Molecular characterization of the genome of a partitivirus from the basidiomycete Rhizoctonia solani.

The bisegmented genome of a double-stranded (ds) RNA virus from the fungus Rhizoctonia solani isolate Rhs 717 was characterized. The larger segment, dsRNA 1, is 2363 bases long whereas the smaller segment, dsRNA 2, has 2206 bases. The 5' ends of the coding strands of dsRNA 1 and dsRNA 2 are highly conserved (100% identity over 47 bases), and contain inverted repeats capable of forming stable stem-loop structures. Analysis of the coding potential of each of the two segments showed that dsRNAs 1 and 2 could code for polypeptides of 730 aa (bases 86-2275; molecular mass 86 kDa) and 683 aa (bases 79-2130; molecular mass 76 kDa), respectively. The 86 kDa polypeptide has all the motifs of dsRNA RNA-dependent RNA polymerases (RDRP), and has significant homology with putative RDRPs of partitiviruses from Fusarium poae and Atkinsonella hypoxylon. The 76 kDa protein shows homology with the putative capsid proteins (CP) of the same viruses. Northern blot analysis revealed no subgenomic RNA species, consistent with the fact that the long open reading frames encoding the putative RDRP and CP cover the entire length of the respective dsRNAs.

A virulence-associated, 6.4-kb, double-stranded RNA from Rhizoctonia solani is phylogenetically related to plant bromoviruses and electron transport enzymes.

We have recently shown that acquisition of a 6.4-kb, double-stranded (ds) RNA (M1) by hyphal anastomosis is associated with enhanced vigor and virulence, whereas its removal by hyphal tipping correlates with diminished virulence in the plant-pathogenic basidiomycete Rhizoctonia solani. The M1 dsRNA is not encapsidated by a typical nucleocapsid, has a circular and/or concatemeric form, and is associated with the mitochondrial and cytosolic fractions. M1 possesses six open reading frames (ORFs) the longest of which (ORF 2) is located on the (+) strand, and encodes a putative polypeptide consisting of 1,747 amino acids or 199.4 kDa. This polypeptide has a significant amino acid sequence similarity, including six conserved helicase domains and an ATP/GTP binding motif, with the 1A protein of broad bean mottle virus (BBMV) and other bromoviruses. ORF 5, which is located on the (-) strand of M1 and is complementary to a region of ORF 2, codes for a putative polypeptide that has a significant amino acid sequence similarity with the cytochrome c oxidase assembly factor. This complementarity provides direct evidence suggesting that the long-standing hypothesis of viruses evolving from cellular genes may be valid.

A double-stranded RNA element from a hypovirulent strain of Rhizoctonia solani occurs in DNA form and is genetically related to the pentafunctional AROM protein of the shikimate pathway.

M2 is a double-stranded RNA (dsRNA) element occurring in the hypovirulent isolate Rhs 1A1 of the plant pathogenic basidiomycete Rhizoctonia solani. Rhs 1A1 originated as a sector of the virulent field isolate Rhs 1AP, which contains no detectable amount of the M2 dsRNA. The complete sequence (3,570 bp) of the M2 dsRNA has been determined. A 6.9-kbp segment of total DNA from either Rhs 1A1 or Rhs 1AP hybridizes with an M2-specific cDNA probe. The sequences of M2 dsRNA and of PCR products generated from Rhs 1A1 total DNA were found to be identical. Thus this report describes a fungal host containing full-length DNA copies of a dsRNA element. A major portion of the M2 dsRNA is located in the cytoplasm, whereas a smaller amount is found in mitochondria. Based on either the universal or the mitochondrial genetic code of filamentous fungi, one strand of M2 encodes a putative protein of 754 amino acids. The resulting polypeptide has all four motifs of a dsRNA viral RNA-dependent RNA polymerase (RDRP) and is phylogenetically related to the RDRP of a mitochondrial dsRNA associated with hypovirulence in strain NB631 of Cryphonectria parasitica, incitant of chestnut blight. This polypeptide also has significant sequence similarity with two domains of a pentafunctional polypeptide, which catalyzes the five central steps of the shikimate pathway in yeast and filamentous fungi.

Multimeric non-radioactive cRNA probes improve detection of potato spindle tuber viroid (PSTVd).

