Genetic diversity and host range studies of turnip curly top virus.

Turnip curly top virus (TCTV) is a unique geminivirus that has recently been characterised as infecting turnips in Iran. The genome of TCTV shares <68 % pairwise identity with other geminiviruses and has a genome organisation similar to that of curtoviruses and topocuvirus. The replication-associated protein (Rep) bears the highest similarity to curtovirus Reps (48.5-69.0 %); however, in the case of the capsid protein (CP), the extent of similarity is only 39.5-44.5 %. We constructed an agroinfectious clone of TCTV and undertook host range studies on ten plant species; in three species (turnip, sugar beet and cowpea), we detected infection which presents curly top symptoms in turnip and sugar beet. The efficiency of TCTV infection in agroinoculated turnip plants was 71.7 %, and the infection was successfully transmitted to 80 % of the healthy turnip plants used in the insect transmission studies by Circulifer haematoceps under greenhouse conditions. We also determined the genome sequence of 14 new TCTV isolates from southern Iran isolated from turnips. We observed ~13 % diversity amongst all the TCTV isolates and found evidence of recombination in the CP- and Rep-coding regions of the genomes.

Molecular characterization of the Rep protein of the blackgram isolate of Indian mungbean yellow mosaic virus.

The complete nucleotide sequence of the blackgram isolate of mungbean yellow mosaic virus, IMYMV-Bg, which infects legumes in India, was determined and compared at the amino acid level with those of other whitefly-transmitted geminiviruses. The genome organization of IMYMV-Bg was similar to that of the begomoviruses. A unique feature of the genome organization was the sequence divergence of the common region (CR) between DNA-A and DNA-B. In order to understand the mechanism of viral DNA replication, the replication initiator protein, Rep, of IMYMV-Bg was overexpressed in E. coli. The recombinant and refolded Rep bound to CR-sequences of IMYMV-Bg in a specific manner. In this study, evidence is presented for ATP-upregulated cleavage function and ATP-mediated conformational change of Rep. It is hypothesized that, although ATP is not required for cleavage, ATP-mediated conformational changes may result in better access of Rep to the DNA-cleavage site. Evidence is also presented for a site-specific topoisomerase function of Rep, which has not been demonstrated before. The Rep protein can be classified as a type-I topoisomerase because of its nicking activity and sensitivity towards camptothecin, a topoisomerase type-I inhibitor.

Phenotypic recombination between bean common mosaic and bean common mosaic necrosis potyviruses in vivo.

The US-5 strain of bean common mosaic virus (BCMV) and the NL-8 strain of bean common mosaic necrosis virus (BCMNV) are both seedborne potyviruses in common bean (Phaseolus vulgaris L). They have contrasting and highly stable biological characteristics which are genetically controlled. BCMV strain US-5 belongs to pathogenicity group IV. BCMNV strain NL-8 belongs to pathogenicity group III. The two strains have contrasting serological characteristics: NL-8 is serotype A; US-5 is serotype B. When these two strains were maintained separately or as a mixture for more than three years (39 serial transfers) or in more than 100 plants of either of two susceptible hosts, we were unable to isolate a single virus strain that exhibited mutant-like or recombinant-like characteristics. However, within 28 days (during the 1st passage) after these 2 strains were inoculated to opposite primary leaves of bean plants that were susceptible to one virus and resistant to the other, we were able to recover 17 strains that clearly possessed recombinations of various phenotypic characteristics from each of the two "parental" viruses. Three types of phenotypic characteristics were recombined singly or in combination during a single passage in vivo: 1) Biological characteristics known to be controlled by genes for pathogenicity; 2) Serotype; and 3) Temperature-induced hypersensitive vascular necrosis. Each of the phenotypic recombinant strains contained only pathogenicity genes or serological characteristics found in one or both parents. In no case did we isolate a strain that could be described as a random mutation or one that contained pathogenicity or serological characteristics which were not found in at least one parent strain. This is the first known demonstration of phenotypic recombinations between distinct potyviruses in vivo. Implications for the evolution of new virus strains through the use of resistant cultivars and its impact on breeding programs and bean seed production are discussed.

