Functional analysis of a multicopy host-selective ACT-toxin biosynthesis gene in the tangerine pathotype of Alternaria alternata using RNA silencing

Y. Miyamoto, A. Masunaka, T. Tsuge, Mikihiro Yamamoto, K. Ohtani, T. Fukumoto, K. Gomi, T. L. Peever, Kazuya Akimitsu

Research output: Contribution to journalArticle

37 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)1591-1599
Number of pages9
JournalMolecular Plant-Microbe Interactions
Volume21
Issue number12
DOIs
Publication statusPublished - Dec 2008

Fingerprint

tangerines
Alternaria
Alternaria alternata
pathotypes
RNA Interference
RNA interference
toxins
biosynthesis
Genes
genes
RNA
Fungal Genome
ethyl-2-methylthio-4-methyl-5-pyrimidine carboxylate
genome
homologous recombination
gene targeting
hydrolases
Homologous Recombination
Hydrolases
plasmids

ASJC Scopus subject areas

  • Physiology
  • Agronomy and Crop Science

Cite this

Functional analysis of a multicopy host-selective ACT-toxin biosynthesis gene in the tangerine pathotype of Alternaria alternata using RNA silencing. / Miyamoto, Y.; Masunaka, A.; Tsuge, T.; Yamamoto, Mikihiro; Ohtani, K.; Fukumoto, T.; Gomi, K.; Peever, T. L.; Akimitsu, Kazuya.

In: Molecular Plant-Microbe Interactions, Vol. 21, No. 12, 12.2008, p. 1591-1599.

Research output: Contribution to journalArticle

Miyamoto, Y. ; Masunaka, A. ; Tsuge, T. ; Yamamoto, Mikihiro ; Ohtani, K. ; Fukumoto, T. ; Gomi, K. ; Peever, T. L. ; Akimitsu, Kazuya. / Functional analysis of a multicopy host-selective ACT-toxin biosynthesis gene in the tangerine pathotype of Alternaria alternata using RNA silencing. In: Molecular Plant-Microbe Interactions. 2008 ; Vol. 21, No. 12. pp. 1591-1599.
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