Molecular characterization of spiroplasma viruses and the mechanism of resistance of Spiroplasma citri lines to infection by the virus SVTS2

Scope and method of study. The total protein profiles of S. citri M200H and two SVTS2 virus-resistant lines derived from it were compared by 2-D gel electrophoresis. The virus binding site was investigated by blocking tests with anti-membrane protein serum. The three lines were transformed with viral DNA by electroporation to determine whether viral DNA could replicate. The SVTS2 genome was cloned and used as a DNA probe to screen the genome of spiroplasma. The virus was characterized by developing a restriction map and its partial DNA sequencing. Host modification of viral DNA by methylation was investigated. A previously undescribed virus, isolated from S. citri BR3, was characterized.; Findings and conclusions. Membrane proteins P1 and P2 were missing or significantly reduced in both virus-resistant lines, MR2 and MR3, compared to the susceptible M200H. Membrane proteins of 55 kd, 77 kd and 89 kd are not involved in virus attachment or invasion. SVTS2 DNA which is present in extrachromosomal DNA and also integrated into the genome of virus-resistant lines, MR2 and MR3, but not M200H, may function as a viral incompatibility element and provide immunity to superinfection by SVTS2. Differences in host range and restriction maps, and only 56% identity of DNA sequences, indicated that SVTS2 is different from but closely related to known virus SpV1-R8A2B. Methylation of one of the Sau3AI sites of SVTS2 RF DNA in S. citri M200H was observed. A virus designated SVBR3, isolated from S. citri BR3-3X, was rod-shaped and contained single-stranded DNA of 8.6 kb. No DNA sequence identity between SVBR3 and SVTS2 was detected under our conditions. SVBR3 DNA, integrated into the genome of S. citri BR3 lines, may cause disruption or rearrangement of genes required for insect transmission of the spiroplasma.