PMC

Origin and neutral phylogeny of a world collection of 45 X. campestris pv. campestris strains. (A) Countries of origin for the different X. campestris pv. campestris strains of the collection are in dark red. (B) A neighbor-joining tree based on Dice similarity indices inferred from 1,942 AFLP markers shows the phylogenetic relationships among X. campestris pv. campestris strains. Bootstrap values higher than 70% are indicated. X. campestris pv. campestris strains with genome sequences available are highlighted in black. On the right, lines indicate AFLP-based clades of X. campestris pv. campestris strains. X. campestris pv. raphani strain CFBP5828 was used to root the tree. XccA to -G, X. campestris pv. campestris of the A to G clades, respectively.

Southern blot hybridization analyses of total DNAs from 20 bacteria. Samples of about 3 μg of EcoRI-digested genomic DNAs were analyzed on an agarose gel, transferred to a nylon membrane, and hybridized with the IS476A probe (A), IS1478A probe (B), or IS1479A probe (C). DNAs from X. campestris pv. campestris 11A (lane 1), X. campestris pv. campestris 11 (lane 2), X. campestris pv. campestris 17 (lane 3), X. campestris pv. campestris 2 (lane 4), X. campestris pv. campestris 6 (lane 5), X. campestris pv. campestris 85 (lane 6), X. campestris pv. campestris 88 (lane 7), X. campestris pv. mangiferaeindicae 38 (lane 8), X. campestris pv. glycinea 69 (lane 9), X. campestris pv. dieffenbachiae 65 (lane 10), X. campestris pv. vesicatoria 64 (lane 11), X. campestris pv. phaseoli 73 (lane 12), X. campestris pv. citri (lane 13), X. campestris pv. begoniae (lane 14), X. campestris pv. oryzae 1 (lane 15), Agrobacterium tumefaciens LBA4404 (lane 16), Erwinia carotovora subsp. carotovora ZL4 (lane 17), Pseudomonas solanacearum RD4 (lane 18), Rhizobium leguminosarum 128C53 (lane 19), and E. coli DH5α (lane 20) were analyzed. The sizes of HindIII-digested λ DNA fragments are indicated to the left of the gels.

Oxidative burst reactions in heterologous N. tabacum cell suspension cultures after elicitation with supernatants of X. campestris pv. campestris co-incubated with plant cell wall material. The production of hydrogen peroxide was quantified by means of an H₂O₂-dependent chemiluminescence reaction (A). For each measurement, 200 μl of the respective supernatants were added to the cell cultures. The hydrogen peroxide formation was monitored at different time intervals upon the addition of supernatants of C. annuum cell wall material (✶), supernatants of X. campestris pv. campestris cultures (▲), supernatants of X. campestris pv. campestris cultures co-incubated with C. annuum cell wall material (●), and for a negative control of 200 μl water (♦). There was a clear oxidative burst upon the addition of a supernatant of X. campestris pv. campestris co-incubated with cell wall material, but an almost similar explicit reaction when a supernatant of X. campestris pv. campestris was added that had not been co-incubated with cell wall material. (B) Supernatants of X. campestris pv. campestris cultures were treated with polymyxin B agarose to remove LPS. Then the effect of the purified supernatants on N. tabacum cell suspension cultures was analyzed. The formation of H₂O₂ was monitored upon the addition of supernatants of X. campestris pv. campestris cultures (▲), supernatants of X. campestris pv. campestris cultures co-incubated with C. annuum cell wall material (●), supernatants of X. campestris pv. campestris cultures purified from LPS (■), supernatants of X. campestris pv. campestris cultures co-incubated with C. annuum cell wall material and purified from LPS (✶), and after adding 200 μl water as a negative control (♦). Removing the LPS reduced the response to X. campestris pv. campestris supernatant to the level of the water control. In contrast to this, the removal of LPS reduced the amplitude of the cell culture response to X. campestris pv. campestris co-incubated with cell wall material, but this supernatant still evoked a clear oxidative burst reaction.

Secretion assays with X. campestris pv. vesicatoria and X. campestris pv. campestris strains revealed differences in T2S substrate specificities. (A) Secretion of XCV0536, XCV0965, XCV4358, and XCV4360 from X. campestris pv. vesicatoria was type II independent in X. campestris pv. campestris. X. campestris pv. campestris strains LMG 568 (wt), LMG 568ΔxpsD (ΔxpsD), LMG 568ΔxcsD (ΔxcsD), and LMG 568ΔxpsDΔxcsD (ΔxpsDΔxcsD) carrying XCV0536–c-Myc, XCV0965–c-Myc, XCV3671–c-Myc, XCV4358–c-Myc, or XCV4360–c-Myc as indicated were incubated in NYG medium. Total cell extracts (TE) and culture supernatants (SN) were analyzed by immunoblotting using a c-Myc epitope-specific antibody. For the analysis of extracellular protease and xylanase activities, bacteria were incubated on NYG agar plates containing milk proteins or RBB xylan. Halo formation was documented 2 dpi. (B) Efficient secretion of the predicted xylanase XCC0857 in X. campestris pv. campestris depends on xpsD and xcsD. X. campestris pv. campestris strain LMG 568 and deletion derivatives thereof lacking xpsD (ΔxpsD), xcsD (ΔxcsD), or both xpsD and xcsD (ΔxpsDΔxcsD) and carrying XCC0857–c-Myc, XCC4115–c-Myc, or XCC4118–c-Myc as indicated were incubated in NYG medium. TE and SN were analyzed as described for panel A. (C) PghAxc and PghBxc were secreted independently of the T2S systems of X. campestris pv. campestris strain LMG 568. X. campestris pv. campestris strain LMG 568 and T2S mutants thereof carrying PghAxc–c-Myc and PghBxc–c-Myc as indicated were incubated in NYG medium. TE and SN were analyzed as described for panel A. The blot was reprobed with an antibody directed against the putative translocon protein XopA, which unspecifically detects intracellular proteins in X. campestris pv. campestris. One representative signal is shown.