| In the nature, plant suffers from the pathogen or the pathogenic exciton attack frequently, but they can use their own innate immune system to form a set complex and effective defense mechanism for resisting these kinds of violation, including hypersensitive cell death (HCD) and system acquired resistance (SAR). HCD is also called hypersensitive response (HR), is one of the typical symptoms for the plant disease-resistant response. The HR production are related with two types of pathogen interacted with the plant. One kind is the interaction between the pathogen and the host like the avr-R. Another kind is the interaction between the pathogen and the non-host plant. Previous researches indicate that elicitins are the main fungi which can cause HR to the non-host plant. ParAl is one kind of elicitin produced by Phytophthra parasitica var nicotianae. The tobacco leaf trichomes treated by ParA1 may cause a series of HCD correlation response. This indicated that there are a set of molecular mechanism for receiving the ParA1 signal in the tobacco leaf trichomes. However, how does the plant recognize the ParA1? How does the trichome development protein NtTTG1 participate in the HCD process induced by the ParA1?Transcription factor plays a vital role in plant growth and development process to answering much kinds of biological and non-biological intimidate. According to binding the cis-element of downstream gene promoter, or forming homologous or heterogenous dimmers, or interacting with other proteins, transcription factor can regulate the expression of target genes, or participate in the ethylene, salicylic acid (SA), jasmine acid (JA) and abscisic acid (ABA) signal transductions, so they can form the complex gene expression regulation network and enhance the ability for the plant to adapt to the environment. HrpNEa is one kind of protein produced by Erwinia amylovora, which can stimulate different signal pathways to cause many kinds of advantageous phenotypes. EIN2 (ethylene insensitive2) is the key regulation factor in the ethylene signal pathway, however, how the relationship between the transcription factor and EIN2? What does the transcription factor play in the ethylene signal transduction pathway?This study analyzes the tobacco trichome protein NtTTGl and the Arabidopsis transcription factor AtMYB44 function to regulate transduction of two distinct defensive signals (ParA1 and HrpNEa) via the membrane and in the plant cell.1、Production of the loss of function protein C51S and its functional analysis compared with ParAlAccording to the previous studies about ParA1, it is a kind of proteinous elicitor with low molecular weight derived from Phytophthora parasitica var. nicotianae. Infiltrated in the tobacco leaves, ParA1 can induce hypersensitive cell death (HCD). Sprayed on the tobacco leaves, it may induce the microscopic hypersensitive response (micro-HR), companied with ROS burst and chromatin condensation (Cc) in the corresponding plant cells. The leaves were excised at 12h post treatment (hpt) and stained with green fluorescence dye 2’, 7’-dichlorofluorescein diacetate (DCFH-DA) and ROS could be detected, but not the C51S treatment. Cc was studied by 4’,6’-diamino-2-phenylindole (DAPI) fluoluminescence assay. There are 6 conserved cysteines that form three disulfides to maintain the specific structure in ParA1, and this structure is required for the function of ParAl. This study focused on the site direct mutant of ParA1. We replaced the cysteine at the site of 51 by serine, and obtained the mutagenesis protein C51S. Compared with ParA1, C51S lost the function of inducing HR in tobacco leaves. And we could not detect ROS burst、micro-HR and chromatin condensation in trichome cells by further experiments. Our results suggest that C51S has been lost of biological functions.2、Analysis of the interaction between ParA1 and NtTTG1 in tobacco trichome cell membraneAccording to cloning the homologues genes AtGL1, AtGL3, AtTTG1, and AtTTG2, which are essentially involved in the development of Arabidopsis thaliana leaf trichomes, we found only AtTTG1 homologue responded to ParA1 and NtTTG1 was cloned. Also amino acid sequence of NtTTG1 is highly identical with that of AtTTG1. The expression of NtTTG1 was strongly induced by ParA1 in tobacco trichomes rather than leaves. ParA1, but not C51S, could interact with NtTTG1 by yeast two-hybrid assay and pull-down assay. Analysis of ParA1 structure model,51’ cysteine is positioned on the surface of the outer edge of the protein, which is implicated in interacting with other proteins. These also suggested that the importance of 51/95 disulfide bone with 3/71 in ParA1. ParA1-NtTTG1 interaction conformed to subcellular localization of both proteins in tobacco trichomes that transiently expressed NtTTG1 fused to a red-fluorescence protein (RFP) gene. Fluorescence microscopy showed different distribution of these proteins in trichomes. NtTTG1-RFP showed conspicuous localization at the trichome cell membrane. At equivalent locations, ParAl-eGFP was affluent but C51S was little when applied externally and treated with a washing solution. This observation suggested that ParAl was bound tightly but C51S was not. The following experiment about mergence of red and green fluorescence indicated that ParA1-eGFP, instead of C51S-eGFP, bound NtTTG1-RFP, and that the interacting sign clearly localized at the cell membrane.3、Tobacco leaf trichomes