| Auxin and salicylic acid are phytohormones that regulate plant development and resistance to pathogens. Under pathogen attack, plants modulate hormone signaling crosstalk to coordinate development and resistance. How the crosstalk is modulated is largely unclear. Therefore, we identified the WD40-domain protein TTG2and characterized it as a modulator of auxin-salicylic acid crosstalk in tobacco. The results here will provide basic clues to explore the mechanism of the auxin-salicylic acid crosstalk in tobacco mediated plant defense and growth pathways and crosstalk with both of these two signaling pathways.1. Cloning the full-length cDNA of NtTTG2in Nicotiana tabacum, and the analysis of its expressionFor getting more information of the mechanism on development of thichome controlling by tobacco (Nicotiana tabacum). We successfully cloned a new trichome relative gene NtTTG2(Nicotiana tabacum Transparent TESTA GLABRA2) from tobacco NC89by using the technology of RT-PCR accompany with rapid amplification of cDNA ends (RACE). The full length cDNA sequence of NtTTG2we’ve got is1379bp,and the open reading frames (ORF) is1029bp, including putative four WD40-repeat domains, which means NtTTG2has ability to interact with other proteins. The result of sequence analysis showed that the putative protein sequence of NtTTG2has high homology with other reported trichome relative genes, and also AN11, which controls the pigment synthesis in corolla of Petunia×hybrida. The results indicated that the NtTTG2could participate in the foundation of the trichome growth or pigment synthesis in corolla of tobacco. The further study showed that the gene NtTTG2is expressed in all of the roots, stems, leaves, and flowers, especially in the flowers. The expression of TTG2was enhanced in NAA-treated plants but depressed in SA-treated plants. plant inoculation with pathogens also repressed NtTTG2expression. TTG2takes a double location in the cytoplasm and nucleus. 2. NtTTG2activates auxin/ARF signaling but represses SA/NPR1signalingA hairpin-based gene silencing unit made with a plant binary vector was introduced into NC89genome. Created TTG2-silencing (RNAi) NC89(TTG2iNC) plants. In tested TTG2iNC lines, TTG2iNC4was the most effective TTG2silencer. TTG2overexpression was manipulated by using the construct that contained the cauliflower mosaic virus35S promoter. In tested lines of TTG2-Overexpression Transgenic NC89(TOTNC) plant lines, TOTNC1was the most vigorous TTG2expressor. They can use for future studies, such as physiological and biochemical identification, and the gene in-depth study of signal transduction in tobacco. ARF8was TTG2-regulated as its expression was repressed in TTG2iNC4and enhanced in TOTNC1compared to TCNC. Like ARF8, GH3expression was also TTG2-dependent. TTG2iNC had a lower level but TOTNC a higher level of GH3expression in consistence with TTG2production. PR-1a and PR-2a genes, which respond to SA and require NPR1for transcription. Both genes were highly expressed in TTG2iNC but little in TOTNC compared to TCNC.3. NtTTG2performs as an affector of auxin signaling to promotes tobacco growth and developmentAs auxin regulates almost every aspect of plant development, TTG2acting as an auxin signaling activator is likely to play a role in the development of tobacco. To verify this hypothesis, we first investigated plants incubated on medium and in pots, and determined growth of roots and aerial parts in correlation with the expression of TTG2as well as expansin (EXP) protein-encoding genes EXP1and EXP2. The biomass scored as height and weight of fresh plants were also tested. TTG2iNC4was inferior to TCNC in growth appearance, foliar expression of TTG2, EXP1and EXP2and the biomass. NtTTG2was required for DFR and ANS expression with consequent effects on anthocyanin production and flower colorization. TTG2was also required for seed production as TTG2silencing caused acute seed abortion. TOTNC (TTG2overexpression plant) grew faster, and had a greater biomass. In TOTNC, flowers were better colorized with more anthocyanin