Draft Genome Sequence of Xylella fastidiosa subsp. fastidiosa Strain Stag's Leap.

ITALIC! Xylella fastidiosasubsp. ITALIC! fastidiosacauses Pierce's disease of grapevine. Presented here is the draft genome sequence of the Stag's Leap strain, previously used in pathogenicity/virulence assays to evaluate grapevine germplasm bearing Pierce's disease resistance and a phenotypic assessment of knockout mutants to determine gene function.

Effects of citrus and avocado irrigation and nitrogen-form soil amendment on host selection by adult Homalodisca vitripennis (Hemiptera: Cicadellidae).

Host plant water status is thought to influence dispersal of the xylophagous leafhopper Homalodisca vitripennis Germar, especially where plants are grown under high evaporative demand. Preference by adult H. vitripennis for plants grown under different water deficit and nitrogen form fertilization regimens was studied under laboratory conditions. Leafhopper abundance and ovipositional preference were studied on potted 'Washington navel' orange and 'Haas' avocado in cage choice tests, and feeding rate was estimated using excreta produced by insects confined on plants. A similar study compared responses to citrus treated with 1:1 and 26:1 ratios of fertigated nitrate-N to ammonium-N. The insects were more abundant, oviposited, and fed significantly more on surplus-irrigated plants than on plants under moderate continuous deficit irrigation except avocado feeding, which was nearly significant. Plants exposed to drought became less preferred after 3 and 7 d in avocado and citrus, respectively. Citrus xylem fluid tension at this point was estimated at 0.93 MPa. A corresponding pattern of decline in feeding rate was observed on citrus, but on avocado, feeding rate was low overall and not statistically different between treatments. No statistical differences in abundance, oviposition, or feeding were detected on citrus fertigated with 26:1 or 1:1 ratios of nitrate-N to ammonium-N. Feeding occurred diurnally on both plant species. Discussion is provided on the potential deployment of regulated deficit irrigation to manage H. vitripennis movement as part of a multitactic effort to minimize the risk of disease outbreaks from Xylella fastidiosa Wells et al. in southern California agriculture.

Substitution of conserved cysteine residues in wheat streak mosaic virus HC-Pro abolishes virus transmission by the wheat curl mite.

Substitutions in the amino-proximal region of wheat streak mosaic virus (WSMV) HC-Pro were evaluated for effects on transmission by the wheat curl mite (Aceria tosichella Keifer). Alanine substitution at cysteine residues 16, 46 and 49 abolished vector transmission. Although alanine substitution at Cys(20) had no effect, substitution with arginine reduced vector transmission efficiency. Random substitutions at other positions (Lys(7) to Asn, Asn(19) to Ile, and Arg(45) to Lys) did not affect vector transmission. These results suggest that a zinc-finger-like motif (His(13)-X2-Cys(16)-X29-Cys(46)-X2-Cys(49)) in WSMV HC-Pro is essential for vector transmission.

Genetic and Phenotypic Variation of the Pepper golden mosaic virus Complex.

ABSTRACT Three isolates of the bipartite begomovirus Pepper golden mosaic virus (PepGMV) were characterized for genomic and biological properties. The complete nucleotide sequences of the DNA-A and DNA-B components were determined from infectious clones of PepGMV-Serrano (PepGMV-Ser), PepGMV-Mosaic (PepGMV-Mo), and PepGMV-Distortion (PepGMV-D). Nucleotide sequence identity among PepGMV components ranged from 91 to 96% for DNA-A and from 84 to 99% for DNA-B, with each PepGMV component most closely related to the corresponding component of Cabbage leaf curl virus (CaLCV). However, phylogenetic relationships among begomovirus components were incongruent because DNA-A of PepGMV and CaLCV share an inferred evolutionary history distinct from that of DNA-B. The cloned components of PepGMV-Ser, -Mo, and -D were infectious by biolistic inoculation to pepper but differed in symptom expression: PepGMV-Ser exhibited a bright golden mosaic, PepGMV-Mo produced a yellow-green mosaic, and PepGMV-D caused only a mild mosaic and foliar distortion followed by a "recovery" phenotype in which leaves developing after initial symptom expression appeared normal. Differences in symptoms also were observed on tomato, tobacco, and Datura stramonium. Progeny virus derived from clones of PepGMV-Ser and -Mo were transmitted from pepper to pepper by the B biotype of Bemisia tabaci; progeny virus derived from PepGMV-D clones was not transmissible by the B biotype. Reassortant genomes derived from heterologous DNA components of the three isolates were infectious in all possible pairwise combinations, with symptom phenotype in pepper determined by the DNA-B component. Collectively, these results indicate that the three virus isolates examined may be considered distinct strains of PepGMV that have the capacity to exchange genetic material.

