Stellos M. Tavantzis

Association of distinct double-stranded RNAs with enhanced or diminished virulence in Rhizoctonia solani infecting potato

A virulent field isolate of Rhizoctonia solani AG 3, Rhs 1AP, has given rise to three sectors in a period of several years. Subculturing of these sectors resulted in three hypovirulent isolates—Rhs 1A1, Rhs 1A2, and Rhs 1A3. We reported previously that five genetically different double-stranded (ds) RNAs occur in these four isolates, with each isolate containing a unique combination of two or more dsRNAs. We report here that all five dsRNA elements occur in the cytoplasm, and none in the nucleus. The mitochondria contains low to moderate concentrations of the four larger dsRNAs. The four isolates were paired in selected combinations in an attempt to transmit specific dsRNAs to cultures lacking these dsRNAs. This approach generated groups of near-isogenic lines possessing the same dsRNA elements. As many as six cultures possessing the same dsRNA genotype were found to have the same degree of pathogenicity. Consistently, acquisition of a 6.4-kb dsRNA brought about increased virulence, whereas the presence o...

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.

Effect of hypovirulent rhizoctonia solani on rhizoctonia disease, growth, and development of potato plants

Field experiments were conducted to determine whether a hypovirulent, anastomosis group (AG) 3 isolate (Rhs 1A1) of R. solani could control rhizoctonia disease of potato caused by a virulent AG-3 isolate (Rhs 27). A uniform amount of Rhs 1A1, Rhs 27, or both was applied to formaldehyde-disinfected potato seed tubers at planting. In plots inoculated with Rhs 1A1 and Rhs 27, the area of infected stem tissue was significantly (P = 0.05) reduced by 56% as compared with that of plots inoculated with Rhs 27 alone. The number of stolons per plant was decreased by isolate Rhs 27 to one-half that of the uninoculated control, but remained unchanged in plots treated with both Rhs 1A1 and Rhs 27. The type of inoculum did not affect the percentage of infected stems or stolons. Plants inoculated with Rhs 1A1 alone exhibited a significantly (P = 0.05) greater growth response, expressed as a 4-fold increase in the dry weight of stolons, a 1.7-fold increase in the dry weight of stems, and reached full bloom approximately one week earlier than their respective control. Tuber yield increases associated with Rhs 1Al-treated plots were not statistically significant. When untreated seed tubers bearing natural inoculum (sclerotia) of R. solani were used, Rhs 1A1 failed to reduce disease severity or sig-nificantly stimulate plant growth.

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.

Quinic acid induces hypovirulence and expression of a hypovirulence-associated double-stranded RNA in Rhizoctonia solani.

Abstract. A double-stranded (ds)RNA, designated as M2, is associated with hypovirulence, conversion of the quinic acid pathway from inducible to constitutive and downregulation of the shikimic acid pathway in the Rhizoctonia solani culture Rhs 1A1. In this study, we report that in the virulent, M2-lacking isolate Rhs 1AP, which is isogenic to Rhs 1A1, quinic acid reduces virulence dramatically and induces synthesis of an M2-encoded polypeptide and its respective mRNA. The full-length sense strand of M2 is detected in untreated Rhs 1AP only after a second 30-cycle amplification, using nested primers. Quinate-induced Rhs 1AP contains low concentrations of both full-length sense and complementary strand of M2. The quinic acid-induced hypovirulence in Rhs 1AP cannot be overturned by the end-product of the shikimic acid pathway, chorismic acid, which enhances the virulence of Rhs 1AP dramatically when used alone. In addition to its apparent applications, this study confirms the strong association between the M2 dsRNA and hypovirulence in R. solani.

dsRNA genetic elements: concepts and applications in agriculture, forestry, and medicine.

