Identification and Characterization of Expansins from Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae)

Ilsong Institute of Life Science, Hallym University, Anyang 431-060, Korea(Received on August 7, 2012; Accepted on August 16, 2012)We identified two novel expansin (EXP) genes in theexpressed sequence tag database of Bursaphelenchusxylophilus, designated as Bx-EXPB2 and -EXPB3.Novel Bx-EXPBs encoded 150 amino acids and theirsimilarities in coding sequence were 70.7−84.0% to thepreviously reported EXPB1 of B. xylophilus. Bx-EXPB2and Bx-EXPB3 were clustered with Bx-EXPB1 and Bm-EXPB1, respectively, forming the independent phylo-geny with other nematode EXPs. All identified Bx-EXPBscontained the signal peptide and were only expressedduring the propagative stage, suggesting that they aresecreted to facilitate nematode migration through hostsby loosening cell walls during infection. Quantitativereal-time PCR analysis showed that the relative ac-cumulation of Bx-EXPB3 mRNAs was the highestamong the three Bx-EXPs examined and the order ofmRNA accumulation was as follows: Bx-EXPB3 > Bx-EXPB2 >> Bx-EXPB1. Homology modeling of Bx-EXPBs showed that the structurally optimum templatewas EXLX1 protein of Bacillus subtilis, whichsharedresidues essential for catalytic activity with Bx-EXPB1and Bx-EXPB2 except for Bx-EXPB3. Taken together,Bx-EXPB1 and Bx-EXPB2 may be involved migrationthrough plant tissues and play a role in pathogenesis.Keywords : Bursaphelenchus xylophilus, infections and propagation in host trees. expansin, homo-logy protein modeling, pathogenicity, pine wilt disease The pinewood nematode, Bursaphelenchus xylophilus isthe known agent causative of pine wilt disease (Mamiya,1975; Lee et al., 2011). Bursaphelenchus xylphilus areknown to be transferred from infected pine trees to newones by the pine sawyer beetle, Monochamus alternates(Mamiya, 1975). To express their potent phytopathogeni-city that is associated with infection and maintenance inthe host trees, B. xylophilus must overcome the physicalbarriers such as plant cell walls, which are mainly com-posed of polysaccharides, lipids, and proteins. The architectof plant cell wall is determined by cellulose, made of re-peating glucose molecules joined by β-1,4 glycosidic bonds(McQueen-Mason and Cosgrove, 1994; van den Brink andde Vries, 2011). The cellulose framework of the wall isinterpenetrated by a cross-linked matrix of non-cellulosemolecules, hemicelluloses, or pectin. Even though pinewoodnematode infected trees usually wither and die within acouple of months (Mamiya, 1975), we still do not understandthe molecular and cellular pathophysiology underlying theabilities of pinewood nematodes to infection and propagatein the trees. However, the presence of diverse cell walldegrading enzymes such as endoglucanases (Kikuchi et al.,2004; Smant et al., 1998), pectate lyases (Kikuchi et al.,2006; Popeijus et al., 2000) and expansins (EXPs) (Kikuchiet al., 2009) that to play crucial roles in initiation andprogression of the plant diseases are present in B. xylophilusgenomes suggested that secreted cell wall degrading enzymesfrom B. xylophilus may be important factors involved withEXPs in plants play pivotal roles in a wide variety ofdevelopmental processes by loosening of their cell walls byweakening non-covalent interactions (McQueen-Mason andCosgrove, 1994; McQueen-Mason and Cosgrove, 1995;Whitney et al., 2000), which makes cell walls susceptible todegradation and remodeling by other cell wall degradingenzymes (Cosgrove, 2000; van den Brink and de Vries,2011). While most EXPs were identified from plants, recentfindings that plant-parasitic nematodes express functionalEXPs suggested that they could be one of the major factorsregulating plant-parasite interactions (Qin et al., 2004; vanden Brink and de Vries, 2011). However, compared withEXPs in plants, the understanding of in vivo functions of