Genome sequences of Agropyron mosaic virus and Hordeum mosaic virus support reciprocal monophyly of the genera Potyvirus and Rymovirus in the family Potyviridae.

Assignment of mite-transmitted species to the genus Rymovirus (family Potyviridae) has changed several times, and the status of the genus has been questioned. To address this issue, complete genome sequences of the rymoviruses Agropyron mosaic virus (AgMV) and Hordeum mosaic virus (HoMV) were determined. AgMV (9540 nucleotides) and HoMV (9463 nucleotides) each encode a single polyprotein with proteinase cleavage sites demarcating protein products characteristic of monopartite species of the family Potyviridae. Of the described species of Potyviridae, AgMV and HoMV are most closely related to each other (68.5% nucleotide and 71.6% amino acid sequence identity) and equidistant (about 53% nucleotide and about 49% amino acid sequence identity) from a third rymovirus, Ryegrass mosaic virus (RGMV). Phylogenetic analyses by neighbor joining, maximum parsimony, and Bayesian inference each grouped the three Rymovirus species in an exclusive clade distinct from a clade containing 34 species of the genus Potyvirus. Because AgMV, HoMV, and RGMV share a reciprocal monophyletic relationship with species of the genus Potyvirus and are divergent in sequence and type of vector, the genus Rymovirus should be retained as a taxonomic unit within the family Potyviridae.

Complete nucleotide sequence of Oat necrotic mottle virus: a distinct Tritimovirus species (family Potyviridae) most closely related to Wheat streak mosaic virus.

The RNA genome (9346 nucleotides) of Oat necrotic mottle virus (ONMV) was cloned and sequenced. Complete genome comparisons indicated that ONMV, currently classified as a rymovirus, was most closely related ( approximately 73% nucleotide, approximately 79% amino acid identity) to the tritimovirus Wheat streak mosaic virus (WSMV). ONMV encoded a single polyprotein, with proteinase cleavage sites very similar to those of WSMV. Pairwise comparison of ONMV and WSMV cistrons revealed that P3 was most conserved ( approximately 79% nucleotide, approximately 86% amino acid), whereas HC-Pro was most divergent ( approximately 67% nucleotide, approximately 69% amino acid). In contrast, the ONMV sequence was distantly related ( approximately 40% nt, approximately 26% amino acid identity) to that of the rymoviru s Ryegrass mosaic virus, with highest sequence conservation noted within the NIb cistron ( approximately 47% nucleotide, approximately 41% amino acid identity). These results firmly establish that ONMV is not a rymovirus but is instead a distinct species of the genus Tritimovirus.

Structure and temporal dynamics of populations within wheat streak mosaic virus isolates.

