We investigated the mechanism of resistance to demethylation inhibitors (DMI) in Penicillium digitatum by isolating the CYP51 gene, which encodes the target enzyme (P450 14DM ) of DMI, from three DMI-resistant and three DMI-sensitive strains. The structural genes of all six strains were identical, but in the promoter region, a unique 126-bp sequence was tandemly repeated five times in the DMI-resistant strains and was present only once in the DMI-sensitive strains. Constitutive expression of CYP51 in the resistant strains was about 100-fold higher than that in the sensitive strains. We introduced CYP51, including the promoter region, from a DMI-resistant strain into a DMI-sensitive strain, which rendered the transformants DMI resistant and increased CYP51 expression. We also found that if the number of copies of the repeat was reduced to two, resistance and CYP51 expression also decreased. These results indicate that the 126-bp unit acts as a transcriptional enhancer and that a tandem repeat of the unit enhances CYP51 expression, resulting in DMI resistance. This is a new fungicide resistance mechanism for filamentous fungi.Demethylation inhibitors (DMIs) are widely used as fungicides in agriculture and medicine. In the 1970s, the development of resistance to DMI fungicides under practical conditions was thought to be unlikely (9, 16). However, in practice, DMI-resistant strains occur widely in several important plant and animal pathogens, such as Erysiphe graminis, Sphaerotheca fuliginea, Pyrenophora teres (12), and Candida albicans (11), causing acute problems in crop production and in the treatment of candidiasis of AIDS patients. Determining the mechanism of resistance is, therefore, quite important.We previously showed that an ATP-binding cassette (ABC) transporter gene, PMR1, is involved in DMI resistance in Penicillium digitatum by disrupting the PMR1 gene and concomitantly increasing sensitivity to DMI fungicides (21). PMR1 expression is strongly induced by fungicide treatment in both DMI-sensitive and DMI-resistant strains, but the constitutive expression level of PMR1 in the resistant strain was relatively higher than that in the sensitive strain (21). ABC transportermediated resistance to toxicants in yeast and human cells is a consequence of increased constitutive expression of the ABC transporter gene (3, 7), so we thought that the higher level of constitutive expression of the PMR1 gene could be responsible for the higher DMI resistance of the resistant strains. However, introduction of the PMR1 coding region under the control of a strong constitutive promoter, PgpdA, into a DMIsensitive strain had no observable effect on DMI resistance (H. Hamamoto, O. Nawata, K. Hasegawa, R. Nakaune, Y. J. Lee, Y. Makizumi, K. Akutsu, and T. Hibi, submitted for publication), suggesting that the constitutive expression level of PMR1 is not the determinative factor for DMI resistance. The coding sequences of this gene from three DMI-sensitive and three DMI-resistant strains were also identical. These results sugges...

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Rice blast is the most devastating plant disease in Japan. Our goal is to create new rice varieties which show enhanced resistance against blast, regardless of the race of blast. By an Agrobacterium-mediated transformation method, we reintroduced a rice class-I chitinase gene, Cht-2 or Cht-3, under the control of the enhanced CaMV 35S promoter and a hygromycin phosphotransferase gene, as a selection marker into the Japonica rice varieties Nipponbare and Koshihikari, which have retained the best popularity over a long period in Japan. In regenerated plants (R(0)), the Cht-2 product was found to accumulate intracellularly whereas the Cht-3 product was found to be targeted extracellularly. The transgenic rice plants which constitutively expressed either chitinase gene showed significantly higher resistance against the rice blast pathogen Magnaporthe grisea races 007.0 and 333. Both high-level expression of the chitinase and blast-resistance were stably inherited by the next generation in several lines.

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