We have developed a pHANNIBAL-like silencing vector, pSilent-1, for ascomycete fungi, which carries a hygromycin resistance cassette and a transcriptional unit for hairpin RNA expression with a spacer of a cutinase gene intron from the rice blast fungus Magnaporthe oryzae. In M. oryzae, a silencing vector with the cutinase intron spacer (147 bp) showed a higher efficiency in silencing of the eGFP gene than did those with a spacer of a GUS gene fragment or a longer intron (850 bp) of a chitin binding protein gene. Application of pSilent-1 to two M. oryzae endogenous genes, MPG1 and polyketide synthase-like gene, resulted in various degrees of silencing of the genes in 70-90% of the resulting transformants. RNA silencing was also induced by a pSilent-1-based vector in Colletotrichum lagenarium at a slightly lower efficiency than in M. oryzae, indicating that this silencing vector should provide a useful reverse genetic tool in ascomycete fungi.

SummaryWe developed an RNA-silencing vector, pSilent-Dual1 (pSD1), with a convergent dual promoter system that provides a high-throughput platform for functional genomics research in filamentous fungi. In the pSD1 system, the target gene was designed to be transcribed as a chimeric RNA with enhanced green fluorescent protein (eGFP) RNA. This enabled us to efficiently screen the resulting transformants using GFP fluorescence as an indicator of gene silencing. A model study with the eGFP gene showed that pSD1-based vectors induced gene silencing via the RNAi pathway with slightly lower efficiency than did hairpin eGFP RNA-expressing vectors. To demonstrate the applicability of the pSD1 system for elucidating gene function in the rice-blast fungus Magnaporthe oryzae, 37 calcium signalling-related genes that include almost all known calcium-signalling proteins in the genome were targeted for gene silencing by the vector. Phenotypic analyses of the silenced transformants showed that at least 26, 35 and 15 of the 37 genes examined were involved in hyphal growth, sporulation and pathogenicity, respectively, in M. oryzae. These included several novel findings such as that Pmc1-, Spf1-and Neo1-like Ca 2+ pumps, calreticulin and calpactin heavy chain were essential for fungal pathogenicity.

Systematic analysis of RNA silencing was carried out in the blast fungus Magnaporthe oryzae (formerly Magnaporthe grisea) using the enhanced green fluorescence protein (eGFP) gene as a model. To assess the ability of RNA species to induce RNA silencing in the fungus, plasmid constructs expressing sense, antisense, and hairpin RNAs were introduced into an eGFP-expressing transformant. The fluorescence of eGFP in the transformant was silenced much more efficiently by hairpin RNA of eGFP than by other RNA species. In the silenced transformants, the accumulation of eGFP mRNA was drastically reduced, but no methylation of the promoter or coding region was involved in it. In addition, we found small interfering RNAs (siRNAs) only in the silenced transformants. Interestingly, the siRNAs consisted of RNA molecules with at least three different sizes ranging from 19 to 23 nucleotides, and all of them contained both sense and antisense strands of the eGFP gene. To our knowledge, this is the first demonstration in which different molecular sizes of siRNAs have been found in filamentous fungi. Overall, these results indicate that RNA silencing operates in M. oryzae, which gives us a new tool for genome-wide gene analysis in this fungus.

Comprehensive phylogenetic analyses of fungal Argonaute, Dicer, and RNA-dependent RNA polymerase-like proteins have been performed to gain insights into the diversification of RNA silencing pathways during the evolution of fungi. A wide range of fungi including ascomycetes, basidiomycetyes, and zygomycetes possesses multiple RNA silencing components in the genome, whereas a portion of ascomycete and basidiomycete fungi apparently lacks the whole or most of the components. The number of paralogous silencing proteins in the genome differs considerably among fungal species, suggesting that RNA silencing pathways have diversified significantly during evolution in parallel with developing the complexity of life cycle or in response to environmental conditions. Interestingly, orthologous silencing proteins from different fungal clades are often clustered more closely than paralogous proteins in a fungus, indicating that duplication events occurred before speciation events. Therefore, the origin of multiple RNA silencing pathways seems to be very ancient, likely having occurred prior to the divergence of the major fungal lineages.

Dicer is a ribonuclease III-like enzyme playing a key role in the RNA silencing pathway. Genome sequencing projects have demonstrated that eukaryotic genomes vary in the numbers of Dicer-like (DCL) proteins from one (human) to four (Arabidopsis). Two DCL genes, MDL-1 and -2 (Magnaporthe Dicer-like-1 and -2) have been identified in the genome of the filamentous fungus Magnaporthe oryzae. Here we show that the knockout of MDL-2 drastically impaired gene silencing of enhanced green fluorescence protein by hairpin RNA and reduced related small interfering RNA (siRNA) accumulation to nondetectable levels. In contrast, mutating the other DCL, MDL-1, exhibited a gene silencing frequency similar to wild type and accumulated siRNA normally. The silencing-deficient phenotype and loss of siRNA accumulation in the mdl-2 mutant was restored by genetic complementation with the wild-type MDL-2 allele. These results indicate that only MDL-2 is responsible for siRNA production, and no functional redundancy exists between MDL-1 and MDL-2 in the RNA silencing pathway in M. oryzae. Our findings contrast with a recent report in the filamentous fungus Neurospora crassa, where two DCL proteins are redundantly involved in the RNA silencing pathway, but are similar to the results obtained in a more distantly related organism, Drosophila melanogaster, where an individual DCL protein has a distinct role in the siRNA/micro-RNA pathways.The ribonuclease III (RNase III) 1 family, which specifically processes double-stranded RNA (dsRNA) precursors into mature RNAs, is one of the most interesting endonuclease families. RNase III enzymes of the Dicer class are large multidomain proteins and have been shown to play an important role in the post-transcriptional gene silencing pathway triggered by double-stranded RNA. Here, we call the silencing mechanisms collectively, RNA silencing, since this phenomenon has been termed "co-suppression or PTGS" (post-transcriptional gene silencing) in plants, "quelling" in the fungus Neurospora crassa, and "RNA interference" in animals (1). In the RNA silencing pathway, Dicer cleaves long dsRNA into short RNA duplexes of 21-25 nucleotides (small interfering RNAs (siRNAs)) (2-4). Subsequently, these siRNAs are incorporated into the RNA-inducing silencing complex (RISC) to serve as sequence-specific guides for targeting cognate mRNA for destruction (5). Recently, Dicer has been implicated in downstream processes of siRNA production in the silencing pathway. Depletion of Dicer in mammalian cells resulted in a significant reduction in gene silencing efficiency induced by cognate siRNA treatment (6). Supporting this, Dicer has been shown to physically interact with protein components of RISC and siRNAs (7). In addition to the RNA silencing pathway, Dicer also plays an essential role in developmental pathways by generating regulatory 19 -25-nucleotide small RNAs, termed micro-RNAs (miRNAs), from longer partially doublestranded RNA precursors (8 -10). miRNAs negatively regulate gene expression by annealing to the 3Ј-un...