1.2.15
THE EXPRESSION OF 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHASE GENES IN POTATO LEAVES INFECTED WITH PHYTOPHTHORA INFESTANS OR TREATED WITH ELICITORS

K KAWAKITA1, M NAGANO1, D TAKEMOTO1, N DOKE1 and A THEOLOGIS2

1Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan; 2Plant Gene Expression Center, Albany, California 94710, USA

Background and objectives
The phenomenon of enhanced ethylene production by pathogen attack in plants has been observed, while the function of ethylene production is elusive. The plant hormone ethylene controls many aspects of plant growth and development and is produced in response to various biotic and abiotic stresses, such as mechanical strain, wounding, hypoxia and flooding, chilling, soil salinity and infection by pathogens. Ethylene is synthesized from S-adenosyl-L-methionine via 1-aminocyclopropane-1-carboxylic acid (ACC). The rate-limiting step in ethylene production is ACC synthase (ACS), which is encoded by a highly divergent multigene family in a number of plant species. In the potato, five members of the ACS multigene family are known. Genomic clones have been characterized of ST-ACS1A, ST-ACS1B and ST-ACS2 [1] and a partial cDNA sequence is known for the ST-ACS4 and ST-ACS5 [2]. We investigated the expression of ACS genes in potato leaves inoculated with Phytophthora infestans or treated with elicitors.

Materials and methods
We utilized potato leaves of cv. Rishiri (an interspecific hybrid between Solanum tuberosum and S. demissum) carrying the R1 resistance gene to Phytophthora infestans, potato late blight fungus. Fungal elicitor, hyphal wall components (HWC) was prepared from mycelia of P. infestans. Total genomic DNA was isolated from potato leaves and digested with EcoRI and HindIII, and then it was electrophoresed on 0.8% agarose gels and transferred to nylon membranes for Southern hybridization. Total RNA was extracted from potato leaves by the phenol-SDS method and polyA+ RNA was isolated to construct a cDNA library.

Results and conclusions
We examined the induction of PR proteins as one of the resistance reactions in potato leaves treated with various elicitors, salicylic acid, arachidonic acid and HWC. The accumulation of PR-N by the elicitors was repressed by the addition of AVG, an ACS inhibitor, suggesting the involvement of ethylene production in the induction of resistance reactions. Preliminary genomic Southern blot analysis of potato DNA, using the tomato ACS clones as probes, revealed several hybridizing bands, indicating the presence of related members of an ACS gene family in potato. To determine whether the induction of ethylene biosynthesis is correlated with the induction of specific ACS transcripts, RNA from potato leaves treated for various times with the elicitors or inoculated with P. infestans was analysed by use of LE-ACS genes as probes. The transcripts were detected by LE-ACS1A, 1B and 2 in potato leaves after 12 h of treatment with arachidonic acid, whereas they were detectable after 36 h HWC treatment. LE-ACS1A and 1B homologous transcripts were detectable in potato leaves 24 h after inoculation with an incompatible race, while LE-ACS2 homologous transcript was detectable 36 h after inoculation with a compatible race. The result that different ACS genes were expressed between compatible and incompatible combinations suggests different gene regulatory mechanisms among these genes. Next, we constructed the cDNA library from potato leaves 36 h after the treatment with HWC and tried to isolate potato ACS gene(s) using LE-ACS2 as a first step to gain an understanding of the molecular action of ethylene in resistance reactions.

References
1. Destefano-Beltran LJC, Van Caeneghem W, Gielen J et al., 1995. Molecular and General Genetics 246, 496-508.
2. Schlagnhaufer CD, Arteca RN, Pell EJ, 1997. Plant Molecular Biology 35, 683-688.