Roles Of The HPA1Protein In Disease Resistance And In Phtosynthesis After Interacting With Aquaporin PIP1;4in Arabidopsis

Harpin proteins produced by gram-negative phytopathogenic bacteria generally affect virulence to host plants and the hypersensitive reaction (HR) of nonhost plants. When applied to plants, any harpins can induce resistance to pathogens and insects, confer drought tolerance, and enhance plant growth. AQP is a kind of plasma membrane intrinsic protein which could transport water and some other hydrone solute to the cell on the plasma membrane.The Hpa1Xoo protein we used was produced by the rice bacterial leaf blight pathogen Xanthomonas oryzae pv. Oryzae. This study focuses on verification the interaction between Hpa1Xoo and PIP1;4in Arabidopsis membrane, the impact of this interaction and the reason.1. Achievement of transgenic Arabidopsis HETAt and SHETAt and the screening of strains for pathogen resistance in transgenic ArabidopsisGram-negative phytopathogenic bacteria can secrete Harpin proteins,which have been shown to activate plant defense pathways that involve transduction of hydrogen peroxide (H2O2) signal generated in the apoplast. But how a harpin can be recognized by a pathway or how the apoplastic H2O2participate in defense responses are still unknown.We transformed Hpa1Xoo gene and Hpa1Xoo fused to an apoplastic localization signal (S) generated SHpalxoo into Arabidopsis thalianaplants (HETAt and SHETAt). Hpa1Xoo was found to associate with the cytoplasm in HETAt and the apoplast and cytoplasm in SHETAt. We found that the transgenic plants have a stronger resistant to the bacterial pathogens (Pst DC3000and Pcc RL4). Among them, the strongest strains were HETAt4and SHETAt4.So we selected them for the subsequent experiments. 2. The verification of the interaction between Hpalxoo and PIP1;4in Arabidopsis cell membraneThe former research has shown that Hpalxoo can induce plant resistance to pathogenic bacteria and enhance plant growth. In order to understand the acing mechanism of Hpalxoo further, we used Hpalxoo as a bait to screen the Arabidopsis cDNA libraries. Then we found the plant water channel protein PIP1;4can interact with Hpalxoo.In this chapter, we used membrane yeast two-hybrid system, bimolecular fluorescence complementation and fluorescence dye (FM4-64) observation to confirm the interaction between PIP1;4and Hpalxoo. We also observed that the interaction localize at the cell membrane. We set a control group in which we used the ANT protein. ANT protein was HpalXoo protein which was lacking53amino acid. We found the ANT protein can’t interact with Hpaloo.3-The study of the interaction between Hpalxoo and PIP1;4inducing growth enhancement in Arabidopsis thalianaThe last chapter confirmed the interaction between Hpalxoo and PIP1;4. We bought7kinds of atpip1;4Arabidopsis seeds to study the impact of the interaction on plants from TAIR. The mutants were all T2-generation hybrids and we screened out the homozygotes from each mutant with the resistance to kanamycin and basta. We also had molecular verification to keep accuracy. The two proteins Hpalxoo and ANT were successfully expressed and purified. We treat the seeds with the two proteins and water, and then we could observe the phenotype and assay some parameters of photosynthesis. HpalXoo-treated WT plants grew better compared with Hpalxoo-treated atpipl;4Arabidopsis, ANT or H2O-treated WT and ΔNT or H2O-treated atpip1;4Arabidopsis. All the tests suggested that the interaction between Hpalxoo and PIP1;4could enhance the plant growth. We presumed the reason was that the interaction between HpalXoo and PIP1;4could let more CO2come into the intra-cellular through PIP1;4protein. There would be more CO2to participate in the photosynthesis and the growth of the plant was enhanced.