Dilip K. Lakshman

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.

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.

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.

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.

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.

Extrachromosomal Elements and Degree of Pathogenicity in Rhizoctonia Solani

The advent of molecular biology has played a pivotal role in advancing our understanding of the genetics and biology of filamentous fungi such as Neurospora crassa and Aspergillus nidulans. There exists a great need for similar advances in the molecular genetics of other fungi, especially those causing economically important plant diseases. Rhizoctonia solani Kuhn [teleomorph, Thanatephorus cucumeris (Frank) Donk], is an important soilborne pathogen attacking numerous crops, worldwide. Understanding the genetic structure of field soil populations, the functions of the genes involved in virulence expression and genetic recombination is necessary for the design of Rhizoctonia disease management that will be both effective and environmentally sound.

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.