The emergence and dissemination of whitefly-transmitted geminiviruses in Latin America.

The proliferation and rapid dissemination of whitefly-transmitted viruses of important food and industrial crops in Latin America, have been the consequence of drastic changes in traditional cropping systems. Some of the expanding non-traditional cash and export crops, such as soybean and several vegetables, have served as suitable reproductive hosts for the whitefly Bemisia tabaci. This insect pest has been shown to transmit at least 20 different geminiviruses that affect different commercial and basic food crops in Latin America. This review summarizes the existing knowledge on this important group of viruses and their vector in this region.

Genes Ia, II, III, IV and V of Soybean chlorotic mottle virus are essential but the gene Ib product is non-essential for systemic infection.

Soybean chlorotic mottle virus (SbCMV) is the type species of the genus 'Soybean chlorotic mottle-like viruses', within the family Caulimoviridae. The double-stranded DNA genome of SbCMV (8178 bp) contains eight major open reading frames (ORFs). Viral genes essential and non-essential for the replication and movement of SbCMV were investigated by mutational analysis of an infectious 1.3-mer DNA clone. The results indicated that ORFs Ia, II, III, IV and V were essential for systemic infection. The product of ORF Ib was non-essential, although the putative tRNA(Met) primer-binding site in ORF Ib was proved to be essential. Immunoselection PCR revealed that an ORF Ia deletion mutant was encapsidated in primarily infected cells, indicating that ORF Ia is required for virus movement but not for replication. ORF IV was confirmed to encode a capsid protein by peptide sequencing of the capsid. Analysis of the viral transcripts showed that the SbCMV DNA genome gives rise to a pregenomic RNA and an ORF VI mRNA, as shown in the case of Cauliflower mosaic virus.

Complete nucleotide sequence of Sesbania mosaic virus: a new virus species of the genus Sobemovirus.

The complete nucleotide sequence of the Sesbania mosaic virus (SeMV) genomic RNA was determined by sequencing overlapping cDNA clones. The SeMV genome is 4149 nucleotides in length and encodes four potential overlapping open reading frames (ORFs). Comparison of the nucleotide sequence and the deduced amino acid sequence of the four ORFs of SeMV with that of other sobemoviruses revealed that SeMV was closest to southern bean mosaic virus Arkansas isolate (SBMV-Ark, 73% identity). The 5' non-coding regions of SeMV, SBMV and southern cowpea mosaic virus (SCPMV) are nearly identical. However ORF1 of SeMV which encodes for a putative movement protein of M(r) 18370 has only 34% identity with SBMV-Ark. ORF 2 encodes a polyprotein containing the serine protease, genome linked viral protein (VPg) and RNA dependent RNA polymerase domains and shows 78% identity with SBMV-Ark. The N-terminal amino acid sequence of VPg was found to be TLPPELSIIEIP, which mapped to the region 326-337 of ORF2 product and the cleavage site between the protease domain and VPg was identified to be E325-T326. The cleavage site between VPg and RNA dependent RNA polymerase was predicted to be E445-T446 based on the amino acid sequence analysis of the polyprotein from different sobemoviruses. ORF3 is nested within ORF2 in a--1 reading frame. The potential ribosomal frame shift signal and the downstream stem-loop structure found in other sobemoviruses are also conserved in SeMV RNA sequence, indicating that ORF3 might be expressed via--1 frame shifting mechanism. ORF4 encodes the coat protein of SeMV, which shows 76 and 66% identity with SBMV-Ark and SCPMV, respectively. Thus the comparison of the non-coding regions and the ORFs of SeMV with other sobemoviruses clearly revealed that it is not a strain of SBMV. Phylogenetic analysis of six different sobemoviruses, including SeMV, suggests that recombination event is not frequent in this group and that SeMV is a distinct member of the genus sobemovirus. The analysis also shows sobemoviruses infecting monocotyledons and dicotyledons fall into two distinct clusters.

