J. W. Randles

A NAC Domain Protein Interacts with Tomato leaf curl virus Replication Accessory Protein and Enhances Viral Replication

Geminivirus replication enhancer (REn) proteins dramatically increase the accumulation of viral DNA species by an unknown mechanism. In this study, we present evidence implicating SlNAC1, a new member of the NAC domain protein family from tomato (Solanum lycopersicum), in Tomato leaf curl virus (TLCV) REn function. We isolated SlNAC1 using yeast (Saccharomyces cerevisiae) two-hybrid technology and TLCV REn as bait, and confirmed the interaction between these proteins in vitro. TLCV induces SlNAC1 expression specifically in infected cells, and this upregulation requires REn. In a transient TLCV replication system, overexpression of SlNAC1 resulted in a substantial increase in viral DNA accumulation. SlNAC1 colocalized with REn to the nucleus and activated transcription of a reporter gene in yeast, suggesting that in healthy cells it functions as a transcription factor. Together, these results imply that SlNAC1 plays an important role in the process by which REn enhances TLCV replication.

A two-dimensional electrophoretic technique for the detection of circular viroids and virusoids

A new gel electrophoretic technique for the rapid and sensitive detection of circular viroids and virusoids is described. Starting from plant material, a typical multisample analysis requires less than 8 h. Viroid concentrations as low as 60 ng/g tissue can be detected unambiguously without the use of radioactivity or highly specialized laboratory equipment. The technique presented here is compared to earlier methods of gel electrophoresis, nucleic acid fingerprinting, and currently employed hybridization techniques. A number of important technical advantages, including speed, simplicity, and sensitivity, suggest that the methods described here may have wide utility in checking the spread of viroid infections.

Nucleotide sequence of a circular single-stranded DNA associated with coconut foliar decay virus

A circular single-stranded (ss) covalently closed (ccc) DNA associated with coconut foliar decay virus (CFDV) was purified, amplified by the polymerase chain reaction, and subcloned and its sequence established by analysis of overlapping subgenomic cDNA clones. The complete CFDV sequence comprised 1291 nucleotides and contained open reading frames for six proteins of molecular weight larger than 5 kDa. One of these (ORF1, 33.4 kDa) codes for a leucine-rich protein with the nucleoside triphosphate-binding motif GXGKS and may possibly participate in virus replication. The putative viral protein encoded by ORF3 (6.4 kDa) is a positively charged arginine-rich protein with homology to the capsid protein of nuclear polyhedrosis virus, and may represent the CFDV coat protein. CFDV DNA can form a stable stem structure of 10 GC base pairs subtending a loop sequence which in one orientation closely resembles the motif TAATATTAC conserved in a similar structural arrangement within the geminivirus group. Otherwise no sequence homology to DNA-containing plant viruses of the gemini- or caulimovirus groups was found. CFDV therefore represents a new taxonomic group of plant viruses.

A proposed scheme for viroid classification and nomenclature.

Viroids are the only class of autonomously replicating subviral pathogens whose molecular structure is well defined. Their structural and functional properties, as well as their evolutionary origin differ fundamentally from those of viruses (see below), posing specific problems for the classification of these subviral pathogens. For example, some of the criteria suggested by the Executive Committee of the International Committee on Taxonomy of Viruses (ICTV) for the definition of species, which include those regarding morphological characteristics, protein characteristics and antigenic properties, are not applicable to viroids. The purpose of this note is to present a consensus taxonomic proposal (see Fig. 1) that has emerged from consultations among the members of the Viroid Study Group of the Plant Virus Subcommittee of the ICTV. This proposal updates and expands the previous classification, which appeared in the Sixth Report of the ICTV [9], and is open to discussion and suggestions.

