Rapid indexing of the sunblotch disease of avocados using a complementary DNA probe to avocado sunblotch viroid

A routine procedure has been established for the sensitive and specific detection of avocado sunblotch viroid in partially purified nucleic acid extracts of avocado leaves by hybridisation analysis with ³²P-complementary DNA prepared against the purified viroid. Avocado sunblotch viroid was shown to be present in 12 avocado trees that had indexed positive in a biological test for sunblotch disease but was absent from 10 trees that indexed negative. The complete correlation between sunblotch disease and the presence of viroid indicates that the complementary DNA hybridisation assay procedure can be used for the indexing of sunblotch disease. The overall procedure of leaf extraction and hybridisation analysis can be completed in 5 days and is to be compared with up to 2 yr required for indexing by biological methods. The level of avocado sunblotch viroid in partially purified nucleic acid extracts of a number of different sources of sunblotch infected avocado leaves was found to vary 10 000-fold from 0.2% to 2 × 10^-5% by weight. The lower limit of detectability of the viroid by the hybridisation assay is considered to be about 10^-5% by weight; this is at least 10³ times more sensitive than the detection of the viroid by polyacrylamide gel electrophoresis of the leaf nucleic acid extracts followed by staining.     

In situ hybridization localizes avocado sunblotch viroid on chloroplast thylakoid membranes and coconut cadang cadang viroid in the nucleus

Viroids, small single-stranded circular RNA molecules, are the smallest known infectious agents in Nature. The apparent inability of viroids to encode for proteins means that they must rely fully on host functions for their replication. The specific ultrastructural localization of viroids is fundamental to the determination of their replication strategies. In this paper the first in situ hybridization study to localize viroids within the cell at the electron microscope level is reported. Biotin-labelled RNA probes were used with subsequent detection by gold-labelled monoclonal anti-biotin antibodies to localize avocado sunblotch viroid and coconut cadang cadang viroid. Avocado sunblotch viroid was located in chloroplasts, mostly on the thylakoid membranes of cells from infected leaves of avocado (Persea americana). In contrast, coconut cadang cadang viroid was located in the nucleolus and nucleoplasm of cells of infected leaves of oil palm (Elaeis guineensis), with a higher concentration in the nucleolus. The results provide insight on the potential host RNA polymerases involved in the replication of these two viroids.     

Detection of avocado sunblotch viroid (ASBV) by dot-spot self-hybridization with a [32P]-labelled ASBV-RNA

RNA was extracted from plants infected with avocado sunblotch viroid (ASBV) and was analyzed by electrophoresis in polyacrylamide gel. The ASBV related fraction was eluted from the gel, labelled with [³²P] using polynucleotide kinase and used as a probe for hybridization with a purified ASBV-RNA preparation dot spotted on nitrocellulose paper. Positive self-hybridization indicated a high degree of internal complementarity. Dot spots of whole cell RNA and of leaf sap from ASBV infected plants were shown to hybridize with the labelled probe. This hybridization procedure proved to be 16–64 times more sensitive in diagnosing ASBV when compared with polyacrylamide gel analysis.     

Avocado sunblotch viroid: primary sequence and proposed secondary structure.

The sequence of the 247 nucleotide residues of the single strand circular RNA of avocado sunblotch viroid (ASBV) was determined using partial enzymic cleavage methods on overlapping viroid fragments obtained by partial ribonuclease digestion followed by 32p-labelling in vitro at their 5'-ends. ASBV is much smaller than potato spindle tuber viroid (PSTV; 359 residues) and chrysanthemum stunt viroid (CSV; 356 residues). A secondary structure model for ASBV is proposed and contains 67% of its residues base paired. In contrast to the extensive (69%) sequence homology of CSV with PSTV, only 18% of the ASBV sequence is homologous to PSTV and CSV. There are eight potential polypeptide translation products with chain lengths from 4 to 63 amino acid residues coded for by the plus (infectious) strand and four potential translation products (2 to 60 residues) coded for by the minus strand. An improved method is described for the synthesis of gamma-32p-ATP of high specific activity.     

Self-cleavage of plus and minus RNAs of a virusoid and a structural model for the active sites

Virusoids are circular single-stranded RNAs dependent on plant viruses for replication and encapsidation. Virusoid replication appears to involve longer-than-unit-length plus and minus RNAs, indicating that unit-length plus RNA is generated by specific cleavage reactions. Here, we synthesize plus and minus partial-length RNAs of the 324-nucleotide virusoid from lucerne transient streak virus in vitro. Both RNAs self-cleave at a unique site in the presence of magnesium ions to give 5' hydroxyl and 2',3' cyclic phosphodiester termini. Conformations other than the native structures are necessary for cleavage. Similar secondary structures with considerable sequence homology are proposed for the active sites of these and other plant pathogenic RNAs. Our results are consistent with certain rolling-circle replication models.     

