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.     

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.     

Computer analysis identifies sequence homologies between potential gene products of Maize Streak Virus and those of Cassava Latent Virus and Tomato Golden Mosaic Virus

Summary The amino acid sequences of the putative polypeptides of maize streak virus (MSV) have been systematically compared with those of cassava latent virus (CLV) and tomato golden mosaic virus (TGMV) using the programme DIAGON (8).Conserved sequences have been detected between peptides encoded by the complementary (-) sense of MSV and those of CLV and TGMV, viz; the 40 200 M_r polypeptide of CLV-1 (3) and the 40 285 M_r polypeptide of TGMV-A (4) show extensive homologies with the 17 768 M_r and 31 388 M_r polypeptides of MSV (6).Distant and variable homologies have been detected between the putative coat protein of MSV when compared with those of CLV and TGMV. No other relationships between the potential gene products of MSV and those of CLV and TGMV have been detected.The extensive homologies detected between the complementary sense encoded peptides suggest that they are derived from functional genes, and that the directly conserved sequences may contain amino acids essential to the function of these proteins. The less extensive homologies among the putative coat proteins are considered in relation to their possible structures and functions.     

Immunocytochemical investigation of the causal agent of cassava mosaic disease in southern Africa

Cassava mosaic disease (CMD) exists throughout Africa, and cassava latent virus (CLV) has been implicated as the etiological agent in Kenya and West Africa. However, in Southern Africa, the causal agent of CMD was not until recently associated with CLV, and the possibility of a second flexuous virus particle has not been ignored. Attempts to isolate and visualize CLV antigen have been successful with Nicotiana benthamiana, an indicator host plant of CLV, but all efforts to isolate and visualize particles in infected cassava plants have failed. Immunocytochemical studies were undertaken in an attempt to localize virus antigen in infected cassava tissue.Cytochemical staining (light microscope) of infected cassava leaf material revealed the presence of inclusion bodies in epidermal and palaside mesophyll cells, and in epidermal collenchyma and outer parenchyma cells from the petiole and stem. However, transmission electron-microscopical (TEM) investigations revealed electron dense bodies in the cytoplasm, and no characteristic CLV nuclear inclusion bodies were evident. Transmission experiments to N. benthamiana and N. tabacum were attempted and leaves, exhibiting symptoms, examined microscopically. The nuclei appeared swollen (in comparison to uninfected leaves), a characteristic of CLV- infected N. benthamiana. However at the TEM level, no characteristic fibrillar-ring inclusion bodies or particles, could be visualized.Further immunocytochemical investigations were initiated, employing antisera raised against CLV isolated from N. benthamiana, and antisera for cassava common mosaic virus (CCMV), cassava brown streak virus (CBSV) and cassava X virus (CsXV). Goat anti-rabbit IgG-gold was used as a direct stain. No labelling occurred with CCMV and CBSV antisera. Intense gold labelling was located in the cytoplasm of phloem, mesophyll and epidermal cells of infected cassava and to a lesser extent in N. tabacum and N. benthamiana using affinity chromatography purified CLV antiserum. Little labelling was observed in nuclei of infected cells. Inconclusive results were obtained with CsXV antiserum.Immunogold labelling located CLV viral antigens in infected cassava leaf tissue. This observation, together with positive ELISA, transmission and DNA hybridization experiments, proves conclusively that CLV viral antigen is present in infected cassava in Southern Africa. However, most viral antigen in infected cassava, unlike N. benthamiana (fibrillar and granular nuclear inclusions) appears to be in the cytoplasm. This may tentatively suggest that the CLV protein is synthesized in the cytoplasm of its natural host, cassava, even though the virus may assemble in the nucleus at the appropriate time. However, as yet no virus inclusions have been observed in nuclei of infected cassava. Due to previous isolation of a flexuous rod and ambiguous staining results, the possibility of two viruses in cassava cannot be ruled out.     

The nucleotide sequence of an infectious clone of the geminivirus beet curly top virus

A number of infectious clones of a Californian isolate of the leafhopper-transmitted geminivirus beet curly top virus (BCTV) have been constructed from virus-specific double-stranded DNA isolated from infected Beta vulgaris and used to demonstrate a single component genome. The nucleotide sequence of one infectious clone has been determined (2993 nucleotides). Comparison with other geminiviruses has shown that the organisation of the genome closely resembles DNA 1 of the whitefly-transmitted members. The four conserved coding regions of DNA 1 have highly homologous counterparts in BCTV with the exception of the putative coat protein which is more closely related to those of the leafhopper-transmitted geminiviruses suggesting a strong interrelationship between coat protein and insect vector. A BCTV component equivalent to DNA 2 is not required for virus infection or transmission and has not been isolated from infected plants.     