Experimental data showed that multimeric, complementary RNA (cRNA) probes, labelled with non-radioactive digoxigenin (DIG), improved sensitivity of detection of the potato spindle tuber viroid (PSTVd) RNA by 2- to 30-fold as compared with corresponding multimeric cDNA probes. The degree of PSTVd detectability improvement depended upon the type of alkaline phosphatase substrate (colorimetric vs. chemiluminescent) used. Use of hexameric DIG-labelled cRNA probes in combination with chemiluminescent (Lumi-Phos 530) substrate resulted in detection of 0.48 pg of PSTVd RNA. The size of the synthesized cRNA probes corresponded to the size of the respective PSTVd cDNA templates. Interestingly, there was no relationship between the size of the synthesized, DIG-labelled DNA probes and that of the PSTVd cDNA template. This type of anomaly was not observed with other plant viral cDNA templates. Monomeric or multimeric cDNA probes detected both a mild and a severe PSTVd strain in viroid-infected potato leaf extracts diluted 1024 to 2048 times. In comparison, cRNA probes exhibited a much greater dilution end point; PSTVd RNA was detectable in viroid-infected potato leaf tissue diluted up to 16,384 times. Comparable levels of PSTVd sensitivity of detection were obtained with viroid-infected potato tuber tissue.

Primary and secondary structure of a 360-nucleotide isolate of potato spindle tuber viroid.

Full-length complementary DNAs (cDNA) of a mild (KF5) and a severe (S-PSTVd) isolate of potato spindle tuber viroid (PSTVd) were constructed. DNA sequencing of four KF5 cDNA clones (M3, M4, M5, and M7) revealed that KF5 is comprised of 360 nucleotides. By comparison, all three cDNA clones (S2, S9, and S10) of S-PSTVd possess 359 nucleotides. Sequence microheterogeneity was observed among the KF5 cDNA clones. Clone M5 differs from mild PSTVd isolate KF6 by a U-to-A transversion at position 303 followed by an A addition at the lower half of the "virulence-modulating" (VM) region. These changes modified the PSTVd consensus sequence of the VM region from 5' UCUAUCU 3' to 5' UCAAAUCU 3'. Additionally, clones M4 and M7 have a G-to-A transition at position 65 of the pathogenic domain, and M3 has a G-to-A transition at position 133 of the variable domain. The sequence of the three cDNA clones of S-PSTVd was identical to that of PSTVd isolate 440-1. An improved computer program was used to predict the secondary structure of the above two sequence variants as well as that of other PSTVd variants of which the structure has been reported previously. The data provides support for the hypothesis that increasing thermodynamic instability of the VM region is correlated with increasing virulence of the respective naturally occurring PSTVd isolate.

A viroid from Nematanthus wettsteinii plants closely related to the Columnea latent viroid.

A viroid was isolated from symptomless Nematanthus wettsteinii plants using the return-PAGE method for analysis of low M(r) nucleic acids. The RNA was transmitted to tomato, three cultivars of potato, and Scopolia sinensis plants by mechanical inoculation or by grafting. Infected solanaceous plants developed symptoms similar to those caused by potato spindle tuber viroid (PSTVd). The Nematanthus viroid consists of 372 nucleotides, 214 G+C, 158 A+U, with a G+C/A+U ratio of 1.35. One of seven cDNA clones showed a sequence heterogeneity (G to A) at position 73. The most stable secondary structure of this viroid has 78 G:C, 37 A:U and 11 G:U base pairs with a minimum free energy of -456.9 kJ. The viroid is closely related to the 370 nucleotide Columnea latent viroid. The Nematanthus viroid possesses regions of 100% sequence identity with six viroids belonging to the PSTVd and apple scar skin viroid groups. The viroid also replicated in tomato plants when mixed with PSTVd. Tomato plants were cross-protected against PSTVd when preinfected with the viroid from N. wettsteinii.

A rapid and versatile method for cloning viroids or other circular plant pathogenic RNAs.

We surveyed the occurrence of unique restriction sites on the cDNAs of viroids, virusoids, and plant viral satellite RNAs that have a circular RNA as an intermediate of replication and found that four such sites would linearize their circular cDNAs. A rapid and simple method was then developed for cloning a naturally occurring viroid from Nematanthus wettsteinii plants. First-strand cDNA was synthesized using random hexanucleotide DNA primers and M-MuLV reverse transcriptase (Superscript RT). Second-strand DNA was synthesized by employing the replacement synthesis method using Escherichia coli RNase H, E. coli DNA polymerase I, E. coli DNA ligase, and beta-NAD+. The circular double-stranded DNA was analyzed for the presence of commonly available, unique restriction sites and subsequently linearized with a selected restriction enzyme. The linear cDNA was ligated to dephosphorylated plasmid vector pGEM 3Z f(+) and cloned in E. coli strain DH5 alpha. This cDNA cloning procedure is suitable for cloning sequence variants of well-characterized viroids, virusoids, certain plant viral satellite RNAs, and new such pathogens of unknown sequence.

