Cassava latent virus infections mediated by the Ti plasmid of Agrobacterium tumefaciens containing either monomeric or dimeric viral DNA

Plant infections with cassava latent virus (CLV) were mediated by the Ti plasmid of Agrobacterium tumefaciens containing either monomeric or dimeric copies of the virus genome. The CLV DNAs caused typical symptoms when they were inoculated in Agrobacterium strains C58, LBA4404 and a virE mutant A1026, but not other Agrobacterium strains with mutations in other vir loci or an E. coli polA strain. Virus-specific DNA forms characteristic of normal CLV infections were found after such infection. Characterization of progeny CLV DNA from selected plants identified several infectious mutants. These were found to be small insertions and/or deletions in the coat protein gene of DNA 1 and in the intergenic region of DNA 2.     

Characterisation of DNA forms associated with cassava latent virus infection.

In addition to the major encapsidated DNA species found in preparations of cassava latent virus (genomic DNAs 1 and 2) there are minor DNA populations of twice (dimeric) and approximately half genome length. Both minor species resemble the genomic DNAs in that they are composed of predominantly circular single-stranded DNA. All of these size groups have a corresponding covalently-closed circular double-stranded DNA form in infected tissue. Infectivity studies using cloned DNAs 1 and 2 show that dimeric DNA routinely appears, suggesting it to be an intermediate in the DNA replicative cycle that can be encapsidated at low efficiency. In contrast, half unit length DNA has not yet been detected after multiple passaging of virus derived from the cloned DNA inoculum. Half unit length DNAs appear to be derived exclusively from DNA 2 and consist of a population of molecules exhibiting a relatively specific deletion. As they have an inhibitory effect on virus multiplication, their encapsidated forms are analogous to defective interfering particles associated with other eukaryotic DNA containing viruses. Small primer molecules associated with the genomic single-stranded DNAs, as reported for another geminivirus, have not been detected in CLV.     

Molecular characterisation of subgenomic single-stranded and double-stranded DNA forms isolated from plants infected with tomato golden mosaic virus.

A subgenomic single-stranded DNA present in particles of the geminivirus, tomato golden mosaic virus, has been shown by electron microscope heteroduplex mapping and Southern hybridisation analysis to consist of circular molecules, ca. 1.2 kb in size, derived from the smaller of the two genomic DNA components, DNA B, by deletion of open reading frame (ORF) BR1 and the C-terminal portion of ORF BL1. A covalently closed circular, supercoiled, double-stranded form of the subgenomic DNA has been isolated from virus-infected plants and cloned into pEMBL9. Analysis of the sequence of 22 clones across the deletion boundaries revealed only four different deletion boundaries, derived from four different left hand borders and three different right hand borders. Each border was within a region of 11 nucleotides and gave rise to a narrow size range (1248-1261 nucleotides) for the population of 22 subgenomic DNAs. However apparently smaller subgenomic DNAs were sometimes formed when plants were inoculated with cloned subgenomic DNA, or a construct derived from a subgenomic DNA in which a neomycin phosphotransferase gene had been inserted, together with the genomic DNA components. Mechanisms to account for the size, specificity and formation of the subgenomic DNA are discussed.     

The Cysteine Pairs in CLV2 are Not Necessary for Sensing the CLV3 Peptide in Shoot and Root Meristems

Receptor-like proteins (RLPs) are involved in both plant defense and developmental processes. Previous genetic and biochemical studies show that the leucine-rich repeat (LRR) receptor-like protein CLAVATA2 (CLV2) functions together with CLAVATA1 (CLV1) and CORYNE (CRN) in Arabidopsis to limit the stem cell number in shoot apical meristem, while in root it acts with CRN to trigger a premature differentiation of the stem cells after sensing the exogenously applied peptides of CLV3p, CLE19p or CLE40p. It has been proposed that disulfide bonds might be formed through two cysteine pairs in the extracellular LRR domains of CLV1 and CLV2 to stabilize the receptor complex. Here we tested the hypothesis by replacing these cysteines with alanines and showed that depletions of one or both of the cysteine pairs do not hamper the function of CLV2 in SAM maintenance. In vitro peptide assay also showed that removal of the cysteine pairs did not affect the perception of CLV3 peptides in roots. These observations allow us to conclude that the formation of disulfide bonds is not needed for the function of CLV2.     

