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Applied and Environmental Microbiology, July 2004, p. 3960-3967, Vol. 70, No. 7
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.7.3960-3967.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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

A. Boutareaud, J. L. Danet, M. Garnier,{dagger} and C. Saillard*

UMR Génomique Développement et Pouvoir Pathogène, INRA et Université Victor Ségalen Bordeaux 2, 33883 Villenave d'Ornon cedex, France

Received 29 September 2003/ Accepted 5 April 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.

* Corresponding author. Mailing address: UMR GDPP, IBVM-Centre INRA de Bordeaux, BP 81, 33883 Villenave d'Ornon cedex, France. Phone: 33 5 57 12 23 62. Fax: 33 5 57 12 23 69. E-mail: saillard{at}bordeaux.inra.fr.

{dagger} Deceased.

Applied and Environmental Microbiology, July 2004, p. 3960-3967, Vol. 70, No. 7
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.7.3960-3967.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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