RNA progeny of an infectious two-base deletion cDNA mutant of potato spindle tuber viroid (PSTV) acquire two nucleotides in Planta.

Deletion mutations were generated in four structural domains of a potato spindle tuber viroid (PSTV) complementary DNA (cDNA) clone. Deletions of 3 to 5 nucleotides at the central conserved domain (CCGGG, positions 94 to 98), variable domain (GCCG, positions 146 to 149) and pathogenicity domain (CGA, positions 286 to 288) abolished infectivity of dimeric or trimeric cDNA constructs, or their in vitro transcripts. By contrast, a clone (St4) with a deletion of two nucleotides (UU, positions 339 and 340), located at the left terminal domain, retained infectivity when DNA or in vitro transcribed (+)RNA was used as inoculum. Sequencing of four cDNA clones of such viroid progeny demonstrated that two nucleotides were added at the deletion site. Two of the viroid progeny contained a CG addition. A third clone possessed a GU addition, whereas the fourth clone had a UU addition which represents a true reversion to full-length wild-type PSTV RNA. Ribonuclease protection assay of viroid progeny from St4-infected tomato plants suggested that only a negligible proportion of the St4 progeny were true revertants.

Coat protein and protease activity as in vitro translation products of potato carlavirus M RNA.

Genomic-size RNA isolated from purified potato carlavirus M (PVM) was translated in both the reticulocyte and the wheat germ cell-free, messenger-dependent systems. The PVM RNA translated the same set of major products in both in vitro systems. The Mr values of the most prominent polypeptides observed consistently were 185,000 (P185), 147,000 (P147), 94,000 (P94), 87,000 (P87), 72,000 (P72), 67,000 (P67), 52,000 (P52), 46,000 (P46), 35,000 (P35) and 25,000 (P25). Relatively low amounts of a translation product of Mr 200,000 (P200) were often detectable in both systems. The P35 polypeptide displayed the same molecular weight and one-dimensional peptide map as the virus coat protein (CP), and was precipitated by antibodies raised against PVM and PVM CP. The kinetics of appearance of the in vitro synthesized polypeptides suggested that primary translation products of high molecular weight undergo post-translational proteolytic cleavage.

The maize En-1/Spm element transposes in potato.

The maize transposable element En-1 has been introduced into a diploid potato line via transformation with Agrobacterium tumefaciens. The element is transcriptionally active in potato. Numerous En specific RNAs are observed, including a 6 kb transcript characteristic of an active En-1 element in maize. In contrast to maize, where the 6.0 kb transcript is hardly detectable, this transcript is very abundant in the transgenic potato. Transposition of En-1 in the potato clone was analysed by Southern blot hybridization and confirmed by molecular isolation of En-1 excision and integration events. Sequence data of excision and integration sites revealed footprints and target site duplications similar to the ones described for En-1 in maize.

Physicochemical properties of potato virus M.

Potato virus M (PVM), a member of the carlavirus group was found to have a sedimentation coefficient (S20,w) of 157 S, a buoyant density in CsCl of 1.322 g.cm(-3), an extinction coefficient, at 260 nm, of 2.88 cm(2).mg(-1), and a nucleic acid content of 6.0%. The PVM genome is a polyadenylate-containing single-stranded RNA with a molecular weight (MW) of 2.5 x 10(6). Virus-specific, double-stranded RNA is consistently associated with PVM infection. PVM possesses a single capsid polypeptide with a MW of 35.7 x 10(3).

Purification and partial characterization of a bacilliform virus from Agaricus bisporus: a single-stranded RNA mycovirus.

A bacilliform virus from the cultivated mushroom, Agaricus bisporus, has been extensively purified and shown to be a unique mycovirus in that the genome is single-stranded (ss) RNA. The purification procedure entailed extraction at pH 6, followed by polyethylene glycol-NaCl precipitation, and differential, rate-zonal, and equilibrium centrifugation. Mushroom bacilliform virus (MBV) has a buoyant density of 1.317 g/cm(3) in Cs2SO4, A260/A280 nm absorbance ratio of 1.67, and contains approximately 20% nucleic acid by weight. SDS-polyacrylamide gel electrophoretic analyses revealed that MBV contains a single major nucleic acid species with a molecular weight of 1.4 x 108 and a single capsid polypeptide of molecular weight 24,400. The nucleic acid was hydrolyzed by pancreatic ribonuclease A in high ionic strength buffer and by 0.4 M KOH, but not by pancreatic deoxyribonuclease I. Further, the kinetics of melting of MBV RNA upon thermal denaturation closely paralleled that of the ssRNA of tobacco mosaic virus. Based on this evidence, we propose that MBV is the first mycovirus shown to possess a ssRNA genome.