Variation within the Type and Sidney 81 strains of wheat streak mosaic virus was assessed by single-strand conformation polymorphism (SSCP) analysis and confirmed by nucleotide sequencing. Limiting-dilution subisolates (LDSIs) of each strain were evaluated for polymorphism in the P1, P3, NIa, and CP cistrons. Different SSCP patterns among LDSIs of a strain were associated with single-nucleotide substitutions. Sidney 81 LDSI-S10 was used as founding inoculum to establish three lineages each in wheat, corn, and barley. The P1, HC-Pro, P3, CI, NIa, NIb, and CP cistrons of LDSI-S10 and each lineage at passages 1, 3, 6, and 9 were evaluated for polymorphism. By passage 9, each lineage differed in consensus sequence from LDSI-S10. The majority of substitutions occurred within NIa and CP, although at least one change occurred in each cistron except HC-Pro and P3. Most consensus sequence changes among lineages were independent, with substitutions accumulating over time. However, LDSI-S10 bore a variant nucleotide (G(6016)) in NIa that was restored to A(6016) in eight of nine lineages by passage 6. This near-global reversion is most easily explained by selection. Examination of nonconsensus variation revealed a pool of unique substitutions (singletons) that remained constant in frequency during passage, regardless of the host species examined. These results suggest that mutations arising by viral polymerase error are generated at a constant rate but that most newly generated mutants are sequestered in virions and do not serve as replication templates. Thus, a substantial fraction of variation generated is static and has yet to be tested for relative fitness. In contrast, nonsingleton variation increased upon passage, suggesting that some mutants do serve as replication templates and may become established in a population. Replicated mutants may or may not rise to prominence to become the consensus sequence in a lineage, with the fate of any particular mutant subject to selection and stochastic processes such as genetic drift and population growth factors.

Contributions of genetic drift and negative selection on the evolution of three strains of wheat streak mosaic tritimovirus.

Genome sequences of three Wheat streak mosaic virus (WSMV) strains were compared. The Type and Sidney 81 strains of WSMV from the American Great Plains were closely related, with sequence identities of 97.6% (nucleotide) and 98.7% (amino acid). In contrast, the El Batán 3 strain from central Mexico was divergent, and shared only 79.2-79.3% (nucleotide) and 90.3-90.5% (amino acid) sequence identity with Type and Sidney 81. All three WSMV strains were serologically related, however the El Batán 3 capsid protein (CP) had 15 fewer amino acid residues. Phylogenetic analysis of the CP cistron indicated that Type, Sidney 81, and nine other American isolates of WSMV were closely related and distinct from the El Batán 3 sequence. Nucleotide substitutions among the WSMV strains were not randomly distributed across the genome with more variation within P1, HC-Pro, and CP, and less within P3. One 400-nucleotide region of the genome, corresponding to the 3'-end of P3, was strikingly deficient in silent substitutions. Nonetheless, the ratio of synonymous to non-synonymous substitutions throughout the genome was essentially the same for all three WSMV strains. Collectively, our data indicate that both genetic drift and negative selection have contributed to the evolution of WSMV strains.

Three distinct mechanisms facilitate genetic isolation of sympatric wheat streak mosaic virus lineages.

Cross-protection and vector transmission bottlenecks have been proposed as mechanisms facilitating genetic isolation of sympatric viral lineages. Molecular markers were used to monitor establishment and resolution of mixed infections with genetically defined strains of wheat streak mosaic virus (WSMV). Two closely related WSMV strains from the U.S. (Type and Sidney 81) exhibited reciprocal cross-protection in wheat, confirming this classic phenomenon as a mechanism of genetic isolation. In contrast, cross-protection between either U.S. strain and the divergent El Batán 3 strain from Mexico was unilateral, erratic, and only partially effective. Distribution of WSMV strains within individual leaves of plants supporting a mixed infection of Type and Sidney 81 was spatially nonuniform. Strain distribution among individual tillers of coinfected plants also was heterogeneous, with some containing either Type or Sidney 81 alone and some containing both. Transmission by wheat curl mites, acquiring virus from source plants simultaneously infected with both Type and Sidney 81, often resulted in test plants bearing only a single WSMV strain. Spatial subdivision of virus strains within coinfected plants likely contributed to vector transmission bottlenecks during acquisition. Collectively, these three distinct mechanisms enhance genetic isolation of individual viral lineages, and together with stochastic processes, may explain generation and maintenance of genetic diversity in field populations.

A plant virus vector for systemic expression of foreign genes in cereals.

