Lignin and Suberin are Deposited Near the Wound Boundary When inoculation sites were microscopically observed, an autofluorescent lining that followed the borders of the wound was consistently discerned from the second or third day after the moment of inoculation and onwards, in both mock and P. s. pv. aesculi inoculated stems. The autofluorescence was confined to the cell walls of a continuous layer of parenchyma cells, several cells wide. Sections of sampled time points were stained with histochemical dyes to define the cell wall components that make up the observed lining near the wound boundary. Using phloroglucinol-HCl staining it was determined that the localiza- tion and timing of the observed layer of autofluorescent cells corresponded with distinct appearance of lignin (Fig. 1D). The distribution of lignin was restricted to several cells wide for the inwards flank of the wound (Fig. 1D, arrowhead) and more diffusely spread in the disjointed part of the wound (Fig. 1D, arrow). Often, the parenchyma cells that participated in lignin impregnation were located at a variable distance from the wound surface, suggesting a signal was relayed from the wound surface to these cells. Altogether, these findings indicate that lignin is a major component of the early wound repair responses in A. hippocastanum bark. Besides lignin another histological compound with autofluor- escent properties that could potentially be of importance in the barrier lining the wound is suberin [13,14,17]. To simultaneously visualize suberin and lignin, Sudan IV stained sections were observed under fluorescence conditions whereupon suberin lamellae appeared in dim red, while lignin autofluorescence was unaltered (See materials and methods). From the sixth day after inoculation and onwards suberin was found as a lamella against the cell wall of the cells that exhibited lignification earlier (Fig. 1E/ F). The deposition was uniform and did not occlude pit fields (Fig. 1G), supporting that the lining of lignified and suberized cells around the wound perimeter functions in sealing off the wound from healthy tissues. No comprehensive differences regarding the timing and extent of suberin deposition were detectable between P. syringae and mock inoculated wounds. This hints that the observed layer of cells with lignified and suberized walls is an intrinsic response of A. hippocastanum to wounding, which also takes place in the presence of P. s. pv. aesculi. Initial Plant Responses do not Prevent Bacterial Spread The effectiveness of the cellular response in restraining pathogen invasion after wounding can be assessed when related to observations on bacterial proliferation. Using immunolabelling, the spread of inoculated P. s. pv. aesculi was monitored. During the first 2 days after inoculation, the bacteria were still mainly localized on the wound surface, with some individual bacteria present several cell layers deeper (data not shown). At the fourth day after inoculation several scattered bacteria were found behind the developing barrier zone. These first signs of bacterial ingress were clearly discernible in samples taken 6 days post inoculation, in which small clusters of bacterial cells were found in intercellular spaces of parenchyma cells, evidently inward from the lignified and suberized lining (Fig. 2A, arrowheads). Besides the coloniza- tion of living tissues, bacterial clusters remained in the wound fissure (Fig. 2A, arrows). During the days after the first bacterial clusters were observed behind the histological barrier, the first disease effects were visible on the cellular level. Individual parenchyma cells were found to be ablated and replaced by a large cluster of bacteria (Fig. 2B). Other, solitary bacteria were often found in the intercellular spaces of surrounding cells. One week post inoculation, bacteria were also P. syringae in Aesculus, Infection and Control found in the cambial region, although signs of destructive nature were not yet recorded in this tissue. Interestingly, the bacteria were found distributed along the sclerenchyma fibres of the phloem adjacent to the wound surface (Fig. 2C). Bacterial Spread on the Long Term Bacterial behaviour was further monitored up to 18 weeks after inoculation. Whereas mock inoculated plants exhibited wound healing and continued secondary growth at the inoculation site over a period of 15 weeks, infected plants showed reduced secondary growth (Fig. 2D–H) and necrosis that expanded in longitudinal direction. Twelve days after inoculation, clusters of bacteria were still found in ablated cells in the living cortex (cf Fig. 2 I and J) and at 2 and 3 weeks post inoculation, bacteria were observed in the phloem close to the cambium and incidentally in the xylem, although exclusively in the vicinity of the inoculation site (Fig. 2K). Three to four weeks after inoculation many bacteria were observed in the intercellular space of phloem parenchyma (Fig. 2L), ray parenchyma (Fig. 2M), around phloem fibres (Fig. 2N), and between phloem cells close to the cambium region (Fig. 2O). As such, intercellular space might facilitate the longitudinal and radial spread of bacteria. As a reaction upon infection, phloem fibres decorated with bacteria were often surrounded by periderm (Fig. 2P). In samples taken 10 and 18 weeks after inoculation, bacteria were found at the edges of the expanding lesions, ex planta in degenerated bark (Fig. 2Q), at the open surface of the wounded area (Fig. 2R), and in enclosures of necrotic tissue adjacent to the cambium but surrounded by periderm (Fig. 2S–T). Thus, bacteria were found in both degenerated as well as still degenerating bark tissue during the whole period of investigation. Expression of GFP in the aesculi Pathovar To be able to study the bacteria in planta more accurately and uncover possible dynamic behaviour, an approach to constitutively express GFP in P. s. pv. aesculi was undertaken. To facilitate GFP expression in plant-associated, Gram-negative bacteria Bloemberg et al. [18] constructed plasmid pMP4655, which features the eGFP-encoding gene driven by the lac promoter (Table 1). Replication functions of this plasmid are derived from the Pseudomonas stable plasmid pVS1, known for high stability in absence of selection pressure [19], which is required when bacteria are associated with their host. The two P. s. pv. aesculi strains used in this study (Table 1) appeared to be extremely refractory to transformation methods promoting physical displacement of plasmid DNA across the cell membrane (i.e. electroporation and heat-shock methods; data not shown). Also, using plasmid DNA prepared from methyl- ation deficient E. coli strain GM119 (Table 1) did not improve transformation efficiency, as was described for other P. syringae strains [20]. A triparental conjugation protocol was therefore employed, using carbenicillin as selective agent to favour P. syringae growth after conjugation (See materials and methods for details). Transconjugants were obtained for both P. s. pv. aesculi strains and plasmid borne expression of GFP appeared adequate for visualization by fluorescence microscopy. The expression of GFP did not significantly alter growth rates when strains were grown in LB (Fig. S1). The plasmid was stably maintained for at least 30 generations when subculturing in medium without antibiotic selection pressure, as was reported for this plasmid when maintained in other hosts [18,21]. When the P. s. pv. aesculi PD4818-pMP4655 transformant was inoculated on the stems of PLoS ONE | www.plosone.org 3 July 2012 | Volume 7 | Issue 7 | e39604