Robert H. Symons

Cucumber mosaic virus contains a functionally divided genome.

Abstract The four main RNA species isolated from purified Q strain of cucumber mosaic virus (CMV) were separated on 2.4% polyacrylamide gels. Using 16S and 23S Escherichia coli ribosomal RNA as molecular weight standards, the molecular weights of species 1–4 of CMV RNA were estimated in aqueous gels to be 1.30 × 10 6 , 1.13 × 10 6 , 0.78 × 10 6 , and 0.34 × 10 6 daltons and in gels run in 98% formamide to be 1.35 × 10 6 , 1.16 /sx 10 6 , 0.85 × 10 6 , and 0.35 × 10 6 daltons. The three largest species were essential for infectivity as determined by local lesion assay on cowpeas ( Vigna sinensis ). The variation of infectivity with the concentration of CMV and of CMV RNA has shown single hit kinetics even though lesion formation probably requires the initial simultaneous infection of each cell by the three largest RNA species.

Self-cleavage of RNA in the replication of small pathogens of plants and animals

The ability of certain small, circular, pathogenic RNAs of plants and animals to self-cleave at specific sites in vitro in the complete absence of protein most likely plays a central role in their replication in vivo by a rolling circle mechanism. The self-cleavage of an RNA transcript from a satellite DNA of the newt indicates that this reaction is not limited to pathogenic RNAs. Further, the site-specific self-cleavage in trans by two separate RNA molecules suggests that such reactions may be important in gene regulation in normal cells as well as in the genesis of symptom expression on infection by RNA pathogens.

Comparison of multimeric plus and minus forms of viroids and virusoids

SummaryIn order to investigate the mechanism of replication of viroids and virusoids, we have compared the replication intermediates of three members of each group in nucleic acid extracts of infected plants. Viroids were avocado sunblotch viroid (ASBV), citrus exocortis viroid (CEV) and coconut cadang cadang viroid (CCCV). Virusoids were from velvet tobacco mottle virus (VTMoV), solanum nodiflorum mottle virus (SNMV) and lucerne transient streak virus (LTSV). Analysis of intermediates was by the Northern hybridization technique with single-strand DNA and RNA probes prepared from recombinant DNA clones. The results obtained are discussed in terms of current models of viroid and virusoid replication.The plus RNA species consisted of an oligomeric series up to decamers based on the unit of full-length viroid or virusoid, which was always the major component, except for CEV where only monomer and dimer species were found. In the case of ASBV and the virusoids of VTMoV and SNMV, a minor, multimeric series of components (X-bands) was superimposed on the main oligomeric series.The complementary minus species proved more difficult to detect and characterise, with each viroid and virusoid exhibiting a unique pattern on Northern hybridization. However, they all had greater than unit-length minus species. In addition, minus species analogous to the plus X-bands were found in ASBV and CEV. The experimental difficulties encountered in this work are discussed in terms of the problem of detecting minus species by Northern analysis in the presence of excess complementary plus species.

Fractionation of cucumber mosaic virus RNA and its translation in a wheat embryo cell-free system

Abstract Cucumber mosaic virus (CMV) contains four major RNA species designated RNAs 1–4 in order of decreasing molecular weight. We report the preparative fractionation of CMV-RNA and the in vitro translation of the purified RNA species in a wheat embryo cell-free system. CMV-RNA was fractionated by 2–3 cycles of centrifugation in 10–40% sucrose density gradients employing a heat denaturation step after the first centrifugation. This method yielded a pure mixture of RNA 1 and RNA 2 (RNA 1 + 2), pure RNA 3 and pure RNA 4. The heat denaturation step was found necessary for elimination of hidden breaks and possible RNA aggregates in the final purified RNA samples. The purified CMV-RNAs were translated in a wheat embryo cell-free system in the presence of 3 H-labeled amino acids and the 3 H-labeled products were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. With RNA 1 + 2 as messenger, four major and approximately eight minor products were found (molecular weight range 13,000–90,000). RNA 3 yielded CMV coat protein and one or two other products of molecular weight 26,000–28,000. Coat protein was the single translation product of both RNA 4 and unfractionated CMV-RNA. The identity of the 3 H-labeled coat protein product was confirmed by cyanogen bromide cleavage and comparison of the peptides with those obtained from marker 14 C-labeled coat protein.

Translation of the four major RNA species of cucumber mosaic virus in plant and animal cell-free systems and in toad oocytes.

Abstract Cucumber mosaic virus (CMV) contains four major RNA species, designated RNAs 14 in order of decreasing molecular weight. As a preliminary means of analysing gene content and distribution between these RNA species, each of the RNAs was purified and used to direct protein synthesis in cell-free systems from wheat embryos, wheat germ, and rabbit reticulocytes, and in intact oocytes of the toad, Bufo marinus. In vitro translation products of the CMV RNAs (Q strain) were analysed by electrophoresis on discontinuous slab polyacrylamide gels in the presence of sodium dodecyl sulphate. RNA 1 and RNA 2 proved untranslatable in oocytes but both directed synthesis of a large number of products in the wheat and rabbit reticulocyte cell-free systems. The highest molecular weight products obtained, about 105,000 for RNA 1 and 120,000 for RNA 2, represent most of the coding potential of these RNAs. Thus, RNA 1 and RNA 2 may be monocistronic messengers for the 105,000 and 120,000 MW products, respectively. Translation of RNA 3 in the wheat cell-free systems yielded three major products of molecular weights 34,000–39,000. In contrast, RNA 3 directed synthesis of essentially one product of molecular weight about 34,000 in both the rabbit reticulocyte system and oocytes. RNA 3 did not direct synthesis of coat protein in any of the systems, contrary to our earlier results and the known presence of the coat protein gene on RNA 3. RNA 4 directed synthesis of coat protein and higher- and lower molecular weight minor products in the cell-free systems and in oocytes. A product corresponding to the entire coding potential of RNA 4 was observed in the rabbit reticulocyte system, but not in the wheat systems or toad oocytes. The conflicting results obtained for translation of RNA 3 and RNA 4 in the different systems illustrate the danger in analysing genetic material by in vitro translation. The discussion considers which of the in vitro products are most likely to be synthesized in vivo and a tentative model of the CMV genome is proposed.

