KADO Clarenc カリフォルニア大学, デービス校(米国), 教授
ICHINOSE Yuki Faculty of Agriculture, Okayama University, Associate Professor, 農学部, 助教授 (50213004)
SHIRAISHI Tomonori Faculty of Agriculture, Okayama University, Associate Professor, 農学部, 助教授 (10033268)
OKU Hachiro Faculty of Agriculture, Okayama University, Professor, 農学部, 教授 (20033144)
KADO Clarence I. Department of Plant Pathology, University of California, Professor
|Budget Amount *help
¥3,000,000 (Direct Cost : ¥3,000,000)
Fiscal Year 1991 : ¥3,000,000 (Direct Cost : ¥3,000,000)
The organization and the structure of the genes encoding phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) were determined and the effects of elicitor and suppressor produced by a pea pathogen, Mycosphaerella pinodes, were examined by transforming a chimeric DNA containing the PAL- and CHS-promoter fragments connected to a reporter gene into tobacco or pea plant with particle gun, Agrobacterium-mediated transformation, or electroporation.
1. Analysis of PAL genes. Chimeric DNA containing PAL promoter fragment connected to luciferase gene (lux), beta-glucuronidase (GUS) or chloramphenicol acetyltransferase (CAT) gene as a reporter were constructed. Chimeric DNA (PAL-GUS) in the vector, pBI101, were introduced into tobacco and pea by Agrobacterium mediated- transformation. Km^R-transgenic tobacco plants were regenerated from the callus. GUS activities were induced by the treatment with pathogen or elicitors in some of the transgenic plants. Since the regeneration line of pea p
lant has not been established from the single cell lines, we obtained Km^R calli and the induction of GUS activities were examined by the treatment with fungal elicitor and the infection of pathogens. Considerable induction of GUS activity was observed in some of the callus. The integration of the chimeric DNA into the plant chromosomes are currently under investigation. Gene transfer by using a particle gun (Dupont Inc.) into these plants were performed at UCD, Davis. Cytological studies showed that plants with Lux or GUS gene connected to 35S promoter as a control exhibited the distinctive GUS atibity in the cells possibly carrying the chimeric DNA, however those with PAL promoter did not exhibit GUS or Lux activities even by the treatment with fungal elicitor. Furthermore, the sequentially deleted promoter fragment of PSPAL1 connected to a plant expression vector containing the reporter gene (CAT) was introduced into pea protoplasts by electroporation, and induction by elicitor and suppression by orthovanadate was examined. Cis-element responsible for these regulations exists between -340--140 of PSPAL1.
2. Analysis of CHS gene. The structure and the organization of three major CHS genes were determined from 38 CHS-cDNAs prepared from elicitor treated pea epicotyl tissues. Two of the major pea CHS genes (PSCHS1, PSCHS2) from a cluster in a tandem repeat with a spacer, and an identical 31 bp sequence containing the consensus motif of box I located 5'-upstream from the putative TATA box of both CHS genes. Buth were highly expressed even without the treatment with fungal elicitor by transient transformation assay mentioned above, which indicate that the activator elements exist in the promoter-distal region (PCHS1 : -1493 - +80, and PCHS2 : - 1889 - +83).
3. Isolation of signalling substances from the pathogen and study of the signal transduction pathway Prof. C. I. Kado visited our laboratory on December, 1991, and discussed about the signaling substances produced by the pathogens. We particularly focused on the substances that blocked the induction of defense reactions. Since M. pinodes suppressor specifically inhibits pea plasma membrane ATPase, he examined bacterial signalling molecules from the aspect of blocking PM-ATPase. Less