Identification and characterization of the a-L-arabinofuranosidase B of Fusarium oxysporum f. sp. dianthi Carlos A. Chaco ´n-Martı ´nez a , Juan M. Anzola a , Andre ´s Rojas a , Freddy Herna ´ndez a , Howard Junca a,b , Walter Ocampo a , Patricia Del Portillo a, * a Corporacio ´n CorpoGen, Department of Molecular Biotechnology, Carrera 5, No. 66A-34, Bogota ´ D.C., Colombia b Department of Environmental Microbiology, GBF-German Research Centre for Biotechnology, Mascheroder Weg 1, D-38124 Braunschweig, Germany Accepted 26 August 2004 Abstract The gene encoding a-L-Arabinofuranosidase B (abfB) from the phytopathogenic fungus Fusarium oxysporum f. sp. dianthi (Fod) was identified, cloned, sequenced and heterologously expressed. AbfB consists in an intronless open reading frame of 1500-bp coding for a protein of 499 aminoacid residues. The partially purified fusion protein has an apparent molecular mass of approximately 58 kDa and exhibits a-L-arabinofuranosidase activity. The specific activity of the recombinant enzyme was 1.07 units mg K1 protein. Optimal activity is attained at pH 4.0 and 50 8C. Interestingly, the abfB gene is actively transcribed in carnation plants infected with Fod. Its upregulation during the infection process suggests a possible role of this gene as virulence factor in the phytopathogenicity of this fungus. q 2004 Elsevier Ltd. All rights reserved. Keywords: Carnation; Fusarium wilt; Phytopathogenic fungus; Hemicellulase; Arabinase; a-L-arabinofuranosidase 1. Introduction The main structural components of plant cell walls are cellulose, hemicellulose, lignin and pectin, compounds also acting as protective barriers against phytopathogenic organisms [12]. Fungal plant pathogens are able to produce a variety of enzymes hydrolysing these compounds, facilitating the penetration and colonization of their hosts [34]. Xylan is a predominant hemicellulose polysaccharide composed of a backbone of b-1,4-xylopyranosyl residues, some of which are substituted with arabinosyl, acetyl and glucuronosyl residues [29,30,35]. L-Arabinosyl residues are widely distributed in some hemicelluloses, such as arabinan, arabinoxylan, arabic gum, and arabinogalactan [30]. The complete breakdown of these compounds requires the cooperative action of several microbial enzymes, particularly endo-b-xylanases and arabinosi- dases [11]. Arabinosidases comprise the a-L-arabinofura- nosidases (a-L-arabinofuranoside arabinofuranohydrolase, EC 3.2.1.55, ABF), enzymes able to hydrolyse terminal non-reducing a-L-1,2-, a-L-1,3- and a-L-1,5-a-L-arabinofur- anosyl linkages from a-L-arabinofuranosides, arabinans, arabinoxylans and arabinogalactans [25]. The ABFs have been classified into four families of glycanases (glycosyl hydrolase families 43, 51, 54, and 62) on the basis of aminoacid (aa) sequence similarities [18]. Glycosyl hydrolase families 51 and 54 comprise those glycanases showing a preferential activity against arabinose-containing polysaccharides. Enzymes degrading hemicellulose polysaccharides use to be present in plant pathogens, e.g. xylanases have been isolated from a wide variety of fungal plant pathogens [34]. For this reason, attempts to interpret their functions as virulence factors have been a common trend in phytopathol- ogy, despite that the specific roles of these enzymes in pathogenicity have not been clearly established yet. As an example, disruptions of two xylanase genes in Cochliobolus 0885-5765/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.pmpp.2004.08.005 Physiological and Molecular Plant Pathology 64 (2004) 201–208 www.elsevier.com/locate/pmpp Abbreviations: AP-PCR, arbitrary priming-polymerase chain reaction; ABF, a-L-arabinofuranosidase; abfB, a-L-arabinofuranosidase B gene; Fod, Fusarium oxysporum f. sp. dianthi; Fox, Fusarium oxysporum; PNP-A, para-nitrophenyl-a-L-arabinofuranoside; PNP, p-nitrophenol. * Corresponding author. Tel.: C57 1 3484606/8; fax: C57 1 3484607. E-mail address: corpogen@etb.net.co (P. Del Portillo).