Copy number-dependent DNA methylation of the Pyricularia oryzae MAGGY retrotransposon is triggered by DNA damage.

Transposable elements are common targets for transcriptional and post-transcriptional gene silencing in eukaryotic genomes. However, the molecular mechanisms responsible for sensing such repeated sequences in the genome remain largely unknown. Here, we show that machinery of homologous recombination (HR) and RNA silencing play cooperative roles in copy number-dependent de novo DNA methylation of the retrotransposon MAGGY in the fungus Pyricularia oryzae. Genetic and physical interaction studies revealed that RecA domain-containing proteins, including P. oryzae homologs of Rad51, Rad55, and Rad57, together with an uncharacterized protein, Ddnm1, form complex(es) and mediate either the overall level or the copy number-dependence of de novo MAGGY DNA methylation, likely in conjunction with DNA repair. Interestingly, P. oryzae mutants of specific RNA silencing components (MoDCL1 and MoAGO2) were impaired in copy number-dependence of MAGGY methylation. Co-immunoprecipitation of MoAGO2 and HR components suggested a physical interaction between the HR and RNA silencing machinery in the process.

Effects of Modified Atmosphere Packaging, Food Life Extenders and Temperature on the Shelf Life of Ready-Made Dishes.

The combined effect of several microbial control factors including gas barrier of containers, modified atmosphere packaging, food life extenders and storage temperature was discussed in order to determine the possibility for improving the shelf life for hamburger steak and deepfried chicken, representative ready-made dishes sold at convenience stores in Japan. Multiple measures including cold storage were effective in improving the shelf life of ready-made dishes. It was also suggested that storage tests for ready-made dishes should be conducted at 10℃, a practical temperature, to confirm the storable period, as well as at 15℃, an adequate abuse temperature, to confirm the effects of various microbial control factors. In the present study, the test group 4 (nitrogen + barrier containers + pH modifier) performed most favorably at both temperatures, indicating the efficacy of multiple means including "cold storage" in improving the shelf life (extending the consume-by date) of ready-made dishes. All strains isolated from the tested hamburger steak and deep-fried chicken were common food contaminant bacterial species.

Exogenous proteinogenic amino acids induce systemic resistance in rice.

BACKGROUND: Plant immune responses can be induced by endogenous and exogenous signaling molecules. Recently, amino acids and their metabolites have been reported to affect the plant immune system. However, how amino acids act in plant defense responses has yet to be clarified. Here, we report that treatment of rice roots with amino acids such as glutamate (Glu) induced systemic disease resistance against rice blast in leaves. RESULTS: Treatment of roots with Glu activated the transcription of a large variety of defense-related genes both in roots and leaves. In leaves, salicylic acid (SA)-responsive genes, rather than jasmonic acid (JA) or ethylene (ET)-responsive genes, were induced by this treatment. The Glu-induced blast resistance was partially impaired in rice plants deficient in SA signaling such as NahG plants expressing an SA hydroxylase, WRKY45-knockdown, and OsNPR1-knockdown plants. The JA-deficient mutant cpm2 exhibited full Glu-induced blast resistance. CONCLUSIONS: Our results indicate that the amino acid-induced blast resistance partly depends on the SA pathway but an unknown SA-independent signaling pathway is also involved.

Is the fungus Magnaporthe losing DNA methylation?

The long terminal repeat retrotransposon, Magnaporthe gypsy-like element (MAGGY), has been shown to be targeted for cytosine methylation in a subset of Magnaporthe oryzae field isolates. Analysis of the F1 progeny from a genetic cross between methylation-proficient (Br48) and methylation-deficient (GFSI1-7-2) isolates revealed that methylation of the MAGGY element was governed by a single dominant gene. Positional cloning followed by gene disruption and complementation experiments revealed that the responsible gene was the DNA methyltransferase, MoDMT1, an ortholog of Neurospora crassa Dim-2. A survey of MAGGY methylation in 60 Magnaporthe field isolates revealed that 42 isolates from rice, common millet, wheat, finger millet, and buffelgrass were methylation proficient while 18 isolates from foxtail millet, green bristlegrass, Japanese panicgrass, torpedo grass, Guinea grass, and crabgrass were methylation deficient. Phenotypic analyses showed that MoDMT1 plays no major role in development and pathogenicity of the fungus. Quantitative polymerase chain reaction analysis showed that the average copy number of genomic MAGGY elements was not significantly different between methylation-deficient and -proficient field isolates even though the levels of MAGGY transcript were generally higher in the former group. MoDMT1 gene sequences in the methylation-deficient isolates suggested that at least three independent mutations were responsible for the loss of MoDMT1 function. Overall, our data suggest that MoDMT1 is not essential for the natural life cycle of the fungus and raise the possibility that the genus Magnaporthe may be losing the mechanism of DNA methylation on the evolutionary time scale.

