Downloaded from www.microbiologyresearch.org by IP: 54.162.133.179 On: Fri, 19 Feb 2016 01:00:56 Identification of a Spiroplasma citri hydrophilic protein associated with insect transmissibility Nabil Killiny, Brigitte Batailler, Xavier Foissac and Colette Saillard Correspondence Colette Saillard saillard@bordeaux.inra.fr UMR 1090 Ge ´ nomique De ´ veloppement et Pouvoir Pathoge ` ne, INRA et Universite ´ Victor Segalen Bordeaux 2, Centre INRA de Bordeaux, 71 avenue Edouard Bourlaux, BP 81, 33883 Villenave d’Ornon cedex, France Received 18 October 2005 Revised 20 December 2005 Accepted 21 December 2005 With the aim of identifying Spiroplasma citri proteins involved in transmission by the leafhopper Circulifer haematoceps, protein maps of four transmissible and four non-transmissible strains were compared. Total cell lysates of strains were analysed by two-dimensional gel electrophoresis using commercially available immobilized pH gradients (IPGs) covering a pH range of 4–7. Approximately 530 protein spots were visualized by silver staining and the resulting protein spot patterns for the eight strains were found to be highly similar. However, comparison using PDQuest 2-D analysis software revealed two trains of protein spots that were present only in the four transmissible strains. Using MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry and a nearly complete S. citri protein database, established during the still-ongoing S. citri GII-3-3X genome project, the sequences of both proteins were deduced. One of these proteins was identified in the general databases as adhesion-related protein (P89) involved in the attachment of S. citri to gut cells of the insect vector. The second protein, with an apparent molecular mass of 32 kDa deduced from the electrophoretic mobility, could not be assigned to a known protein and was named P32. The P32-encoding gene (714 bp) was carried by a large plasmid of 35?3 kbp present in transmissible strains and missing in non-transmissible strains. PCR products with primers designed from the p32 gene were obtained only with genomic DNA isolated from transmissible strains. Therefore, P32 has a putative role in the transmission process and it could be considered as a marker for S. citri leafhopper transmissibility. Functional complementation of a non-transmissible strain with the p32 gene did not restore the transmissible phenotype, despite the expression of P32 in the complemented strain. Electron microscopic observations of salivary glands of leafhoppers infected with the complemented strain revealed a close contact between spiroplasmas and the plasmalemma of the insect cells. This further suggests that P32 protein contributes to the association of S. citri with host membranes. INTRODUCTION The first-cultured and most-studied spiroplasma is Spiro- plasma citri, the causal agent of citrus stubborn disease, one of the three plant-pathogenic, sieve-tube-restricted, and leafhopper-vector-transmitted mollicutes (Bove ´ et al., 1989; Bove ´ & Garnier, 2003). The main vector of S. citri in the Mediterranean area and the Near East is the leafhopper Circulifer haematoceps (Fos et al., 1986), and Circulifer tenel- lus is thought to be the most important natural vector in California (Oldfield et al., 1976; Kaloostian et al., 1979). Spiroplasmas ingested via phloem-sap feeding traverse the insect gut wall and move into the haemolymph, where they multiply and circulate. They eventually invade the salivary glands, where they multiply further (Liu et al., 1983; Kwon et al., 1999). Probably delivered by exocytosis into the sali- vary duct, they are introduced with saliva into the phloem of a new host plant (Fletcher et al., 1998). Thus, S. citri cells undergo a series of molecular and cellular interactions with the insect vector that are required for transmission to a plant. The detailed mechanisms by which these events take place remain to be elucidated (Fletcher et al., 1998; Kwon et al., 1999). Although many S. citri strains multiply within the haemocoel, the ability to cross insect gut and salivary gland barriers is lost by some strains maintained for a long time in vitro or in planta without passage through an insect host (Bove ´ et al., 1989; Wayadande & Fletcher, 1995). Attachment of bacteria to host cells is thought to be a critical step leading to colonization of a particular tissue, and bacterial pathogens typically express adhesins, i.e. bacterial surface proteins that promote host cell attachment. In the case of human and animal mycoplasmas, adhesins play an important role in invasion and pathogenicity (Rottem, Abbreviations: IPG, immobilized pH gradient; MALDI-TOF, matrix- assisted laser desorption/ionization time-of-flight ScARP, S. citri adhesion-related protein. 0002-8602 G 2006 SGM Printed in Great Britain 1221 Microbiology (2006), 152, 1221–1230 DOI 10.1099/mic.0.28602-0