Fig. 3

Screening of CDIPs by the optimized prokaryotic expression system. A Flowchart of screening CDIPs with the optimized prokaryotic expression system. Proteins of interest were preliminarily selected from secretomes. ORFs of the genes of interest without a signal peptide were homologously recombined into the pCold-TF vector. Escherichia coli harboring the recombined vectors were induced by 0.5 mM IPTG to express proteins overnight at 15 ℃, and SDS-PAGE was performed to examine the protein expression. The supernatant from disrupted cells was used for protein purification. Purified proteins were infiltrated into tobacco leaves at the same concentration, and the necrosis-inducing activities were monitored. B Representative Nicotiana benthamiana leaves with different cell death symptoms. Photos were taken 3 d after infiltration. C Functional classification of candidate proteins