The nucleotide sequence of cloned wheat dwarf virus DNA

Restriction analysis and cloning of virus-specific double-stranded DNA isolated from plants infected with wheat dwarf virus (WDV) indicated that the virus genome, like that of maize streak virus (MSV), consists of a single DNA circle. The complete nucleotide sequence of cloned WDV DNA (2749 nucleotides) has been determined. Comparison of the potential coding regions in WDV DNA with those in the DNA of two strains of MSV suggests that these viruses encode at least two functional proteins, the coat protein read in the virion (+) DNA sense and a composite protein, formed from two open reading regions, in the complementary (-) DNA sense. Although WDV and MSV are serologically unrelated their coat proteins showed 35% direct amino acid sequence and their DNAs showed 46% nucleotide sequence homology. There was too little homology between the DNAs of WDV and those of two geminiviruses with bipartite genomes, cassava latent virus (CLV) and tomato golden mosaic virus (TGMV), to align the sequences. However comparison of the amino acid sequences of predicted proteins of WDV, MSV, TGMV and CLV revealed clear relationships between these viruses and suggested that the monopartite and the bipartite geminiviruses have a common ancestral origin. Four inverted repeat sequences which have the potential to form hairpin structures of deltaG >/= -14 kcal/mol were detected in WDV DNA. The sequence TAATATTAC present in the loop of one of these hairpins is conserved in similar putative structures in MSV DNA and in both DNA components of CLV and TGMV and may function as a recognition sequence for a protein involved in virus DNA replication.     

DNA forms of the geminivirus African cassava mosaic virus consistent with a rolling circle mechanism of replication.

We have analysed DNA from African cassava mosaic virus (ACMV)-infected Nicotiana benthamiana by two-dimensional agarose gel electrophoresis and detected ACMV-specific DNAs by blot-hybridisation. ACMV DNA forms including the previously characterised single-stranded, open-circular, linear and supercoiled DNAs along with five previously uncharacterised heterogeneous DNAs (H1-H5) were resolved. The heterogeneous DNAs were characterised by their chromatographic properties on BND-cellulose and their ability to hybridise to strand-specific and double-stranded probes. The data suggest a rolling circle mechanism of DNA replication, based on the sizes and strand specificity of the heterogeneous single-stranded DNA forms and their electrophoretic properties in relation to genome length single-stranded DNAs. Second-strand synthesis on a single-stranded virus-sense template is evident from the position of heterogeneous subgenomic complementary-sense DNA (H3) associated with genome-length virus-sense template (VT) DNA. The position of heterogeneous virus-sense DNA (H5), ranging in size from one to two genome lengths, is consistent with its association with genome-length complementary-sense template (CT) DNA, reflecting virus-sense strand displacement during replication from a double-stranded intermediate. The absence of subgenomic complementary-sense DNA associated with the displaced virus-sense strand suggests that replication proceeds via an obligate single-stranded intermediate. The other species of heterogeneous DNAs comprised concatemeric single-stranded virus-sense DNA (H4), and double-stranded or partially single-stranded DNA (H1 and H2).     

RPK2 is an essential receptor-like kinase that transmits the CLV3 signal in Arabidopsis

The shoot apical meristem (SAM) is the fundamental structure that is located at the growing tip and gives rise to all aerial parts of plant tissues and organs, such as leaves, stems and flowers. In Arabidopsis thaliana, the CLAVATA3 (CLV3) pathway regulates the stem cell pool in the SAM, in which a small peptide ligand derived from CLV3 is perceived by two major receptor complexes, CLV1 and CLV2-CORYNE (CRN)/SUPPRESSOR OF LLP1 2 (SOL2), to restrict WUSCHEL (WUS) expression. In this study, we used the functional, synthetic CLV3 peptide (MCLV3) to isolate CLV3-insensitive mutants and revealed that a receptor-like kinase, RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2), also known as TOADSTOOL 2 (TOAD2), is another key regulator of meristem maintenance. Mutations in the RPK2 gene result in stem cell expansion and increased number of floral organs, as seen in the other clv mutants. These phenotypes are additive with both clv1 and clv2 mutations. Moreover, our biochemical analyses using Nicotiana benthamiana revealed that RPK2 forms homo-oligomers but does not associate with CLV1 or CLV2. These genetic and biochemical findings suggest that three major receptor complexes, RPK2 homomers, CLV1 homomers and CLV2-CRN/SOL2 heteromers, are likely to mediate three signalling pathways, mainly in parallel but with potential crosstalk, to regulate the SAM homeostasis.     