Inserts bearing the coding sequences of NPT II and beta-glucuronidase (GUS) were placed between the nuclear inclusion b (NIb) and coat protein (CP) domains of the wheat streak mosaic virus (WSMV) polyprotein ORF. The WSMV NIb-CP junction containing the nuclear inclusion a (NIa) protease cleavage site was duplicated, permitting excision of foreign protein domains from the viral polyprotein. Wheat, barley, oat and maize seedlings supported systemic infection of WSMV bearing NPT II. The NPT II insert was stable for at least 18-30 days post-inoculation and had little effect on WSMV CP accumulation. Histochemical assays indicated the presence of functional GUS protein in systemically infected wheat and barley plants inoculated with WSMV bearing GUS. The GUS constructs had greatly reduced virulence on both oat and maize. RT-PCR indicated that the GUS insert was subject to deletion, particularly when expressed as a GUS-NIb protein fusion. Both reporter genes were expressed in wheat roots at levels comparable to those observed in leaves. These results clearly demonstrate the utility of WSMV as a transient gene expression vector for grass species, including two important grain crops, wheat and maize. The results further indicate that both host species and the nature of inserted sequences affect the stability and expression of foreign genes delivered by engineered virus genomes.

Multiple interactions among proteins encoded by the mite-transmitted wheat streak mosaic tritimovirus.

The genome organization of the mite-transmitted wheat streak mosaic virus (WSMV) appears to parallel that of members of the Potyviridae with monopartite genomes, but there are substantial amino acid dissimilarities with other potyviral polyproteins. To initiate studies on the functions of WSMV-encoded proteins, a protein interaction map was generated using a yeast two-hybrid system. Because the pathway of proteolytic maturation of the WSMV polyprotein has not been experimentally determined, random libraries of WSMV cDNA were made both in DNA-binding domain and activation domain plasmid vectors and introduced into yeast. Sequence analysis of multiple interacting pairs revealed that interactions largely occurred between domains within two groups of proteins. The first involved interactions among nuclear inclusion protein a, nuclear inclusion protein b, and coat protein (CP), and the second involved helper component-proteinase (HC-Pro) and cylindrical inclusion protein (CI). Further immunoblot and deletion mapping analyses of the interactions suggest that subdomains of CI, HC-Pro, and P1 interact with one another. The two-hybrid assay was then performed using full-length genes of CI, HC-Pro, P1, P3, and CP, but no heterologous interactions were detected. In vitro binding assay using glutathione-S-transferase fusion proteins and in vitro translation products, however, revealed mutual interactions among CI, HC-Pro, P1, and P3. The failure to detect interactions between full-length proteins by the two-hybrid assay might be due to adverse effects of expression of viral proteins in yeast cells. The capacity to participate in multiple homomeric and heteromeric molecular interactions is consistent with the pleiotropic nature of many potyviral gene mutants and suggests mechanisms for regulation of various viral processes via a network of viral protein complexes.

Chino del tomate virus:Relationships to Other Begomoviruses and Identification of A-Component Variants that Affect Symptom Expression.

Phylogenetic and distance analyses place Chino del tomate virus (CdTV) in the New World clade of begomoviruses and indicate that CdTV and Tomato leaf crumple virus (TLCrV) are closely related strains of the same virus. One cloned CdTV A component (pCdTV-H6), when inoculated to tomato with the B component (pCdTV-B52), produced mild symptoms and low DNA titers. Another cloned CdTV A component (pCdTV-H8), when coinoculated to tomato with the B component, produced moderate leaf curling and veinal chlorosis similar to that of TLCrV. Coinoculation of both CdTV A components and the B component to tomato produced wild-type chino del tomate (CdT) disease symptoms consisting of severe leaf curling, veinal and interveinal chlorosis, and stunting. The two CdTV A components were nearly identical, except at nucleotide positions 1,722 and 2,324. The polymorphism at nucleotide 1,722 resulted in a change at Rep amino acid 261. The second polymorphism at nucleotide 2,324 resulted in changes at Rep amino acid 60 and AC4 amino acid 10. Two chimeric A components constructed by reciprocal exchange of a fragment bearing the polymorphic site at nucleotide 1,722 were evaluated for symptom phenotype. One chimeric A component (pCdTV-H86) produced wild-type CdT symptoms when coinoculated to tomato with the B component. The reciprocal chimeric A component (pCdTV-H68), when coin-oculated to tomato with the B component, also produced severe leaf curling, veinal chlorosis, and stunting. However, pCdTV-H68 induced less obvious interveinal chlorosis than wild-type or pCdTV-H86. Examination of A component genotypes recovered from tomato coinoculated with pCdTV-H6 and pCdTV-H8 indicated that recombination occurred to produce a genotype identical to pCdTV-H86. These results indicate that subtle genotypic variation has significant effects on symptom expression and may explain phenotypic differences observed among isolates and cloned DNAs of CdTV and TLCrV.

