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Bacterial extracellular polysaccharides are required by virtually all pathogenic species for pathogenicity or full virulence. Their roles probably include protection from antimicrobials, UV, desiccation, recognition, adhesion and biofilm formation. However, their role may be much more important in infection. Most of these macromolecules are polyanionic and have the ability to bind cations, especially calcium. Binding of calcium is an effective means of defence suppression because calcium influx from the apoplast is a prerequisite for activation of defence responses and plant pathogenic bacteria are usually confined to the apoplast. Clearly, the effectiveness of EPSs in establishing/maintaining compatability is dependent on timing and amounts produced in the apoplast.
Confirmation of EPS production, levels and timing in planta are clearly relevant to the role of these polymers so we decided to quantify EPS production by pathogenic bacteria in biofilms and in planta during different stages of disease. At first I investigated levels of two EPSs in biofilms on solid media. The in vitro system was intended to mimic the biofilms formed against the host cell walls by plant pathogens during invasion of the apoplast. EPSs were extracted from X. campestris pv. campestris (xanthan) and P. syringae pv. syringae (alginate) biofilms grown on agar based medium at the University of Bath, UK. Biofilm extracts were hydrolysed, then acetylated using pyridine and acetic anhydride and chloroform extracted. After the identification of representative sugars of xanthan and alginate in biofilm extracts their ratios were calculated. GC-MS revealed xanthan from Xcc at 32 mg/ml and the Pss biofilm comprised 22 mg/ml alginate.
EPS production by Xcc was also examined in the Nicotiana benthamiana pathosystem. Extraction of EPSs from infected plants is problematic because they form calcium gels and can intersperse with and possibly interact with host wall polymers. Xanthan was extracted from Xcc and water control (pH 6. 0) infected plants. Plant material was harvested when plants were displaying progressive stages of disease development, and extracts were subjected to derivitisation as described above, and analysed by GC-MS. Xanthan concentration was calculated per dry and fresh weight. Xanthan was revealed at all stages of infection; after 2 dpi (water-soaking symptoms), 4 dpi (chlorosis), and 6 dpi (necrosis). Xanthan levels from GC-MS were 14 (1), 19 (2) and 25 (6) mg/g as dry wt. and fresh wt. (bracketed) respectively. These concentrations of xanthan are sufficient to bind local [Ca2+]apo and suppress signalling. This result supports ultrastructural studies showing bacteria embedded in an EPS matrix from early infection (e.g. after 2 hpi and 8 hpi), and concurs with the need for xanthan production in initial infection by Xcc.
During 3 weeks I had a great opportunity to understand carbohydrate analysis, and would like to thank BSPP for funding this project, my supervisor Richard Cooper, and Antonio Molinaro for providing me this opportunity.