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Applied and Environmental Microbiology, May 2006, p. 3274-3283, Vol. 72, No. 5
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.5.3274-3283.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Stolbur Phytoplasma Genome Survey Achieved Using a Suppression Subtractive Hybridization Approach with High Specificity{dagger}

Agnès Cimerman, Guillaume Arnaud, and Xavier Foissac*

Laboratoire de Biologie Cellulaire et Moléculaire, UMR GDPP, INRA, Université Victor Ségalen Bordeaux 2, 71 avenue Edouard Bourlaux, BP 81, 33883 Villenave d'Ornon, France

Received 24 October 2005/ Accepted 26 February 2006

Phytoplasmas are unculturable bacterial plant pathogens transmitted by phloem-feeding hemipteran insects. DNA of phytoplasmas is difficult to purify because of their exclusive phloem location and low abundance in plants. To overcome this constraint, suppression subtractive hybridization (SSH) was modified and used to selectively amplify DNA of the stolbur phytoplasma infecting a periwinkle plant. Plasmid libraries were constructed, and the origins of the DNA inserts were verified by hybridization and PCR screenings. After a single round of SSH, there was still a significant level of contamination with plant DNA (around 50%). However, the modified SSH, which included a second round of subtraction (double SSH), resulted in an increased phytoplasma DNA purity (97%). Results validated double SSH as an efficient way to produce a genome survey for microbial agents unavailable in culture. Assembly of 266 insert sequences revealed 181 phytoplasma genetic loci which were annotated. Comparative analysis of 113 kbp indicated that among 217 protein coding sequences, 83% were homologous to "Candidatus Phytoplasma asteris" (OY-M strain) genes, with hits widely distributed along the chromosome. Most of the stolbur-specific SSH sequences were orphan genes, with the exception of two partial coding sequences encoding proteins homologous to a mycoplasma surface protein and riboflavin kinase.

* Corresponding author. Mailing address: UMR Génomique Développement et Pouvoir Pathogène, INRA and Université Victor Ségalen Bordeaux 2, 71 avenue Edouard Bourlaux, BP 81, 33883 Villenave d'Ornon, France. Phone: 33 557 122360. Fax: 33 557 122369. E-mail: foissac{at}bordeaux.inra.fr.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, May 2006, p. 3274-3283, Vol. 72, No. 5
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.5.3274-3283.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.





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