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J. Biol. Chem., Vol. 279, Issue 43, 44467-44474, October 22, 2004

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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^*

Naoki Kadotani, Hitoshi Nakayashiki, Yukio Tosa, and Shigeyuki Mayama

From the Laboratory of Plant Pathology, Faculty of Agriculture Kobe University, 1-1 Rokkodaicho, Nada, 657-8501 Kobe, Japan

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.

Received for publication, July 21, 2004 , and in revised form, August 10, 2004.

^* This work is supported in part by Hyogo Science and Technology Association Grant 15W014. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 81-78-803-5867; Fax: 81-78-803-5867; E-mail: hnakaya{at}kobe-u.ac.jp.

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