Application of rapid biochemical methods for detecting avocado sunblotch disease

The method of Palukaitis & Symons (1980) for extracting low molecular weight ribonucleic acids from plant tissue was improved by CF-11-cellulose chromatography and further simplified for use in routine biochemical indexing for avocado sunblotch viroid (ASBV). Extracts were prepared routinely at 10 g dry weight equivalents (DWE) of tissue per ml; more concentrated than previously possible with many avocado cultivars. Conditions for assaying extracts of ASBV were standardised and the lower limits for detection determined as 80–230 ng ASBV/g DWE of tissue for polyacrylamide gel electrophoresis (PAGE) and 1 ng/g DWE of tissue for complementary DNA (cDNA) probe assays. The concentration of ASBV in a single infected tree varied from 5 to 5000 ng/g DWE between branches, but only to a minor degree between mature leaves and young blossoms within branches. Six independent sources of sunblotch disease were examined and all proved positive for ASBV by PAGE. The ASBV extracted from five of these sources hybridised with cDNA prepared from the sixth or standard source (Hass/SB-1), with hybridisation values ranging from 43% to 89%. In a survey of 76 trees intended for propagation in Australia, all of 17 trees previously accepted as healthy on the basis of graft transmission tests were negative for ASBV by PAGE and had cDNA hybridisation values ranging from 1.8% to 12.1%. Amongst 59 trees apparently free of sunblotch symptoms but not previously indexed, only one tree was positive for ASBV by both PAGE and cDNA probe assay. The other 58 trees were negative by PAGE but had hybridisation values ranging from 1.0% to 42.8%. Forty-nine trees had values consistent with known healthy trees (<12% hybridisation), while the results of the remaining nine trees will require confirmation by additional tests before a conclusion about ASBV is made. The cDNA probe assay successfully detected ASBV in avocado seedlings graft inoculated with Hass/SB-1, 1–2 months before symptoms were displayed but not until 6 months after inoculation. Methods for improving the cDNA probe assay still further are discussed.     

The viroid and viroid-like RNA database.

The viroid and viroid-like RNA database is a compilation of all natural sequences published in journals or available from the GenBank and EMBL nucleotide sequence libraries. Several information regarding these RNA species such as the position of their self-catalytic domains and the open reading frame of the human hepatitits delta virus are provided. The database also includes a determination of the likely ancestral sequence of most species and a prediction of the most stable secondary structures of these sequences. This online database is available on the World Wide Web (http://www.callisto.si.usherb.ca/[symbol: see text]jpperra ). It should provide an excellent reference point for further phylogenetic and structure-function studies of these RNA species.     

The viroid and viroid-like RNA database.

This is an online database to facilitate research on viroid, viroid-like RNAs and human hepatitis delta virus (vHDV) by presenting a large number of sequences and related data in a comprehensive and user-friendly format (e.g. position of their self-catalytic domains, open reading frame of the vHDV, prediction of the most stable secondary structures, etc.). Most of these RNA species share a common proposed replication pattern known as a DNA-independent rolling circle mechanism. Together, these species form the 'brotherhood' of the smallest known auto-replicable RNAs. This online database is available on the World Wide Web at http://www.callisto.si.usherb.ca/jpperra     

Open reading frame in the minus strand of two plus type RNA viruses

Inspection of the nucleotide sequences of the RNAs complementary to the coat protein mRNAs from two plant viruses with a tripartite genome: alfalfa mosaic virus and brome mosaic virus, showed the presence of open reading frames for 138 and 118 amino acids, respectively. A third virus (cowpea chlorotic mottle virus) from the same family (1) does not show this phenomenon. This suggests that if a protein is coded for by the open reading frames it may be not essential for virus multiplication. Alternatively the open reading frames have no coding function but result from structural requirements of the RNAs.     

Compilation and analysis of viroid and viroid-like RNA sequences.

We have created a catalogue comprising all viroid and viroid-like RNA sequences which to our knowledge have been either published or were available from on-line sequence libraries as of October 1, 1995. In the development of this catalogue nomenclature ambiguities were removed, the likely ancestral sequence of most species was determined and the most stable secondary structures of these sequences were predicted using the MulFold package. Only viroids of PSTVd-type possessed a rod-like secondary structure, while most other viroids adopted branched secondary structures. Several viroids have predicted secondary structures that include either a Y or cruciform structure reminiscent of the tRNA-like end of virus genomes at an extremity. However, it remains unknown whether or not these predicted structures are adopted in solution, and if they serve a particular function in vivo. Additional information such as the position of the self-catalytic domains are included in the catalogue. An analysis of the data compilated in the catalogue is included. The catalogue will be available on the world wide web (http://www.callistro.si.usherb.ca/jpperra), on computer disk and in printed form. It should provide an excellent reference point for further studies.     

Self-cleavage of a 71 nucleotide-long ribozyme derived from hepatitis delta virus genomic RNA.

Self-cleavage efficiency of ribozymes derived from hepatitis delta virus (HDV) has been shown to depend on the RNA structure, which in turn may be determined by the length of the considered sequences. Here we describe the construction and functional analysis of a 71 nucleotide-long RNA genomic fragment, Rz71, which carries an 18 nucleotide deletion in a very stable GC-rich stem-loop (stem IV), predicted to be present in several computer-derived secondary structure models. Rz71 is able to undergo self-cleavage under non-denaturing conditions (the t1/2 of the reaction at 37 degrees C is 3 min). The deletion, however, is not neutral, since under the same conditions the non-deleted ribozyme cleaves to 50% in less than 15 sec. Therefore, stem-loop IV seems to play a structural role, not being directly involved in the catalytic reaction, but contributing to the correct positioning of the catalytic core of the HDV ribozyme. Rz71 is the smallest self-cleaving sub-fragment of HDV genomic RNA reported so far.