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.
Nakayashiki
Hitoshi
H
Laboratory of Plant Pathology, Kobe University, 1-1 Rokkodaicho, Nada, 657-8501 Kobe, Japan. hnakaya@kobe-u.ac.jp
Hanada
Shugo
S
Nguyen
Bao Quoc
BQ
Kadotani
Naoki
N
Tosa
Yukio
Y
Mayama
Shigeyuki
S
D001483:Base Sequence; D020231:Colletotrichum / Q000235:genetics* / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism*; D020868:Gene Silencing; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D020082:Magnaporthe / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D012275:Oryza / Q000382:microbiology; D010935:Plant Diseases / Q000382:microbiology; D034622:RNA Interference
D005656:Fungal Proteins; D049452:Green Fluorescent Proteins