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The Use of a Dexamethasone-inducible System to Synchronize Xa21 Expression to Study Rice Immunity
使用地塞米松诱导系统同步Xa21表达进行水稻免疫研究

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Abstract

Inducible gene expression systems offer researchers the opportunity to synchronize target gene expression at particular developmental stages and in particular tissues. The glucocorticoid receptor (GR), a vertebrate steroid receptor, has been well adopted for this purpose in plants. To generate steroid-inducible plants, a construct of GAL4-binding domain-VP16 activation domain-GR fusion (GVG) with the target gene under the control of upstream activation sequence (UAS) has been developed and extensively used in plant research.
Immune receptors perceive conserved molecular patterns secreted by pathogens and initiate robust immune responses. The rice immune receptor, XA21, recognizes a molecular pattern highly conserved in all sequenced genomes of Xanthomonas, and confers robust resistance to X. oryzae pv. oryzae (Xoo). However, identifying genes downstream of XA21 has been hindered because of the restrained lesion and thus limited defense response region in the plants expressing Xa21. Inducible expression allows for a synchronized immune response across a large amount of rice tissue, well suited for studying XA21-mediated immunity by genome-wide approaches such as transcriptomics and proteomics. In this protocol, we describe the use of this GVG system to synchronize Xa21 expression.

Keywords: Dexamethasone(地塞米松), Xa21-mediated immunity(Xa21介导的免疫), Xanthomonas oryzae pv. oryzae(Xanthomonas oryzae pv。米曲霉), Glucocorticoid receptor(糖皮质激素受体)

Materials and Reagents

  1. Wild-type rice seeds (Oryza sativa ssp. japonica cv. Kitaake)
  2. Transgenic rice seeds containing pTA7002::Myc::Xa21 (Park et al., 2012)
  3. Transgenic rice seeds containing Ubi::Myc::Xa21 (Park et al., 2010)
  4. Xanthomonas oryzae pv. oryzae (Xoo; Philippines race 6, strain PXO99Az)
  5. Dexamethasone (Sigma-Aldrich, catalog number: D1756 )
  6. Dimethyl sulfoxide (DMSO) (Thermo Fisher Scientific, catalog number: D128-1 )
  7. Tween-20 (Bio-Rad Laboratories, catalog number: 170-6531 )
  8. Sterile H2O (Milli-Q)
  9. TRIzol (Life Technologies, InvitrogenTM, catalog number: 15596-026 )
  10. M-MLV reverse transcriptase (Life Technologies, InvitrogenTM, catalog number: 28025-013
  11. SsoFastEvaGreenSupermix (Bio-Rad Laboratories, catalog number: 172-5203 )
  12. Peptone sucrose agar (PSA) solid media containing 20 µg/ml cephalexin (MP Biomedicals, catalog number: 02150585 ) (see Recipes)
  13. Dexamethasone (see Recipes)
  14. Greenhouse rice growing conditions (see Recipes)
  15. Walk-in growth chamber rice growing conditions (see Recipes)

Equipment

  1. Spray bottle (550 ml) (any supplier) for dexamethasone foliar spray
  2. 1.5 ml Eppendorf tube (any supplier)
  3. Surgical scissors (sharp/sharp, straight, 5 ½ inch or similar) for Xoo clipping inoculation
  4. 5 ½ inch square disposable pots
  5. Supertub (24 inch x 36 inch x 8 inch) (Mac Court Products, model: ST3608 or similar)
  6. Scale suitable for measurements down to 0.0001 g (any manufacturer)
  7. Spectrophotometer suitable for taking optical density measurements at 600 nm (any manufacturer)
  8. Growth chamber (14 h light and 10 h dark photoperiod with 28 °C temperature) (any manufacturer) for rice seed germination
  9. Incubation chamber (28 °C) (any manufacturer) for Xoo preparation
  10. Greenhouse capable of temperature and humidity control for growing rice plants
  11. Walk-in growth chamber (conviron or equivalent) for Xoo inoculation and dexamethasone treatment
  12. qPCR machine (Bio-Rad Laboratories, model: CFX96 Real-Time PCR )

