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.     

Potential gene products of bean golden mosaic virus have higher sequence homologies to those of tomato golden mosaic virus than to those of cassava latent virus

Comparison of the nucleotide sequences of the DNAs of bean golden mosaic virus (BGMV), tomato golden mosaic virus (TGMV) and cassava latent virus (CLV) revealed a fairly close relationship between BGMV DNA1, TGMV DNA1, and CLV DNA1 and a comparatively distant relationship between BGMV DNA2, TGMV DNA2, and CLV DNA2. The 200-base region common to the two DNAs of each virus had little sequence homology, except for a highly conserved 33–36 base sequence potentially capable of forming a stable hairpin structure. All the potential coding regions in the BGMV DNAs had counterparts in the TGMV and CLV DNAs suggesting an overall similarity in genome organization but two potential coding regions in the BGMV DNAs had no counterparts in the TGMV DNAs. The most highly conserved ORFs, BGMV 1R1, TGMV 1R1, and CLV 1R1, are the putative genes for the coat proteins of BGMV, TGMV, and CLV. BGMV 1R1 has 91.9% and 71.6% homology with respect to TGMV and CLV. The ORFs (BGMV 1L1; CLV 1L1; TGMV 1L1) and the two smaller overlapping ORFs (BGMV 1L2, 1L3; TGMV 1L2, 1L3; CLV 1L5, 1L3) are conserved in the three viruses. BGMV 2R1 and BGMV 2L1 have higher homology with respect to TGMV but not with respect to 2R1 and 2L1 in CLV. From this study we conclude that BGMV is more closely related to TGMV than CLV.     

Genetic analysis of tomato golden mosaic virus: ORF AL2 is required for coat protein accumulation while ORF AL3 is necessary for efficient DNA replication

Tomato golden mosaic virus (TGMV) is a geminivirus whose genome is divided between two DNA components, designated A and B. The TGMV genome contains six open reading frames (ORFs) which can encode proteins of greater than 10 kDa. We have used a protoplast transfection system to determine the effects of viral proteins, as defined by these ORFs, on the accumulation of viral DNA in infected cells. The accumulation of cost protein was also examined in leaf discs. Our results indicate that mutations in ORFs AR1 and AL2 do not affect viral double-stranded DNA (dsDNA) levels, although AR1 and AL2 mutants accumulate only small amounts of single-stranded viral DNA (ssDNA). In contrast, a large reduction in both ss- and dsDNA levels is observed when a mutation is introduced into ORF AL3. Mutations within either of the two DNA B ORFs do not affect DNA replication. The AL3, BR1, and BL1 mutants are capable of synthesizing coat protein; however, coat protein is not detected in leaf discs inoculated with AR1 or AL2 mutants. Testable models are proposed to explain the influence of AL2 protein on coat protein accumulation and to account for the stimulation of viral DNA synthesis mediated by the AL3 gene product.     

Inhibition of African cassava mosaic virus systemic infection by a movement protein from the related geminivirus tomato golden mosaic virus.

Plant viruses encode proteins that mediate their movement through the host plant leading to the establishment of a systemic infection. We have analyzed the effect of tomato golden mosaic virus (TGMV) genes BL1 and BR1, which are thought to be involved in the process of virus movement, on the infectivity of African cassava mosaic virus (ACMV) in Nicotiana benthamiana. Recombinant genomes were constructed by replacing the ACMV coat protein coding sequence with those of either BL1 or BR1. Replication of recombinants containing BL1 and BR1 coding sequences in the sense orientation with respect to the coat protein promoter was detected in the inoculated leaves only when the constructs were co-inoculated, suggesting that both genes are being expressed and act in a cooperative manner. Co-inoculated recombinants induced localized symptoms on inoculated leaves but did not spread systematically, either because of a defect in BL1 and/or BR1 expression or due to the inability of the TGMV gene products to functionally complement their ACMV counterparts. Systemic spread of ACMV was inhibited when the recombinant containing the BL1 coding sequence in the sense, but not in the antisense, orientation was co-inoculated with ACMV DNA B. Disruption of the BL1 coding sequence by a frameshift mutation restored the ability of the recombinant to spread systemically, suggesting that the gene product is responsible for the inhibitory effect. The inhibitory phenotype was mimicked by a chimera containing amino-terminal sequences of TGMV BL1 and carboxy-terminal sequences of its ACMV homologue, BC1. The chimera has characteristics of a dominant negative mutant. We suggest that dominant negative mutants of virus movement genes may provide a novel source for virus resistance genes.     

Transactivation in a geminivirus: AL2 gene product is needed for coat protein expression

The beta-glucuronidase (GUS) reporter gene was used to replace the coat protein gene (open reading frame AR1) of tomato golden mosaic virus (TGMV) and transiently expressed in tobacco protoplasts. While these TGMV/GUS genomes gave a high level of GUS activity, genomes which also contained a mutation in the AL2 open reading frame (TGMV/GUS/AL2-) did not express GUS. GUS activity could be restored by cotransfecting protoplasts with the TGMV/GUS/AL2- genome and a wild-type TGMV genome. Thus, the AL2 gene product transactivates expression of TGMV coat protein gene.     

