Abstract
Spiroplasma citri is a plant pathogenic mollicute transmitted by the leafhopper vector Circulifer haematoceps. Successful transmission requires the spiroplasmas to cross the intestinal epithelium and salivary gland barriers through endocytosis mediated by receptor-ligand interactions. To characterize these interactions we studied the adhesion and invasion capabilities of a S. citri mutant using the Ciha-1 leafhopper cell line. S. citri GII3 wild-type contains 7 plasmids, 5 of which (pSci1 to 5) encode 8 related adhesins (ScARPs). As compared to the wild-type strain GII3, the S. citri mutant G/6 lacking pSci1 to 5 was affected in its ability to adhere and enter into the Ciha-1 cells. Proteolysis analyses, Triton X-114 partitioning and agglutination assays showed that the N-terminal part of ScARP3d, consisting of repeated sequences, was exposed to the spiroplasma surface whereas the C-terminal part was anchored into the membrane. Latex beads cytadherence assays showed the ScARP3d repeat domain (Rep3d) to be involved, and internalization of the Rep3d-coated beads to be actin-dependent. These data suggested that ScARP3d, via its Rep3d domain, was implicated in adhesion of S. citri GII3 to insect cells. Inhibition tests using anti-Rep3d antibodies and competitive assays with recombinant Rep3d both resulted in a decrease of insect cells invasion by the spiroplasmas. Unexpectedly, treatment of Ciha-1 cells with the actin polymerisation inhibitor cytochalasin D increased adhesion and consequently entry of S. citri GII3. For the ScARPs-less mutant G/6, only adhesion was enhanced though to a lesser extent following cytochalasin D treatment. All together these results strongly suggest a role of ScARPs, and particularly ScARP3d, in adhesion and invasion of the leafhopper cells by S. citri.
Highlights
Spiroplasma citri is a phloem-limited plant pathogenic bacterium belonging to the class Mollicutes and was the first plant mollicute to be cultivated in cell-free medium [1]
S. citri adhesion-related proteins (ScARPs) are believed to play a critical role in spiroplasma-host interactions by promoting insect transmission because: (i) ScARPs are conserved in all three plant pathogenic spiroplasmas S. citri, S. kunkelii and S. phoeniceum, as well as in the bee pathogen S. melliferum and the rabbit tick spiroplasma sp. 277F [18,26,27,28], (ii) ScARPs are absent in non-insect-transmissible strains of S. citri [13,15] (iii) ScARPs are predicted to localize at the spiroplasma cell surface [14], (iv) ScARPs share homology with S. citri BR3 protein P89, the detection of which was associated to adhesion to insect cells [19], and (v) ScARPs share N-terminal repeat domains with characterized adhesins [20]
Our study focused on the repeat domain of ScARP3d (Rep3d), made of 7 repeats of 38–40 amino acids, which we showed to exhibit adhesin function
Summary
Spiroplasma citri is a phloem-limited plant pathogenic bacterium belonging to the class Mollicutes and was the first plant mollicute to be cultivated in cell-free medium [1]. Transmission of S. citri from infected to healthy plants is mediated by phloem sapfeeding leafhoppers, Circulifer haematoceps in the Mediterranean basin [4] and Circulifer tenellus in the USA and Mediterranean basin [5,6], in a persistent propagative manner. In S. citri GII3, these proteins are encoded by plasmids pSci to 6 [14], which are absent in S. citri 44 [15]. The implication of plasmid-encoded determinants in insect-transmission was further documented by the finding that pSci from S. citri GII3 partially restored insect-transmissibility when introduced into S. citri 44 [16]. At the same time as pSci contains sequences that are essential for transmission, pSci to 5 encode determinants that are required for efficient transmission of S. citri by its leafhopper vector C. haematoceps [16,17]
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