Molecular cloning of the Harvey sarcoma virus circular DNA intermediates. II. Further structural analyses.

Three species of unintegrated supercoiled Harvey sarcoma virus DNA (6.6, 6.0, and 5.4 kilobase pairs) have been molecularly cloned from Harvey sarcoma virus-infected cells. On the basis of restriction enzyme analyses, the 6.6- and 6.0-kilobase pair viral DNAs contain two and one copies, respectively, of a 650-base pair DNA segment which contains sequences present at the 3' and 5' termini of the viral genome. R-loop structures formed between Moloney leukemia virus RNA and the cloned Harvey sarcoma virus DNA indicated that about 500 base pairs of the 650-base pair repeating segment was complementary to the 3' end of the viral RNA. During amplification in the Escherichia coli host, some recombinants containing the 6.6- or the 6.0-kilobase pair Harvey sarcoma virus DNA insert acquired or lost the complete 650-base pair DNA segment. These changes occurred in both recA+ and recA- E. coli.     

Electron microscopy of amplified DNA forms in antifolate-resistant Leishmania

Three independently derived antifolate-resistant Leishmania major cell lines overproduce the bifunctional protein thymidylate synthase-dihydrofolate reductase (TS-DHFR) by amplification of a region of DNA (R-region DNA) that contains the gene for TS-DHFR. On orthogonal-field-alteration gel electrophoresis (OFAGE), the extrachromosomal R-region DNAs are circular molecules, and different forms of R-region DNA within these cell lines are resolved. The R-region DNAs migrate aberrantly on OFAGE with respect to linear DNA and supercoiled plasmid standards. We describe a method for the isolation of these R-region DNA forms from OFAGE. By electron microscopy, we show that the extrachromosomal elements are single supercoiled circular DNA molecules, and are predominantly circular monomers and dimers of the original R-region DNA amplification unit. Using OFAGE, an analysis of cloned isolates shows that individual cells may contain multiple forms of R-region DNA. Furthermore, within a given cell line, certain distinguishable forms appear to have the same size and restriction map, suggesting they may be topoisomers. The multiple forms of R-region DNA are in a dynamic state in the antifolate-resistant populations, and the relative amount of DNA in each form as well as the number of forms within each cell line change through time. As currently understood, the generation of amplified R-region DNA in L. major is summarized.     

A model study of the role of proteins CLV1, CLV2, CLV3, and WUS in regulation of the structure of the shoot apical meristem

In order to elucidate the role of proteins CLV1, CLV2, CLV3, and WUS in the mechanism underlying the maintenance of compartmental structure (spatial arrangement of the zones of biosynthesis of marker proteins) of the shoot apical meristem, a model of such mechanism was developed. Computational experiments led to biologically plausible solutions only when synthesis of substance W in a space between the organizing center and meristem apex was limited by the mechanism based on interaction of CLV3 with membrane receptor CLV1/CLV2 and lower boundary of the zone of W synthesis was determined by isoline of the corresponding threshold level of substance Y concentration. The model of the “reaction-diffusion” type formalizing the role proteins CLV1/CLV2, CLV3, and WUS can describe the basis of the mechanism underlying regulation of the compartmental structure of the shoot apical meristem and positioning of the organizing center in a certain site of the cell ensemble of such meristem.     

Isolation and characterization of faithful and altered clones of the genomes of cauliflower mosaic virus isolates Cabb B-JI, CM4-184, and Bari I

Full-length genomes of cauliflower mosaic virus (CaMV) isolates Cabb B-JI, CM4-184, and Bari I have been cloned in the SalGI site of plasmid pAT 153. The cloned DNAs were characterized by restriction mapping and infectivity assays. All the sites present in the virion DNAs were found in the cloned DNAs. Comparison of restriction maps with those of DNA from two other isolates which have been recently completely sequenced revealed a close relationship among the different isolates. Some of the clones appear to be faithful copies of the viral genomes and these viral inserts are infectious when inoculated into turnip plants. Various clones with deletions in the CaMV DNA have been isolated and characterized. Some of them may correspond to deletions naturally occurring in a subpopulation of the virus whereas others occurred during cloning. None of the deleted fragments are infectious when inoculated into plants. Strikingly, all the deletions overlap one or two of the specific single-stranded breaks characteristic of caulimoviruses, suggesting that sequences surrounding the breaks are not dispensable.     