Procedure

  1. Rice growth conditions
    1. Sow 12-20 rice seeds on filter paper in a petri dish (10 cm diameter) with 10 ml of sterile water and place it in an incubation chamber for one week.
    2. Transplant one-week old seedlings into 5 ½ inch pots (three seedlings per pot) and grow them in the green house for approximately another five weeks. For irrigation, keep individual pots in supertubs filled with fertilized water until four weeks then continue filling super tub with reverse osmosis water.
      Notes:
      1. Water level should be maintained near the soil surface but not higher to prevent green algae from covering the soil in the pots.
      2. A maximum capacity of 24 pots is possible per supertub, but 12 pots are optimum for these experiments to allow spacing between pots.
    3. When the flag leaf is fully extended but before panicle emergence (approximately six weeks after germination) transfer plants to walk-in growth chamber for Xoo inoculation and dexamethasone treatment.
      Notes:
      1. Allow a minimum of three days for plants to equilibrate to chamber conditions before inoculation.
      2. Roots often grow out of the bottom of the pots. Take care to minimize root damage while transferring plants to walk-in chambers.

  2. Rice inoculation with Xoo
    1. Two days before transferring plants to the walk-in growth chamber, transfer 30 μl of Xoo from -80 °C to a PSA solid media containing cephalexin and incubate at 28 °C for three days until a biofilm is formed.
      Note: If needed, after biofilm formation Xoo can be stored at 4 °C until ready to proceed, but should not be stored for more than two weeks.
    2. Two days before inoculating rice plants, subculture approximately 1 cm diameter of Xoo from the original PSA solid media containing cephalexin to a new PSA solid media containing cephalexin and incubate at 28 °C for an additional two days until biofilm has formed.
    3. On the day of inoculation, suspend Xoo from the most recent PSA solid media containing cephalexin in sterile H2O to OD600 = 0.5.
    4. Inoculate rice leaves by dipping scissor tips into the Xoo suspension and cutting the leaf approximately 2-3 cm away from the leaf tip of the 1st and 2nd leaves, or just 2nd leaf. Expected bacterial load immediately after inoculation is approximately 1 x 105 cfu/ml (Song et al., 1995).
      Note: For a non-inoculated control, rice leaves should be clipped with scissors dipped into water alone.
    5. Allow five to seven days after inoculation for Xoo to spread from the inoculated region and form a more even distribution across the rice leaves.
      Note: Five to seven days after inoculation was selected because based on previous experiments (Song et al., 1995), Xoo was shown to be completing log growth phase during that period of time. After seven days, disease symptoms begin to be more visibly pronounced.

  3. Dexamethasone foliar spray
    1. Prepare 30 μM dexamethasone + 0.01% Tween-20 fresh (400 ml) in 550 ml spray bottle on the day of foliar application.
    2. Spray 200 ml of dexamethasone per supertub from a distance of 12-24 inches away from plants, walking around tub as you spray and moving in an up and down motion to ensure spray is being applied evenly to all portions of the plants.
      Notes:
      1. Avoid bumping or disturbing the plants at this step or dexamethasone solution will slide off the leaves.
      2. If walk-in growth chamber has a strong circulating fan, plants should be placed in a less windy area within the growth chamber for dexamethasone application as the fans will cause dexamethasone to evaporate too quickly and dexamethasone uptake may not be as uniform. Plants can be returned after dexamethasone solution is no longer visible on the surface of leaves (after 1 to 2 h).
    3. Harvest tissues at the desired time points after dexamethasone treatment. To harvest tissue, 3 to 5 cm at the inoculated leaf tip were cut with sterilized scissors and immediately frozen in liquid nitrogen.

Representative data



Figure 1. Experimental design using the dexamethasone-inducible system. Six-week old rice plants are inoculated with Xoo. Before dexamethasone treatment, pTA7002::Myc::XA21 plants (A) do not express XA21 and phenocopy the susceptible Kitaake plants (B). Six days after inoculation, dexamethasone is applied to rice plants and XA21 begins to be expressed, inducing a robust immune response. Leaf tissue is harvested at 0 h, 12 h, and 24 h after dexamethasone application and frozen in liquid nitrogen for downstream applications. Ubi::Myc::XA21 plants(C) overexpressing under the control of the maize ubiquitin promoter (Park et al., 2010) are resistant to Xoo and used as a positive control here.


