Spiralin is the most abundant protein at the surface of the plant pathogenic mollicute Spiroplasma citri and hence might play a role in the interactions of the spiroplasma with its host plant and/or its insect vector. To study spiralin function, mutants were produced by inactivating the spiralin gene through homologous recombination. A spiralin-green fluorescent protein (GFP) translational fusion was engineered and introduced into S. citri by using an oriC-based targeting vector. According to the strategy used, integration of the plasmid by a single-crossover recombination at the spiralin gene resulted in the expression of the spiralin-GFP fusion protein. Two distinct mutants were isolated. Western and colony immunoblot analyses showed that one mutant (GII3-9a5) did produce the spiralin-GFP fusion protein, which was found not to fluoresce, whereas the other (GII3-9a2) produced neither the fusion protein nor the wild-type spiralin. Both mutants displayed helical morphology and motility, similarly to the wild-type strain GII-3. Genomic DNA analyses revealed that GII3-9a5 was unstable and that GII3-9a2 was probably derived from GII3-9a5 by a double-crossover recombination between plasmid sequences integrated into the GII3-9a5 chromosome and free plasmid. When injected into the leafhopper vector Circulifer haematoceps, the spiralinless mutant GII3-9a2 multiplied to high titers in the insects (1.1 ؋ 10 6 to 2.8 ؋ 10 6 CFU/insect) but was transmitted to the host plant 100 times less efficiently than the wild-type strain. As a result, not all plants were infected, and symptom production in these plants was delayed for 2 to 4 weeks compared to that in the wild-type strain. In the infected plants however, the mutant multiplied to high titers (1.2 ؋ 10 6 to 1.4 ؋ 10 7 CFU/g of midribs) and produced the typical symptoms of the disease. These results indicate that spiralin is not essential for pathogenicity but is required for efficient transmission of S. citri by its insect vector.

In the plant-pathogenic mollicute Spiroplasma citri, spiralin is the major lipoprotein at the cell surface and is thought to be one of the components involved in the interactions of the spiroplasma with its insect vector. With the aim of identifying surface proteins other than spiralin, monoclonal antibodies (mAbs) were produced by immunization of mice with the spiralin-defective S. citri mutant GII3-9a2. mAb 10G3 was found to react with several polypeptides of 43-47 and 80-95 kDa, all of which were detected in the detergent phase after Triton X-114 partitioning of proteins. Mass spectrometry (MALDI-TOF) analyses of the two major polypeptides P47 and P80 of GII3-9a2, reacting with mAb 10G3, revealed that P47 was a processed product and represented the C-terminal moiety of P80. Search for sequence homologies revealed that P80 shared strong similarities with the S. citri adhesion-related protein P89 (Sarp1) of S. citri BR3, and is one (named Scarp4a) of the eight Scarps encoded by the S. citri GII-3 genome. The eight scarp genes are carried by plasmids pSci1-5. Western immunoblotting of proteins with mAb 10G3 revealed that, in contrast to the insect-transmissible S. citri strain GII-3, the non-insect-transmissible strains ASP-1, R8A2 and 44 did not express Scarps. Southern blot hybridization experiments indicated that these strains possessed no scarp genes, and did not carry plasmids pSci1-5. However, S. citri strain GII3-5, lacking pSci5, was still efficiently transmitted, showing that, in the genetic background of S. citri GII-3, the pSci5-encoded genes, and in particular scarp2b, 3b and 5a, are not essential for insect transmission. Whether plasmid-encoded genes are involved in transmission of S. citri by its leafhopper vector remains to be determined.

The insect-transmissible strain GII-3 of Spiroplasma citri contains plasmids pSci1-6, five of which (pSci1-5) encode adhesin-like proteins and one (pSci6) encodes protein P32, which has been associated with insect transmissibility. In contrast, S. citri strains ASP-1 and 44, which cannot be transmitted via injection into the leafhopper vector Circulifer haematoceps, lack these proteins and also do not carry plasmids pSci1-6. To further study the apparent relationship between the presence of plasmids and insect transmissibility, plasmids from S. citri GII-3 were introduced into the insect-non-transmissible S. citri strain 44 by electrotransformation using the tetM gene as the selection marker. Tetracycline-resistant transformants were shown to carry one, two or three distinct plasmids. Plasmids pSci1-6 were all detected in the transformants, pSci1 being the most frequently found, alone or together with other plasmids. Selected S. citri 44 transformants having distinct plasmid contents were submitted, separately or in combination, to experimental transmission to periwinkle (Catharanthus roseus) plants via injection into the leafhopper vector. The occurrence of symptomatic plants indicated that, in contrast to S. citri 44, spiroplasmal transformants were transmitted to the host plant, in which they multiplied. Spiroplasma cultures isolated from these infected plants all contained pSci6, leading to the conclusion that, under the experimental conditions used, transformation by pSci6 conferred insect transmissibility to S. citri strain 44. This is believed to be the first report of a phenotypic change associated with transformation of S. citri by natural plasmids.