Orthologs of the Arabidopsis CLAVATA1 Gene in the Cultivated Brassicaceae Plants

In Arabidopsis thaliana, the CLAVATA1 (CLV1) gene is involved in maintaining the balance between the stem cells in the central zone of the stem apical meristem and the determined cells at its periphery. However, CLV1 has not been previously characterized in other Brassicaceae. Using the direct amplification of genomic DNA, we obtained a full-length CLV1 ortholog from canola plants (Brassica napus), and also three CLV1 fragments from rape (B. rapa), canola (B. napus), and false flax (Camelina sativa), which corresponded to the transmembrane domain and a part of the kinase domain of the CLAVATA1 protein. The nucleotide and deduced amino acid sequences of the full-size CLV1 ortholog from B. napus were similar by 81 and 87% to the prototype gene from arabidopsis; in the case of shorter gene fragments, the similarity was as high as 91–93 and 98%, respectively. By their primary structure, the CLV1 genes in the Brassicaceae considerably differ from their putative structural homologs beyond this family.     

Delimitation of essential genes of cassava latent virus DNA 2

Insertion and deletion mutagenesis of both extended open reading frames (ORFs) of cassava latent virus DNA 2 destroys infectivity. Infectivity is restored by coinoculating constructs that contain single mutations within different ORFs. Although frequent intermolecular recombination produces dominant parental-type virus, mutants can be retained within the virus population indicating that they are competent for replication and suggesting that rescue can occur by complementation of trans acting gene products. By cloning specific fragments into DNA 1 coat protein deletion vectors we have delimited the DNA 2 coding regions and provide substantive evidence that both are essential for virus infection. Although a DNA 2 component is unique to whitefly-transmitted geminiviruses, the results demonstrate that neither coding region is involved solely in insect transmission. The requirement for a bipartite genome for whitefly-transmitted geminiviruses is discussed.     

The coat protein of potato virus X is a strain-specific elicitor of Rx1-mediated virus resistance in potato

The Rx1 gene in potato confers extreme resistance to potato virus X (PVX). To investigate the mechanism and elicitation of Rx resistance, protoplasts of potato cv. Cara (Rx1 genotype) and Maris Bard (rx1 genotype) were inoculated with PVX and tobacco mosaic virus (TMV). At 24 h post-inoculation in Maris Bard protoplasts there was at least 100-fold more PVX RNA than in protoplasts of Cara. TMV RNA accumulated to the same level in both types of protoplast. However, when the TMV was inoculated together with PVX the accumulation of TMV RNA was suppressed in the Cara (Rx1 genotype) protoplasts to the same extent as PVX. The Rx1 resistance also suppressed accumulation of a recombinant TMV in which the coat protein gene was replaced with the coat protein gene of PVX. It is therefore concluded that Rx1-mediated resistance is elicited by the PVX coat protein, independently of any other proteins encoded by PVX. The domain of the coat protein with elicitor activity was localized by deletion and mutation analysis to the structural core of a non-virion form of the coat protein.     

Orthologs of arabidopsis CLAVATA 1 gene in cultivated Brassicaceae plants

In arabidopsis (Arabidopsis thaliana), the CLAVATA1 (CLV1) gene is involved in maintaining the balance between the stem cells in the central zone of the stem apical meristem and the determined cells at its periphery. However, CLV1 has not been previously characterized in other Brassicaceae. Using the direct amplification of genomic DNA, we obtained a full-length CLV1 ortholog from canola plants (Brassica napus), and also three CLV1 fragments from rape (B. rapa), canola (B. napus), and false flax (Camelina sativa), which corresponded to the transmembrane domain and a part of the kinase domain of the CLAVATA1 protein. The nucleotide and deduced amino acid sequences of the full-size CLV1 ortholog from B. napus were similar by 81 and 87% to the prototype gene from arabidopsis; in the case of shorter gene fragments, the similarity was as high as 91-93 and 98%, respectively. By their primary structure, the CLV1 genes in the Brassicaceae considerably differ from its putative structural homologs beyond this family.     

Negative and positive regulation in trans of gene expression from adeno-associated virus vectors in mammalian cells by a viral rep gene product.

