P. syringae in Aesculus, Infection and Control Figure 3. Behaviour of GFP-expressing P. syringae in planta. (A) An A. hippocastanum seedling inoculated with strain PD4818-pMP4655 photographed after 2 months shows vertical lesion expansion (up to 6 cm in length) and tissue necrosis after removal of the outer tissues of the bark. The inoculation site is indicated with an arrowhead. Note the bending of the infection around the lower node. The scale bar indicates 1 cm. (B) Side view of a longitudinally split piece of stem from a PD4818-pMP4655 inoculated seedling removed 1–2 cm from the inoculation site 4 weeks after inoculation. Necrosis and discolouration is visible in the cambial and phloem areas. (C) A longitudinal section of an infection border in bark tissue after inoculation with PD4818-pMP4655 sampled 4 weeks after inoculation. A parabolic shaped zone with elevated cell wall autofluorescence is discernible (arrowheads) with a P. s. pv. aesculi colony located in the tip of the infected zone, at about 1 cm away from the inoculation site. The boxed area is shown in detail in D. The scale bar indicates 25 mm. (D) Numerous bacteria are clustered near the forefront of an advancing lesion in parenchyma 4 weeks after inoculation. The bacteria observed here are relatively small and densely packed. The scale bar indicates 10 mm. (E) Within the necrotic area near the original site of inoculation several clusters of bacteria are found that occupy the cavities left by dead cells. Image taken from a longitudinal section of a 6 week old infection. The scale bar indicates 25 mm. (F) A large aggregate of PD4818-pMP4655 located in necrotic tissue of a 4 week old lesion. Individual bacteria were spaced apart and did not undergo any type of motility. The scale bar indicates 25 mm. (G) Detail of a PD4818-pMP4655 cluster in necrotic tissue of a 4 week old infection, emphasizing the spacing between individual bacteria. The scale bar indicates 10 mm. (H) Immunodetection of wild-type PD4818 in the necrotic tissue of a 10 week old infection showing a similar spatial arrangement of bacteria to that shown in G. The scale bar indicates 10 mm. doi:10.1371/journal.pone.0039604.g003 PLoS ONE | www.plosone.org 6 July 2012 | Volume 7 | Issue 7 | e39604