J.S. Semancik

Functional distinctions between the ribonucleic acids from citrus exocortis viroid and plant viruses: cell-free translation and aminoacylation reactions

Abstract A low molecular weight RNA having tRNA-like physical characteristics has previously been shown to be the pathogen causing citrus exocortis disease. The ability of this RNA to serve as a messenger molecule, or as an amino acid acceptor, was compared with that of other small RNA molecules from plant viruses. While the viral RNAs showed both of these activities, the citrus exocortis viroid had neither, contrasting the nature of this pathogen with that of plant viruses.

Citrus exocortis viroid: survey of protein synthesis in Xenopus laevis oocytes following addition of viroid RNA.

Abstract Citrus exocortis viroid (CEV) RNA was injected into oocytes of Xenopus laevis under conditions which demonstrated translation of bromegrass mosaic virus (BMV) RNA and rabbit hemoglobin mRNA. The viroid RNA was neither translated nor interfered with protein synthesis. These results were not altered by adenylation of the CEV RNA. BMV-directed protein synthesis could be detected following the injection of a total RNA preparation extracted from infected barley. The absence of evidence for a viroid-specified protein in these studies further supports a regulatory role for the viroid RNA.

Conversion of the electrophoretic forms of cowpea mosaic virus in vivo and in vitro

Abstract The electrophoretic forms of purified cowpea mosaic virus (CPMV) and bean pod mottle virus (BPMV) were examined by disc electrophoresis. The slow-migrating form (S) of CPMV predominated in early infection, and the fast-migrating form (F) predominated in late infection. The reverse relationship was true for BPMV. The transition from S to F in CPMV and F to S in BPMV suggested a precursor-product relationship between the electrophoretic forms. Evidence for this relationship was provided by kinetic studies of 32P incorporation into the forms of CPMV and by short-interval sequential yield data. When separated by density gradient electrophoresis, the F form of both viruses demonstrated a greater specific infectivity than the S form. The S to F conversion of CPMV was simulated in vitro by reaction with a mixture of carboxypeptidases A and B or chymotrypsin. This conversion of the electrophoretic forms of CPMV resulted in an increase in specific infectivity. The F to S conversion of BPMV was observed only after exposure to trypsin.

Application of selected viroids for dwarfing and enhancement of production of ‘Valencia’ orange

SummaryCitrus viroids, CVd-la, CVd-IIa, and CVd-IIIb, representative of three distinct Citrus Viroid Groups, resulted in a statistically significant reduction of 20-50% in tree size of Valencia orange, Citrus sinensis (L.) Osb., on Poncirus trifoliata (L.) Raf. rootstock. Reduction in canopy volume was accompanied by an increase in yield per tree in the presence of CVd-IIa, whereas a net decreases in yield resulted with either CVd-la or CVd-IIIb. Nevertheless, the yield per canopy volume was significantly enhanced in all viroid-containing trees when compared with the viroid-free trees. Analyses of “dwarfing factors” from different citrus growing countries indicated that single viroid isolates with nucleotide sequences similar to CVd-IIIb have been independently selected. Although pathogenic agents in some hosts, selected viroids may also be viewed as transmissible, small, nuclear ribonucleic acids (Tsn-RNAs) in those specific hosts which do not express a disease syndrome.

Exocortis viroid: Alteration in the proteins of Gynura aurantiaca accompanying viroid infection

Abstract Protein alterations following the onset of symptoms of the citrus exocortis disease in Gynura aurantiaca were detected in protein extracts of tissue as well as lysed protoplasts. The principal distinction occurred in low molecular weight proteins of about 15,000 MW (CEV-P 1 ) and an 18,000-MW (CEV-P 2 ) species following exposure to sodium dodecyl sulfate. Postribosomal preparations demonstrate the most marked differences between healthy and citrus exocortis viroid (CEV)-affected tissue, with the P 1 component comprising one of the most metabolically active proteins in this fraction, as determined by the incorporation of labeled amino acids. Since the alteration in the protein profiles appears to constitute a quantitative distinction in a host-specified protein, the functional role of the viroid-related protein may reflect a specific aberration in host regulation and development resulting from viroid infection.

Avocado sunblotch disease: a persistent viroid infection in which variants are associated with differential symptoms.

Variants of avocado sunblotch viroid (ASBVd) of between 247 and 250 nucleotides in length have been recovered from diseased avocado tissues. The sunblotch syndrome covers a complex pattern of disease symptoms which are associated with infection by variants of ASBVd. The viroid species are designated ASBVd-B, ASBVd-V and ASBVd-Sc from their association with bleached, variegated or symptomless carrier tissues respectively. Host-viroid interactions and structural relationships among the variants suggest a transition in sunblotch disease from a severe acute to a persistent mild form of infection.

Exocortis disease: Subcellular distribution of pathogenic (viroid) RNA☆

Abstract The pathogenic (viroid) RNA identified as the causal agent of the exocortis disease is associated not only with nuclei-rich preparations but also with a plasma-membranelike component of the endomembrane system. This distribution of CEV-RNA with subcellular constituents may reflect distinct phases in the host-viroid interaction, namely, a site of viroid-RNA synthesis and a site of accumulation resulting in the pathological condition. Properties of the membrane-associated CEV-RNA resemble those of the transmissible agent associated with the scrapie disease of animals.

A stable 463 nucleotide variant of citrus exocortis viroid produced by terminal repeats

An unusual variant of citrus exocortis viroid (CEV) was detected when an inoculum source from Gynura aurantiaca D.C. was used to infect a hybrid tomato (Lycopersicon esculentum Mill. x L. peruvianum). The 92 nucleotide larger variant, CEV D-92, which displayed the characteristic circular and linear viroid structural forms, contained two repeated sequences spanning the V and T2 domains. A dramatic moderation of symptom expression in Gynura accompanied the incorporation of these repeated sequences. A comparison of the sequence and structure of CEV D-92 with coconut cadang-cadang viroid revealed similarities in the regions generating the naturally occurring terminal repeats suggesting a possible preferred site for RNA recombination between viroids.

Characterization of viroid-like RNAs associated with the citrus exocortis syndrome

Three additional viroid-like RNA species (RNA-I, -II, and -III), smaller than the 371-nucleotide citrus exocortis viroid (CEV), have been identified in citron (Citrus medica) trees which display symptoms of the exocortis disease. The three RNAs migrate on polyacrylamide gels under denaturing conditions in the region between CEV and avocado sunblotch viroid circular molecules, indicating a size range of about 311-335 nucleotide residues in both circular and linear molecular forms. RNA-II is removed from the preparations when chromatographed on CF-11 cellulose. All three RNAs fail to replicate when introduced into Gynura aurantiaca, a herbaceous host of CEV, yet RNA-I and RNA-III can be independently transmitted to citron. Dot-blot hybridization with CEV-cDNA indicates no significant homology between CEV and RNA-I, RNA-II, or RNA-III. The relationship among CEV-RNA and the three viroid-like RNA species in the segregation of expression and intensity of the exocortis disease reaction is discussed.

Nucleotide sequence and structural features of the Group III citrus viroids.

The nucleotide sequence and secondary structure of two representative variants from the Group III citrus viroids. CVd-IIIa (297 bases) and CVd-IIIb (294 bases) were determined. The variants are related to the apple scar skin viroid (ASSVd) family. Although smaller in size than any of the ASSVd-related viroids, the central conserved region as well as most of the terminal conserved region of ASSVd is retained. The rod-like structural configuration (characteristic of ASSVd) of the variants as predicted by minimum free energy analysis is presented.