title: Functional analysis of a multicopy host-selective ACT-toxin biosynthesis gene in the tangerine pathotype of Alternaria alternata using RNA silencing.
abstract: Alternaria brown spot, caused by the tangerine pathotype of Alternaria alternata, is a serious disease of commercially important tangerines and their hybrids. The pathogen produces host-selective ACT toxin, and several genes (named ACTT) responsible for ACT-toxin biosynthesis have been identified. These genes have many paralogs, which are clustered on a small, conditionally dispensable chromosome, making it difficult to disrupt entire functional copies of ACTT genes using homologous recombination-mediated gene disruption. To overcome this problem, we attempted to use RNA silencing, which has never been employed in Alternaria spp., to knock down the functional copies of one ACTT gene with a single silencing event. ACTT2, which encodes a putative hydrolase and is present in multiple copies in the genome, was silenced by transforming the fungus with a plasmid construct expressing hairpin ACTT2 RNAs. The ACTT2 RNA-silenced transformant (S-7-24-2) completely lost ACTT2 transcripts and ACT-toxin production as well as pathogenicity. These results indicated that RNA silencing may be a useful technique for studying the role of ACTT genes responsible for host-selective toxin biosynthesis in A. alternata. Further, this technique may be broadly applicable to the analysis of many genes present in multiple copies in fungal genomes that are difficult to analyze using recombination-mediated knockdowns.
authors.affiliation: United Graduate School and Faculty of Agriculture, Kagawa University, Miki, Kagawa 761-0795, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Miyamoto
authors.forename: A
authors.initials: A
authors.lastname: Masunaka
authors.forename: T
authors.initials: T
authors.lastname: Tsuge
authors.forename: M
authors.initials: M
authors.lastname: Yamamoto
authors.forename: K
authors.initials: K
authors.lastname: Ohtani
authors.forename: T
authors.initials: T
authors.lastname: Fukumoto
authors.forename: K
authors.initials: K
authors.lastname: Gomi
authors.forename: T L
authors.initials: TL
authors.lastname: Peever
authors.forename: K
authors.initials: K
authors.lastname: Akimitsu
mesh_terms: D000528:Alternaria / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity; D002957:Citrus / Q000382:microbiology*; D005656:Fungal Proteins / Q000235:genetics*; D018628:Gene Dosage; D055785:Gene Knockdown Techniques / Q000379:methods; D005800:Genes, Fungal; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D009183:Mycotoxins / Q000096:biosynthesis / Q000235:genetics*; D010957:Plasmids; D034622:RNA Interference; D012331:RNA, Fungal / Q000235:genetics; D014170:Transformation, Genetic
chemical_list: D005656:Fungal Proteins; D009183:Mycotoxins; D012331:RNA, Fungal; D049452:Green Fluorescent Proteins