^^^^^^^^^^^^ ЭКСПЕРИМЕНТАЛЬНЫЕ
СТАТЬИ
581.1
NEW ROLES FOR THE Arabidopsis TAO1 GENE BESIDES DISEASE RESISTANCE1
© 2015 J. Yang*, **, 2, L. Yan*, 2, Y. Song*, C. Chai*, L. Song*, L. Guan*, S. Hou*
*Key Laboratory of Cell Activities and Stress Adaptations, Ministry of Education, School of Life Sciences, Lanzhou University, Lanzhou, China ** Longdong University, Qingyang, China Received November 1, 2014
Target of AvrB Operation (TAO1) is a TIR-NB-LRR disease resistance protein that responds to avirulence gene B (avrB). However, whether TAO1 plays a role in Arabidopsis thaliana development and/or abiotic stress is unknown. Here, we found that TAO1 expression is regulated in a tissue-specific manner, and that TAO1 protein is localized to both the plasma membrane and chloroplasts. Moreover, we showed that three taol T-DNA insertion mutants had no significant abnormal phenotype. However, we found that TAO1 expression was up-regulated by ethylene precursor ACC and auxin NAA, but down-regulated by ethylene inhibitor AgNO3 and auxin inhibitor NPA, suggesting that TAO1 is coordinately regulated by ethylene and auxin. We also found that endogenous ethylene production decreased in TAO1 -overexpression lines, but increased in the tao1-10 T-DNA mutant line compared to the wild type, suggesting that TAO1 may be a negative regulator of ethylene signaling. These novel characteristics shed new light on the role(s) of TAO1 in Arabidopsis independent of its role in disease resistance.
Keywords: Arabidopsis thaliana — TAO1 — expression pattern — subcellular localization — auxin — ethylene
ФИЗИОЛОГИЯ РАСТЕНИЙ, 2015, том 62, № 4, с. 579-587
УДК
DOI: 10.7868/S0015330315040193
INTRODUCTION
Plants use different mechanisms to fight pathogenic infections. One of the main disease resistance mechanisms is mediated by plant resistance proteins encoded by resistance genes (R genes), which comprise an enormous gene family. Most of R proteins contain nucleotide binding site (NB) and leucine-rich repeat (LRR) domains possessing either a coiled-coil (CC) domain or a TIR (Toll/Interleukin-1 receptor) domain at the amino terminus [1, 2]. The TIR-NB-LRR subgroup is dominant in the NB-LRR-containing group [3]. Because of the specificity of the genetic interactions between R genes and their cognate avr genes, NB-LRR proteins are suggested to be receptors for a wide range of ligands, such as pathogen-derived pep-
1 This text was submitted by the authors in English.
2 These authors contributed equally to this work.
Abbreviations'. ACC — 1-aminocyclopropane-1-carboxylic acid; GADPH — glyceraldehyde-3-phosphate dehydrogenase; GFP — green fluorescent protein; GUS — P-glucuronidase; MS — Murashige and Skoog medium; NAA — 1-naphthalene-acetic acid; NPA — 1-N-naphthylphthalamic acid; TAO1 — Target of AvrB Operation; TIR-NB-LRR - Toll/Interleukin-1 receptor, nucleotide binding site and leucine-rich repeat. Corresponding author. Suiwen Hou. Key Laboratory of Cell Activities and Stress Adaptations, Ministry of Education, School of Life Sciences, Lanzhou University, Lanzhou 730000, China; e-mail. housw@lzu.edu.cn
tides and small molecule hormones, that activate a downstream signal transduction cascade [4]. Therefore, in addition to their role in defense responses, NB-LRR proteins could potentially partake in plant development. However, little is known about the roles of NB-LRR proteins in plant development or abiotic stress resistance.
Arabidopsis Target ofAvrB Operation (TAO1), a typical TIR-NB-LRR disease resistance protein, was reported to recognize Pseudomonas syringae type III effector AvrB by inducing pathogenesis-related (PR-1) gene in response to Pto (P. syringae pv. tomato) DC3000 [5, 6]. Unfortunately, there are no other reports of other functions of TAO1 in addition to its role in disease resistance. In this study, we found that TAO1 was strongly expressed in various Arabidopsis tissues and organs in a de-velopmentally regulated pattern. We also found that TAO1 expression was induced by ACC and auxin. Moreover, TAO1 protein localized to both the plasma membrane and chloroplasts. Transgenic plants with overexpressed TAO1 displayed less ethylene emission. These results provide new supporting evidence for potential TAO1 functions in plant development and hormones responses.