Cloned tomato golden mosaic virus back in tomatoes

Clones of tomato golden mosaic virus (TGMV), a key model for geminivirus research, have been transmitted back to their original host tomato for the first time. In contrast to the high pathogenicity in other solanaceous species, TGMV induced only very mild symptoms: a few chlorotic spots on the leaf lamina for the common variant (formerly strain cs), and limited vein yellowing for the yellow vein variant (yv). Symptoms disappeared over time, though viral DNA remained detectable in newly developed leaves. Both TGMV variants invaded phloem and, occasionally, also mesophyll parenchyma cells in tomato. Complete direct sequencing of rolling circle amplification products of the viral progeny in tomato plants revealed the consensus of the DNA populations for the two genome components (DNA-A, DNA-B) of both TGMV variants. The DNA-A components showed 98.5% and 99.9% nucleotide sequence identity, respectively, with the independently cloned TGMV molecule sequenced initially in 1984, confirming the classification of csTGMV and yvTGMV as variants. The results are discussed with reference to the history of the Brazilian "mosaico dourado" disease in tomato, and the odyssey of TGMV passaging through Nicotiana benthamiana plants and bacteria of numerous laboratories worldwide.     

Restriction map and southern analysis of the bean golden mosaic virus genome

A restriction map showing 5'-3' polarity for two species of circular single-stranded (ss) DNA from bean golden mosaic virus (BGMV) is presented. The two viral single-stranded DNAs, which are very similar in size, were separated by prolonged agarose gel electrophoresis at low voltage. Hybridization analysis using probes made from these separated DNAs shows they are largely dissimilar in nucleotide sequence except for a common region of high homology which has been mapped to a unique location on each species. Molecular clones of BGMV DNA from restriction fragments comprising all of one of the two DNAs and part of the other were used to confirm the restriction and hybridization analysis. These results provide further evidence consistent with the hypothesis that BGMV and other geminiviruses possess divided genomes with two components of nearly equal size.     

The composition of bean golden mosaic virus and its single-stranded DNA genome

Bean golden mosaic virus (BGMV) contains predominantly circular single-stranded DNA, but DNA isolation by treating virus with proteinase K results in conversion of the DNA to a linear form. The conversion is inhibited by as little as 0.025% sodium dodecyl sulfate but not by phenylmethylsulfonyl fluoride and does not occur when purified DNA is incubated with proteinase K. Protein is not involved in the covalent structure of BGMV DNA; the conversion appears to be caused by an endonuclease that copurifies with the virus and is resistant to proteinase K. BGMV contains a major capsid protein with an estimated molecular weight of 27,400. Two minor proteins are found, even in the most highly purified virus preparations; their origin and role in virus structure or function are unknown. BGMV particles contain 19% DNA; therefore each geminate particle must contain a single DNA molecule, of which over 90% are circular. The estimated molecular weight of the virus is 3.8 x 10(6).     

African cassava mosaic virus DI DNA interferes with the replication of both genomic components.

Natural infections of the geminivirus African cassava mosaic virus (ACMV) are known to be associated with low levels of defective interfering (DI) DNAs. Recently it has been demonstrated that extrachromosomal copies of the DI DNA, mobilized and amplified from an integrated DI DNA dimer, can ameliorate ACMV symptoms in transformed Nicotiana benthamiana, providing a possible means for the control of cassava mosaic disease. To further understand the molecular basis of the interference phenomenon, we have compared the ability of ACMV and tomato golden mosaic virus (TGMV) genomic components to replicate in leaf discs derived from DI DNA-transformed and control plants. Results indicate that the ACMV DI DNA interferes with the replication of both genomic components of ACMV to a similar extent. TGMV DNA A replicates to normal levels in transformed leaf discs and plants because it is unable to mobilize and amplify ACMV DI DNA. Differences in the relative levels of ACMV genomic components in transformed leaf discs and plants are discussed in terms of DNA replication and the availability of the genomic components for spread throughout the plant.     

Dual interaction of a geminivirus replication accessory factor with a viral replication protein and a plant cell cycle regulator

Geminiviruses replicate their small, single-stranded DNA genomes through double-stranded DNA intermediates in plant nuclei using host replication machinery. Like most dicot-infecting geminiviruses, tomato golden mosaic virus encodes a protein, AL3 or C3, that greatly enhances viral DNA accumulation through an unknown mechanism. Earlier studies showed that AL3 forms oligomers and interacts with the viral replication initiator AL1. Experiments reported here established that AL3 also interacts with a plant homolog of the mammalian tumor suppressor protein, retinoblastoma (pRb). Analysis of truncated AL3 proteins indicated that pRb and AL1 bind to similar regions of AL3, whereas AL3 oligomerization is dependent on a different region of the protein. Analysis of truncated AL1 proteins located the AL3-binding domain between AL1 amino acids 101 and 180 to a region that also includes the AL1 oligomerization domain and the catalytic site for initiation of viral DNA replication. Interestingly, the AL3-binding domain was fully contiguous with the domain that mediates AL1/pRb interactions. The potential significance of AL3/pRb binding and the coincidence of the domains responsible for AL3, AL1, and pRb interactions are discussed.     