Complete sequences and phylogenetic analyses of a Singapore isolate of broad bean wilt fabavirus.

The complete nucleotide (nt) sequence of a Singapore isolate of broad bean wilt fabavirus from Megakepasma erythrochlamys L., designated BBWV-ME, was determined. Its bipartite genome consisted of two positive-sense single-stranded ribonucleic acids (RNA). RNA1 (5951 nt in length) encoded a putative protease cofactor, nucleotide triphosphate (NTP)-binding domain (helicase), viral genome-linked protein (VPg), protease and RNA-dependent RNA polymerase (RdRp). RNA2 (3607 nt in length) encoded a putative movement protein (MP) and coat proteins (CP). Genome organization of BBWV-ME was similar to other viruses in the Comoviridae family. Phylogenetic analyses showed that fabaviruses were more closely related to the comoviruses than the nepoviruses.

Functional equivalence of late gene promoters in bean golden mosaic virus with those in tomato golden mosaic virus.

In the bipartite geminivirus tomato golden mosaic virus (TGMV), the activity of late gene promoters is up-regulated by the multifunctional viral protein AL2. Cis-acting sequences required for AL2-mediated promoter responses have not been well characterized. However, nucleotide sequence analysis has implicated a motif termed the conserved late element (CLE). The CLE is present in TGMV and many other begomoviruses, although it is not ubiquitous. Here we analysed the regulation of late gene expression in bean golden mosaic virus (BGMV), one of the begomoviruses which lacks the CLE. Transient reporter gene assays showed that BGMV late gene promoters were trans-activated in Nicotiana benthamiana protoplasts, both by the homologous BGMV AL2 protein and by the heterologous TGMV AL2 protein. The BGMV AL2 protein also trans-activated TGMV late gene promoters. Consistent with these results, we found that hybrid viruses with the late gene promoters exchanged between BGMV and TGMV were viable in planta.

Site-specific single amino acid changes to Lys or Arg in the central region of the movement protein of a hybrid bromovirus are required for adaptation to a nonhost.

A hybrid Cowpea chlorotic mottle virus (CCMV) [CCMV(B3a)] in which the CCMV 3a movement protein gene is replaced by the 3a (B3a) gene of Brome mosaic virus cannot infect cowpea systemically. Previously, analysis of RNA3 cDNA clones constructed from cowpea-adapted mutants derived from CCMV(B3a) revealed that a single codon change in the B3a gene allowed CCMV(B3a) to infect cowpea systemically. In this study, to extend the analysis of the CCMV(B3a) adaptation mechanism, we directly sequenced B3a gene RT-PCR products prepared from 28 cowpea plants in which cowpea-adapted mutants appeared, and found seven patterns of a codon change localized at five specific positions in the central region (Ser(118), Glu(132), Glu(138), Gln(178), and Ser(180)). All of the patterns involved an amino acid change to Lys or Arg. Mutational analysis of the B3a gene demonstrated that a single codon change resulting in either Lys or Arg at any of the five positions was sufficient for the adaptation of CCMV(B3a) to cowpea. In contrast, CCMV(B3a) variants with a codon change resulting in Lys or Arg at three other positions (137, 155, and 161) in the B3a gene not only showed lack of systemic infection of cowpea but also showed weakened initial cell-to-cell movement in the inoculated leaves and diminished B3a accumulation in protoplasts. These results suggest that adaptive changes in the B3a gene are site-specifically selected in cowpea plants.

Complete nucleotide sequence and host range of South African cassava mosaic virus: further evidence for recombination amongst begomoviruses.