Studies on encapsidated viroid-like RNA I. Characterization of velvet tobacco mottle virus

Velvet tobacco mottle virus (VTMoV) isolated from Nicotiana velutina growing wild in arid Central Australia was transmitted by inoculation to a limited number of plant species of which N. clevelandii was the most convenient experimental host. The virus was also transmitted from field-grown plants toN. velutina and N. clevelandii by the mirid Cyropeltis nicotianae. VTMoV preparations purified by clarification with organic solvents and differential centrifugation contained polyhedral particles about 30 nm in diameter sedimenting as a single component at about 115 S. The particles were shown to be located in the nucleus, cytoplasm, and vacuoles of infected plant cells. Virus dissociated in the presence of mercaptoethanol and sodium dodecyl sulfate (SDS) separated into one major and two minor polypeptides with estimated molecular weights of 33,000, 36,000 and 31,000, respectively. Single-stranded RNA isolated from VTMoV by extraction with phenol was separated into five components with apparent molecular weights of 1.5 x 10(6), 0.63 x 10(6), 0.25 x 10(6), 0.16 x 10(6), and 0.12 x 10(6) referred to as RNAs 1, 1a, 1b, 2, and 3, respectively. It appears that RNAs 1a and 1b are breakdown products of RNA 1, as shown elsewhere, and electron microscopic examination of the other species showed that whereas RNAs 1 and 3 are linear molecules, RNA 2 is circular. The similarity of RNAs 2 and 3 to the RNA of viroids is discussed. VTMoV has been compared with several RNA plant viruses with small polyhedral particles. Only solanum nodiflorum mottle virus appears to share some of its unique features and the two have been shown to be antigenically related.

Some properties of purified cucumber mosaic virus (Q strain).

Abstract Strain Q of cucumber mosiac virus (QCMV) from Queensland, Australia, contains 18% ribonucleic acid (RNA) which has a base ratio (moles %) of adenine 22.4%, guanine 24.7%, cytosine 22.8%, and uracil 30.1%. The molecular weight of the virus is approximately 5.8 × 10 6 , based on a sedimentation coefficient of 98.6 S and a diffusion coefficient of 1.20 × 10 7 cm 2 per second. Hence the virus contains about 1.0 × 10 6 molecular weight units of RNA. The electrophoretic mobility of QCMV is −7.2 × 10 −5 cm 2 per volt per second at pH 9 and −4.0 × 10 −5 cm 2 per volt per second at pH 7.4. Electron micrographs of QCMV preparations show particles about 28 mμ in diameter when stained in phosphotungstic acid (PTA) or uranyl acetate and about 42 mμ when shadowed with gold-palladium. Partially purified QCMV in 0.005 M borate buffer, pH 9, remained infectious for several months when stored at 0–4°C. Such preparations retained some infectivity after heating between 70 and 80° for 10 minutes whereas freshly extracted sap from infected cucumber plants was completely inactivated after 10 minutes at 60°. QCMV precipitated in the pH range 4.3–5.5. At 0°C, QCMV was unaffected by the presence of NaCl at concentrations up to about 0.25 M , but was rapidly precipitated on mild heating. QCMV is unstable and slowly disrupts in 2% PTA, pH 7. It is also degraded in 1 M CaCl 2 and 2 M LiCl and the RNA precipitates quantitatively leaving most of the protein in solution. CaCl 2 -precipitated QCMV-RNA could not be dissolved in neutral buffers, but the RNA precipitated with LiCl was readily resuspended and shown to be several times more infectious than phenol-extracted RNA. QCMV has been shown to be antigenically similar to the Y strain of CMV (CMV-Y) from North America. However, these two strains appear to differ in some respects which are discussed.

