After the unexpected appearance of lethal symptoms on tomato plants infected with the PSTVd strain Intermediate Di, viroids were isolated and sequenced. It was found that a new strain, named RG 1, had been generated spontaneously in our greenhouse. In a different series of plant passages two new strains, named QF A and QF B, were detected which coexisted with the wild-type strain Di. Strains QF A and QF B showed intermediate symptoms when inoculated separately. In order to confirm the working hypothesis that the more pathogenic strain outcompetes the less pathogenic strain but strains of similar pathogenicity might coexist in the host, strains of different pathogenicity were mixed for inoculation in a ratio from 1:1 to 1:100 (more pathogenic:less pathogenic). The concentrations of the individual strains were determined 6 weeks postinfection with the method of nondenaturing polyacrylamide gel electrophoresis, and the working hypothesis was confirmed. The total concentrations of viroids in infected plants were very similar, irrespective of whether severe, intermediate, or mild strains or mixtures of different strains were present. The mutations in all new strains (3 in RG 1, 2 in QF A, 3 in QF B) were located in the so-called virulence-modulating region. The mutations of strain RG 1 influenced dramatically the thermodynamic stability of the native rod-like structure, as determined experimentally by temperature-gradient gel electrophoresis. Since during replication a multihairpin structure is generated transiently which is transformed afterwards into the rod-like structure, a lower thermodynamic stability of the rod-like structure leads to a higher accumulation of the transient structure. It is assumed that the transient structure, which is active in replication as shown earlier, is essential also in pathogenesis. This model explains the experimentally determined interdependence between pathogenicity and replicability of PSTVd strains.
SUMMARYComplexes of potato spindle tuber viroid (PSTV) with plant nuclear proteins were investigated by in vitro reconstitution of purified viroids and proteins from nuclear extracts, on nitrocellulose filters and in solution. Well defined complexes were formed under both conditions. Competition for complex formation was achieved by greater than a 1000-fold excess of 5S RNA. Linear PSTV transcripts form complexes with protein that are different from those of circular PSTV. Natural viroid-protein complexes were crosslinked covalently by u.v. radiation in nuclei isolated from infected plant tissue. They have a sedimentation coefficient of approximately 10S, and exhibit defined bands in SDS-PAGE gels which are similar to those of viroid-protein complexes reconstituted in solution. A 43K protein was isolated from the cellular complexes by RNase digestion. Complexes crosslinked by u.v. radiation eluted together with non-crosslinked PSTV RNA in anion-exchange HPLC on Nucleogen columns. Using HPLC of non-irradiated samples a 43K protein eluted at a KCI concentration of 0-61 M, but only in samples from infected material. The relevance of the viroid-protein complexes for viroid function is discussed. INTRODUCTIONViroids are an independent class of plant pathogens. They are circular ssRNAs composed of 240 to 370 nucleotides. They differ from viruses by the lack of a protein coat and their smaller size. The sequences of more than a dozen viroid species are known (for reviews see S/inger, 1982; Riesner & Gross, 1985;Diener, 1987). Intramolecular base pairing leads to a secondary structure in which short double helices and internal loops form an unbranched, rod-like structure. From the fact that viroids are infectious as naked RNA molecules and do not code for a translational product, one cannot infer that viroids are present in the cell as free RNA molecules. It is reasonable to assume that they are protected against nuclease degradation by being complexed with proteins or other cellular components. Furthermore several steps in viroid replication and their pathogenic action require well defined interactions of viroids and host cell components.As a particular aspect of viroid-host cell interactions the location of viroids in the infected plant cell was investigated. Earlier studies based on infectivity tests suggested that viroids were associated primarily with nuclei (Diener, 1971; Sanger, 1972; Takahashi & Diener, 1975; Takahashi et al., 1982) and/or membranes (Semancik et al., 1976). More recent studies (Schumacher et al., 1983) with highly purified nuclei from tomato green leaf tissue infected with potato spindle tuber viroid (PSTV) and fractionation of the nuclear components showed that more than 90~ of the viroid molecules are associated with the nucleoli. Incubation of infected nucleoli with buffers of increasing ionic strength abolished these interactions. This dependence strongly indicates that viroids are complexed with proteins within the nucleoli. The subfractionation of nucleoli after DNase I treatment and so...
Plasmalemmasomes were observed in leaves of healthy, PSTVd (potato spindle tuber vroid) infected and artifically stunted tomato plants. Their shape and distribution could not be clearly related to infection with PSTVd as previously suggested. These membranous proliferations were suggested to represent one of the normal physiological responses of the plants to a multitude of possible exogenous influences.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.