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

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.

Comparative analysis of putative pathogenesis-related gene expression in two Rhizoctonia solani pathosystems.

Rhizoctonia solani, teleomorph Thanatephorus cucumeris, is a polyphagous necrotrophic plant pathogen of the Basidiomycete order that is split into 14 different anastomosis groups (AGs) based on hyphal interactions and host range. In this investigation, quantitative real-time PCR (qRT-PCR) techniques were used to determine potential pathogenicity factors of R. solani in the AG1-IA/rice and AG3/potato pathosystems. These factors were identified by mining for sequences of pathogen origin in a library of rice tissue infected with R. solani AG1-IA and comparing these sequences against the recently released R. solani AG3 genome. Ten genes common to both AGs and two specific to AG1-IA were selected for expression analysis by qRT-PCR. Results indicate that a number of genes are similarly expressed by AG1 and AG3 during the early stages of pathogenesis. Grouping of these pathogenicity factors based on relatedness of expression profiles suggests three key events are involved in R. solani pathogenesis: early host contact and infiltration, adjustment to the host environment, and pathogen proliferation through necrotic tissue. Further studies of the pathogenesis-associated genes identified in this project will enable more precise elucidation of the molecular mechanisms that allow for the widespread success of R. solani as a phytopathogen and allow for more targeted, effective methods of management.

Detection of double-stranded RNA elements in the plant pathogenic fungus Rhizoctonia solani.

Many species of fungi have been shown to harbor double-stranded RNA (dsRNA) elements. A single fungal isolate of Rhizoctonia solani may have as many as five different dsRNA elements within them. The presence of specific dsRNA elements influence pathogenicity in host plants.

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 (AG-A, 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.

Characterization of the arom gene in Rhizoctonia solani, and transcription patterns under stable and induced hypovirulence conditions.

The quinate pathway is induced by quinate in the wild-type virulent Rhizoctonia solani isolate Rhs 1AP but is constitutive in the hypovirulent, M2 dsRNA-containing isolate Rhs 1A1. Constitutive expression of the quinate pathway results in downregulation of the shikimate pathway, which includes the pentafunctional arom gene in Rhs 1A1. The arom gene has 5,323 bp including five introns as opposed to a single intron found in arom in ascomycetes. A 199-bp upstream sequence has a GC box, no TATAA box, but two GTATTAGA repeats. The largest arom transcript is 5,108 nucleotides long, excluding the poly(A) tail. It contains an open reading frame of 4,857 bases, coding for a putative 1,618-residue pentafunctional AROM protein. A Kozak sequence (GCGCCATGG) is present between +127 and +135. The 5'-end of the arom mRNA includes two nucleotides (UA) that are not found in the genomic sequence, and are probably added post-transcriptionally. Size and sequence heterogeneity were observed at both 5'- and 3'-end of the mRNA. Northern blot and suppression subtractive hybridization analyses showed that presence of a low amount of quinate, inducer of the quinate pathway, resulted in increased levels of arom mRNA, consistent with the compensation effect observed in ascomycetes.

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

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.

Expression of a hypovirulence-causing double-stranded RNA is associated with up-regulation of quinic acid pathway and down-regulation of shikimic acid pathway in Rhizoctonia solani.

We reported previously that a 3.6-kb double-stranded RNA, designated as M2, is associated with hypovirulence in the Rhizoctonia solani isolate Rhs 1A1 and proposed that the M2-encoded putative polypeptide A (pA) might interfere with the regulation of the quinate and shikimate pathways. In this study, Western blot analysis showed that a protein band of the predicted size (83 kDa) binds antibodies specific to a pA epitope and is detectable in M2-containing but not in M2-lacking cultures. A mRNA, associated with Rhs 1A1 polysomes immunoprecipitated with anti-pA antibodies, has a sequence basically identical to that of the sense-strand of M2. The normally inducible quinate pathway was constitutively expressed, whereas the shikimate pathway was down-regulated in the M2-containing, hypovirulent Rhs 1A1. Finally, the relative concentration of phenylalanine, precursor of the virulence determinant phenylacetic acid, was correlated with the degree of pathogenicity in the virulent Rhs 1AP but not in the hypovirulent Rhs 1A1.