Studies On The Inhibitory Effect Of Hydroxide Radicals On Para1-Induced Hypersensitive Resistance In Tobacco

Elicitins are a family of10kD small proteins which are secreted by Oomycetes of the genera Phytophthora and some Pythium. They are thought to be major determinants, mediating the basic resistance response of tobacco, since purified elicitins, alone and at low concentrations, can induce vigorous defense responses in tobacco, such as hypersensitive resistance (HR) and systemic acquired resistance (SAR), that is resistance against subsequent pathogen attack. Previous studies have proved that the above effects are oxygen burst-independent. ParA1, an elicitin secreted by P. parasitica, has a high degree of conservation in the amino acid sequences of elicitins, and purified, alone ParAl can also induce HR and SAR in tobacco. Many species of the Oomycetes that are parasites of a diverse range of species, almost show more than60%sequence of elicitins identity with each other, and also similar biologic activations. Based on these, in this study, we expressed and purified ParAl in P. pastoris for further researches.Riboflavin (Vitamin B2), is a water-soluble vitamin essential for normal cellular functions in organisms. It is generally believed that photoexcited riboflavin can generate ractive oxygen species (ROS) in the light. Under biologic stress, ROS is one of the earliest events in resist stress, which regulate many processes, interconnecting branch pathways that amplify and specify the physiological response through transcriptional and metabolic changes. Here, we reported that riboflavin can inhibit HR development induced by ParAl in tobacco leaves, and the primary elucidation of this phenomenon.1. The expression and purification of ParA1proteinIn our lab, the initial trials were to express the ParAl protein in E.coli, but they were hard to get sufficient soluble protein for purification. Even though different ways available had been used, the expressed proteins were in low concentrations and difficult for further researches. Previously, the Pichia pastoris expression system had been taken to express cryptogein, another elicitins successfully. Thus, we took an expression system based on the yeast P. pastoris (wide type stain X-33, mutant type stain KM71H) to express ParA1, with pPICZ(α)A vector. After choosing and optimizing the expression condications, we select KM71H (pPICZA::ParA1) to express the ParA1. Using Ni2+affinity columns, we obtained about75ng/μl (about6,500nM) ParA1protein, which can induce HR at6000x diluted in tobacco leaves in24hours.2. The antagonistic effect of riboflavin in the hypersensitive resistance induced by ParA1in the lightIt is known that ParAl can induce HR in tobacco, and here the HR development was suppressed by photoilluminated riboflavin in the continuous light, but not in the dark. Because of producing ROS by riboflavin in the light, comparing to the zone infiltrated by distilled water, the relative content of H2O2in the riboflavin infiltrated zone was increased about1.4fold in the light1.5h after treatment, and the same result could be found with3,3’-diaminobenzidine staining. Different ROS scavengers, such as SOD, catalase, ascorbic acid, reduced glutathione were used to coinfiltrated with ParAl and riboflavin, all of them, except SOD, could complement the HR phenotype. When different concentrations of H2O2donor coinfiltrated with ParAl, the suppression could not be found. More interestingly, adenine and thiourea, scavengers of hydroxyl radical, could complement the HR phenotype, and exogenous hydroxyl radical had the inhibitory effect on ParA1-induced HR. Since the production of hydroxyl radical was downstream of H2O2, considering with the above results, we hypothesized that the hydroxyl radical was the HR suppressor.