Infection of the Circulifer haematoceps cell line Ciha-1 by Spiroplasma citri: the non-insect-transmissible strain 44 is impaired in invasion

Successful transmission of Spiroplasma citri by its leafhopper vector requires a specific interaction between the spiroplasma surface and the insect cells. With the aim of studying these interactions at the cellular and molecular levels, a cell line, named Ciha-1, was established using embryonic tissues from the eggs of the S. citri natural vector Circulifer haematoceps. This is the first report, to our knowledge, of a cell line for this leafhopper species and of its successful infection by the insect-transmissible strain S. citri GII3. Adherence of the spiroplasmas to the cultured Ciha-1 cells was studied by c.f.u. counts and by electron microscopy. Entry of the spiroplasmas into the insect cells was analysed quantitatively by gentamicin protection assays and qualitatively by double immunofluorescence microscopy. Spiroplasmas were detected within the cell cytoplasm as early as 1 h after inoculation and survived at least 2 days inside the cells. Comparing the insect-transmissible GII3 and non-insect-transmissible 44 strains revealed that adherence to and entry into Ciha-1 cells of S. citri 44 were significantly less efficient than those of S. citri GII3.

Sequences Essential for Transmission of Spiroplasma citri by Its Leafhopper Vector, Circulifer haematoceps, Revealed by Plasmid Curing and Replacement Based on Incompatibility

ABSTRACT Spiroplasma citri GII3 contains highly related low-copy-number plasmids pSci1 to -6. Despite the strong similarities between their replication regions, these plasmids coexist in the spiroplasma cells, indicating that they are mutually compatible. The pSci1 to -6 plasmids encode the membrane proteins known as S. citri adhesion-related proteins (ScARPs) (pSci1 to -5) and the hydrophilic protein P32 (pSci6), which had been tentatively associated with insect transmission, as they were not detected in non-insect-transmissible strains. With the aim of further investigating the role of plasmid-encoded determinants in insect transmission, we have constructed S. citri mutant strains that differ in their plasmid contents by developing a plasmid curing/replacement strategy based on the incompatibility of plasmids having identical replication regions. Experimental transmission of these S. citri plasmid mutants through injection into the leafhopper vector Circulifer haematoceps revealed that pSci6, more precisely, the pSci6_06 coding sequence, encoding a protein of unknown function, was essential for transmission. In contrast, ScARPs and P32 were dispensable for both acquisition and transmission of the spiroplasmas by the leafhopper vector, even though S. citri mutants lacking pSci1 to -5 (encoding ScARPs) were acquired and transmitted at lower efficiencies than the wild-type strain GII3.

Entry of Spiroplasma citri into Circulifer haematoceps Cells Involves Interaction between Spiroplasma Phosphoglycerate Kinase and Leafhopper Actin

ABSTRACT Transmission of the phytopathogenic mollicutes, spiroplasmas, and phytoplasmas by their insect vectors mainly depends on their ability to pass through gut cells, to multiply in various tissues, and to traverse the salivary gland cells. The passage of these different barriers suggests molecular interactions between the plant mollicute and the insect vector that regulate transmission. In the present study, we focused on the interaction between Spiroplasma citri and its leafhopper vector, Circulifer haematoceps. An in vitro protein overlay assay identified five significant binding activities between S. citri proteins and insect host proteins from salivary glands. One insect protein involved in one binding activity was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as actin. Confocal microscopy observations of infected salivary glands revealed that spiroplasmas colocated with the host actin filaments. An S. citri actin-binding protein of 44 kDa was isolated by affinity chromatography and identified by LC-MS/MS as phosphoglycerate kinase (PGK). To investigate the role of the PGK-actin interaction, we performed competitive binding and internalization assays on leafhopper cultured cell lines (Ciha-1) in which His6-tagged PGK from S. citri or purified PGK from Saccharomyces cerevisiae was added prior to the addition of S. citri inoculum. The results suggested that exogenous PGK has no effect on spiroplasmal attachment to leafhopper cell surfaces but inhibits S. citri internalization, demonstrating that the process leading to internalization of S. citri in eukaryotic cells requires the presence of PGK. PGK, regardless of origin, reduced the entry of spiroplasmas into Ciha-1 cells in a dose-dependent manner.

Characterization of a Phytoplasma Associated with Cabbage Yellows in Iran

In 2001, a disease tentatively named Iranian cabbage yellows (ICY) was observed in cabbage fields of Zarghan (Fars Province, Iran). The major symptoms of the disease were yellowing, little leaves, plant stunting, opening of the head, and proliferation of the buds at the base of the stem into a witches'-broom. Among leafhoppers collected in cabbage fields, only Circulifer haematoceps transmitted the ICY agent. The disease agent was transmitted by the leafhopper from cabbage to cabbage, cauliflower, rape, and periwinkle, causing phytoplasma-type symptoms in these plants. Polymerase chain reaction (PCR) using phytoplasma-specific primer pair P1/P7 and nested PCR using P1/P7 and R16F2n/R16R2 primer pairs amplified products of expected size (1.8 and 1.2 kb, respectively) from symptomatic cabbage plants. Both restriction fragment length polymorphism (RFLP) of nested PCR products (1.2 kb) and phylogenetic analyses of 16S–23S rDNA spacer region sequence indicated that the ICY phytoplasma had the closest relationship to subgroup A members of the clover proliferation group, including beet leafhopper-transmitted virescence agent, ‘Candidatus Phytoplasma trifolii’, Columbia Basin potato purple top phytoplasma, and vinca virescence phytoplasma. Cabbage is reported as a new natural host to the 16SrVI group of phytoplasmas.

Plasmid pSci6 from Spiroplasma citri GII-3 confers insect transmissibility to the non-transmissible strain S. citri 44

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.