A model study of the role of proteins CLV1, CLV2, CLV3, and WUS in regulation of the structure of the shoot apical meristem

In order to elucidate the role of proteins CLV1, CLV2, CLV3, and WUS in the mechanism underlying the maintenance of compartmental structure (spatial arrangement of the zones of biosynthesis of marker proteins) of the shoot apical meristem, a model of such mechanism was developed. Computational experiments led to biologically plausible solutions only when synthesis of substance W in a space between the organizing center and meristem apex was limited by the mechanism based on interaction of CLV3 with membrane receptor CLV1/CKV2 and lower boundary of the zone of W synthesis was determined by isoline of the corresponding threshold level of substance Y concentration. The model of the "reaction-diffusion" type formalizing the role proteins CLV1, CLV2, CLV3, and WUS can describe the basis of the mechanism underlying regulation of the compartmental structure of the shoot apical meristem and positioning of the organizing center in a certain site of the cell ensemble of such meristem.     

Demonstration of the bipartite nature of the genome of a single-stranded DNA plant virus by infection with the cloned DNA components

Linear double-stranded (ds)DNA, obtained by excision of the cloned A and B components of tomato golden mosaic virus (TGMV) from recombinant plasmids, was found to infect plants and to elicit symptoms identical to those obtained with TGMV or TGMV DNA. Progeny virus isolated from plants infected with cloned DNA was infective and indistinguishable from TGMV on the basis of (a) its circular single-stranded (ss)DNA genome, (b) its capsid polypeptide, (c) its particle morphology and (d) serological identity. Southern blot analysis of DNA extracted from cells infected with cloned DNA, or TGMV DNA, revealed the same intracellular ss and dsDNA species, represented in both A and B components, except for a subgenomic, possibly defective, DNA, which was not detected in infections with cloned DNA. Infection with cloned DNA was achieved when cloned A and B components were both present, but not with either cloned A or B components separately. TGMV is the first DNA virus for which unequivocal proof of a bipartite genome has been obtained.     

Infectious mutants of cassava latent virus generated in vivo from intact recombinant DNA clones containing single copies of the genome.

Intact recombinant DNAs containing single copies of either component of the cassava latent virus genome can elicit infection when mechanically inoculated to host plants in the presence of the appropriate second component. Characterisation of infectious mutant progeny viruses, by analysis of virus-specific supercoiled DNA intermediates, indicates that most if not all of the cloning vector has been deleted, achieved at least in some cases by intermolecular recombination in vivo between DNAs 1 and 2. Significant rearrangements within the intergenic region of DNA 2, predominantly external to the common region, can be tolerated without loss of infectivity suggesting a somewhat passive role in virus multiplication for the sequences in question. Although packaging constraints might impose limits on the amount of DNA within geminate particles, isolation of an infectious coat protein mutant defective in virion production suggests that packaging is not essential for systemic spread of the viral DNA.     

Regulation of CLV3 expression by two homeobox genes in Arabidopsis

The ability of meristems to continuously produce new organs depends on the activity of their stem cell populations, which are located at the meristem tip. In Arabidopsis, the size of the stem cell domain is regulated by two antagonistic activities. The WUS (WUSCHEL) gene, encoding a homeodomain protein, promotes the formation and maintenance of stem cells. These stem cells express CLV3 (CLAVATA3), and signaling of CLV3 through the CLV1/CLV2 receptor complex restricts WUS activity. Homeostasis of the stem cell population may be achieved through feedback regulation, whereby changes in stem cell number result in corresponding changes in CLV3 expression levels, and adjustment of WUS expression via the CLV signal transduction pathway. We have analyzed whether expression of CLV3 is controlled by the activity of WUS or another homeobox gene, STM (SHOOT MERISTEMLESS), which is required for stem cell maintenance. We found that expression of CLV3 depends on WUS function only in the embryonic shoot meristem. At later developmental stages, WUS promotes the level of CLV3 expression, together with STM. Within a meristem, competence to respond to WUS activity by expressing CLV3 is restricted to the meristem apex.