production, seeds seemed plump and each capsule produced more seeds. Therefore, The developmental role of TTG2has been shown as the repressive effect of the gene silencing and the promoting effect of the overexpression on vegetative growth, floral synthesis of anthocyanin, flower colorization, and seed production. The content of free auxin (indole-3-acetic acid) varied greatly with organs but was close in the same organs of TCNC, TTG2iNC4or TOTNC4. Auxin concentrations unchanged by TTG2silencing and overexpression were regarded as basal levels in different organs. NtTTG2performs as an affector of auxin signaling to promotes tobacco development.4. NtTTG2represses resistance by inhibiting the SA/NPR1-regulated defenseTo test if NtTTG2plays a role in tobacco resistance to pathogens, we compared TTG2iNC4, TOTNC1and TCNC for responses to leaf inoculation with cucumber mosaic virus (CMV) and tobacco mosaic virus (TMV) or Pectobacterium carotovora subsp. carotovora (Pcc) compared to a buffer used in mock inoculation. The symptoms, multiplications and PR gene expression were tested. It was consistent that symptoms were less severe in TTG2iNC4but more severe in TOTNC1compared to TCNC. suggesting that NtTTG2is a resistance repressor in the plant.By using protocols similarly as for NC89, we generated TTG2-Overexpression Transgenic Xanthi (TOTX), Transgenic Control Xanthi (TC-Xanthi), and VIGS-mediated TTG2-silencing Xanthi (TTG2iX) plants. Based on inoculation tests, resistance repression was a consistent character of tested TOTX lines. Performances of these Xanthi genotypes confirm the role of NtTTG2in repressing plant resistance. NahG, a transgenic Xanthi (NN) plant that can not accumulate SA and is therefore highly susceptible to pathogens. TTG2-silencing NahG (TTG2i NahG) plants were compared with NahG and its parent Xanthi in terms of responses to CMV, TMV and Pcc. As anticipated, TTG2i NahG behaved similarly to Xanthi but was more resistant than NahG to pathogens. We further tested the interaction between NtTTG2and NPR1in resistance. VIGS was used to manipulate NPR1silencing in TC-Xanthi and TTG2iX, and generated NPR1-silencing (NPRli) and concurrent TTG2-NPR1-silencing (TTG2i NPR1i) plants. TTG2i NPRli1was more resistant than NPRlil but more susceptible than Xanthi or TTG2iX1based on pathogen growthand symptom severities. Thus, NtTTG2and NPR1antagonized mutually in regulating resistance. Together, data detailed above suggest that TTG2represses resistance to pathogens through repressing the SA/NPR1-regulated defense in the plant.5. NtTTG2modulates nuclear localization of ARF8and NPRl to determine gene expression downstream of auxin signaling and SA signaling pathwaysTTG2is characteristic of a typical WD40-domain protein, it does not interacts with ARF8and NPR1in yeast and in planta. Based on the genetic design for fluorescence imaging, TCNC should produce only RFP, TOTNC1should produce the TTG2-RFP fusion protein, and TTG2iNC4produces little TTG2. In transient expression assays, ARF8was fused to a yellow-fluorescent protein (YFP) and NPR1to a green-fluorescent protein (GFP); fluorescent proteins in plant roots were visualized by laser confocal microscopy. Variations of TTG2production impacted subcellular localization of ARF8and NPR1in TCNC, TTG2iNC4and TOTNC1. Nuclear localization of ARF8-YFP was visualized in TOTNC1but the protein was little in TTG2iNC4nuclei. TTG2iNC4emitted strong fluorescence indicating nuclear localization of NPR1-GFP while the protein was detected only in the cytoplasm of TOTNC1, suggest that TTG2promotes nuclear localization of ARP8but sequestrates NPR1in the cytoplasm. Transient expression of ARF8increased in TOTNC1and decreased in TTG2iNC4but NPR1changed little, implying that manipulation of TTG2(TTG2) only affected NPR1protein but affected both ARF8gene and ARF8protein. The expression of PR-1a and PR-2a was induced in TTG2iNC4but nullified in TOTNC1. Inversely, GH3expression was induced in TOTNC1but nullified in TTG2iNC4. |
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