Figure 2. Expression of Xa21 after dexamethasone application. pTA7002::Myc::XA21 rice leaves were harvested at the indicated time points after application of dexamethasone. RNA was extracted using TRIzol with standard protocol; cDNA used for quantitative PCR was transcribed with M-MLV reverse transcriptase; and quantitative PCR was performed with SsoFastEvaGreenSupermix on a PCR machine. The gene expression of Xa21 was normalized using the rice ubiquitin gene (LOC_Os06g46770) as an internal control, and the expression level in the Ubi::Myc::XA21 plants (labeled as XA21) was set as 1.0.


Figure 3. Dexamethasone treatment inhibits the elongation of lesion lengths in pTA7002::Myc::XA21 plants. Six week old rice plants were inoculated with Xoo. At six days after inoculation (6 DAI) disease progression was measured (red line) before dexamethasone (DEX) was applied to the plants. Until 6 DAI, pTA7002::Myc::XA21 plants did not express Xa21 and the observed variation in disease development between with DEX and without DEX treated plants was not statistically significant. Disease progression was allowed to continue until 12 days after inoculation (12 DAI, 6 days after the DEX treatment), and lesion length was measured as indicated (blue line). pTA7002::Myc::XA21 plants treated with dexamethasone showed significantly less disease progression compared to non-dexamethasone treated plants indicating that DEX treatment was successful in inducing expression of Xa21 and that XA21-mediated immunity was triggered.

Notes

  1. “Leaky” expression in the absence of hormone has previously been reported in the chemical-inducible systems (Padidam, 2003; Zuo and Chua, 2000). In our system, about half of the independently transformed rice lines displayed weak or moderate leaky expression (Park et al., 2012). Therefore, it is critical to identify the transgenic lines that display the least leaky expression while maintaining highly inducible expression. To confirm non-leaky expression of Xa21, selected lines should be fully susceptible to Xoo in the absence of DEX and resistant to Xoo after DEX is applied. Additionally, Xa21 gene expression and protein abundance should be low or not detectable by quantitative RT-PCR and western blot analysis before DEX treatment and should be induced by DEX application (Park et al., 2012).
  2. To ensure dexamethasone treatment is successful, inoculate additional plants and maintain without harvesting leaves to measure lesions 12 to 14 days after inoculation.
  3. In our experience, when we used separate walk-in growth chambers for dexamethasone and non-dexamethasone treatments, we observed variation in Xoo lesion lengths between Kitaake (dexamethasone treated) and dexamethasone-inducible XA21 plants (non-dexamethasone induced) even when identical settings were applied to both walk-in growth chambers (data not shown). Therefore, we recommend limiting experiments to a single growth chamber in which dexamethasone is applied to all plants and Kitaake wild type rice are used as controls.
  4. Plants were transferred to walk-in growth chambers for inoculation with Xoo due to University biosafety protocols. The authors suspect that Xoo inoculation and dexamethasone application should be successful in the greenhouse as well, although these conditions were not tested.
  5. In our pre-experiments, we combined a 30 μM dexamethasone foliar application with 10 μM dexamethasone irrigation. However we didn’t see additional Xa21 expression compared to foliar application alone (data not shown).

