P. syringae in Aesculus, Infection and Control remain in the original inoculation cut (arrows). The image shown is taken from a longitudinal section. (B) A bacterial accumulation in a cortex parenchyma cell observed in a longitudinal section of an infection site sampled 14 days after inoculation. Besides the large cluster, several individual bacteria are scattered in intercellular spaces. (C) A longitudinal section of phloem tissue 6 days post inoculation showing the decoration of bark fibres by P. s. pv. aesculi seemingly following the vertical orientation of these elements. (D, E) Close-up and micrograph of transversal section of a mock- inoculated site on a horse chestnut plant 7 weeks after inoculation. The asterisk points to site of wounding during inoculation. (F, G, H) Close up and transversal sections of inoculation sites 15 weeks after inoculation. Note partially restored development of secondary xylem despite infection. Asterisks point to site of wounding during inoculation. (I, J) Transversal sections of cortex in mock-inoculated (I) and infected (J) horse chestnut 12 days after inoculation. Note ablated plant cells filled with bacteria in J. (K) Clusters of bacteria in a longitudinal section of the xylem close to the inoculation site, 4 weeks after inoculation. (L–N) Longitudinal sections of the cambium region with bacteria in intercellular spaces of phloem parenchyma (L), ray parenchyma (M) and along phloem fibres (N), 3 weeks after inoculation. (O) P. s. pv. aesculi in intercellular spaces observed in a transversal section of the cambial zone at 4 weeks after inoculation. (P) Bacteria decorating a bundle of phloem fibres which is surrounded by newly formed periderm (*) and degenerating parenchyma 4 weeks after inoculation. (Q) Bacteria colonizing necrotic bark tissue at 10 weeks after inoculation. (R-T) Transversal sections of enclosures surrounded by periderm with bacteria ex planta at 4, 10 and 18 weeks after inoculation, respectively. doi:10.1371/journal.pone.0039604.g002 A. hippocastanum seedlings, typical, elongated necrotic lesions were formed underneath the periderm (Fig. 3A), mainly in the cambium and phloem (Fig. 3B). Given the high plasmid stability and the retained virulence towards A. hippocastanum the GFP-expressing transformants were considered useful to enable accurate re- isolation and further studies on bacterial localization and behaviour in situ. P. syringae pv. aesculi is Embedded in an Extracellular Matrix in planta Lesions formed by GFP-expressing PD4818 were analysed 4 and 6 weeks after inoculation. The vertical expansion of an infection was sometimes observed as a parabolic shaped row of cells exhibiting elevated cell wall fluorescence, containing a cluster of small, densely packed bacteria in the tip (Fig. 3C/D). Especially the borders of necrotic tissue housed many bacteria. Clustered bacteria occupied empty plant cells (Fig. 3E) and large aggregates of bacteria were found in voids within the necrotic host tissue (Fig. 3F). Individual bacteria in these aggregates were spaced at some distance from their neighbours and did not seem to undergo any type of motion, including Brownian (Fig. 3G). A similar distribution pattern was also observed in lesions formed by wild- type bacteria using immunolabelling (Fig. 3H). The spatial arrangement and motionlessness of these bacteria suggests that formation of a mucoid matrix occurs within host tissues, which Table 1. Bacterial strains and plasmids used in this study. might serve a role in the infection and/or survival strategy of the bacteria. To explore whether the matrix is of bacterial origin, P. s. pv. aesculi PD4818 was grown axenically in a liquid minimal medium. When cultures grown under these conditions were analysed by scanning electron microscopy, cell clusters wrapped in a fibrillar matrix were observed, which was not encountered when cultures were grown under nutrient-rich conditions (Fig. 4A). Repetition of these experiments using the GFP- expressing derivative of strain PD4818 indeed revealed the presence of aggregates of bacterial cells up to approximately 100 mm in size among a population of motile bacteria. Clusters contained both live and dead bacteria. Analysis of optical sections and the orthogonal planes along the z-axis revealed the 3D spacing of bacteria (Fig. 4B). Cells inside a cluster were completely static, although some individuals at the cluster margin sometimes exhibited oscillatory motion (Fig. 4C). These findings support that a bacterium derived matrix could indeed enwrap the individuals within the observed bacterial aggregates in planta. To identify what type of exopolysaccharide could potentially contribute to the observed matrix, P. s. pv. aesculi was grown in liquid minimal medium with additional salt stress for 2 days. An m-hydroxydiphenyl assay revealed the presence of uronic acids in the exopolysaccharide obtained after salt stress (data not shown). High Performance Anion Exchange Chromatography Strain or plasmid P. syringae pv. aesculi PD4818 PD5126 E. coli DH5a GM119 Cel40 plasmids pMP4655 pRK2013 Relevant characteristicsa Reference or source A. hippocastanum isolate, Winssen, the Netherlands, 2004, CbR A. hippocastanum isolate, the Netherlands, 2005, CbR F2, w80DlacZM15, recA1, endA1, hsdR17, phoA, supE44, gyrA96, relA1, l2 F2, dam2, dcm2, metB1, lacY1, galK2, galT22, tonA31, tsx-78, supE44, mtl-1 Tra+ (pRK2013), KmR oriBBR1, oriVS1, oriT (p15A), lac::eGPF, TcR oriColE1, Tra (RK2), KmR Plantenziektenkundige Dienst, Wageningen, the Netherlands Plant Research International, Wageningen, the Netherlands [48] [18,19] [49] Dr. M. G. Marinus (University of Massachusetts medical school) E. L. Lagendijk (Molecular Microbiology Leiden University) aAbbreviations used: Cb, carbenicillin; Km, Kanamycin; Tc, Tetracycline; Tra, conjugal transfer functions. doi:10.1371/journal.pone.0039604.t001 PLoS ONE | www.plosone.org 5 July 2012 | Volume 7 | Issue 7 | e39604