NtTTG1 And NtARP1 Predicted As Egulators Of Leaf Trichome And Plant Growth Regulate Hypersensitive Response And Pathogendefense In Tobacco

Hypersensitive response (HR) and systemic acquired resistance (SAR) are reported to be the two main responses in the plant pathogen resistant response.Many elicitors have been shown to be able to induce these two reactions, and several hormones signalling pathways enrolled in plant development have also been reported to participate in the plant pathogen resistant responses. Elicitins secreted by Phytophthora are a kind of proteinous elicitors. Theses mall proteins can induce HR and SAR in tobacco plants. ParAl is a kind of elicitins produed by Phytophthra parasitica var nicotianae. Studies on ParAl showed that ParAl infiltrated into tobacco leaves could induce dramatically HR, and when sprayed on the leaves, ParAl could also induce micro-HR and a series of defense response. Further studies in our lab showed that when spayed on the tobacco leave trichomes, ParAl could also induced a series of micro-HR related responses. This indicates that there is a set of molecular mechanism in leave trichomes to receive or to interact with ParA1 protein. How ParAl was received? How leave trichomes participate in the micro-HR induced by ParAl? These questions are not still to be answered until now. Otherwise, the tobacco dwarf mutant cesl-1 produced by somaclonal variation protocol was shown to represent constitutive SAR. Whether is there a regulator in this mutant in charge of regulating both development and plant defense? So, our study focused on analysis of the role and mechanism of factors participating in both development and defense sidnalling.1. Tobacco leaf trichomes are enrolled in parA1 induced HR signalingParA1 derived from Phytophthora parasitica var. nicotianae, is a kind of proteinous elicitor with low molecular weight. Infiltrated in the tobacco leaves, ParA1 can induce hypersensitive cell death (HCD). Sprayed on the tobacco leaves, it may induce the micro scopic hypersensitive response (micro-HR), companied with ROS burst and chromatin condensation in the corresponding plant cells. As the most external barriers of the plant, trichomes always touch the ParAl elicitor at the earliest time, and also are enrolled in the induced micro-HR. We began began with determining whether trichomes respond to ParA1, with H2O2 and chromatin condensation being detected. After localized treatment of trichomes, the leaves were excised at 6h posttreatment (hpt) and stained with green fluorescence dye 2,7-dichlorofluorescein diacetate (DCFH-DA), apparently, H2O2 had spread from the trichome to leaf epidermal cells and the mesophyll in 6 hpt. Cc were studied by 4,6-diamino-2-phenylindole (DAPI) fluoluminescence assay. Typical Cc was detected in trichomes and mesophylls following the use of ParAl 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 ParAl-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. HR marker genes PAL, hinl, and hsr203 were expressed evidently in trichomes 12 hpt with ParA1. Mesophylls were inferior to trichomes in time and extents of the gene expression. These results depict HR signal transduction from trichomes to mesophylls.2. Production of the loss of function protein C51S and its functional analysisAccording to the previous studies, there are 6 conserved cysteines harbored in ParA1, and they form three disulfides to maintain the specific structure of ParA1. And this structure is required for the function of ParAl. This study focused on the site direct mutagenesis of ParAl. In this way, the cysteine at the site of 51 was replaced by serine, and the mutant protein C51S was obtained. On the basis of functional analysis, C51S was tested to be lost of functions of inducing HR,micro-HR and ROS burst in tobacco leaves. Further experiments also showed that C51S could not induce ROS burst and chromatin condensation in trichome cells, either. These results indicate that C51S has been lost of biological functions.3. Cloning of NTTTG1 and the interaction between NtTTG1 and ParA1Initially, we cloned homologues of Arabidopsis thaliana genes AtGL1, AtGL3, AtTTG1, and AtTTG2, which are essentially involved in the development of leaf trichomes. Only AtTTGl homologue responded to ParAl and was designated as NtTTGl based on its function in the HR pathway. Amino acid sequence of predicted NtTTGl is highly identical with that of AtTTG1. The expression of NtTTGl was strongly induced by ParA1 in trichomes rather than leaves. In yeast two-hybrid assay, NtTTGl interacted with ParAl but not C51S, consistent with the importance of 51/95 disulfide bone and with a structural model of ParAl as well. In the modelling,51' cysteine is positioned on the surface of the outer edge of the protein and the position is implicated in interacting with other proteins. Consistently, ParAl did not interact with NtTTGl mutant protein S94F created through the substitution of 94'serine with phenylalanine in NtTTGl. Similar replacement nullifies functions of AtTTG1 in Arabidopsis. In AtTTG1 and NtTTG1, the serine residue is close to protein-binding WD40 domain. This position is thought important to the molecular interaction and explains our yeast two-hybrid assay result. A pull-down assay confirmed the physical interaction between ParAl and NtTTG1 but not in else combinations. NtTTG1-ParA1 interaction conformed to subcellular localization of both proteins in trichomes of plants that transiently expressed NtTTG1 fused to a red-fluorescence protein (RFP) gene. Fluorescence microscopy showed different distribution of these proteins in trichomes. NtTTGl-RFP showed conspicuous localization to the trichome cell membrane. At equivalent locations, ParAl-GFP 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. Mergence of red and green fluorescence indicated that NtTTG1-RFP bound ParAl-eGFP, instead of C51S-eGFP, and that the interacting sign clearly localized to the cell membrane.4. Virus induced gene silencing of NTTTG1 and detection of silencing effectsWe further corroborated the role of NtTTG1 in activation of the HR pathway. When NtTTGl was silenced through virus-mediated RNA interference (RNAi), the gene was no longer induced by ParAl to express in trichomes and leaves, neither hinl, suggesting a compromise in the HR pathway. Compared to control plants, RNAi plants were 3-h and～5-h later in Cc, H2O2 burst, and cell death in trichomes and mesophylls, respectively, and extents of these events decreased significantly (one-way F tests, P< 0.01) at each time point. Therefore, NtTTG1 is critical for trichomes to precede mesophylls in activation of the HR pathway. The pathway was also activated preferentially in trichomes versus mesophylls when leaves are sprayed with a ParAl solution, confirming the importance of leaf trichomes in sensing of an extrinsic signal.5. Constitutive expression of systemic acquired resistance in a tobacco dwarf mutant is regulated by an auxin-repressed proteinTo study coordinate regulation of plant growth and systemic acquired resistance (SAR), we applied a somaclonal variation protocol to tobacco (Nicotiana tabacum) and identified the mutant constitutive expresser of SAR 1-1 (cesl-1). Compared to wild-type (WT) plant, ces1-1 dwarfs but resists infection by a fungal pathogen. The mutant constitutively accumulates transcripts of plant defense response genes but fails to express expansin genes involved in the plant growth pathway. Genetic analyses of the backcross reveal that ces1-1 represents a disruption at a single gene and is dominant in heredity over the WT locus. The profile of differentially expressed cDNA shows that a transcript highly identical with the NtARP1 gene encoding an auxin-repressed protein is present in ces1-1 but absent in WT. We cloned NtARPl by rapid amplification of cDNA end and tested effects of auxin on expression of the gene and production of NtARP1 protein in the plant. NtARP1 is expressed and NtARP1 localizes to nuclei in cesl-1 rather than WT. NtARPl expression and NtARP1 production are both inhibited by treating ces1-1 with the auxin indole-3-acetic acid (IAA). We further characterized the functions of NtARPl in regulation of tobacco growth and defense responses. When NtARP1 is silenced, ces1-1 grows like WT but loses constitutive SAR characters. Our results suggest the presence of an auxin signaling pathway that oppositely regulates growth and defense in the plant.