A viral double-stranded RNA up regulates the fungal virulence of Nectria radicicola.

Double-stranded RNAs (dsRNAs) are widespread in plant pathogenic fungi, but their functions in fungal hosts remain mostly unclear, with a few exceptions. We analyzed dsRNAs from Nectria radicicola, the causal fungus of ginseng root rot. Four distinct sizes of dsRNAs, 6.0, 5.0, 2.5, and 1.5 kbp, were detected in 24 out of the 81 strains tested. Curing tests of individual dsRNAs suggested that the presence of 6.0-kbp dsRNA was associated with high levels of virulence, sporulation, laccase activity, and pigmentation in this fungus. The 6.0-kbp dsRNA-cured strains completely lost virulence-related phenotypes. This 6.0-kbp dsRNA was reintroduced by hyphal anastomosis to a dsRNA-cured strain marked with hygromycin resistance, which resulted in the restoration of virulence-related phenotypes. These results strongly suggest that 6.0-kbp dsRNA up regulates fungal virulence in N. radicicola. Sequencing of several cDNA clones derived from 6.0-kbp dsRNA revealed the presence of a RNA-dependent RNA polymerase (RDRP) gene. Phylogenetic analysis showed that this gene is closely related to those of plant cryptic viruses. Biochemical analyses suggested that the 6.0-kbp dsRNA may regulate fungal virulence through signal-transduction pathways involving cyclic AMP-dependent protein kinase and protein kinase C.

Molecular characterization of the cDNA encoding an acidic isoform of PR-1 protein in rice.

Rice cDNA encoding an acidic type of pathogenesis-related protein-1 (PR-1a) was cloned and characterized. The deduced PR-1a protein consisted of 168 amino acid residues, including 24 hydrophobic signal sequences at the N-terminus. The predicted molecular mass of the PR-1a was 15,728 Da with a theoretical pI of 4.5, an indication of an acidic protein. The PR-la showed high homology to an acidic PR-1 of Zea mays (74%) and a previously identified basic type PR-1 of rice (64%). Both rice PR-1 and PR-1a genes were found to exist as small gene families through Southern blot hybridization analyses. The PR-1 mRNA was accumulated only in leaves, while the PR-1a transcript was accumulated throughout the plant at a low level. Expression of both PR-1 genes was induced by infections of the rice blast fungus, Magnaporthe grisea, or the bacterial leaf blight pathogen, Xanthomonas oryzae pv. oryzae, and the treatment of benzo (1, 2, 3) thiadiazole-7-carbothioic acid S-ethyl ester, H2O2, or CuSO4. The expression of both PR-1 genes was higher and more rapidly induced in an incompatible interaction than in a compatible interaction in the rice M. grisea interactions.

Analysis of genes expressed during rice-Magnaporthe grisea interactions.

Expressed sequence tag (EST) analysis was applied to identify rice genes involved in defense responses against infection by the blast fungus Magnaporthe grisea and fungal genes involved in growth within the host during a compatible interaction. A total of 511 clones was sequenced from a cDNA library constructed from rice leaves (Oryza sativa cv. Nipponbare) infected with M. grisea strain 70-15 to generate 296 nonredundant ESTs. The sequences of 293 clones (57.3%) significantly matched National Center for Biotechnology Information database entries; 221 showed homologies with previously identified plant genes and 72 with fungal genes. Among the genes with assigned functions, 32.8% were associated with metabolism, 29.4% with cell/organism defense or pathogenicity, and 18.4% with gene/protein expression. cDNAs encoding a type I metallothionein (MTs-1) of rice and a homolog of glucose-repressible gene 1 (GRG1) of Neurospora crassa were the most abundant representatives of plant and fungal genes, comprising 2.9 and 1.6% of the total clones, respectively. The expression patterns of 10 ESTs, five each from rice and M. grisea, were analyzed. Five defense-related genes in rice, including four pathogenesis-related genes and MTs-1, were highly expressed during M. grisea infection. Expression of five stress-inducible or pathogenicity-related genes of the fungus, including two hydrophobin genes, was also induced during growth within the host. Further characterization of the genes represented in this study would be an aid in unraveling the mechanisms of pathogenicity of M. grisea and the defense responses of rice.