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.
Laboratory of Plant Pathology, Faculty of Agriculture Kobe University, 1-1 Rokkodaicho, Nada, 657-8501 Kobe, Japan.
Naoki
N
Kadotani
Hitoshi
H
Nakayashiki
Yukio
Y
Tosa
Shigeyuki
S
Mayama
D000483:Alleles; D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D004331:Drosophila melanogaster; D005656:Fungal Proteins / Q000502:physiology; D020868:Gene Silencing; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D016681:Genome, Fungal; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007091:Image Processing, Computer-Assisted; D020082:Magnaporthe / Q000378:metabolism*; D008957:Models, Genetic; D009154:Mutation; D009492:Neurospora crassa / Q000235:genetics; D009690:Nucleic Acid Conformation; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D034622:RNA Interference; D012331:RNA, Fungal; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D043244:Ribonuclease III / Q000235:genetics* / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D014908:Triticum / Q000382:microbiology
D005656:Fungal Proteins; D012331:RNA, Fungal; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins; D012313:RNA; D043244:Ribonuclease III