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.

Eighty-five Pyricularia isolates were collected from 29 host species of Gramineae, Bambusideae and Zingiberaceae plants sampled in Brazil, Uganda, Ivory Coast, India, Nepal, China, Indonesia and Japan. These isolates were compared on the basis of pathogenicity, mating ability and restriction fragment length polymorphisms with single-copy DNA probes. Based on the pathogenicity to eight differential gramineous plants, these isolates were classified into seven pathotypes : finger millet type, foxtail millet type, common millet type, rice type, crabgrass type, Italian ryegrass/ weeping lovegrass type, and non-cereal/grass type. Genetic variation among these isolates was assessed by RFLP analysis with two restriction enzymes and nine single-copy DNA probes isolated from a finger millet strain. An UPGMA dendrogram based on the RFLPs revealed that the 86 isolates could be classified into seven major groups. Isolates from cereal crops (finger millet, foxtail millet, common millet, wheat and rice) and a grass, Brachiaria plantaginea, were clustered into a single group. They were further divided into six subgroups corresponding to the pathotypes. Among cereal crop isolates only an isolate from pearl millet was located into a different group. The remaining isolates were clustered into five groups designated as the crabgrass group, the buffelgrass and jungle rice group, the rice cutgrass, knotroot bristlegrass and Setaria tomentosa group, the bamboo and bamboo grass group and the Zingiber mioga group. The isolates from cereal crops were generally capable of mating with finger millet strains and constituted a closed mating compatibility group. These results suggested that the isolates from cereal crops form a single group with a common ancestor although they are pathogenic to taxonomically diverse plants. A combined analysis of the pathogenicity and genetic similarity suggested that the transmission of M. grisea isolates occurs in natural agroecosystems between finger millet and Eleusine africana, goosegrass or Bambusa arundinacea, between foxtail millet and green bristlegrass, and between rice and tall fescue, Italian ryegrass, sweet vernalgrass, reed canarygrass or Oryza longistaminata.

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.