Fully biologically active in vitro transcripts of the eriophyid mite-transmitted wheat streak mosaic tritimovirus.

ABSTRACT Infectious RNA of wheat streak mosaic virus (WSMV) has been produced using a full-length cDNA clone as a template for in vitro transcription with SP6 RNA polymerase. Infectivity was dependent on the use of template plasmid DNA that had not undergone spontaneous rearrangement during amplification in Escherichia coli. The presence of WSMV in systemically infected wheat plants inoculated with in vitro transcripts was confirmed by reverse-transcription polymerase chain reaction of the WSMV P3 gene and by accumulation of WSMV coat protein as detected by immunoblotting. Maintenance of the full-length WSMV cDNA in the high copy number plasmid pUC18 was problematic because of spontaneous rearrangement of WSMV sequences during growth in liquid media for more than 8 h or if the clone was subcultured. Stability of the WSMV cDNA clone was improved by the use of the low copy number plasmid pACYC177, and it could be grown in large scale volumes (up to 1 liter) of liquid culture for 14 h without noticeable rearrangements. Both the original WSMV culture and the progeny virus derived from infectious in vitro transcripts were efficiently transmitted by the natural eriophyid mite vector Aceria tosichella. This is the first report of infectious in vitro transcripts for any eriophyid mite-transmitted plant virus and represents the only monopartite member of the family Potyviridae infecting monocotyledonous hosts for which infectious in vitro transcripts are available.

Biotic, molecular, and phylogenetic characterization of bean calico mosaic virus, a distinct begomovirus species with affiliation in the squash leaf curl virus cluster.

ABSTRACT Bean calico mosaic virus (BCMoV), a whitefly-transmitted geminivirus from Sonora, Mexico, was purified, and the genome components were cloned and sequenced. Purified viral fractions and cloned genome components were infectious by biolistic inoculation to bean, completing Koch's postulates for both. The B biotype of the whitefly Bemisia tabaci efficiently transmitted both native virus and progeny virus derived from cloned DNA inoculum. Host ranges of native virus and of progeny virus derived from cloned DNA were identical based upon whitefly and biolistic mediated transmission, respectively. BCMoV has a relatively wide experimental host range among begomoviruses known to infect bean, encompassing genera and species within the Fabaceae, Malvaceae, and Solanaceae. BCMoV has a bipartite genome, as do other New World begomoviruses. BCMoV DNA-A shared highest nucleotide sequence identities with squash leaf curl virus-E strain (SLCV-E) and cabbage leaf curl virus (CaLCV) at 80.1 and 80.7%, respectively. BCMoV DNA-B shared highest nucleotide sequence identity with SLCV-E at 70.7%. The common region (CR) sequences of BCMoV and SLCV-E are 73 to 76% identical; however, modular cis-acting elements within the CR involved in replication origin function and recognition are 100% conserved. Phy-logenetic analysis indicated that BCMoV DNA-A shares a most recent common ancestor with the DNA-A of two viruses that also occur in the Sonoran Desert, SLCV-E and Texas pepper virus (TPV-TAM), and CaLCV from Florida. In contrast, a phylogenetic analysis indicated that BCMoV DNA-B shares a most recent common ancestor with SLCV-E; whereas DNA-B of CaLCV clustered in a separate clade with pepper hausteco virus. Collectively, biological and molecular characteristics indicate that BCMoV is a distinct begomovirus species with the northernmost distribution of any begomovirus isolated from bean in the Americas. Furthermore, the phylogenetic relationships of begomovirus cognate components are not necessarily identical, suggesting that DNA-A and DNA-B of some begomoviruses may have different evolutionary histories.

Replication Specificity Elements of the Worland Strain of Beet Curly Top Virus Are Compatible with Those of the CFH Strain But Not Those of the Cal/Logan Strain.

