Identification of a Spiroplasma citri hydrophilic protein associated with insect transmissibility

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.