Complete nucleotide sequences of the DNA-A (2800 nt) and DNA-B (2760 nt) components of a novel cassava-infecting begomovirus, South African cassava mosaic virus (SACMV), were determined and compared with various New World and Old World begomoviruses. SACMV is most closely related to East African cassava mosaic virus (EACMV) in both its DNA-A (85% with EACMV-MH and -MK) and -B (90% with EACMV-UG2-Mld and EACMV-UG3-Svr) components; however, percentage sequence similarities of less than 90% in the DNA-A component allowed SACMV to be considered a distinct virus. One significant recombination event spanning the entire AC4 open reading frame was identified; however, there was no evidence of recombination in the DNA-B component. Infectivity of the cloned SACMV genome was demonstrated by successful agroinoculation of cassava and three other plant species (Phaseolus vulgaris, Malva parviflora and Nicotiana benthamiana). This is the first description of successful infection of cassava with a geminivirus using Agrobacterium tumefaciens.

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.

Bromovirus movement protein conditions for the host specificity of virus movement through the vascular system and affects pathogenicity in cowpea.

Previously, we reported that CCMV(B3a), a hybrid of bromovirus Cowpea chlorotic mottle virus (CCMV) with the 3a cell-to-cell movement protein (MP) gene replaced by that of cowpea-nonadapted bromovirus Brome mosaic virus (BMV), can form small infection foci in inoculated cowpea leaves, but that expansion of the foci stops between 1 and 2 days postinoculation. To determine whether the lack of systemic movement of CCMV(B3a) is due to restriction of local spread at specific leaf tissue interfaces, we conducted more detailed analyses of infection in inoculated leaves. Tissue-printing and leaf press-blotting analyses revealed that CCMV(B3a) was confined to the inoculated cowpea leaves and exhibited constrained movement into leaf veins. Immunocytochemical analyses to examine the infected cell types in inoculated leaves indicated that CCMV(B3a) was able to reach the bundle sheath cells through the mesophyll cells and successfully infected the phloem cells of 50% of the examined veins. Thus, these data demonstrate that the lack of long-distance movement of CCMV(B3a) is not due to an inability to reach the vasculature, but results from failure of the virus to move through the vascular system of cowpea plants. Further, a previously identified 3a coding change (A776C), which is required for CCMV(B3a) systemic infection of cowpea plants, suppressed formation of reddish spots, mediated faster spread of infection, and enabled the virus to move into the veins of inoculated cowpea leaves. From these data, and the fact that CCMV(B3a) directs systemic infection in Nicotiana benthamiana, a permissive systemic host for both BMV and CCMV, we conclude that the bromovirus 3a MP engages in multiple activities that contribute substantially to host-specific long-distance movement through the phloem.

The I gene of bean: a dosage-dependent allele conferring extreme resistance, hypersensitive resistance, or spreading vascular necrosis in response to the potyvirus Bean common mosaic virus.

The resistance to the potyvirus Bean common mosaic virus (BCMV) conferred by the I allele in cultivars of Phaseolus vulgaris has been characterized as dominant, and it has been associated with both immunity and a systemic vascular necrosis in infected bean plants under field, as well as controlled, conditions. In our attempts to understand more fully the nature of the interaction between bean with the I resistance allele and the pathogen BCMV, we carefully varied both I allele dosage and temperature and observed the resulting, varying resistance responses. We report here that the I allele in the bean cultivars we studied is not dominant, but rather incompletely dominant, and that the system can be manipulated to show in plants a continuum of response to BCMV that ranges from immunity or extreme resistance, to hypersensitive resistance, to systemic phloem necrosis (and subsequent plant death). We propose that the particular phenotypic outcome in bean results from a quantitative interaction between viral pathogen and plant host that can be altered to favor one or the other by manipulating I allele dosage, temperature, viral pathogen, or plant cultivar.

Bipartite geminivirus host adaptation determined cooperatively by coding and noncoding sequences of the genome.

