Xanthomonas bacteria secrete transcription activator-like effector (TALE) proteins into host cells to activate plant disease susceptibility genes to cause disease, and the process is dependent on interaction between bacteria TFB domain of TALEs and host plant basal transcription factor IIA gamma subunit (TFIIAγ). The key domain or residues of plant TFIIAγ and core residues of bacteria TFB domain that are indispensable for TFIIAγ-TALEs interaction in the process of TALE-carrying Xanthomonas invasion plants are unknown. Here, we showed that the third α-helix domain of OsTFIIAγ5/Xa5, especially the 38th, 39th, 40th and 42th residues were key sites for capturing by TALEs of Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of rice bacterial blight disease. The latter segment of Xoo TFB domain harboring seventy-two amino acid residues was vital for TALE specific binding with host plant OsTFIIAγ5/Xa5. Substitution of some residues in this core region of TFB domain completely compromised capacity of TALEs capturing rice OsTFIIAγ5/Xa5. The rich and conserved arginine residues in this core region of TFB domain were responsible for TALE-dependent plant susceptibility gene activation and virulence of Xoo. These results provide a potential strategy for improving resistance to TALE-carrying pathogens in plants by site-specific modification of key residues of host plant TFIIAγ. 

Organophosphate insecticide residues on vegetable, fruit, tea and even grains are primary cause of food poisoning. Organophosphate compounds can cause irreversible inhibition of the activity of acetylcholinesterase and butyrylcholinesterase (BChE, EC 3.1.1.8), which are both candidates for rapid detection of organophosphate pesticides. To develop an easy-tohandle method for detecting organophosphate pesticides using BChE, BChE from human was optimized according to the codon usage bias of Pichia pastoris and successfully expressed in P. pastoris GS115. The codon-optimized cDNA shared 37.3% of the codon identity with the native one. However, the amino acid sequence was identical to that of the native human butyrylcholinesterase gene (hBChE) as published. The ratio of guanine and cytosine in four kinds of bases ((G+C) ratio) was simultaneously increased from 40 to 47%. The recombinant hBChE expression reached a total protein concentration of 292 mg mL–1 with an activity of 14.7 U mL–1, which was purified 3.2×103-fold via nickel affinity chromatography with a yield of 68% and a specific activity of 8.1 U mg–1. Recombinant hBChE was optimally active at pH 7.4 and 50°C and exhibited high activity at a wide pH range (>60% activity at pH 4.0 to 8.0). Moreover, it had a good adaptability to high temperature (>60% activity at both 50 and 60°C up to 60 min) and good stability at 70°C. The enzyme can be activated by Li+, Co+, Zn2+ and ethylene diamine tetraacetic acid (EDTA), but inhibited by Mg2+, Mn2+, Fe2+, Ag+ and Ca2+. Na+ had little effect on its activity. The values of hBChE of the Michaelis constant (Km) and maximum reaction velocity (Vm) were 89.4 mmol L–1 and 1 721 mmol min–1 mg–1, respectively. The bimolecular rate constants (Ki) of the hBChE to four pesticides were similar with that of electric eel AChE (EeAChE) and higher than that of horse BChE (HoBChE). All values of the half maximal inhibitory concentration of a substance (IC50) for hBChE were lower than those for HoBChE, but most IC50 for hBChE were lower than those for EeAChE except dichlorvos. The applicability of the hBChE was further verified by successful detection of organophosphate insecticide residues in six kinds of vegetable samples. Thus, hBChE heterologously over-expressed by P. pastoris would provide a sufficient material for development of a rapid detection method of organophosphate on spot and produce the organophosphate detection kit.