Purification and characterization of the circular and linear forms of chrysanthemum stunt viroid.

Summary A new and rapid extraction procedure is described for the isolation and partial purification of low mol. wt. RNA as well as DNA from 500 g batches of chrysanthemum stunt viroid-infected chrysanthemum. The chrysanthemum stunt viroid was then purified to homogeneity from this extract by one cycle of non-denaturing polyacrylamide slab gel electrophoresis followed by one cycle of denaturing slab gel electrophoresis. The covalently closed circular form and the linear form of the viroid co-migrated on the first gel but were well separated on the second. The yield of circular chrysanthemum stunt viroid was 200 µg/kg of infected chrysanthemum shoots and of the linear form was 35 µg/kg. 32P-labelled complementary DNA prepared against the circular form was used to show, by hybridization analysis, that the circular and linear forms contained identical nucleotide sequences. The circular and linear forms were equally infectious when inoculated onto Gynura aurantiaca. The linear viroid contained all four nucleotides at the 5′-terminus, about 72% of which had a 5′-phosphate.

Replication of in vitro constructed viroid mutants: location of the pathogenicity-modulating domain of citrus exocortis viroid

Sequence variants from field isolates of citrus exocortis viroid (CEV) that cause either mild or severe symptoms on tomato plants have previously been classified into two groups, A and B. These groups differ primarily in two domains, PL and PR, of the proposed native structure. Infectivity studies with full‐length cDNA clones of variants from each class have now directly confirmed the original correlation between Class A sequences and the severe phenotype and between Class B sequences and the mild phenotype. Direct evidence for this correlation could only be obtained by using individual sequence variants since field isolates of CEV have been shown to contain a mixture of RNA species. The construction and infectivity of chimaeric cDNA clones derived from mild and severe sequence variants of CEV has demonstrated that novel, infectious viroid molecules can be generated in vitro, and that PL is the pathogenicity‐modulating domain. The role of the PR domain is not known but infectivity experiments with one chimaeric cDNA clone suggest that it may influence the efficiency of the infection or replication process of the viroid in the plant.

Characterization of a satellite RNA associated with cucumber mosaic virus.

Abstract Two types of RNA, each with a molecular weight of approximately 0.12 × 10 6 , designated RNA 5 and satellite RNA, have been found in purified cucumber mosaic virus (CMV) preparations and have been characterized by molecular hybridization analysis using 32 P-labeled complementary DNA probes transcribed from these RNAs. RNA 5 usually makes up about 5% or less by weight of the total viral RNA and was shown to consist of specific cleavage products of CMV RNAs 1–4. Its nucleotide complexity was equivalent to about three times its molecular weight. By contrast, satellite RNA could form up to about 50% by weight of virion RNA and had the following properties: (1) It contained a unique nucleotide sequence with no homology with CMV RNAs, (2) CMV and tomato aspermy virus, but not alfalfa mosaic virus or tobacco ringspot virus, could function as helper viruses for its replication and encapsidation, (3) its nucleotide sequence was independent of the host plant and the helper virus used for its propagation and it was not derived from a host plant RNA, and (4) it was not a negative copy of any of the CMV RNA species. We concluded that this RNA is a true satellite RNA and has no relationship to the RNA found in defective interfering particles of animal viruses.

Differential virulence by strains of Cucumber mosaic virus is mediated by the 2b gene

The approximately 12-kDa 2b protein, encoded by all cucumoviruses, had been shown to play an important role in viral long-distance movement, hypervirulence, and suppression of post-transcriptional gene silencing. The role of the 2b gene in the hypervirulence of Cucumber mosaic virus (CMV) and whether hypervirulence was linked to movement were analyzed using a hybrid virus (CMV-qw), generated by replacing the 2b gene in a subgroup II strain, Q-CMV, with the 2b gene from a subgroup IA strain, WAII-CMV. CMV-qw was more virulent than Q-CMV or WAII-CMV on most of the host plant species tested. Northern blot and nucleotide sequence analyses demonstrated that CMV-qw was stably maintained during the course of infection and upon passage. Kinetic studies revealed that the hypervirulence induced by the hybrid virus was associated with neither increased viral RNA accumulation nor more rapid viral movement per se, suggesting that other functions of the 2b protein are important in determining the hypervirulence.

Citrus exocortis viroid: nucleotide sequence and secondary structure of an Australian isolate

Exocortis or ‘scaly butt disease’ of citrus is distributed world-wide [I]. The causative agent is citrus exocortis viroid (CEV) [2,3] which is a member of that unique group of plant pathogens, the viroids, of which only 8 have been described [ 1,4,5]. Like other members of the group, CEV consists of a singlestranded covalently closed circular RNA molecule which is highly base-paired, rod-like, infectious and non-encapsidated [ 1,4]. The primary sequence and proposed secondary structure of only 3 viroids have been reported so far: potato spindle tuber viroid (PSTV) with 359 residues [6]; chrysanthemum stunt viroid (CSV) with 356 residues [7]; and avocado sunblotch viroid (ASBV) with 247 residues [8]. We report here the sequence and proposed secondary structure of the 371 residues of an Australian isolate of CEV and discuss the significance of the extensive sequence homology which exists between PSTV, CSV and CEV.