Bipartite geminiviruses are small, plant-infecting viruses with genomes composed of circular, single-stranded DNA molecules, designated A and B. Although they are closely related genetically, individual bipartite geminiviruses frequently exhibit host-specific adaptation. Two such viruses are bean golden mosaic virus (BGMV) and tomato golden mosaic virus (TGMV), which are well adapted to common bean (Phaseolus vulgaris) and Nicotiana benthamiana, respectively. In previous studies, partial host adaptation was conferred on BGMV-based or TGMV-based hybrid viruses by separately exchanging open reading frames (ORFs) on DNA A or DNA B. Here we analyzed hybrid viruses in which all of the ORFs on both DNAs were exchanged except for AL1, which encodes a protein with strictly virus-specific activity. These hybrid viruses exhibited partial transfer of host-adapted phenotypes. In contrast, exchange of noncoding regions (NCRs) upstream from the AR1 and BR1 ORFs did not confer any host-specific gain of function on hybrid viruses. However, when the exchangeable ORFs and NCRs from TGMV were combined in a single BGMV-based hybrid virus, complete transfer of TGMV-like adaptation to N. benthamiana was achieved. Interestingly, the reciprocal TGMV-based hybrid virus displayed only partial gain of function in bean. This may be, in part, the result of defective virus-specific interactions between TGMV and BGMV sequences present in the hybrid, although a potential role in adaptation to bean for additional regions of the BGMV genome cannot be ruled out.

Structure of native and expanded sobemoviruses by electron cryo-microscopy and image reconstruction.

Rice yellow mottle virus (RYMV) and southern bean mosaic virus, cowpea strain (SCPMV) are members of the Sobemovirus genus of RNA-containing viruses. We used electron cryo-microscopy (cryo-EM) and icosahedral image analysis to examine the native structures of these two viruses at 25 A resolution. Both viruses have a single tightly packed capsid layer with 180 subunits assembled on a T=3 icosahedral lattice. Distinctive crown-like pentamers emanate from the 12 5-fold axes of symmetry. The exterior face of SCPMV displays deep valleys along the 2-fold axes and protrusions at the quasi-3-fold axes. While having a similar topography, the surface of RYMV is comparatively smooth. Two concentric shells of density reside beneath the capsid layer of RYMV and SCPMV, which we interpret as ordered regions of genomic RNA. In the presence of divalent cations, SCPMV particles swell and fracture, whereas the expanded form of RYMV is stable. We previously proposed that the cell-to-cell movement of RYMV in xylem involves chelation of Ca(2+) from pit membranes of infected cells, thereby stabilizing the capsid shells and allowing a pathway for spread of RYMV through destabilized membranes. In the context of this model, we propose that the expanded form of RYMV is an intermediate in the in vivo assembly of virions.

Development of clover yellow vein virus as an efficient, stable gene-expression system for legume species.

A highly infectious cDNA clone of clover yellow vein virus (pClYVV) was tested as a viral vector, especially for legume species. The genes for green fluorescent protein (GFP) and soybean glutamine synthetase (GS) were inserted between the genes for P1 and HC-Pro on pClYVV to create three recombinant plasmids: pClYVV-GFP, pClYVV-GFP-GS, and pClYVV-GFP:GS. In the former two constructs all the junctions between the inserted proteins contained the sequences of protease cleavage recognition sites, whereas the third construct expressed a fusion of GFP and GS. Western blot analyses showed that GFP and GS appeared to have been precisely excised from the viral polyprotein with the viral proteases (P1 and NIa). Under UV irradiation, green fluorescence was detected in infected broad bean, kidney bean, and soybean plants. The stability of the constructs in the symptomatic tissues was confirmed by RT-PCR and Western blot analyses. The plants expressing GS together with GFP became tolerant to the herbicide glufosinate, and flowered early. As the GS gene, one of the nodulin genes for nitrogen fixation, is expressed in legume species, this system will be useful for examining the function of genes important to legume plants.

Refined structure of desmodium yellow mottle tymovirus at 2.7 A resolution.

