Table 2. Effect of heat-treatment on recovery of bacteria from lesions in inoculated A. hippocastanum saplings. (0/14) (0/4) Control (no treatment)a 396C for 48 ha Inoculation PD4818-pMP4655 Mock (PBS) (11/11) (0/5) aThe figures are given as number of successful re-isolations per no. of lesions assessed. doi:10.1371/journal.pone.0039604.t002 shed some light on the origins of pathogenesis, the histopathology of this interaction remained unknown. In this work the histological events after host entry of P. s. pv. aesculi and its behaviour in planta were investigated. Histological responses upon bark injury and bacterial dispersal and clustering were analysed. The histochemically observed lining of cells with lignified and suberized cell walls in A. hippocastanum bark has been described for various other tree species [13–16]. Also, in agreement with these earlier reports, the time window in which it is detected in horse chestnut is in the order of several days. However, direct comparison of the speed of its construction is difficult due to a strong correlation with environmental temperature [29]. Both in presence and absence of P. s. pv aesculi, the wounded bark tissue showed lignification and suberization of the parenchy- ma cells bordering a wound to a similar extent, suggesting this process is part of an intrinsic defence response of A. hippocastanum. However, subtle differences may be present as is reported for some plants in the presence of fungal pathogens [17,30]. Nevertheless, the observed response is presumably broadly defensive [31], such that a possible specific relation to pathogen presence is obscured. When monitoring bacterial ingress, it appeared that the lignified and suberized barrier was insufficient or constructed too late to shield off underlying healthy tissue (Fig. 2A). Remarkably, the barrier sometimes seemed a bit fainter near invading bacteria, hinting at pathogen-mediated degeneration (Fig. 2A). This could be facilitated by the various pathways for degradation of lignin- related compounds uncovered in the P. s. pv aesculi genome [8]. Although not determinative in restraining P. s. pv. aesculi invasion, presence of the observed barrier might still serve a beneficial, protective role. For instance, it could prevent water and mineral flow towards the bacteria, prevent diffusion of bacterial toxins and effectors to healthy plant tissues, reduce tissue digestibility and give rigidity, preventing further tissue collapse. Rather than systemic spread through vascular elements, advance of the bacterial infection mostly took place in parenchyma tissue of the bark and presumably through intercellular space (Fig. 2L, M). Under the assumption that this type of tissue is less conductive for bacterial spread, this observation can explain the relatively slow rate of lesion expansion, which is measured to be in the order of 100 mm/day [1] until 1000 mm/day (Our unpub- lished data). Along with spread throughout parenchyma, bacteria were often found on sclerenchyma fibres (Fig. 2C, N). While the localization and longitudinal orientation of these elements coincides with the site of lesion expansion (Fig. 3B), no function in pathogenicity or bacterial spread could be unequivocally assigned to this apparent association. However, the development of periderm around infected phloem fibres (Fig. 2P) constrains lateral rather than longitudinal spread, potentially explaining the elongated nature of lesions. With the conductive tissue for lesion expansion appearing to be bark parenchyma and/or sclerenchy- ma, it will be interesting to uncover the means by which the bacterial cells migrate through these tissues. Of particular interest P. syringae in Aesculus, Infection and Control is the potential function of intercellular space in bacterial spread. In mature trees, besides the elongated lesions, laterally spreading lesions are observed as well [11], suggesting different modes of colonization can prevail in individual infections. The bacterial cells found near the forefront of expanding infections were small and densely clustered (Fig. 3D). Similarly, disease symptoms on the cellular level included occasional colonization of single phloem/cortex parenchyma cells by a large bacterial cluster (Fig. 2B, J). These local bacterial accumulations might be important in the infection process, since expression of pathogenic traits often requires a certain, quorum sensing coordinated, cell density [32]. That such a quorum sensing system may act within the observed bacterial clusters in horse chestnut tissues is supported by the quorum size described for P. syringae (down to ,20 individuals [33]) and the ability of Indian P. s. pv. aesculi to produce N-acyl-homoserine lactones [34]. In later disease stages (i. e. several weeks after infection) bacteria were still found in necrotic tissues (Fig. 2Q–T; Fig. 3E, F). The ex planta persistence of bacteria in disrupted tissue might aid spread and reinfection later in time. Individuals were non-motile and spaced at some distance from each other, indicative of a mucoid gel wherein the bacteria were contained (Fig. 3G, H). Similar observations were done on P. savastanoi pv. savastanoi during its endophytic lifestyle in olive stems [35]. Such a gel may aid the bacterium to create a stress tolerant microenvironment within the host, for example enhancing resistance to dehydration and toxic compounds [26,36,37]. Similarly, it may also contribute to bacterial survival on outer tissues [38,39]. High epiphytical fitness might lengthen the window of opportunity for P. s. pv. aesculi to gain access to host tissues. Besides contributing to the resistance to environmental stress, the observed matrix may play a direct role in pathogenicity. One such function may be the suppression of host defence signalling through Ca2+ chelation [40]. Another potential mechanism through which a mucoid gel can contribute to virulence has been described for fire blight of pear caused by Erwinia amylovora, where the extracellular matrix is hypothesized to drive lesion expansion [41,42]. Under a rising water potential the matrix can exert a pressure on host tissues, which might even facilitate breaching of defensive barriers constructed by the host plant [41]. If a similar mechanism operates during P. s. pv. aesculi infection, such pushing forces may also in part explain the seasonal exudate formation typically associated with active lesions (Fig. 1A). Scanning electron microscopy revealed that the intervening space within an axenically grown bacterial cluster consists of fibrillar material (Fig. 4A). Extracellular polymeric substances produced by P. syringae strains include levan and alginate [22,24,26,43]. Our results demonstrate the presence of mannuro- nic and guluronic acid after hydrolysis of extracellular polysac- charides produced by cell cultures under salt stress. Alginate is thus likely a component of the extracellular matrix, as is found for many phytopathogenic pseudomonads [23,25]. While isolates of P. s. pv. aesculi have been reported positive for levan production [9], the HPAEC results of hydrolysed exopolysaccharides in our study did not show the presence of fructose so excluding the production of levan under the investigated conditions. The presence of glucose, rhamnose and glucuronic acid together with alginates in the extracellular matrix has been reported for P. syringae pv. syringae [22] and is in agreement with our results. The capability of P. s. pv. aesculi to produce alginate is supported by a full complement of biosynthetic and regulatory genes (Table S1). Additionally, uncharacterised components may contribute to the matrix embedding the bacterial cells [43]. Also, factors that coordinate its production require further attention. PLoS ONE | www.plosone.org 8 July 2012 | Volume 7 | Issue 7 | e39604