Figure 6 Clustering the three-dimensional structures of pectin lyases. The pectin lyase dataset was clustered by the un-weighted pair group method using the arithmetic

mean (UPGMA) [53] with a similarity matrix obtained by the Voronoi contact method [51] using the ProCKSI-Server [52]. The tree image was generated using Dendroscope software [77]. A. Three-dimensional MLN2238 mw structure of PEL B from A. niger [PDB:1QCX]. B-C. Three-dimensional structures of the PNLs from C. lindemuthianum [GenBank: JN034039] and P. carotovorum [GenBank: AAA24856] respectively, predicted by homology modeling using the Swiss-Model Server [48]. Expression analysis of Clpnl2 Analysis of the Clpnl2 transcript in cells grown with glucose as the carbon source showed similar low basal levels of expression in the 0 and 1472 races (Figure 7C). When grown on cell walls, levels of Clpnl2 transcript in the pathogenic race, 1472, increased quickly

after 2 h, reached a peak after 6 h, started to decrease and then again increased, giving a maximal value after 12 h of incubation (Figure 7B and 7C). Race 0 exhibited different expression kinetics: the amount of transcript peaked after 6 h and then fell to undetectable levels after 10 h (Figure 7A and 7C). At all time points between 2 and 8 h, expression levels were lower than those observed in the pathogenic race. The transcript was expressed again after 12 h but

at levels that reached GANT61 nmr only 23% of those observed in the pathogenic race. Figure 7 Analysis of the relative gene expression of Clpnl2 in races 0 and 1472 of C. lindemuthianum. A-B. Gel-like images showing the expression of Clpnl2 in races 0 and 1472, respectively, on the different carbon sources tested. C. Semi-quantitative data for the expression of Clpnl2 in both races on the carbon sources. Total RNA was isolated from induced mycelia and amplified by RT-PCR with specific primers to yield the cDNA of Clpnl2. Amplification products were checked and quantified on a Bioanalyzer (2100 Agilent Bioanalyzer). The data were normalized using 18S rRNA as a control, and the results are expressed in μg/μl of amplified product. The differences between the two races P-type ATPase were much more noticeable when 92% esterified pectin was used as the sole carbon source. Transcript expression in the pathogenic race started to increase rapidly, reached the highest levels after 4-6 h and then started to decline, giving a still significant increase at the end of the experimental period (Figure 7B and 7C). The maximum transcript levels on this substrate were clearly higher than those observed on glucose. In contrast, the levels of the Clpnl2 transcript in the non-pathogenic race remained undetectable after 8 h of incubation.