ABSTRACT Cloned genomes of the CFH, Worland, and Cal/Logan strains of beet curly top virus (BCTV) served as helper viruses to trans-replicate defective (D) DNAs that are incapable of self-replication due to deletions within the C1 open reading frame encoding the replication initiator (Rep) protein. The Logan Rep protein could trans-replicate a Logan-derived D DNA in a transient replication assay conducted in Nicotiana benthamiana leaf disks. However, the Logan Rep protein was unable to trans-replicate D DNAs derived from the CFH or Worland strains. In contrast, the Rep proteins of the CFH and Worland strains could trans-replicate CFH or Worland D DNAs, but not a Logan D DNA. These results indicate that the cis- and trans-acting replication specificity elements of the CFH and Worland strains are compatible and that the three strains of BCTV may be divided into two groupings based upon replication specificity determinants. A comparison of amino acid sequences of the Rep protein for the three BCTV strains suggests that the trans-acting replication specificity element may reside in one or more of 12 amino acid residues that are identical; in two amino acid residues that are chemically similar among the CFH and Worland Rep proteins, yet are different in the Logan Rep protein; or in both. Properties including replication specificity, nucleotide sequence identity, and symptom expression were used as criteria to propose separate species designations for each of the three BCTV strains. In this proposal, the Cal/ Logan strain retains the name BCTV, CFH and the closely related Iranian isolate are designated beet severe curly top virus, and Worland is designated beet mild curly top virus.

Phylogenetic relationships within the family potyviridae: wheat streak mosaic virus and brome streak mosaic virus are not members of the genus rymovirus.

ABSTRACT The complete nucleotide sequence of wheat streak mosaic virus (WSMV) has been determined based on complementary DNA clones derived from the 9,384-nucleotide (nt) RNA of the virus. The genome of WSMV has a 130-nt 5' leader and 149-nt 3'-untranslated region and is polyadenylated at the 3' end. WSMV RNA encodes a single polyprotein of 3,035 amino acid residues and has a deduced genome organization typical for a member of the family Potyviridae (5'-P1/HC-Pro/P3/6K1/CI/6K2/VPg-NIa/NIb/CP-3'). Because WSMV shares with ryegrass mosaic virus (RGMV) the biological property of transmission by eriophyid mites, WSMV has been assigned to the genus Rymovirus, of which RGMV is the type species. Phylogenetic analyses were conducted with complete polyprotein or NIb protein sequences of 11 members of the family Potyviridae, including viruses of monocots or dicots and viruses transmitted by aphids, whiteflies, and mites. WSMV and the monocot-infecting, mite-transmitted brome streak mosaic virus (BrSMV) are sister taxa and share a most recent common ancestor with the whitefly-transmitted sweet potato mild mottle virus, the type species of the proposed genus "Ipomovirus." In contrast, RGMV shares a most recent common ancestor with aphid-transmitted species of the genus Potyvirus. These results indicate that WSMV and BrSMV should be classified within a new genus of the family Potyviridae and should not be considered species of the genus Rymovirus.

Genotypic diversity of beet curly top virus populations in the Western United States.

ABSTRACT The genotypic diversity of beet curly top virus (BCTV) present in the western United States has been examined by the analysis of 58 field isolates and eight laboratory or nursery isolates of the virus. Full-length clones for each isolate have been characterized for genotype by restriction endonuclease mapping. The results indicate that most of the genotypes examined may be classified as variants of the CFH, Worland, or Cal/Logan strains of BCTV. Two genotypes were recovered that appear to share certain genotypic markers of both Worland and CFH strains. Genotypic variants of the CFH and Worland strains and the two genotypes sharing markers of both strains were recovered from field isolates collected during 1994 and 1995. In contrast, the Cal/Logan strain was recovered only from isolates maintained in laboratories or nurseries. Comparisons of restriction endonuclease maps of cloned BCTV genomes revealed considerable variability both within and between strains. Although a total of 43 distinct genotypes of BCTV were identified, only 36 (84%) were recovered from field isolates. Of 37 field isolates for which more than a single clone was recovered, 16 (43%) contained more than a single genotype of one strain, whereas 4 (11%) harbored mixed infections of the CFH and Worland strains. A phylogenetic analysis using 43 characters derived from restriction endonuclease mapping data supported the grouping of 41 genotypes into three taxa consistent with the three currently recognized strains of BCTV. The relationships of the two genotypes sharing genotypic markers of both the Worland and CFH strains to other BCTV genotypes was unresolved in the phylogenetic analysis. Based on the mild symptom phenotype of the isolates from which these two genotypes were recovered and the presence of Worland genotypic markers in portions of the genome containing both cis- and trans-acting elements determining replication specificity, these two genotypes were tentatively considered as variants of the Worland strain.

