Stolbur phytoplasma genome survey achieved using a suppression subtractive hybridization approach with high specificity

Abstract

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.

FIG. 1.

Electrophoretic pattern of SSH products for RsaI-SSH (A) and HincII-SSH (B). Lanes M, size marker GeneRuler DNA Ladder Mix (MBI Fermentas); lanes 1, unsubtracted control; lanes 2, SSH product; lanes 3, reverse SSH; lanes 4, double SSH (composite picture of different lanes from the same gel). Black arrows indicate the 500-bp band.

FIG. 2.

Hybridization screening of SSH libraries. Screening of SSH clones using an individual insert hybridization probe (A) and large-scale negative dot blot screening using a reverse SSH probe for RsaI-SSH (B) and double RsaI-SSH plasmid libraries (C). (A) Column 1, stolbur phytoplasma-infected periwinkle DNA; column 2, healthy periwinkle DNA; column 3, homologous hybridization plasmid control. Clone numbers are indicated on the left. (B) Spots A01 to H05: plasmid DNA of 89 RsaI-SSH clones; spot H12, healthy periwinkle DNA. (C) Spots A01 to D10, plasmid DNA of 40 double RsaI-SSH clones; spots E09 and E10, healthy periwinkle DNA.

FIG. 3.

PCR amplification of SSH orphan sequences. PCRs were performed on healthy periwinkle DNA (lane 1) and stolbur phytoplasma-infected periwinkle DNA (lane 2) for HincII-SSH libraries and RsaI-SSH libraries. Clone names are indicated according to libraries. SH, HincII-SSH; SDH, double HincII-SSH; SR, RsaI-SSH; SDR, double RsaI-SSH.

FIG. 4.

Homology of stolbur phytoplasma sequences with “Ca. Phytoplasma asteris” (OY-M) sequences and comparison of functional distributions of CDS. (A) Proportion of stolbur phytoplasma SSH sequences with homology to “Ca. Phytoplasma asteris” (OY-M) sequences (gray) or other bacterial sequences (black). White indicates orphan sequences. (B) Functional distribution of CDS detected in stolbur PO SSH sequences by comparison of the functional distribution of “Ca. Phytoplasma asteris” (OY-M) CDS (C). Functional classes were macromolecule metabolism (white), small molecule metabolism (gray), cellular process (black), element of external origin (hatched), or unknown function (dotted).