Cloning and expression analysis of NhL1 , a gene encoding an extracellular lipase from the fungal pea pathogen Nectria h
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O R I GI N A L P A P E R
A. Nasser Eddine á F. Hannemann á W. SchaÈfer
Cloning and expression analysis of NhL1, a gene encoding an extracellular lipase from the fungal pea pathogen Nectria haematococca MP VI (Fusarium solani f. sp. pisi ) that is expressed in planta Received: 7 July 2000 / Accepted: 25 October 2000 / Published online: 5 January 2001 Ó Springer-Verlag 2001
Abstract The ®lamentous fungus Nectria haematococca (anamorph Fusarium solani f. sp. pisi) resides in soil, and attacks pea seedlings in the area of the underground epicotyl and upper tap root, causing foot rot disease. We detected lipase activity during in vitro growth of N. haematococca. Subsequently, a lipase gene was cloned and functionally characterised by heterologous expression in Saccharomyces cerevisiae. The full-length cDNA of 1152 bp was cloned using a 3¢ RACE-PCR approach coupled with cDNA library screening. The genomic clone, comprising an ORF of 999 bp interrupted by two introns of 56 and 64 bp, was isolated from a newly constructed lambda phage library. Analysis of the deduced protein sequence revealed the presence of a typical signal peptide at the N-terminus, and of the three conserved amino acids forming the active site of lipases. The lipase of N. haematococca has a low degree of similarity to the lipases from Humicola lanuginosa (37.2%), Rhizomucor miehei (21.6%), Rhizopus delemar (23.1%), Rhizopus niveus (25.9%), and to mono- and diacylglycerol lipase from Penicillium camembertii (30.8%), and very high similarity (94.6%) to a lipase from Fusarium heterosporum. The lipase from N. haematococca shows maximal activity at 37 °C and pH 8.0. Based on Southern analysis, the lipase clone represents a singlecopy gene in N. haematococca. Expression analysis was performed by RT-PCR. In vitro, the lipase gene shows a low basal expression, but is highly inducible by lipase substrates, and repressed by glucose. During plant infection, transcripts of this fungal lipase gene were detected 4, 8, and 10 days after infection. Communicated by C. A. M. J. J. van den Hondel A. N. Eddine á F. Hannemann á W. SchaÈfer (&) Institute of General Botany, Molecular Phytopathology and Genetics, University of Hamburg, Ohnhorststrasse 18, 22609 Hamburg, Germany E-mail: [email protected] Tel.: +49-40-42816266 Fax: +49-40-42816513
Key words Lipase gene á Nectria haematococca á Pisum sativum á Fungal virulence á Gene regulation
Introduction Fungal plant pathogens secrete an array of extracellular enzymes capable of degrading various components of the plant cell wall during invasion of the host. As the aerial parts of plants are covered by the cuticle, which is mainly composed of cutin, fungal cutinases have been considered as the main enzymes implicated in the enzymatic penetration of the plant integument (Kolattukudy 1984; KoÈller and Parker 1989; Trail and KoÈller 1990). However, this theory has been regarded as controversial (SchaÈfer 1998). In several fungal pathogens targeted disruption of cutinase genes produces mutants which show unaltere
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