Desmodium yellow mottle virus is a 28 nm diameter, T=3 icosahedral plant virus of the tymovirus group. Its structure has been solved to a resolution of 2.7 A using X-ray diffraction analysis based on molecular replacement and phase extension methods. The final R value was 0.151 (R(free)=0.159) for 134,454 independent reflections. The folding of the polypeptide backbone is nearly identical with that of turnip yellow mosaic virus, as is the arrangement of subunits in the virus capsid. However, a major difference in the disposition of the amino-terminal ends of the subunits was observed. In turnip yellow mosaic virus, those from the B and C subunits comprising the hexameric capsomeres formed an annulus about the interior of the capsomere, while the corresponding N termini of the pentameric capsomere A subunits were not visible at all in electron density maps. In Desmodium yellow mottle tymovirus, amino termini from the A and B subunits combine to form the annuli, thereby resulting in a much strengthened association between the two types of capsomeres and an, apparently, more stable capsid. The first 13 residues of the C subunit were invisible in electron density maps. Two ordered fragments of single-stranded RNA, seven and two nucleotides in length, were observed. The ordered water structure of the virus particle was delineated and required 95 solvent molecules per protein subunit.

Complete nucleotide sequence and infectious cDNA clone of the RNA1 of a Chinese isolate of broad bean wilt virus 2.

The nucleotide sequence of the RNA1 of broad bean wilt virus 2 (BBWV2) isolate B935 has been determined from overlapping cDNA clones. It contains 5956 nucleotides in length excluding the 3' terminal poly(A) tail and contains a single long open reading frame (ORF) of 5613 nucleotides extending from nucleotide 234 to 5846. A repeated motif has been found in the 5' non-coding region. The predicted polyprotein encoded by the long ORF is 1870 amino acid in length with a molecular weight of 210 K. Amino acid sequence comparisons between portions of the BBWV2 RNA1-encoded polyprotein and proteins encoded by several species in Comoviridae revealed the putative functions of BBWV2 RNA1-encoded proteins and the same general genetic organization as that of comoviruses and nepoviruses. Based on the determined sequence, full-length cDNA clone of RNA1 designated as pU1FL was constructed. Together with transcripts from full-length cDNA clone of RNA2 (pU2FL), transcripts from pU1FL infected Chenopodium quinoa successfully.

[Cytopathology of plants infected with viruses. Ultrastructure of leguminous leaves infected with red clover mottle virus]. / Tsitopatologiia rastenii pri virusnoi infektsii. Ul'trastruktura kletok list'ev bobovykh, zarazhennykh virusom krapchatosti krasnogo klevera.

We studied ultrathin sections of clover, pea, and cowpea leaves from intact plants and after infection by red clover mottle virus (RCMV, comovirus, 28 nm isometric virions) under an electron microscope. Significant pathological changes were observed in the leaf cells after the infection: RCMV virions accumulated in the cytoplasm and vacuoles, tubular structures containing the virion-like particles, as well as vesicular aggregates were observed. Large membrane-containing inclusions with long tubules (approximately 35 nm in diameter) were found in the cytoplasm of the infected clover chlorenchyma cells. Apparently, these novel inclusions correspond to virus-induced protein synthesis.

Effect of silver reflective mulch and a summer squash trap crop on densities of immature Bemisia argentifolii (Homoptera: Aleyrodidae) on organic bean.

Polyethylene mulch with a reflective silver stripe and a yellow summer squash, Cucurbita pepo L., trap crop were tested alone and in combination as tactics to reduce densities of Bemisia argentifolii Bellows & Perring eggs and nymphs, and incidence of bean golden mosaic geminivirus on snap bean, Phaseolus vulgaris L. Egg densities were consistently higher on squash than on bean, but egg densities and virus incidence were not lower on bean grown with squash than on bean grown in monoculture. Silver reflective mulch reduced egg densities compared with bean grown on bare ground during the first week after crop emergence for 2 of the 3 yr that the study was conducted. However, egg suppression by silver mulch was not enhanced by the presence of a squash trap crop when both tactics were combined. The obstacles to suppressing B. argentifolii through the use of trap crops are discussed.