Figure 1.
Gene organization of rdr1, rdr2, rdr3 and rdr4.
Positions of exons (boxes) and introns (solid lines) were determined by comparisons of genomic and cDNA sequences for each gene (see text). Black boxes indicate regions corresponding to the RdRP domains in the encoded RDR proteins. Three alternatively spliced sequences were identified in rdr4, each predicted to result in a truncated RdRP domain because of premature stop codons indicated by asterisks. Lengths are indicated above and below exons and introns, respectively.
Table 1.
Primers and probes used in this study.
Figure 2.
Conserved RdRP domains and amino acid sequences of C. parasitica rdr genes.
A). The RdRP domains were identified by searching NCBI’s Conserved Domains database (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) and are indicated by grey boxes; solid lines represent full-length proteins. E-values of each RdRP domain match and the range of amino acids included in the match are indicated next to and below the domain name, respectively. The longest predicted RDR4 protein is indicated here and contains an RdRP domain that is truncated due to a premature stop codon in its gene transcript and that lacks a conserved DPBB domain. B). The three C. parasitica RDRs containing complete RdRP domains were included in an alignment with QDE-1 from N. crassa, an RdRP involved in RNA silencing (Quelling); highly conserved DPBB domains and residues in QDE-1 [47] are indicated by boxes and are highlighted in red, respectively.
Figure 3.
Sequence relationships of C. parasitica RDR-like proteins with putative and known RDRs in other eukaryotes.
RdRP domains were extracted from each sequence and aligned for tree reconstruction as described in the Methods. The four RDR-like C. parasitica sequences are shown in shaded boxes; two alternatively spliced forms of RDR4, identified by cDNA sequencing (resulting in a truncated RdRP domain) are shown. The RDRs in N. crassa are indicated by asterisks and were used to name the three fungal RDR clades, indicated on the right, along with the clades containing RDR sequences in other eukaryotes. Sequence accessions and species names are the following: An1 = AN2717 (Aspergillus nidulans); An2 = AN4790 (A. nidulans); AtRdRP1 = NP_172932.1, RNA-dependent RNA polymerase 1 (Arabidopsis thaliana); AtRdRP3 = NP_179581.2, RNA-dependent RNA polymerase-like protein (A. thaliana); BfRDR1 = AAQ10792.1, RNA-directed RNA polymerase-like protein (Branchiostoma floridae); CeEGO-1 = NP_492132.1, Protein EGO-1 (Caenorhabditis elegans); CnRdRP = ACJ04634.1, RNA-dependent RNA polymerase (Cryptococcus neoformans var. grubii); Cprdr1 = HF912382, putative RNA-dependent RNA polymerase (Cryphonectria parasitica); Cprdr2 = HF912383, putative RNA-dependent RNA polymerase (C. parasitica); Cprdr3 = HF912384, putative RNA-dependent RNA polymerase (C. parasitica); Cprdr4 = HF912385, putative rdr4 pseudogene (C. parasitica); DdDosA = AF117611_1, DosA protein (Dictyostelium discoideum); DdRdRP = EAL62541.1, RNA-directed RNA polymerase (D. discoideum); Mcrdrp-1 = JGI scaffold_05∶2640291–2644233 (Mucor circinelloides); Mcrdrp-2 = JGI scaffold_05∶245898–249911 (M. circinelloides); Mo1 = EHA51817.1, RNA-dependent RNA polymerase (Magnaporthe oryzae); Mo2 = EHA52286.1, RNA-dependent RNA polymerase (M. oryzae); Mo3 = EHA46264.1, RNA-dependent RNA polymerase 1 (M. oryzae); MtRdRP = XP_003608944.1, RNA-dependent RNA polymerase (Medicago truncatula); NcQDE-1 = AJ133528, qde-1 gene (Neurospora crassa); NcRRP-3 = XP_963405, hypothetical protein (N. crassa); NcSAD1 = AAK31733, suppressor of ascus dominance (N. crassa); Os701 = NP_918046.1, putative RNA-directed RNA polymerase (Oryza sativa); Os702 = NP_913147.1 (O. sativa); PtRDRP1 = CAI39057.1, RNA-dependent RNA polymerase (Paramecium tetraurelia); SpRdp1 = CAB11093.1, RNA-directed RNA polymerase Rdp1 (Schizosaccharomyces pombe); SlRdRP = CAA71421.1, RNA-directed RNA polymerase (Solanum lycopersicum).
Figure 4.
Accumulation of C. parasitica rdr transcripts in response to infection by hypovirus CHV-1/EP713 and the CHV-1/EP713 mutant Δp29 that lacks the p29 suppressor of RNA silencing.
The relative levels of rdr1–4 gene transcripts were measured by quantitative real-time RT-PCR (2−ΔΔCT method) for uninfected (grey bars), CHV-1/EP713-infected (open bars) and Δp29-infected (black bars) C. parasitica strain EP155. The induction patterns for dcl2 and agl2 transcript accumulation has been previously reported [20], [21] and were also measured in parallel here. The identities of the transcript being measured are indicated at the bottom of the histogram. The fold differences in transcript accumulation are indicated on the Y axis and were calculated using the comparative threshold cycle (ΔΔCT) normalized against tubβ transcript levels and calibrated against the normalized value in the wild-type strain, with standard deviations, based on three independent measurements of two independent RNA preparations, indicated by the error bars. Transcript levels for uninfected C. parasitica were set to a value of one.
Figure 5.
Accumulation of rdr transcripts in response to virus infection in dcl2 and agl2 disruption mutant strains.
Panel A. Transcript levels for rdr1–4 and agl2 genes were measured in uninfected (grey bars), CHV-1/EP713- (open bars) and Δp29- (black bars) infected Δdcl2 disruption strain as described for Figure 4. Panel B. Transcript levels for rdr1–4 and dcl2 genes were measured in uninfected (grey bars), CHV-1/EP713- (open bars) and Δp29- (black bars) infected Δagl2 disruption strain as described for Figure 4.
Figure 6.
Effect of rdr gene disruption and hypovirus CHV-1/EP713 on C. parasitica colony growth and morphology.
The colonies of uninfected parental strain DK80 and gene disruption mutant strains are shown in the left column while the corresponding hypovirus CHV-1/EP713- infected strains are shown in the right column. The uninfected triple rdr mutant strain Δrdr1/2/3 and uninfected Δdcl2 strains are shown at the bottom of the left column while the corresponding CHV-1/EP713-infected strains are shown in corresponding positions in the right column. Note the severe growth and morphological changes exhibited by the CHV-1/EP713-infected Δdcl2 strain. Cultures were grown for 7 days on PDA.
Table 2.
Colony diameter (cm) in DNA-damage sensitivity assay measured after 3 days of growth.