Pseudomonas savastanoi pv. savastanoi contains two iaaL paralogs, one of which exhibits a variable number of a trinucleotide (TAC) tandem repeat

Abstract

In this study, Pseudomonas savastanoi pv. savastanoi isolates were demonstrated to contain two iaaL paralogs, which are both chromosomally located in most strains. Comparative analysis of iaaL nucleotide sequences amplified from these two paralogs revealed that one paralog, iaaL(Psn), is 100% identical to iaaL from P. savastanoi pv. nerii, while the other paralog, iaaL(Psv), exhibited 93% identity to iaaL from Pseudomonas syringae pv. tomato (iaaL(Pto)). A 3-nucleotide motif (TAC) comprised of 3 to 15 repeats, which remained stable after propagation of the strains in olive plants, was found in iaaL(Psv). Based on the observed nucleotide sequence variations, a restriction fragment length polymorphism assay was developed that allowed differentiation among iaaL(Psn), iaaL(Psv), and iaaL(Pto)(.) In addition, reverse transcriptase PCR on total RNA from P. savastanoi pv. savastanoi strains demonstrated that both iaaL(Psv) and iaaL(Psn) containing 14 or fewer TAC repeats are transcribed. Capillary electrophoresis analysis of PCR-amplified DNA fragments containing the TAC repeats from iaaL(Psv) allowed the differentiation of P. savastanoi pv. savastanoi isolates.

FIG. 1.

Detection of iaaL gene sequences in P. savastanoi strains. Shown is Southern blot analysis of BglII-digested total DNA using an iaaL probe. Lane 1 (Psn) is P. savastanoi pv. nerii ITM 519, and lane 2 (Pto) is P. syringae pv. tomato DC3000. Lanes 3 to 17 (Psv) are P. savastanoi pv. savastanoi strains. Lanes: 3, CFBP 2074; 4, PVF 1; 5, IVIA 1628-3; 6, NCPPB 1344; 7, IVIA 1624-b1; 8, LSV C2.01; 9, IVIA 1637-B3; 10, IVIA 1637-C1; 11, IVIA 1629-1a; 12, IVIA 1651-c15; 13, NCPPB 64; 14, IVIA 1657-b8; 15, IVIA 2733-1a; 16, IVIA 2743-3; and 17, IVIA 2445-4. L, HindIII-digested λ phage DNA. The positions (in kilobases) of the molecular size markers are indicated on the right of the gel.

FIG. 2.

PCR-RFLP differentiation of the iaaL gene sequences from P. syringae and P. savastanoi strains. (A) Schematic representation of iaaL DNA fragments amplified by PCR from the indicated strains using primers IAAL-F and IAAL-R. Target positions for HaeIII are indicated by arrowheads. iaaL_Psn, P. savastanoi pv. nerii EW-2009; iaaL_Pto, P. syringae pv. tomato DC3000; and iaaL_Psv, P. savastanoi pv. savastanoi ITM 317. (B and C) Gel electrophoresis (3% agarose) of iaaL amplicons and HaeIII-digested amplicons, respectively. Lane 1, P. syringae pv. tomato DC3000; lane 2, P. savastanoi pv. nerii strain LPVM Psn2. Lanes 3 to 12 are P. savastanoi pv. savastanoi strains. Lanes: 3, CFBP 2074; 4, IVIA 1628-3; 5, IVIA 1624-b1; 6, IVIA 1649-1; 7, LSV C2.01; 8, IVIA 1637-B3; 9, IVIA 1629-1a; 10, IVIA 1637-C1; 11, IVIA 1651-c15; and 12, IVIA 1657-b8. Lane C, negative control without template. A fragment of approximately 60 bp, most likely formed by nonspecific primer annealing, is visible in all samples, including C. Lane L, 50-bp DNA step ladder (Promega Co.); the positions (in base pairs) of the molecular size markers are indicated on the left of the gel.

FIG. 3.

Transcriptional analysis of iaaL_Psn and iaaL_Psv in P. savastanoi pv. savastanoi. Gel electrophoresis (3% agarose) of RT-PCR amplicons of iaaL genes (A) and HaeIII-digested iaaL amplicons shown in panel A (B) are shown. Lane 1, P. syringae pv. tomato DC3000. Lanes 2 to 6 correspond to P. savastanoi pv. savastanoi strains. Lanes: 2, ITM 317; 3, NCPPB 3335; 4, NCPBB 639; 5, IVIA 1637-B3; 6, IVIA 1637-C1; and 7, NCPPB 64. Lane C+, positive control RT-PCR amplification reaction with total DNA of NCPPB 3335 as the template. Lane L, 50-bp DNA ladder (Promega Co.); the positions (in base pairs) of the molecular size markers are indicated on the left of the gel.