Recipes

  1. Peptone sucrose agar (PSA) solid media containing cephalexin (1 L)
    Preparation of cephalexin stock: Dissolve cephalexin hydrate to 10 mg/ml in 50% ethanol and store 2 ml aliquots at -20 °C until ready to prepare media
    Dissolve 10 g peptone, 10 g sucrose and 1 g of L-glutamic acid in 900 ml in sterile H2O
    Adjust the pH of the medium to 7.0 using 1 N NaOH and bring volume up to 1 L
    Add 16 g agar and autoclave to sterilize
    Allow solution to cool to 55 °C, and add 2 ml of stock cephalexin to a final concentration of 20 µg/ml
  2. Dexamethasone, 30 μM solution containing 0.01% (w/v) Tween-20 (400 ml)
    For two supertubs, dissolve 0.0048 g of dexamethasone into 400 μl DMSO in a 1.5 ml eppendorf tube
    After dissolving, add it into 400 ml of sterile H2O containing 40 μl of tween-20
  3. Greenhouse rice growing conditions
    Greenhouse (26-28 °C light, 18-20 °C dark, 40-60% humidity)
    From April to September, natural light
    From October to March, light supplemented with 1,000 W metal halide bulbs from 6 am to10 pm (14 h light and 10 h dark photoperiod)
  4. Walk-in growth chamber rice growing conditions
    Walk-in growth chamber (14 h light, 28 °C, 80% humidity; 10 h dark, 24 °C, 85% humidity)
  5. Fertilized water composition
    Fertilized water kept at EC: 0.8-1
    Theoretical breakdown of PPM when injected at 1:200:
    N
    173.58
    P
    45.39
    K
    165.64
    Ca
    60.05
    Mg
    38.88
    S
    146.42
    Fe
    5.23
    Cu
    0.18
    B
    0.4
    Mn
    1.20
    Mo
    0.05
    Zn
    0.37

Acknowledgments

This protocol was modified from Park et al. (2012).
This research was supported by the National Institute of Health (NIH, GM55962), the National Science Foundation (NSF, IOS-0817738), and the Monsanto's Beachell-Borlaug International Scholars Program.

References

  1. Aoyama, T. and Chua, N. H. (1997). A glucocorticoid-mediated transcriptional induction system in transgenic plants. Plant J 11(3): 605-612.
  2. Padidam, M. (2003). Chemically regulated gene expression in plants. Curr Opin Plant Biol 6(2): 169-177.
  3. Park, C. J., Canlas, P. E. and Ronald, P. C. (2012). Establishment of glucocorticoid-mediated transcriptional induction of the rice XA21 pattern recognition receptor. J Plant Biol 55(1): 43-49.
  4. Park, C. J., Lee, S. W., Chern, M., Sharma, R., Canlas, P. E., Song, M. Y., Jeon, J. S. and Ronald, P. C. (2010). Ectopic expression of rice Xa21 overcomes developmentally controlled resistance to Xanthomonas oryzae pv. oryzae. Plant Sci 179(5): 466-471.
  5. Song, W. Y., Wang, G. L., Chen, L. L., Kim, H. S., Pi, L. Y., Holsten, T., Gardner, J., Wang, B., Zhai, W. X., Zhu, L. H., Fauquet, C. and Ronald, P. (1995). A receptor kinase-like protein encoded by the rice disease resistance gene, Xa21. Science 270(5243): 1804-1806.
  6. Zuo, J. and Chua, N. H. (2000). Chemical-inducible systems for regulated expression of plant genes. Curr Opin Biotechnol 11(2): 146-151.

