5.4.2
CHARACTERIZATION AND PARTIAL SEQUENCING OF AN ANTI-RALSTONIA SOLANACEARUM PROTEIN FROM RESISTANT POTATO

LY HE, FH YUAN and J FENG

State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, 2 West Yuan Ming Yuan Road, Beijing 100094, China

Background and objectives
One of the major limiting factors for potato production in China and some other countries of the world is bacterial wilt caused by Ralstonia solanacearum, and the complex biology and ecology of the pathogen make it difficult to control. Breeding for resistance and application of it in practice is attractive and is expected to reduce damage due to this disease. But to get a resistant variety of crop by traditional breeding methods is both laboursome and time-consuming. Plant genetic engineering techniques developed in recent years provide new approaches and prospects to solve this problem. One of the key steps in respect of this work is to obtain genes, ideally from the host plant whose products can resist infective diseases. Naturally, plants have a variety of constitutive and inducible mechanisms for protecting themselves against infections by various pathogens [1]. But constitutive resistant proteins were rarely reported. In recent years, we have found that there are some kinds of antimicrobial proteins which may be involved in potato resistance to bacterial wilt. In this article, we will present the results of preliminary characterization and partial sequencing of an anti-R. solanacearum protein from BW-resistant potato.

Results and conclusions
It was observed that the crude protein extracts from plants of potato resistant varieties or clones, MS-42.3, MS-1C.2, 898006, 381064.12 and AVRDC-1287.19 inhibited growth of the potato strain of Ralstonia solanacearum PO41, but those from susceptible varieties Jinguan, Favorita and Zhongshu No. 3 had no inhibitory activity or had only a very weak effect on PO41. When these potato varieties or clones were investigated for differences of soluble protein content in leaves, a 32-kDa protein band appeared only in SDS-PAGE patterns of resistant potatoes, but not in those of susceptible ones, whether inoculated or not. This protein was purified by improved routine purification procedures and is designated as antimicrobial protein 1 (AP1). Results from isoelectric focusing electrophoresis indicated that the pI value of AP1 was 6.0. Amino acid composition analysis demonstrated that this protein was rich in Gly, Phe and Glx. The stability of AP1 treated by heat, enzyme and different pH revealed that this protein was quite stable at 20-80C and at pH 6-9, and sensitive to proteinase K and trypsin. The characteristics of AP1 are different from previously reported pathogenesis-related proteins, 32 kDa chitinase and beta-1,3-glucanases [2]. A cDNA expression library of potato-resistant cultivars was successfully constructed using the system of mRNA microprep and cDNA quick synthesis with phage gt11 vector. A positive clone was obtained from approximately 3x105 recombinant clones using an antibody probe against AP1 . Truthfulness of the positive clone was identified with Western blot hybridization. Positive recombinant lgt11 DNAs were preparated and purified, and the target DNA was directly subcloned into pGEM-7Zf(+) plasmid. Three recombinant clones carrying 0.5-0.6 kb DNA were detected with gel electrophoresis. Nucleotide sequences of the cDNA insert were determined using T7 and SP6 primers. A DNA sequence of 538 kbp near the 3' poly(A) tail was obtained and 126 amino acids were translated. This result provides a basis for cloning the whole gene encoding AP1 protein.

References
1. Brokaert WF, Terras FRG, Cammue BPA, Osborn RW, 1995. Plant Physiology 108, 1353-1358.
2. Leah R, Tommerup H, Svendsen I, Mundy J, 1991. Journal of Biological Chemistry 266, 1564-1573.