Figure 2.

PbChia1 is characterized as a chitinase. (a) Heatmap visualization of 17 predicted chitinase genes at different life stages of P. brassicae. A coloured bar indicates normalized reads per log10 (FPKM) in the expression profile at three life stages. IN indicates cortical infection; PZ stands for primary zoospores and RS indicates resting spores. Genes and samples were clustered according to their expression profiles. (b) Schematic illustration of PbChia1. (c) PbChia1 contains a functional signal peptide. By using a yeast signal-sequence trap system, yeast expressing the plasmid pSUC2-SPPbChia1 with the expression of PbChia1 signal peptide driven by the pSUC2 promoter could produce a red similar colour to that of the positive control. Yeast expressing the empty pSUC2 vector was used as a negative control, and yeast expressing pSUC2-SPAvr1b containing the signal peptide from the Phytophthora sojae effector Avr1b was the positive control. (d) and (e) Chitinase activity of PbChia1. PbChia1 was fused with the His tag fusion protein in pEt28a (d), or with the GST fusion protein in vector Pgex-6P1, GST alone served as a control (e). The chitinase enzyme activity of affinity-purified His-PbChia1, GST-PbChia1 and GST protein was analysed using chitin as substrate. The reaction of DNS (3,5-dinitrosalicylic acid) and GlcNAc was monitored at OD565 nm. One unit of chitinase activity was defined as the amount of enzyme required to release 1 mmol of N-acetyl-D-glucosamine per hour. Data are mean ± s.D. (f) and (g) Affinity-purified His-PbChia1 (f) and GST- PbChia1 (g) protein had the ability to bind chitin. Purified recombinant proteins were incubated with colloid chitin for 1 h under constant shaking, and the chitin-associated His-PbChia1 (f) and GST- PbChia1 (g) were detected using immunoblotting.