contribute to ParAl-induced signalling and cell deathAs the first and the most external barriers of the plant, trichomes always touch the ParAl elicitor at the earliest time, and also contribute to the ParA1-induced hypersensitive cell death (HCD). We have showed that trichomes could respond to ParA1, with ROS burst and chromatin condensation being detected. After localized treatment of trichomes, the leaves were excised at 6h post treatment (hpt) and stained with green fluorescence dye 2’, 7’-dichlorofluorescein diacetate (DCFH-DA), apparently, ROS had spread from the trichome to leaf epidermal cells and the mesophyll in 6 hpt. Simultaneity, we proved that H2O2 occupies a prominent part of the ROS production that is induced by ParA1 by applying with diphenyleneiodonium (DPI) or catalase (Cat). Typical Cc was detected in trichomes and mesophylls following the use of ParA1 versus EVP. Nuclei in cells of control trichomes and in cells of ParAl-treated trichomes within 1 hpt had a clear central nucleolus surrounding by a uniform stained chromatin; whereas, in cells of ParA1-treated trichomes over 1 hpt, chromatin had a swollen aspect and nuclei were lobated. In one-way F tests (P< 0.01), trichomes significantly exceeded mesophylls in levels of cell death at each time point since 1 hpt. HCD marker genes PAL, hin1, and hsr203 were expressed evidently in trichomes 12 hpt with ParA1. Mesophylls were inferior to trichomes in time and extents of the gene expression. All these results suggested that HCD signal transduct from tobacco trichomes to mesophylls.4、Thirty-seven transcription factor genes differentially respond to a harpin protein and affect resistance to the green peach aphid in ArabidopsisThe harpin protein HrpNEa induces Arabidopsis resistance to the green peach aphid by activating the ethylene signaling pathway and by recruiting EIN2, an essential regulator of ethylene signaling, into defense response in the plant. Here we investigated 37 ethylene-inducible Arabidopsis transcription factor genes for effects on the activation of ethylene signaling and insect defense. In response to HrpNEa,22 genes increased in transcription levels with AtMYB44 being the most inducible,6 genes had decreased transcript levels, and 9 remained unchanged. When Arabidopsis mutants defected at the 37 genes were surveyed,24 mutants were similar to the wild-type (WT) plant while 4 mutants were more resistant and 9 mutants were more susceptible than WT to aphid infestation. The atmyb44 mutant with a defect in the AtMYB44 gene was most susceptible to aphid infestation and most compromised in HrpNEa-induced resistance. Resistance accompanied the expression of PDF 1.2, a an ethylene signaling marker gene that requires EIN2 for transcription, in WT but not in atmyb44 and other 4 aphid-susceptible mutants, suggesting a disruption of ethylene signaling in the five mutants. However, atmyb44 was an mutant with an abrogation in HrpNEa-induced EIN2 expression, suggesting a close relationship between AtMYB44 and EIN2.5、Analysis of the interaction between the transcription factor AtMYB44 and ethylene signaling regulator EIN2AtMYB44 is a transcription factor responsive to ethylene and implicated in Arabidopsis thaliana defense response. Plant defense is often subject to ethylene signaling that recruits EIN2 as a central regulator. Previously we have shown that EIN2 plays a critical role during the development of insect defense in Arabidopsis treated with HrpNEa, a harpin protein produced by bacterial plant pathogen. Regulatory targets of AtMYB44 and regulation of EIN2 activation have not been characterized. Through genetic, molecular, and pharmacological studies, we have shown that AtMYB44, EIN2, and ethylene are required for HrpNEa to induce Arabidopsis resistance against the green peach aphid. In this study, we testified that AtMYB44 could combine the specific consensus MYB recognition sequence 5’-TAACTG-3’ in the promoter of EIN2 by gel mobility shift assay (GMSA) and chromatin immunoprecipitation assay (ChIP).6、Production and identification of the two transgenic double mutant ein2-1 atpp2-a1 and ein2-1 abi2-1AtPP2 class phloem protein belongs to a unique tissue-specific agglutinin combinating with chitin in Arabidopsis thaliana, and its amino acid sequence is highly conserved. It has a special defense capability on the invasion of sucking insects and plays an important role during the plant phloem-related defense (PRD) processes. Abscisic acid (ABA) as the stress hormone can modulate many important events in plant growth and development including stoma closing, resistance to stresses, promoting seeds maturity and dormancy. Ethylene is the most important endogenic signal in plant growth and development and mediate the basal plant defense and disease resistant signal pathways. EIN2 is a central regulator in ethylene signal pathway. The interaction between PRD and ethylene or ABA and ethylene in plant growth and development is not very clear at present. We constructed AtPP2-A1-silencing and ABI2-silencing AtPP2-A1vector, which could be able to silence AtPP2-A1 and ABI2 in ein2-1, and obtained the two transgenic double mutant ein2-1 atpp2-a1 and ein2-1 abi2-1. This study will provide further materials and evidences for research of relationship between PRD and ethylene or ABA and ethylene pathways in the plant development and signal transduction. |
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