The Relationship of Spiroplasma citri and Circulifer tenellus

et al. 2003

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.

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Spiralin Is Not Essential for Helicity, Motility, or Pathogenicity but Is Required for Efficient Transmission of Spiroplasma citri by Its Leafhopper Vector Circulifer haematoceps

Duret

Berho

et al. 2003

“…It also infects many other plants, including the Madagascar periwinkle (Catharanthus roseus), an experimental host plant in which it induces symptoms such as stunting, leaf yellowing, and wilting, eventually leading to plant death. In nature, S. citri is transmitted in a circulative, persistent manner by the leafhoppers Circulifer haematoceps (Hemiptera, Cicadellidae) (in the Mediterranean area and the Near East) (16) and Circulifer tenellus (in the United States) (28).To complete their transmission cycle, spiroplasmas ingested by leafhopper vectors must cross two physical barriers, the gut epithelium (to move from the lumen to the hemocoel) and then the salivary gland-associated membranes (to reach the salivary duct) (24,27). Mainly on the basis of electron microscopy observations, a hypothetical model in which spiroplasmas pass through these two barriers by an endocytosis-exocytosis process has been proposed (13).…”

mentioning

confidence: 99%

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

Breton

Duret

et al. 2010

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.Phytoplasmas and spiroplasmas, two groups of pathogenic mollicutes, are associated with many diseases affecting economically important crops, such as ornamentals, vegetables, fruit trees, and grapevine (6,10,26,36). Whereas most plantpathogenic bacteria colonize the apoplast of plant tissues, phytoplasmas and spiroplasmas are restricted to the phloem sieve tubes and are transmitted from plant to plant by phloem sapsucking insects (31, 42), which are therefore responsible for the spread of the diseases. Despite the fact they share common habitats with phytoplasmas, spiroplasmas are distinguishable in that they display a characteristic helical morphology and can be cultured in vitro. Spiroplasma citri is the etiological agent of citrus stubborn disease (33). It also infects many other plants, including the Madagascar periwinkle (Catharanthus roseus), an experimental host plant in which it induces symptoms such as stunting, leaf yellowing, and wilting, eventually leading to plant death. In nature, S. citri is transmitted in a circulative, persistent manner by the leafhoppers Circulifer haematoceps (Hemiptera, Cicadellidae) (in the Mediterranean area and the Near East) (16) and Circulifer tenellus (in the United States) (28).To complete their transmission cycle, spiroplasmas ingested by leafhopper vectors must cross two physical barriers, the gut epithelium (to move from the lumen to the hemocoel) and then the salivary gland-associated membranes (to reach the salivary duct) (24,27). Mainly on the basis of electron microscopy observations, a hypothetical model in which spiroplasmas pass through these two barriers by an endocytosis-exocytosis pro...

“…In leafhoppers, spiroplasmas ingested from the phloem sieve tubes traverse the insect gut wall and move into the hemolymph, where they multiply and circulate. They eventually invade the salivary glands, where they multiply further (22,23). From there, they are introduced with saliva into the phloem of a new host plant.…”

mentioning

confidence: 99%

Disruption of a Gene Predicted To Encode a Solute Binding Protein of an ABC Transporter Reduces Transmission of Spiroplasma citri by the Leafhopper Circulifer haematoceps

Boutareaud

Garnier

et al. 2004

Spiroplasma citri is transmitted from plant to plant by phloem-feeding leafhoppers. In an attempt to identify mechanisms involved in transmission, mutants of S. citri affected in their transmission must be available. For this purpose, transposon (Tn4001) mutagenesis was used to produce mutants which have been screened for their ability to be transmitted by the leafhopper vector Circulifer haematoceps to periwinkle plants. With one mutant (G76) which multiplied in leafhoppers as efficiently as S. citri wild-type (wt) strain GII-3, the plants showed symptoms 4 to 5 weeks later than those infected with wt GII-3. Thirty to fifty percent of plants exposed to leafhoppers injected with G76 remained symptomless, whereas for wt GII-3, all plants exposed to the transmission showed severe symptoms. This suggests that the mutant G76 was injected into plants by the leafhoppers less efficiently than wt GII-3. To check this possibility, the number of spiroplasma cells injected by a leafhopper through a Parafilm membrane into SP4 medium was determined. Thirty times less mutant G76 than wt GII-3 was transmitted through the membrane. These results suggest that mutant G76 was affected either in its capacity to penetrate the salivary glands and/or to multiply within them. In mutant G76, transposon Tn4001 was shown to be inserted into a gene encoding a putative lipoprotein (Sc76) In the ABCdb database Sc76 protein was noted as a solute binding protein of an ABC transporter of the family S1_b. Functional complementation of the G76 mutant with the Sc76 gene restored the wild phenotype, showing that Sc76 protein is involved in S. citri transmission by the leafhopper vector C. haematoceps.