Plant Growth And Defense Regulation In Response To A Riboflavin Receptor Protein And A Harpin Protein (HpaG_Xoo)

Riboflavin (vitamin B₂) participates in many physiological processes, particularly those characterized by oxidation and reduction. The vitamin is phosphorated and combined with nucleotides to form flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), which are coenzymes of many enzymes, making flavoproteins, play vital roles in many reactions, especially the production of reactive oxygen intermediates (ROIs). ROIs are essential for the hypersensitive cell death, growth, and defense against pathogens and insects, as well as many aspects of responses to environmental stress. Levels of riboflavin in plants are believed to be important to these processes. As such, if riboflavin levels could be manipulated, effects of the compound on the important processes could be monitored and controlled as a consequence. This would result in great improvement of plant defense and productivity. This also could provide us with a particular insight into studying coordination of plant defense with growth regulation, which may be affected by levels of riboflavin.Gene encoding riboflavin receptor protein, named RIR, has been cloned from soft-shelled turtle (Trionyx sinensisjaponicus), and transformed into wide type Arabidopsis Col-0. The properties of RIR-transgenic Arabidopsis (RIRA), such as riboflavin concentration,plant height,plant model,the time of floral transition have changed. We constructed RIR-silencing vector, which was able to silence RIR in RIRA, for further study on the roles of riboflavin in plant development. The silencing line we obtained was named SiRB. Then distinctions in Col-0, RIRA and SiRB were detected to study the function of RIR in Arabidopsis. The endogenous flavins in RIRA, including riboflavin, FMN and FAD, were much higher than those in the wild type. And this increase brings many benefits. The transgenic line RIRA showed much healthier development, larger individuals and somewhat advanced the floral transition which were confirmed by results of molecular analyses on flowering-relevant genes. After silencing, these better symptoms disappeared. Whether the increase in endogenous riboflavin plays a role in disease resistance of Arabidopsis was determined too. We here used Pseudomonas syringae pv. tomato DC3000 to detect it. When determined at 3d post inoculation, bacterial population in RIRA leaves was much lower than that in wild type. Necrosis symptoms also were reduced in plants, which had more riboflavin, compared to Col-0 and SiRB. These results suggest that increase in endogenous riboflavin can enhance both plant growth and disease resistance.The presence and function of RIR in plants is an absolute new area. What we did here is a primary attempt to dissect the problem. Polymerase chain reaction was conducted to amplify the gene encoding RIR in Arabidopsis, and we named it AtRIR. The gene was inserted into the pET30a(+) vector, and pET3Oa(+):: AtRIR was transformed into Escherichia coli BL21(DE3). The bacterium was induced by isopropyl-Beta-D-thiogalactopyranoside (IPTG) and its expressed products were loaded directly onto sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), on which approximately 35 kD exogenous protein was observed. Then the his-tagged AtRIR fusion protein was purified by HisTrap HP columns (pre-charged with Ni²⁺), and the bioactivity was detected by quenching test in the end.HpaG_Xoo, encoded by the gene hpaG_Xoo in Xanthomonas oryzae pv. oryzae, belongs to the harpin group. HpaG_Xoo induces resistance to diseases caused by pathogens like fungi, viruses and bacteria in plants. Whether HpaG_Xoo has similar effect on oomycetes remains unknown. We cloned the hpaG_Xoo gene to the pET30a(+) vector and introduced the recombined plasmid into Escherichia coli cells, resulting the BLHR1 strain, which produced active HpaG_Xoo. HpaG_Xoo induced tobacco hypersensitive response with a concentration threshold of 5μg/ml. HpaG_Xoo did not cause evident effects on the growth of Phytophthora nicotianae, the oomycetal pathogen that causes black shank in tobacco. Expression of parAl, a gene that is essentially involved in pathogenicity, was similar in HpaG_Xoo-treated and control plants. Tobacco defense related genes NPR1, GST1, Chia5, and PR-1b were upregulated coordinately after HpaG_Xoo treatment. In HpaG_Xoo-treated plants, resistance to black shank and leaf necrosis caused by the pathogen were markedly decreased compared to control. These results suggest that HpaG_Xoo elicits plant defense response to enhance resistance to the oomycetal pathogen.