Apoplastic H₂O₂and Aquaporin PIP1;4Regulate HPA1_xoo-induced Pathogen Defense In 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. The diverse effects are attributable to the activation of distinct signaling pathways that involve transduction of phytohormones, reactive oxygen species （ROS） signals, ion channels, programmed cell death, systemic acquired resistance （SAR） or salicylic acid （SA） signal pathway and protein kinase cascade, etc. Currently, harpins have been identified from almost all the Gram-negative phytopathogenic bacteria. Harpin have been shown to activate plant growth and defense signaling pathways, but the recognition of a harpin connecting with a pathway in plants, what a plant receptor of a particular harpin protein is, the transduction of the signal to a cellular pathway and what plant translocator （s） for plant-cytoplasmic trafficking of bacterial effector proteins is （are）, especially type-â…¢ effectors have long been in question.The Hpalxoo protein produced by the rice bacterial leaf blight pathogen Xanthomonas oryzae pv. oryzae belongs to the harpin group of proteins. This study focuses on analysis of the relationship between cellular location of Hpalxoo and subsequent responses in plants, the apoplastic H2O2participation in defenses, the Hpalxoo recognition by a plant receptor and the impact of perception.1. Apoplastic location of harpin protein HpalXoo induces apoplastic generation and cytoplasmic translocation of H2O2required for pathogen resistance in ArabidopsisHarpin proteins secreted by Gram-negative phytopathogenic bacteria have been shown to activate plant defense pathways that involve transduction of hydrogen peroxide （H2O2） signal generated in the apoplast, but how a harpin is recognized connecting with a pathway and how the apoplastic H2O2anticipates in defenses are to be elucidated. Here we study if the cellular location of Hpalxoo, a harpin protein produced by rice bacterial leaf blight pathogen Xanthomonas oryzae pv. oryzae, impacts H2O2production and pathogen resistance in Arabidopsis thaliana. Transformation with the Hpalxoo gene and Hpalxoo fused to an apoplastic localization signal （SHpal Xoo） generated HpalXoo-and SHpal Xoo-expressing transgenic A. thaliana （HETAt and SHETAt） plants, respectively. HpalXoo was found to associate with the cytoplasm in HETAt and the apoplast in SHETAt, accompanying H2O2accumulation in cytoplasts and in apoplasts of SHETAt4as well. Apoplastic H2O2production via nicotinamide adenine dinucleotide phosphate （NADPH） oxidase （NOX） located in the plasma membrane has been shown as a common feature of plant defenses. In SHETAt, H2O2was generated in apoplasts in a NOX-dependent manner but accumulated to a greater extent in apoplasts than in cytoplasts. When applied to the parent plant, Hpalxoo located and induced H2O2generation in apoplasts but caused H2O2accumulation in both apoplasts and cytoplasts. In SHETAt and HpalXoo-treated parent plants, however, inhibition of apoplastic H2O2generation abrogated cytoplasmic H2O2accumulation and plant resistance to bacterial pathogens. These results suggest that the apoplastic H2O2is subject to a cytoplasmic translocation for participation in the regulation of pathogen resistance in the plant.2. Growth enhancement of Hpalxoo transgenic Arabidopsis is not related to Hpalxoo-induced H2O2The Hpalxoo protein produced by the rice bacterial leaf blight pathogen Xanthomonas oryzae pv. oryzae belongs to the harpin group of proteins secreted by Gram-negative phytopathogenic bacteria. In molecule mass, Hpal is smaller than other harpins, only15.6KD. harpin have been shown to activate plant growth signaling pathways, but how a harpin is recognized connecting with the pathway and how the harpin-induced H2O2participates in the pathway have long been in question. In the first chapter, transgenic plants HETAt and SHETAt were generated. In this chapter, HpalXoo and SHpalXoo Expression Transgenic Arabidopsis thaliana （HETAt and SHETAt） displayed growth and growth related genes enhancement. In SHETAt, H2O2accumulated in dependence of the outer membrane-associated NADPH oxidase activity, which didn’t impact growth. In HETAt, the H2O2content elevated but was not related to NADPH oxidase, which also didn’t contribute to growth. When Hpalxoo was applied to the parent plant, plant growth was enhanced, and H2O2of apoplasts and cytoplasts was not related to growth. These results suggested that growth pathways were activated in HETAt and SHETAt, and the pathway was not related to HpalXoo-induced H2O2.3. Analysis of the interaction between Hpalxoo and PIP1;4in Arabidopsis membraneThe first two chapters have shown that Hpalxoo located in the extracellular and intracellular region, induced plant resistance to pathogenic bacteria Pst DC3000and Pcc RL4, enhanced plant growth and induced expression of defense and growth related genes in the transgenic plants. In this chapter, to resolve the acing mechanism of HpalXoo based on these valuable phenotypes of Hpalxoo, the Arabidopsis cDNA libraries were screened using HpalXoo as a bait and six interacting proteins were found including plant water channel protein PIP1;4. HpalXoo, but not ANT （deletion mutant, removed HpalXoo N-terminal53amino acids）, could interact with PIP1;4by yeast two-hybrid assay, membrane yeast two-hybrid system, pull-down assay, bimolecular fluorescence complementation and fluorescence dys observation. And the interaction obviously localized at the cell membrane.4. The preliminary study of the interaction between Hpalxoo and PIP1;4wich inducing growth enhancement and pathogen defenses in ArabidopsisAfter confirming the interaction between HpalXoo and PIP1;4, eight kinds of atpip1;4Arabidopsis （CS803583, CS870828, CS872202, CS876999, CS879846, CS879691, CS870571and SALK₁47568） seeds were purchased from the Arabidopsis Information Resource to study the impact of the interaction on plants and find the factors of plant affected. These mutants were T2-generation hybrids, and homozygotes were screened out from each mutant under the conditions of kanamycin and basta resistance. The two proteins Hpalxoo and ANT were successfully expressed and purified by prokaryotic expression system and protein purification kit. Hpalxoo-treated WT plants grew best compared with Hpalxoo-treated atpipl;4Arabidopsis,â–³NT or H2O-treated WT, and ANT or H2O-treated atpipl;4Arabidopsis. Hpalxoo-treated WT plants were resistant to Pst DC3000, but Hpalxoo-treated atpipl;4Arabidopsis, ANT or H2O-treated WT, and ANT or H2O-treated atpipl;4Arabidopsis were susceptible to Pst DC3000. All these tests suggested the interaction between Hpalxoo and PIP1;4could enhance plant growth and induce plant resistance to plant pathogens.