1.8.12
CLONING OF A CUTINASE GENE FROM SPILOCAEA OLEAGINA, A FUNGAL PATHOGEN CAUSING THE PEACOCK EYE-SPOT DISEASE OF OLIVE

S OUCHI1,2, Y MATSUDA2, H TOYODA1, T KATO1, N MORII1 and A GRANITI3

1Faculty of Agriculture, and 2Institute for Comprehensive Agricultural Sciences, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan; 3Facolta di Agraria, Universita degli studi di Bari, Via Amendola 165/a, 70126 Bari, Italy

Background and objectives
Fungal cutinases have been implicated in their penetration into host cells during the primary interactions. Spilocaea oleagina is a pathogen of the olive tree, causing peacock eye spot on leaves. Characteristically the fungus parasitizes the sub-cuticlar layer of the leaves and never enters the host cells. The fungus may utilize cutin components for its pathogenicity and survival. The objective of this study was to determine the essential role of cutinases in the parasitism of this fungus.

Results and conclusions
The fungus, S. oleagina, was inoculated into a Czapek medium containing 0.05% (w/v) juvenic acid, an inducer for the production of cutinases in fungal cells, and shake-cultured at 26C for several days. Cutinase activity of both fungal mycelia and culture filtrate was determined on a daily basis. The enzyme was isolated from mycelia by acetone extraction and from the culture filtrate by ammonium sulfate precipitation. The extracted proteins were dissolved in 50 mM sodium phosphate buffer (pH 7.0) and examined for cutinase activity using a model substrate, p-nitrophenylbutyrate. The results indicated that cutinase activity was initially detected after 3-4 days and rapidly increased, reaching a maximum after 8-9 days of incubation. The enzyme was purified by two-dimensional electrophoresis; firstly by electrofocusing and subsequently by SDS-PAGE. Finally, the enzymatically active protein of approximately 20 kDa was obtained from both mycelial and culture-filtrate fractions. This protein was electrophoretically eluted from gels and blotted onto a PVDF-membrane to analyse the N-terminal amino-acid sequence. On the basis of the determined sequence of the purified enzyme (N-SDLVELQKDY-), degenerative DNA primers were synthesized for subsequent cDNA cloning of the cutinase mRNA. Total mRNAs were extracted from mycelial cells cultured in the presence of the inducer, and cDNAs were artificially synthesized using the poly-T primer linked with the anchor primer (3' AGCTACAGCTGAGCTCAG 5'). The cDNAs were PCR-amplified by the use of Taq DNA polymerase, dNTP and the anchor and degenerative primers. The PCR product was then subcloned and used for hybridization analysis and DNA sequence determination. Alternatively, the cutinase genes were directly screened on genomic DNAs of S. oleagina by the PCR amplification method. For this purpose, the nucleotide sequences of cutinase genes which had been reported in different cutinolytic phytopathogenic fungi such as Fusarium solani f. sp. pisi, Colletotrichum capsici, Alternaria brassicicola and Magnaporthe grisea were analysed on the database and highly conserved sequences were utilized to construct primers for PCR amplification of chromosomal DNA of S. oleagina. Consequently, some DNA fragments were obtained with the synthesized PCR primers and their sequences were determined.