Identification of a novel receptor-like protein kinase that interacts with a geminivirus nuclear shuttle protein

Despite extensive studies in plant virus-host interactions, the molecular mechanisms of geminivirus movement and interactions with host components remain largely unknown. A tomato kinase protein and its soybean homolog were found to interact specifically with the nuclear shuttle protein (NSP) of Tomato golden mosaic virus (TGMV) and Tomato crinkle leaf yellows virus (TCrLYV) through yeast two-hybrid screening and in vitro protein binding assays. These proteins, designated LeNIK (Lycopersicon esculentum NSP-Interacting Kinase) and GmNIK (Glycine max NIK), belong to the LRR-RLK (leucine rich-repeat receptor-like kinase) family that is involved in plant developmental processes and/or resistance response. As such, NIK is structurally organized into characteristic domains, including a serine/threonine kinase domain with a nucleotide binding site at the C-terminal region, an internal transmembrane segment and leucine-rich repeats (LRR) at the N-terminal portion. The potential significance of the NSP-NIK interaction is discussed.     

Geminivirus pathogenicity protein C4 interacts with Arabidopsis thaliana shaggy-related protein kinase AtSKeta, a component of the brassinosteroid signalling pathway

Beet curly top virus (BCTV) C4 interacted with two members of the shaggy-related protein kinase family (AtSKeta and AtSKzeta) and a putative leucine-rich repeat receptor-like kinase (LRR-RLK) in a yeast two-hybrid assay. Tomato golden mosaic virus (TGMV) AC4 also bound with similar efficiency to AtSKeta and AtSKzeta but was unable to interact with the LRR-RLK. BCTV C4 interaction with AtSKeta was confirmed using an in vitro binding assay. The protein kinases were capable of autophosphorylation in vitro and AtSKeta phosphorylated BCTV C4 at threonine and serine residues. AtSKeta phosphorylation of TGMV AC4 was significantly less efficient. The LRR-RLK did not efficiently phosphorylate BCTV C4. BCTV C4 localisation to the cell periphery in Nicotiana benthamiana was dependent on an intact N-terminal myristoylation motif, consistent with plasma membrane targeting. The intact motif was also required to produce the wild-type disease phenotype. Transient expression of BCTV C4 and TGMV AC4 derivatives in N. benthamiana identified additional amino acids within a central domain that contribute to the phenotype. The interaction with AtSKeta indicates that BCTV C4 interacts with the brassinosteroid signalling pathway.     

The size and topology of single-stranded DNA from bean golden mosaic virus

The single-stranded DNA (ssDNA) of bean golden mosaic virus (BGMV) was resolved into two components by polyacrylamide gel (2.5%) electrophoresis in 8 M urea and by rate-zonal sedimentation in 5-20% alkaline sucrose density gradients. Viral DNA, either unfractionated or fractionated and electroeluted from polyacrylamide gels, was spread for electron microscopic examination. Unfractionated DNA and the slower of the two fractionated components contained predominantly circular molecules. The faster component consisted entirely of linear molecules. Circular molecules had contour lengths corresponding to 8.0 x 10(5) daltons.     

Chromosomal location of endogenous geminivirus-related DNA sequences inNicotiana tabacum L.

TheN. tabacum (tobacco) nuclear genome carries approximately 25 multiple direct repeats of a geminivirus-related DNA (GRD) sequence that probably arose by illegitimate recombination, following geminivrus infection, duringNicotiana evolution. Each GRD repeat carries sequences similar to the geminiviralAL1 gene of the tomato golden mosaic virus (TGMV), encoding a protein required for viral DNA replication, plus thecis-essential replication origin. Using a cloned 14-kb GRD repeat sequence as a probe for fluorescencein situ hybridization (FISH), we identified a unique tobacco chromosome carrying GRD. Translocations between chromosomes of the tobacco S and T genomes were used as physical markers by sequentially hybridizing chromosomes with labelled GRD and total genomic DNA fromN. sylvestris (equivalent to the S genome). The 25S, 18S and 5.8S ribosomal gene clusters were detected in double-labelling experiments for use as additional markers to identify the chromosomal location of GRD. GRD occupies one site on a homologous pair of small submetacentrics from the T genome characterized by a lack of either translocated segments from the S genome or ribosomal genes. GRD provides an additional marker for the small chromosomes of the T genome and a useful phylogenetic tool.