B components and their infectivity was tested by biolistic inoculation of tomato and pepper plants. The inoculated tomatoes developed a mild mottle in the younger leaves, whereas no symptoms were visible on the inoculated pepper plants. However, the presence of viral DNA was confirmed in both tomatoes and peppers by Southern blot hybridization analysis with A- and B-specific probes. Partial sequences of both components were obtained and their analysis showed that both components shared a 170-bases common region with a 95% identity. In addition, the nucleotide sequences of two open reading frames, one in each component (AC1 and BC1), were determined and compared with geminivirus sequences deposited in Gen-Bank. A dendogram generated with the CLUSTAL program and obtained with the AC1 and BC1 amino acid sequences, placed the new geminivirus in a cluster with tomato mottle virus (ToMoV; accession nos. L14460, L14461), Abutilon mosaic virus (AbMV; X15983, X15984), potato yellow mosaic virus (PYMV; D00940, D00941), and bean dwarf mosaic virus (BDMV; M88179, M88180). The percentages of identity obtained with the amino acid sequences were as follows. For AC1: ToMoV, 87%; PYMV, 79.5%; BDMV, 78.7%; and AbMV, 78%. For BC1 protein: BDMV, 92.8%; ToMoV, 89.1%; PYMV, 88.1%; and AbMV, 67.5%. In addition, the sequences were compared with partial nucleotide sequences (AC1, coat protein [CP], and common region) of a bipartite geminivirus affecting tomatoes in Jamaica (accession nos. U83855, U83854, and U83850). Interestingly, the common regions showed a higher percentage of identity (88%) than the CP and AC1 partial nucleotide sequences (86 and 74%, respectively). These data suggest that the virus reported here is a new geminivirus and the first bipartite geminivirus reported in Cuba. Thus, the name of Taino tomato mottle virus is proposed. (Taino refers to the name of the inhabitants of Cuba at the time of Columbus's arrival in the Caribbean).

Nucleotide sequence of a new bipartite geminivirus isolated from the common weed Sida rhombifolia in Costa Rica.

The nucleotide sequence of infectious clones of a geminivirus from Costa Rica that infects Sida rhombifolia was determined. Sida golden mosaic virus (SiGMV-Co) has a bipartite genome (DNAs A and B). Computer analysis showed that the bipartite genome of SiGMV-Co resembles that of other whitefly-transmitted geminiviruses. The DNA A (2605 nt) and DNA B (2587 nt) components have little sequence homology other than within the common region (CR). Analysis of DNAs A and B showed that SiGMV-Co is closely related to bean dwarf mosaic virus (BDMV). SiGMV-Co was introduced via agroinoculation into seven plant species, including tomato and bean.

Increased pathogenicity in a pseudorecombinant bipartite geminivirus correlates with intermolecular recombination.

Most whitefly-transmitted geminiviruses possess bipartite DNA genomes, and this feature may facilitate viral evolution through pseudorecombination and/or recombination. To test this hypothesis, the DNA-A and DNA-B components of the geminiviruses bean dwarf mosaic virus (BDMV) and tomato mottle virus (ToMoV) were exchanged, and the resultant pseudorecombinants were serially passaged through plants. Both pseudorecombinants were infectious in Nicotiana benthamiana but induced attenuated symptoms and had reduced DNA-B levels. Serial passage experiments revealed that the BDMV DNA-A plus ToMoV DNA-B pseudorecombinant could not be maintained beyond three passages. In contrast, the ToMoV DNA-A plus BDMV DNA-B pseudorecombinant was maintained during serial passage through N. benthamiana and Phaseolus vulgaris and, after three to five passages, became highly pathogenic. Furthermore, the increased pathogenicity of this pseudorecombinant was consistently associated with an increased level of DNA-B, which eventuated in equivalent levels of both components. Sequence analysis of the DNA-B component of the more pathogenic pseudorecombinant revealed that intermolecular recombination had taken place in which most of the BDMV DNA-B common region was replaced with the ToMoV DNA-A common region. This recombinant DNA-B component, which contained the ToMoV origin of replication, was the predominant DNA-B component associated with the more pathogenic pseudorecombinant. These results provide the first demonstration of recombination between distinct bipartite geminiviruses and establish that the bipartite genome can facilitate viral evolution through pseudorecombination and intermolecular recombination.

Complete Nucleotide Sequence of a Nonvector-Transmissible Strain of Abutilon Mosaic Geminivirus in Hawaii

The complete nucleotide sequence of a geminivirus infecting lantern 'ilima (Abutilon hybridum) in Hawaii was determined. Sequence analysis showed that this geminivirus was a strain of Abutilon mosaic geminivirus (AbMV-HI). The genome of AbMV-HI consisted of two circular, single-stranded DNA molecules of 2,634 bases (DNA-A) and 2,571 bases (DNA-B). AbMV-HI shared 95 and 91% nucleotide sequence identity with the DNA-A and DNA-B components, respectively, of a West Indian strain of AbMV (AbMV-WI). AbMV-HI also shared 90% identity with the deduced amino acid sequences of the coat proteins of tomato mottle virus, bean dwarf mosaic virus, and potato yellow mosaic virus. Results of transmission experiments showed that AbMV-HI was not transmissible by the sweet potato whitefly Bemisia tabaci. The derived amino acid sequences of the coat proteins of two nonwhitefly-transmissible strains of AbMV were compared with those of 19 whitefly-transmissible geminiviruses. Five amino acids in the N-terminal region of the coat proteins were identical for two strains of AbMV, but were different from vector-transmissible geminiviruses. These results suggested that some or all of the five amino acids at the N-terminal end of the coat protein may have been involved in whitefly transmission of bipartite geminiviruses.