Primers used in this study
Primer Sequence Used for
TAO1-L P1 CTTTGACCACATTATCTACG tao1-10 genotyping
TAO1-R P2 CCATCTGGAGAATCAGTAAT tao1-10 genotyping
TAO1-L P3 CTAGCAAATAGAGCAGTTAC tao1-11 genotyping
TAO1-R P4 TTACAACTCCTTAGCTGAAC tao1-11 genotyping
TAO1-L P5 CCTCATATCTACAGACTTCT tao1-12 genotyping
TAO1-R P6 AACCCTCCTCTTTTGCTAAT tao1-12 genotyping
T-DNA-LB CAGGATTTTCGCCTGCTGGGGC tao1 genotyping
GAPC-F ACCACACGGGAACTGTAACC qRT-PCR
GAPC-R GGCTATCAAGGAGGAATCCG qRT-PCR
TAO1-F TGATGAGTTCCTTACACTGG qRT-PCR
TAO1-R AGAGTTGCAAATGGGTCATG qRT-PCR
GFP-F CGGCGGCGGTCACGAACTC transgenic line detection
GFP-R CACCTACGGCAAGCTGACCCTGAA transgenic line detection
pTAO1-F AAAAAAGCAGGCTTCGTCTGGTAATGTTTTCAGGTGG promotor cloning
pTAO1-R CAAGAAAGCTGGGTTGAAGGGGGAGATGAAGAAGATAG promotor cloning
TAO1-F AAAAAAGCAGGCTTCCCGTTGTGATCTTGATCGTCTT gDNA cloning
TAO1-R CAAGAAAGCTGGGTTCTGATTGATGCTTGGCATTGGA gDNA cloning
MATERIALS AND METHODS
Growth and identification of T-DNA insertion mutants of taol alleles. The Columbia ecotype (Col-0) of Arabidopsis thaliana was used as wild type, and tao1 (At5g44510) T-DNA insertion mutants tao1-10 (SALK_124245C), tao1-11 (SALK_016670) [6] and tao1-12 (named in this study, SALK_003383C) were obtained from the Arabidopsis Biological Resource Center (ABRC). Seeds were sterilized and vernalized on half-strength Murashige and Skoog (MS) plates containing 1% (w/v) sucrose and 1% (w/v) agar for 3 days at 4°C, then the plates were either incubated at 23°C in a growth chamber for 14 days under 16 h light/8 h dark or for 5 days under absolute dark. Alternatively, vernalized seeds were grown in soil pots in a greenhouse under the normal photoperiod condition. The T-DNA left border primer and TAO1 gene-specific primers (see the table) were used to amplify genomic DNA to genotype the mutants.
RNA extraction and RT-PCR analysis. Total RNA was extracted from Arabidopsis seedlings using a kit (E.Z.N.A. Plant RNA Kit, "Omega Bio-Tek", USA), and genomic DNA was removed with RNase-free DNase I ("Promega", USA). cDNAs were synthesized with 1 ^g purified total RNA using M-MLV Reverse Transcriptase ("Promega") according to the manufacturer's instructions. PCR was performed with the primers listed in the table, using the cytosolic glyc-eraldehyde-3-phosphate dehydrogenase C subunit (GAPC) gene as an internal standard. For RT-PCR
analysis, SYBR Premix ExTaq ("TaKaRa Bio Inc.", Japan) was used in a Bio-Rad CFX-96 Real-Time System. Each sample was analyzed in triplicate. All experiments were performed with three replicates.
Plasmid construction and plant transformation. The
2-kb, 5' upstream sequence and the full genomic DNA sequence of TAO1 were fused in frame with the P-glucuronidase (GUS) and the green fluorescent protein (GFP) in the modified Gateway binary vectors pBIB-GUS and pBIB-35S-GFP ("Invitrogen", USA) [7], respectively. The constructs pTAO1-GUS and p35S-TAO1-GFP were transformed to Agrobacte-rium tumefaciens strain GV3101 and verified by colony PCR using the GFP-specific primers listed in the table. Col-0 Arabidopsis was transformed using the A. tumefaciens-mediated floral dip method [8]. Seeds of transformed Arabidopsis were vernalized by soaking in deionized water for 3 days at 4°C, and then grown in soil pots and kept in greenhouse conditions (16 h light period, 23°C). Seedlings were selected by spraying 0.02% BASTA. Three independent lines of the T2 generation were randomly chosen for further analysis.
GUS histochemical assay. Histochemical staining of plant tissue for GUS activity was performed as follows: pTAO1-GUS seedlings were washed with 1 M phosphate buffer, pH 7.0, and transferred to incubation buffer (50 mM sodium phosphate, pH 7.0, 10 mM EDTA, 0.1% Triton X-100, 0.5 mM potassium ferricyanide, 0.5 mM ferrocyanide, and 1 mM 5-bromo-4-chloro-
3-indolyl-P-D-glucuronide), vacuum-infiltrated for 1 min, and incubated overnight at 37°C. Stained tissues were decolorized in 8 . 3 . 1 (w/v/v) chloral hydrate . glycerol . water. GUS staining patterns were recorded using a light microscopy (80i, "Nikon").
Subcellular localization of TAO1-GFP proteins. To
detect the TAO1 localization, the roots of 10-day-old p35S-TAO1-GFP seedlings were soaked in the 0.8 M mannitol for 3 h to induce plasmolysis [9]. In addition, the protoplasts isolated from leaves of 10-day-old p35S-TAO1-GFP seedlings were observed with the untransformed protoplasts from the wild type and protoplasts transformed with the empty vector pBIB-35S-GFP serving as controls. The roots and protoplasts were observed as follows. GFP and chlorophyll were excited at 488 nm, while GFP and chlorophyll fluorescence were detected at 505 to 515 nm and 650 nm, respectively, using an Olympus FV1000MPE2 confocal microscope.
Exogenous hormone application and analysis of transgenic plants. The pTAO1-GUS Arabidopsis seeds were grown on MS medium containing 0.5 ^M auxin 1-naphthaleneacetic acid (NAA), 0.1 ^M auxin inhibitor 1-N-naphthylphthalamic acid (NPA), 5 ^M immediate ethylene precursor 1-aminocyclopropane-1-car-boxylic acid (ACC), or 0.5 ^M ethylene inhibitor AgNO3, respectively, with untreated pTAO1-GUS transgenic seeds as a control. Plates were placed at 4°C for 3 days, then incubated in a growth chamber with long-day conditions (16 h light/8 h dark) for 14 days. cDNAs from 14-day-old seedlings were isolated and used for real-time PCR analysis. These experiments were repeated three times independently.
Ethylene production measurement. Surface-sterilized seeds were grown in vials containing 2 mL MS media for 3 days at 4°C to break dormancy. Each vial contained 50 seedlings. After 3 days, vials wer
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