Michio Masuko

Transcriptional activation of the alternative oxidase gene of the fungus Magnaporthe grisea by a respiratory-inhibiting fungicide and hydrogen peroxide

Alternative oxidase (AOX) is dramatically induced when the fungus Magnaporthe grisea is incubated with the fungicide SSF-126, which interacts with the cytochrome bc1 complex in the electron transport system of mitochondria. A full-length cDNA for the alternative oxidase gene (AOX) was obtained, and the deduced amino acid sequence revealed marked similarity to other AOXs, but lacks two cysteine residues at corresponding sites which are conserved in plant AOXs and play essential roles in the post-translational regulation. Northern blot experiments showed that treatment of M. grisea cells with SSF-126 induces accumulation of AOX mRNA in a dose-dependent manner, and the level was correlated with the activity of alternative respiration. H2O2 also induced the accumulation of the transcript with a short half-life (<15 min). Nuclear run-on experiments showed that the AOX gene was transcribed constitutively in unstimulated cells. Cycloheximide did not change the basal level of transcription, but induced the accumulation of the transcript, indicating that active degradation of the transcript occurs by factor(s) sensitive to cycloheximide. On the other hand, SSF-126 enhanced the transcriptional activity of AOX gene threefold compared to that of control cells, and H2O2 was also potent for enhancement of the transcription. From these results, it is concluded that the respiratory inhibitor-dependent activation of the transcription is a primary determinant for the induction of alternative respiration in M. grisea. Because we have previously shown that SSF-126 treatment of M. grisea mitochondria induced the generation of superoxide, active oxygen species are thought to be signal mediators to activate AOX gene transcription in M. grisea.

Effect of the methoxyiminoacetamide fungicide, SSF129, on respiratory activity in Botrytis cinerea

(E)-2-Methoxyimino-N-methyl-2-[2-(2,5-dimethylphenoxymethyl)phenyl]acetamide (SSF129) has been developed as a broad-spectrum systemic fungicide for control of cereal and fruit diseases. This compound inhibited NADH-oxidation by submitochondrial particles from mycelial cells of Botrytis cinerea, with an EC 50 value of 14.5 nM, due to blockage of electron transport through the cytochrome bc 1 complex in the mitochondrial respiratory chain. However, SSF129 did not suppress, but rather increased, oxygen consumption by mycelial cells of the fungus. This was because mycelial cells contain an alternative oxidase protein and the cells have the ability to rapidly switch electron flux from the main cytochrome pathway to the alternative pathway on blockage of the former by SSF129. The alternative pathway of the mycelia seems not to be operative when the cytochrome pathway is functional. Naturally occurring flavonoids inhibited the alternative oxidase of the mycelial cells in a dose-dependent manner, with EC 50 values of 68.4 μM for flavone and 63.7 μM for flavanone. These observations suggested that plant components play an important role in control of gray mould by SSF129.

Control of Rice Blast Disease by Submerged Treatment with SSF-126

SSF-126の水面施用は, イネ葉身上におけるイネいもち病菌の胞子発芽, 付着器形成および葉身内部への侵入には影響を及ぼさなかった. 一方, 同処理は圃場試験においてイネいもち病に対して予防的かつ治療的防除効果を示した. 以上の結果から, 本剤はイネの組織内部に侵入した菌糸の伸長を抑制すると考えられる. さらに, 本剤はイネいもち病が感染する主要な部位の一つであるイネの葉身内に蓄積するという, イネいもち病防除剤として有利な特性をもっている.