The complete genome sequence of Pantoea ananatis AJ13355, an organism with great biotechnological potential.

Pantoea ananatis AJ13355 is a newly identified member of the Enterobacteriaceae family with promising biotechnological applications. This bacterium is able to grow at an acidic pH and is resistant to saturating concentrations of L-glutamic acid, making this organism a suitable host for the production of L-glutamate. In the current study, the complete genomic sequence of P. ananatis AJ13355 was determined. The genome was found to consist of a single circular chromosome consisting of 4,555,536 bp [DDBJ: AP012032] and a circular plasmid, pEA320, of 321,744 bp [DDBJ: AP012033]. After automated annotation, 4,071 protein-coding sequences were identified in the P. ananatis AJ13355 genome. For 4,025 of these genes, functions were assigned based on homologies to known proteins. A high level of nucleotide sequence identity (99%) was revealed between the genome of P. ananatis AJ13355 and the previously published genome of P. ananatis LMG 20103. Short colinear regions, which are identical to DNA sequences in the Escherichia coli MG1655 chromosome, were found to be widely dispersed along the P. ananatis AJ13355 genome. Conjugal gene transfer from E. coli to P. ananatis, mediated by homologous recombination between short identical sequences, was also experimentally demonstrated. The determination of the genome sequence has paved the way for the directed metabolic engineering of P. ananatis to produce biotechnologically relevant compounds.

Transcriptional control and protein specialization have roles in the functional diversification of two dicer-like proteins in Magnaporthe oryzae.

Quantitative RT-PCR and overexpression studies of two Dicer-like proteins, MoDcl1 and MoDcl2, in Magnaporthe oryzae indicated that the functional diversification of the MoDcl1 and MoDcl2 proteins in RNA-mediated gene silencing pathways was likely to have arisen from both transcriptional control and protein specialization.

Systematic functional analysis of calcium-signalling proteins in the genome of the rice-blast fungus, Magnaporthe oryzae, using a high-throughput RNA-silencing system.

We 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.

siRNA-dependent and -independent post-transcriptional cosuppression of the LTR-retrotransposon MAGGY in the phytopathogenic fungus Magnaporthe oryzae.

The LTR-retrotransposon MAGGY was introduced into naive genomes of Magnaporthe oryzae with different genetic backgrounds (wild-type, and MoDcl1 [mdl1] and MoDcl2 [mdl2] dicer mutants). The MoDcl2 mutants deficient in MAGGY siRNA biogenesis generally showed greater MAGGY mRNA accumulation and more rapid increase in MAGGY copy number than did the wild-type and MoDcl1 mutants exhibiting normal MAGGY siRNA accumulation, indicating that RNA silencing functioned as an effective defense against the invading element. Interestingly, however, regardless of genetic background, the rate of MAGGY transposition drastically decreased as its copy number in the genome increased. Notably, in the MoDcl2 mutant, copy-number-dependent MAGGY suppression occurred without a reduction in its mRNA accumulation, and therefore by a silencing mechanism distinct from both transcriptional gene silencing and siRNA-mediated RNA silencing. This might imply that some mechanism possibly similar to post-transcriptional cosuppression of Ty1 retrotransposition in Saccharomyces cerevisiae, which operates regardless of the abundance of target transcript and independent of RNA silencing, would also function in M. oryzae that possesses the RNA silencing machinery.

Evolution and diversification of RNA silencing proteins in fungi.

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.

RNA silencing as a tool for exploring gene function in ascomycete fungi.

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.

One of the two Dicer-like proteins in the filamentous fungi Magnaporthe oryzae genome is responsible for hairpin RNA-triggered RNA silencing and related small interfering RNA accumulation.

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.

RNA silencing in the phytopathogenic fungus Magnaporthe oryzae.

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.