annuum plants C. annuum (cultivar California Wonder) plants derived from seedlings were grown in the greenhouse at 21°C with 12/12 day/night hours. Cell wall material was isolated from 6 weeks old plants. Analysis of enzyme activity Extracellular pectate lyase activity was monitored by an agar plate test and quantified in a photometric assay [38]. For the pectate lyase assay, X. campestris pv. campestris cultures were grown for 24 h in M9 medium supplemented with pectate and

FeSO4. The resulting find more values were calibrated to the activity of glucose-6-phosphate dehydrogenase. For the tests on agar plates [92], X. campestris pv. campestris strains were cultivated for 2 days on M9 medium supplemented with pectate Milciclib supplier and FeSO4 as described elsewhere [93]. Genome analysis and recombinant DNA procedures Genome

data were analyzed and visualized by means of the GenDB RGFP966 molecular weight annotation system [94]. The EDGAR software [95] was employed to compare complete Xanthomonas genomes that were available from public databases [42, 43, 45, 46, 96–99]. For the analysis of genes encoding polysaccharide-degrading enzymes, information provided by the CAZy database (http://​www.​cazy.​org/​) has been considered [100]. All cloning was performed applying standard methods [101] and as described previously [64, 66]. An 11.1 kb chromosomal BamHI fragment of X. campestris pv. campestris 8004 carrying the pglI gene in cosmid pIJ3051 [39] was inserted into the plasmid vector pHGW31 to obtain plasmid pHGW260. A 3.8 kb BamHI-ClaI sub-fragment

with the pglI gene was then transferred to the cloning vectors pBCKS+ and pBCSK+, resulting in the plasmids pHGW261 and pHGW262, respectively. In pHGW262, pglI was constitutively expressed in E. coli from the lac promoter of the pBCSK+ multiple cloning site. To express pglI also in X. campestris pv. campestris, pHGW267 was constructed by cloning the 3.8 kb BamHI-ClaI sub-fragment with the X. campestris pv. campestris 8004 pglI gene into the multiple cloning site of pUC6S (Apr) [90], where it was under the control of the constitutive Pout promoter of the Dapagliflozin aacC1 gene from pMS246 [91], which was cloned as a 1 kb BamHI fragment into the BamHI site upstream of pglI. Isolation of plant cell wall material Leafs of C. annuum were employed to obtain cell wall material. Leafs (30 g) were homogenized in 150 ml sodium acetate (50mM, pH 5) for 3 min and filtered with a fluted filter. After the filtration, the cell wall material was washed with 1 l sodium acetate (4°C), 1 l ethanol (4°C) and with 1 l acetone (−20°C). The washed material was then air dried at room temperature and stored under inert atmosphere at -20°C. Co-incubation of X. campestris pv. campestris and C. annuum cell wall material 5 ml X. campestris pv. campestris over-night liquid culture was centrifuged.