We previously described use of the human parvovirus, adeno-associated virus (AAV), as a vector for transient expression in mammalian cells of the gene for chloramphenicol acetyltransferase (CAT). In the AAV vector, pTS1, the CAT gene is expressed under the control of the major AAV promoter p40. This promoter is embedded within the carboxyl-terminal region of an open reading frame (orf-1) which codes for a protein (rep) required for AAV DNA replication. We show here that the rep product has additional trans-acting properties to regulate gene expression. First, deletion or frame-shift mutations in orf-1, which occurred far upstream of p40, increased expression of CAT in human 293 (adenovirus-transformed) cells. This increased CAT expression was abolished when such mutant AAV vectors were transfected into 293 cells together with a second AAV vector which could supply the wild-type AAV rep product in trans. Thus, an AAV rep gene product was a negative regulator, in trans, of expression of CAT in uninfected 293 cells. In adenovirus-infected 293 cells, the function of the AAV rep product was more complex, but in some cases, it appeared to be a trans activator of the expression from p40. In HeLa cells, only trans activation by rep was seen in the absence or presence of adenovirus. Neither activation nor repression by the rep product required replication per se of the AAV vector DNA. Thus, trans-acting negative or positive regulation of gene expression by the AAV rep gene is modulated by factors in the host cell and by the helper adenovirus.     

Function of the 30 kd protein of tobacco mosaic virus: involvement in cell-to-cell movement and dispensability for replication

We have investigated the function of the 30 kd protein of tobacco mosaic virus (TMV) by a reverse genetics approach. First, a point mutation of TMV Ls1 (a temperature-sensitive mutant defective in cell-to-cell movement), that causes an amino acid substitution in the 30 kd protein, was introduced into the parent strain, TMV L. The generated mutant showed the same phenotype as TMV Ls1, and therefore the one-base substitution in the 30 kd protein gene adequately explains the defectiveness of TMV Ls1. Next, four kinds of frame-shift mutants were constructed, whose mutations are located at three different positions of the 30 kd protein gene. All the frame-shift mutants were replication-competent in protoplasts but none showed infectivity on tobacco plants. From these observations the 30 kd protein was confirmed to be involved in cell-to-cell movement. To clarify that the 30 kd protein is not necessary for replication, two kinds of deletion mutants were constructed; one lacking most of the 30 kd protein gene and the other lacking both the 30 kd and coat protein genes. Both mutants replicated in protoplasts and the former still produced the subgenomic mRNA for the coat protein. These results clearly showed that the 30 kd protein, as well as the coat protein, is dispensable for replication and that no cis-acting element for replication is located in their coding sequences. It is also suggested that the signal for coat protein mRNA synthesis may be located within about 100 nucleotides upstream of the initiation codon of the coat protein gene.     

Conversion in the requirement of coat protein in cell-to-cell movement mediated by the cucumber mosaic virus movement protein

Plant viruses have movement protein (MP) gene(s) essential for cell-to-cell movement in hosts. Cucumber mosaic virus (CMV) requires its own coat protein (CP) in addition to the MP for intercellular movement. Our present results using variants of both CMV and a chimeric Brome mosaic virus with the CMV MP gene revealed that CMV MP truncated in its C-terminal 33 amino acids has the ability to mediate viral movement independently of CP. Coexpression of the intact and truncated CMV MPs extremely reduced movement of the chimeric viruses, suggesting that these heterogeneous CMV MPs function antagonistically. Sequential deletion analyses of the CMV MP revealed that the dispensability of CP occurred when the C-terminal deletion ranged between 31 and 36 amino acids and that shorter deletion impaired the ability of the MP to promote viral movement. This is the first report that a region of MP determines the requirement of CP in cell-to-cell movement of a plant virus.     

The Arabidopsis CLAVATA2 gene encodes a receptor-like protein required for the stability of the CLAVATA1 receptor-like kinase.

The CLAVATA2 (CLV2) gene regulates both meristem and organ development in Arabidopsis. We isolated the CLV2 gene and found that it encodes a receptor-like protein (RLP), with a presumed extracellular domain composed of leucine-rich repeats similar to those found in plant and animal receptors, but with a very short predicted cytoplasmic tail. RLPs lacking cytoplasmic signaling domains have not been previously shown to regulate development in plants. Our prior work has demonstrated that the CLV1 receptor-like kinase (RLK) is present as a disulfide-linked multimer in vivo. We report that CLV2 is required for the normal accumulation of CLV1 protein and its assembly into protein complexes, indicating that CLV2 may form a heterodimer with CLV1 to transduce extracellular signals. Sequence analysis suggests that the charged residue in the predicted transmembrane domain of CLV2 may be a common feature of plant RLPs and RLKs. In addition, the chromosomal region in which CLV2 is located contains an extremely high rate of polymorphism, with 50 nucleotide and 15 amino acid differences between Landsberg erecta and Columbia ecotypes within the CLV2 coding sequence.