Protein composition of tomato spotted wilt virus

Abstract Analysis of the protein composition of tomato spotted wilt virus (TSWV), purified by an improved procedure, by polacrylamide gel electrophoresis, revealed three major structural proteins (of MW 84,000, 50,000, and 29,000d) and a minor one of MW 220,000d. The three major proteins constitute about 98% of the total viral protein and all three were shown to be glycoproteins. One of the major proteins (MW 29,000d) and the minor protein were shown to be associated with subviral particles isolated by treatment of virus with the nonionic detergent Nonidet P-40. Only traces of the other two proteins were detected in the subviral particles. Synthesis of virus-induced proteins in TSWV-infected tobacco leaves was studied by labeling infected and healthy tissue with [3H]and [14C]valine, respectively. The labeled tissues were then fractionated into crude subcellular fractions and protein patterns of healthy and infected tissues were compared by coelectrophoresis on polyacrylamide gels. Only one virus-specific protein (of MW 49,000d) was detected in the virus-enriched fractions; this corresponded with the viral structural protein of MW 50,000d.

Transcriptional Silencing of Geminiviral Promoter-Driven Transgenes Following Homologous Virus Infection

Promoters isolated from the Tomato leaf curl virus (TLCV) drive both constitutive and tissue-specific expression in transgenic tobacco. Following systemic TLCV infection of plants stably expressing TLCV promoter:GUS transgenes, transgene expression driven by all six TLCV promoters was silenced. Silencing in the TLCV coat protein promoter:GUS plants (V2:GUSdeltaC) was characterized in more detail. Transgene silencing observed in leaf, stem, and pre-anthesis floral tissue occurred with the continued replication of TLCV in host tissues. Infection of the V2:GUSdeltaC plants with heterologous geminiviruses did not result in transgene silencing, indicating that silencing was specifically associated with TLCV infection. Nuclear run-on assays indicated that silencing was due to the abolition of transcription from the V2:GUSdeltaC transgene. Bisulfite sequencing showed that silencing was associated with cytosine hypermethylation of the TLCV-derived promoter sequences of the V2:GUSdeltaC transgene. Progeny derived from V2:GUSdeltaC plants silenced by TLCV infection were analyzed. Transgene expression was silenced in progeny seedlings but was partially reactivated in the majority of plants by 75 days postgermination. Progeny seedlings treated with the nonmethylatable cytosine analog 5-azacytidine or the histone deacetylase inhibitor sodium butyrate exhibited partial reactivation of expression. This is the first report of the hypermethylation of a virus-derived transgene associated with a DNA virus infection.

Host Responses to Transient Expression of Individual Genes Encoded by Tomato leaf curl virus

The six open reading frames of Tomato leaf curl virus (TLCV) were expressed in host Nicotiana species using a Tobacco mosaic virus vector. Each of the genes, except that encoding the viral coat protein, produced a phenotypic effect when expressed in planta, but the corresponding untranslatable mutant genes were asymptomatic. The C1 (Rep) gene invoked a hypersensitive response in Nicotiana clevelandii that restricted the viral construct to sites of infection. Expression of the C2 gene in N. benthamiana produced necrotic lesions on inoculated leaves as well as severe veinal necrosis on systemically infected leaves. This gene was also able to suppress post-transcriptional gene silencing in N. tabacum. C4 induced viruslike symptoms in host plants tested, providing further evidence for the involvement of this gene in symptom expression. Expression of the V1 and C3 genes caused severe stunting of N. benthamiana plants, indicating they may also have a role in symptom development. These results reveal that a complex set of interactions between the TLCV gene products and host factors occurs in planta, and these are discussed in relation to our current understanding of TLCV gene function.

Circularity of the ribonucleic acids associated with cadang-cadang disease

The ribonucleic acids, ccRNA-1 and ceRNA-2, associated with cadang-cadang disease are circular single-stranded molecules comprising 310 +/- 3 nucleotides and 438 +/- 5 nucleotides, respectively; their molecular weights are estimated to be 1.05 and 1.49 x 10(5) daltons. Therefore ceRNA-2 appears not to be a dimer of ccRNA-1, although it is known to have nucleotide sequences in common with ccRNA-1. Differences in the native structure of the two RNAs as determined by length measurements may account for previously described differences in their properties. Both RNAs resemble viroids, but ccRNA-1 is smaller, and ceRNA-2 is larger, than the viroids which have been characterized.