Regulation And Induction Of Plant Defense By Thioredoxin And Bioelicitor

Thioredoxins in plants can regulate enzymatic activities through adjusting celluar redox environments and consequently play important roles in many processes, such as regulation of redox, growth, development and defense response in plants. In this study we carried out experiments to determine the function of NtTRXh, which was specially induced by paraquat and TMV. We studied comprehensively the structure and function of NtTRXh in eliminating excessive reactive oxygen species (ROS) and enhancing plant resistance to TMV through a series of biochemical, molecular biological and genetic methods. HrpNEa is a harpin protein produced by the bacterial plant pathogen Erwinia amylovora. When applied to aerial parts of plants, the protein can induce systemic acquired resistance throughout the plants. However, does it act on plant roots? Therefore, we choose to study HrpNEa effects on Chinese cabbage (Brassica camperstris ssp.pekinens) roots and E. carotovora subsp. carotovora (Ecc), the pathogen of soft rot disease in cruciferous plants.1. Gene cloning, expression, and function analysis of tobacco thioredoxin proteinHere, we reported that only NtTRXh (AF435818) was specially induced by paraquat and TMV among three TRXh genes [NtTRXh (AF435818), NtTRXhl (X58527) and NtTRXhl (Z11803)] in Nicotiana tabacum and then cloned the gene. The analyzation of sequence alignment compared with the other sequences of thioredoxin family showed that NtTRXh has "WCGPC", a typical motif, and belongs to subfamilyⅢof h type thioredoxins. Subsequently, NtTRXh was expressed in Escherichia coli and then was purified using metal ion chelate affinity chromatography (Ni column). NtTRXh solution with a concentration of 125μg/ml did not elicit hypersensitive reaction in tobacco leaves. This indicated that NtTRXh was not harmful to plant cells. Furthermore, we determined the activity of NtTRXh based on ability to catalyze disulfide bond of insulin. Meanwhile, genetic modification generated the protein mutants C13T, C68T and C13TC68T by replacing cysteines with threonines and then the mutants were expressed, purified using Ni column. The catalysis activities of the three mutants and wild NtTRXh were determined synchronously. As a result, beside the two cysteines in "WCGPC" motif, the cysteine also participated in the catalyzation of disulfide bond of insulin. In conclusion, we cloned the gene encoding NtTRXh, which may be involved in oxidative stress and resistance against TMV, and analyzed the activity and motif of the protein.2. NtTRXh oppositely regulates plant defense and growthPlants contain several genes encoding thioredoxins h (TRXh), which play important role in development, growth and defense, especially redox regulation in plant cells, but the functions of most TRXh are pooly known. We found that NtTRXh was induced by paraquat and TMV in previous study. In order to study the function of NtTRXh, we fused NtTRXh to an inducible promoter PPP1, which can be induced by HpaGxoo, to regulate the excessive expression of NtTRXh and empolyed virus-induced gene silencing (VIGS) to silence the transcription of NtTRXh in tobacco. As a result, the leaves of transgenic tomato plants overexpressing NtTRXh (TRXOT1) produced more reactive oxygen species (ROS) than that of empty transgenic tobacco (ETT) when the two type plants were treated with paraquat. Furthermore, TMV caused more serious lesion in ETT than TRXOT1 and the expression of genes associated defense in TRXOT1 inoculated with TMV was stronger than that of genes in ETT inoculated with TMV. In addition, we found that overexpression of NtTRXh inhibited the growth of TRXOT1 accompanyed by a lower level expression of genes related to growth. So, NtTRXh is involved in eliminating ROS, enhancing resistance of plant against TMV and inhibiting plant growth. Meanwhile, we carried out the same experiments in the silenced plants (SiTRX) with wild type plants (WT) as control. Although the difference between SiTRX and WT was smaller, the resulting data proved also the function of NtTRXh. In conclusion, we determined the function of NtTRXh, which was involved in alleviating oxidative stress, enhancing resistance to TMV and suppressing plant growth, through excessive expression and silencing gene.3. Subcelluar localization of NtTRXh in tobaccoProteins are the direct executants for biological function. Order distributions of proteins are indispensable to growth and development for organism. Localization signals in amino acid sequences of proteins encoded by plant genes lead the proteins to localize to given position via accurate mechanisms. The proteins play different roles because of different subcelluar localizations. So, it is necessary for understanding comprehensively the function of proteins in plant cells to determine the sublocalization of proteins. We found that NtTRXh was induced by paraquat in last chapter. Paraquat absorbed by plant chlorenchyma elicits the production of superoxides, which stop photosynthesis and then damage seriously plant chlorenchyma. Consequently, we guessed that NtTRXh may be localized in chloroplast though there are few reports about h type TRXs, which localized in chloroplast. Subsequently, we analyzed the sublocalization NtTRXh through bioinformatics methods, but there was not definite signal peptide in the acid sequences of NtTRXh. However, amino acid composition predictes that NtTRXh may be localized to chloroplast, cytoplasm or mitochondrion. Furthermore, we studied the subcelluar localization NtTRXh in tobacco cells via immunological methods. First, NtTRXh was expressed in E.coli and then was purified via his tag. Afterwards, we used the purified NtTRXh to immunize rabbits to prepare polyclonal antibody. Finally, we determined the subcelluar localization NtTRXh using western blot and immunogold labelling. As a result, we found that NtTRXh distributed in chloroplast, cytoplasm and mitochondrion. Moreover, chloroplast had more NtTRXh than cytoplasm and mitochondrion. This was consistent with our prediction about the subcelluar localization NtTRXh. Furthermore, NtTRXh may participate in many physiological and chemical processes and play important roles in plants because of diverse subcelluar localization.4. The bacterial type-Ⅲprotein HrpNEa induces Chinese cabbage resistance against root invasion by the soft rot pathogenHrpNEa is a harpin protein produced by the bacterial plant pathogen Erwinia amylovora. When applied to aerial parts of plants, the protein can induce systemic acquired resistance throughout the plants. Herein, we report that treating Chinese cabbage roots with HrpNEa stimulates the plant to resist infection by E. carotovora subsp. carotovora (Ecc), the bacterial pathogen of soft rot disease in cruciferous plants. Treatment of Chinese cabbage roots with HrpNEa markedly changed the pattern of Ecc attachment to root surfaces and reduced numbers of Ecc cells in polar attachment mode. Total numbers of the bacterial cells attached to root surfaces were also significantly reduced on HrpNEa-treated roots compared to those on control plants. Root treatment with HrpNEa evidently inhibited Ecc cells to produce quorum-sensing (QS) signals that function to modulate bacterial population-dependent pathogenicity of the pathogen. Moreover, HrpNEa treatment induced the expression of several resistance-related genes in Chinese cabbage roots. As a result, severity of soft rot was significantly alleviated in roots treated with HrpNEa relative to that in control roots. Thus, the use of HrpNEa in Chinese cabbage roots induces plant resistance coincidently with interfering Ecc attachment, inhibiting the bacterial QS signals, and inducing expression of resistance-related genes in roots. Our results imply a novel defensive mechanism induced in plant roots.