The strain-specific cis-acting element of beet curly top geminivirus DNA replication maps to the directly repeated motif of the ori.

Strains of beet curly top geminivirus (BCTV) possess distinct cis- and trans-acting replication specificity elements which are not separately interchangeable among strains. Analysis of the replication competency of chimeric BCTV genomes, in which portions of the origin of DNA replication (ori) were derived from heterologous BCTV strains, have permitted identification of an essential cis-acting element governing strain-specific replication in a subgroup II geminivirus. Our studies indicate that the cis-acting element responsible for strain-specific replication properties resides within the directly repeated motif of the BCTV ori. Transient replication assays conducted in leaf disks and complementation experiments conducted in whole plants indicated that the trans-acting replication specificity element, residing within the amino-terminal region of the C1 Rep protein, may recognize and replicate a chimeric BCTV genome containing a heterologous ori so long as all or portions of the core element of the directly repeated motif are derived from the same strain as the Rep protein. As Rep protein binding to the core element of the directly repeated motif has been demonstrated by others to be essential for replication of subgroup III geminiviruses, our results support the hypothesis that replication specificity of subgroup II viruses is governed by processes similar to that of subgroup III viruses. However, a second cis-acting element of the ori, which appears to contribute to subgroup III virus replication specificity, does not seem to be required for replication specificity among the subgroup II viruses examined. Nonetheless, a potential role for a second cis-acting element in the BCTV ori contributing to maximal replication cannot be excluded.

Horseradish curly top virus is a distinct subgroup II geminivirus species with rep and C4 genes derived from a subgroup III ancestor.

The complete nucleotide sequence (3080 nt) of an infectious DNA clone derived from the geminivirus horseradish curly top virus (HrCTV) has been determined. The relationship of HrCTV to other geminiviruses was examined using dot matrix plots of nucleotide sequence similarities, and by phylogeny of predicted amino acid sequences of individual ORFs based upon parsimony or neighbour-joining methods. These analyses indicate that the V1 and V2 virion sense ORFs of HrCTV are most closely related to, yet distinct from, the corresponding ORFs of the subgroup II geminivirus beet curly top virus (BCTV). HrCTV also encodes a third virion sense ORF (V3) which is similar (72-74 percent amino acid identity) to the BCTV V3 ORF; however, the HrCTV V3 ORF has diverged in sequence to a greater extent relative to that observed among isolates of BCTV (98-100% amino acid identity). The HrCTV genome encodes only three complementary sense ORFs (Cl, C2 and C4) and lacks a C3 ORF which is conserved among all other subgroup II and III geminiviruses characterized to date. Although the neighbour-joining analysis indicated that the HrCTV C2 ORF was distantly related to the C2 ORF of BCTV, the predicted amino acid sequence deduced from the HrCTV C2 ORF lacks the characteristic zinc-finger domain present in the transcriptional activating protein (TrAP) encoded by the subgroup III ORF AC2, which is also retained within the TrAP-related product of the BCTV C2 ORF. Surprisingly, the rep and C4 proteins encoded by HrCTV share a closer phylogenetic relationship to the corresponding proteins of the subgroup III geminivirus squash leaf curl virus (SLCV) than to BCTV. These results suggest that the HrCTV genome may have arisen by a recombination event between a BCTV-like subgroup II virus ancestor and an SLCV-like subgroup III virus ancestor. Possible mechanisms that may explain recombination events among geminiviruses are discussed.