| Harpin and Avr proteins are representative type III effectors secreted by Gram-negative phytopathogenic bacteria, that regulate the pathogenicity and the plant defense response to pathogens. Avr proteins are pathogenic effectors and must be translocated from bacteria into plant cell to pathopoiesis. Harpin protein function as a translocator to translocate the effectors. Whether the Harpin or Avr proteins are double effects on the pathogenicity and plant defense response to pathogens, and also the Harpin can induce plant growrh enhancement, however, the molecular mechanism is unclear. In this study, we chose the Hpal protein, focusing on analysis of the molecular mechanism that plant growth enhancement and pathogen defense response induced by Hpal and the effects of the protein structure and subcellular localization on its function. We also analysis the function of the type III effector protein AvrBs2produced by X. oryzae, and focusing on analysis of its effects on pathogenicity.1. Expansin gene expression and plant growth regulated by ethylene and gibbrellin in response to HpalIn early studys, ethylene signal and regulator of EIN5can induce plant growth enhancement in response to Harpin and the expansin protein act importantly on the effects. Expansins act to loose cell walls and modulate growth of the cell and plant mediated by some hormones. However, it’s not clear whether a hormone is specific and distinct hormones interact to regulate EXP activity. In this study, we report the effect that expansin gene expression and plant growth enhancement (PGE) in response to Hpal, the type-Ⅲ effector produced by X. oryzae pv. oryzae and the roles of ethylene and gibbrellin in both responses. Our results show that when applied to Arabidopsis thaliana, tomato, tobacco and rice, Hpal can markedly increase these plants growth concomitantly with the leaf nitrogen, chlorophylls and chlorophyll a/b ratio, which can basally affect plant biosynthesis and productivity. RT-PCR assay of EXP genes revealed that eleven of organ-specific EXPs expression was affected in response to Hpal. Four of eleven EXPs behaved in consistence with the Hpal signaling process and the other four were newly identified as specifically expressed in leaves of tobacco or rice. Using genetic and chemical methods to block ET perception can arrested EXP expression and PGE in the four plants in response to Hpal.In rice, Hpal simulated GA3to induce EXP expression and PGE. Whether disrupting either of ET perception or GA biosynthesis, both effects were impaired. These results suggest that both events of EXP expression and PGE require GA and ET in plants responding to Hpal.2. The Hpal Harpin needs nitroxyl terminus to promote vegetative growth and leaf photosynthesis in ArabidopsisHpal is a Harpin protein produced by Xanthomonas oryzae, an important bacterial pathogen of rice, and has the growth-promoting activity in plants. To understand the molecular basis for the function of Hpal, we generated an inactive variant protein, HpalΔNT, by deleting the nitroxyl-terminal region of the Hpal sequence and compared HpalΔNT with the full-length protein in terms of the effects on vegetative growth and related physiological responses in Arabidopsis. When Hpal was applied to plants, it acted to enhance the vegetative growth but did not affect the floral development. Enhanced plant growth was accompanied by induced expression of growth-promoting genes and cell growth in plant leaves. The growth-promoting activity of Hpal was further correlated with a physiological consequence shown as promotions in stomatal and mesophyll CO2conductance and leaf photosynthesis. On the contrary, plant growth, growth-promoting gene expression, and the physiological consequence changed little in response to the HaplΔNT treatment. These analyses suggest that Hpal requires the nitroxyl-terminus to facilitate CO2transport inside leaf cells and promote leaf photosynthesis and vegetative growth of the plant.3. Apoplastic and cytoplasmic location of Harpin protein Hpal plays different roles in H2O2generation and pathogen resistance in Arabidopsis.Harpins produced by Gram-negative phytopathogenic bacteria have been shown to activate plant defense pathways which involve the production of the reactive oxygen species, especially transduction of hydrogen peroxide signal generated in the apoplast. But how a Harpin is recognized in the pathway and how the apoplastic H2O2anticipates in defense responses are not clear. Here we use Hpal, a Harpin protein that produced by rice bacterial leaf blight pathogen X. oryzae pv. oryzae, to study if the cellular location of it impacts H2O2production and how the H2O2regulates pathogen resistance in nonhost Arabidopsis thaliana. Hpal gene and Hpal fused to an apoplastic localization signal (S) were transformed into Arabidopsis thaliana using the Agrobacterium tumefaciens and generated HETAt (Hpal-expressing transgenic Arabidopsis thaliana) and SHETAt (SHpal-expressing transgenic Arabidopsis thaliana) transgenic plants, respectively. Our results show that the HETAt and SHETAt plants are resistant to pathogen Pseudomonas syringae pv. tomato DC3000and Pectobacterium carotovora subsp. carotovora RL4. In HETAt, Hpal was found to associate with the cytoplasm and in SHETAt it was found in the apoplast and cytomembrane, which accompany with H2O2accumulation in cytoplasts in HETAt and in apoplasts of SHETAt as well. This study also demonstrate that H2O2was generated in cytoplasm in HETAt and was generated in a NOX-dependent manner in apoplasts in SHETAt. When applied to Arabidopsis, Hpal located and induced H2O2generation in apoplasts and caused H2O2accumulation in both apoplasts and cytoplasts. Treatment with the SHETAt and Hpal-treated parent plants using DPI, however, can inhibit apoplastic H2O2generation accompanying with less cytoplasmic H2O2accumulation and reduce plant resistance to bacterial pathogens. These results suggest that the apoplastic H2O2generation induced by Hpal is subject to translocation into cytoplasm for participation in the regulation of pathogen resistance in the plant.4. The function study of Avrbs2protein that secreted through the type III secretion system of the Xanthomonas oryzae pv. oryzicolaXanthomonas oryzae is the causal agent of bacterial disease in rice which can secrete many effectors into plant cells through the type III secretion apparatus. More and more type III effectors including Avr proteins have been indentificated, however, the function studys of many of them are unclear. AvrBs2is the first type III effector that has been shown to enhance bacterial multiplication within host tissue, a virulence contribution only observed in the strains of X. campestris pv.vesicatoria. However, the function of other Xanthomonads AvrBs2proteins is unclear though they are widely conserved in many Xanthomonads especially in Xanthomonas oryzae. Here, we examined the function of AvrBs2by inserted mutation in X. oryzae pv. oryzicola RS105. The mutation of avrBs2xoc resulted in reduced pathogenicity on host rice plants IR24and enhanced the expression of pathogen-related1a and1b protein relative to the wild-type strain RS105, but not affect the hypersensitive response (HR) in non-host Nicotiana benthamiana. These results suggest that the AvrBs2xoc effector protein is a virulence factor and suppress the host defence response in plant. |
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