RNAi and Cosuppression: Double-Stranded RNA as an Agent of Sequence-Specific Genetic Silencing in Animals and Plants, Jeffrey A. Norman and Mary K. Montgomery The Double-Stranded RNA-Activated Protein Kinase PKR, Randal J. Kaufman The Double-Stranded RNA Viruses of Saccharomyces Cerevisiae, Reed B. Wickner, Juan Carlos Ribas, and Anjanette Searfoss The Double-Stranded RNA Viruses of Ustilago Maydis and Their Killer Toxins, Jeremy Bruenn Molecular Basis of Symptom Expression by the Cryphonectria Hypovirus, Patricia M. McCabe and Neal K. Van Alfen Engineering Hypoviruses for Fundamental and Practical Applications, Donald L. Nuss, Baoshan Chen, Lynn M. Geletka, Todd B. Parsley, and Nobuhiro Suzuki Viruses of the Dutch Elm Disease Fungi, Kenneth W. Buck and Clive M. Brasier Double-Stranded RNA Elements Modulating Virulence in Rhizoctonia Solani, Stellos M. Tavantzis, Dilip K. Lakshman, and Chunyu Liu Molecular Genetics of the Viruses Infecting the Plant Pathogenic Fungus Helminthosporium Victoriae, Said A. Ghabrial, Ana I. Soldevila, and Wendy M. Havens Unraveling the Viral Complex Associated with La France Disease of the Cultivated Mushroom, Agaricus Bisporus, C.P. Romaine and M.M. Goodin Large dsRNA Genetic Elements in Plants, Pierre Pfeiffer Index

Phylogenetic relatedness of the M2 double-stranded RNA in Rhizoctonia fungi

Isolates from closely related fungi in the Rhizoctonia species complex were examined for the occurrence of the M2 double-stranded RNA (dsRNA) by amplifying a conserved 1000 nucleotide region of the dsRNA with reverse transcription PCR. The M2 dsRNA was detected in representative isolates belonging to three anastomosis groups (AG) of R. solani (AG-1-IA, AG-4 and AG-6; teleomorph = Thanatephorus) and four AGs of binucleate Rhizoctonia (AGA, AG-F, AG-R and AG-U; teleomorph = Ceratobasidium). Amplified PCR products from the 3′ region of the M2 dsRNA from a representative sample of 12 isolates from eight different AGs were sequenced and subjected to parsimony analysis and coalescent simulations to infer ancestral lineages and to reconstruct the ancestral history of haplotypes. Seven dsRNA haplotypes were inferred from the sample of 12 isolates. One haplotype was composed of only isolates of Ceratobasidium belonging to different AGs. The rooted gene genealogies from coalescent simulations suggested that the ancestral M2 dsRNA haplotype most likely evolved in Thanatephorus (anamorph = R. solani AG-1-IA) and has been acquired recently by isolates of Ceratobasidium. Reconstruction of the ancestral history of haplotypes with a parsimony-based approach that assumes both mutation and recombination suggested that four haplotypes recombined before coalescing to their most recent common ancestor, while three haplotypes coalesced without recombination in the recent past. There was no unique association of haplotype within a specific AG of either Ceratobasidium or Thanatephorus to support co-evolution of the M2 dsRNA within the fungal host. To our knowledge this is the first report of a dsRNA occurring in Ceratobasidium that also is present in Thanatephorus.

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

SummaryFull-length complementary DNAs (cDNA) of a mild (KF 5) and a severe (S-PSTVd) isolate of potato spindle tuber viroid (PSTVd) were constructed. DNA sequencing of four KF 5 cDNA clones (M 3, M 4, M 5, and M 7) revealed that KF 5 is comprised of 360 nucleotides. By comparison, all three cDNA clones (S 2, S 9, and S 10) of S-PSTVd possess 359 nucleotides. Sequence microheterogeneity was observed among the KF 5 cDNA clones. Clone M 5 differs from mild PSTVd isolate KF 6 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 M 4 and M 7 have a G-to-A transition at position 65 of the pathogenic domain, and M 3 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.

Draft Genome Sequence of the Plant-Pathogenic Soil Fungus Rhizoctonia solani Anastomosis Group 3 Strain Rhs1AP.

ABSTRACT The soil fungus Rhizoctonia solani is a pathogen of agricultural crops. Here, we report on the 51,705,945 bp draft consensus genome sequence of R. solani strain Rhs1AP. A comprehensive understanding of the heterokaryotic genome complexity and organization of R. solani may provide insight into the plant disease ecology and adaptive behavior of the fungus.

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).