Appendix

  1. Nucleotide sequence of Myc::Xa21. The underlined region defines the N-terminal Myc tag.The Myc tag was inserted in domain B of the Xa21 gene following the putative signal peptide (domain A) as previously described (Park et al., 2010).
    atgatatcactcccattattgctcttcgtcctgttgttctctgcgctgctgctctgcccttcaagcagtgacgacgatggtgatgctgccggcgacgaactcgcgctgctctctttcaagtcatccctgctataccaggggggccagtcgctggcatcttggaacacgtccggccacggccagcactgcacatgggtgggtgttgtgtgcggccgccgccgccgccggcacccacacagggtgGAGCAAAAGCTGAACCCACAACACACGCCGGCGGCGGCGGCGGCCGTGGGTGTGTCCCACCTCGTTTTCGACTTTTCTGAGGAGGATCTGCTGGAGAAGCACAGGCTGgtgaagctgctgctgcgctcctccaacctgtccgggatcatctcgccgtcgctcggcaacctgtccttcctcagggagctggacctcggcgacaactacctctccggcgagataccaccggagctcagccgtctcagcaggcttcagctgctggagctgagcgataactccatccaagggagcatccccgcggccattggagcatgcaccaagttgacatcgctagacctcagccacaaccaactgcgaggtatgatcccacgtgagattggtgccagcttgaaacatctctcgaatttgtacctttacaaaaatggtttgtcaggagagattccatccgctttgggcaatctcactagcctccaggagtttgatttgagcttcaacagattatcaggagctataccttcatcactggggcagctcagcagtctattgactatgaatttgggacagaacaatctaagtgggatgatccccaattctatctggaacctttcgtctctaagagcgtttagtgtcagagaaaacaagctaggtggtatgatccctacaaatgcattcaaaacccttcacctcctcgaggtgatagatatgggcactaaccgtttccatggcaaaatccctgcctcagttgctaatgcttctcatttgacagtgattcagatttatggcaacttgttcagtggaattatcacctcggggtttggaaggttaagaaatctcacagaactgtatctctggagaaatttgtttcaaactagagaacaagatgattgggggttcatttctgacctaacaaattgctccaaattacaaacattgaacttgggagaaaataacctggggggagttcttcctaattcgttttccaatctttccacttcgcttagttttcttgcacttgaattgaataagatcacaggaagcattccgaaggatattggcaatcttattggcttacaacatctctatctctgcaacaacaatttcagagggtctcttccatcatcgttgggcaggcttaaaaacttaggcattctactcgcctacgaaaacaacttgagcggttcgatcccgttggccataggaaatcttactgaacttaatatcttactgctcggcaccaacaaattcagtggttggataccatacacactctcaaacctcacaaacttgttgtcattaggcctttcaactaataaccttagtggtccaatacccagtgaattattcaatattcaaacactatcaataatgatcaatgtatcaaaaaataacttggagggatcaataccacaagaaatagggcatctcaaaaatctagtagaatttcatgcagaatcgaatagattatcaggtaaaatccctaacacgcttggtgattgccagctcttacggtatctttatctgcaaaataatttgttatctggtagcatcccatcagccttgggtcagctgaaaggtctcgaaactcttgatctctcaagcaacaatttgtcaggccagatacccacatccttagcagatattactatgcttcattccttgaacctttctttcaacagctttgtgggggaagtgccaaccattggtgctttcgcagctgcatccgggatctcaatccaaggcaatgccaaactctgtggtggaatacctgatctacatctgcctcgatgttgtccattactagagaacagaaaacatttcccagttctacctatttctgtttctctggccgcagcactggccatcctctcatcactctacttgcttataacctggcacaagagaactaaaaagggagccccttcaagaacttccatgaaaggccacccattggtctcttattcgcagttggtaaaagcaacagatggtttcgcgccgaccaatttgttgggttctggatcatttggctcagtatacaaaggaaagcttaatatccaagatcatgttgcagtgaaggtactaaagcttgaaaatcctaaggcgctcaagagtttcactgccgaatgtgaagcactacgaaatatgcgacatcgaaatcttgtcaagatagttacaatttgctcgagcattgataacagagggaacgatttcaaagcaattgtgtatgacttcatgcccaacggcagtctggaagattggatacaccctgaaacaaatgatcaagcagaccagaggcacttgaatctgcatcgaagagtgaccatactacttgatgttgcctgcgcactggactatcttcaccgccatggccctgaacctgttgtacactgtgatattaaatcaagcaatgtgctgttagattctgatatggtagcccatgttggagattttgggcttgcaagaatacttgttgatgggacctcattgatacaacagtcaacaagctcgatgggatttatagggacaattggctatgcagcaccagagtatggcgttgggctcattgcatcaacgcatggagatatttacagctatggaattctagtgctggaaatagtaaccgggaagcggccaactgacagtacattcagacccgatttgggcctccgtcagtacgttgaactgggcctacatggcagagtgacggatgttgttgacacgaagctcattttggattctgagaactggctgaacagtacaaataattctccatgtagaagaatcactgaatgcattgtttggctgcttagacttgggttgtcttgctctcaggaattgccatcgagtagaacgccaaccggagatatcatcgacgaactgaatgccatcaaacagaatctctccggattgtttccagtgtgtgaaggtgggagccttgaattctga
  2. pTA7002::Myc::Xa21 construct map.
    SP signal peptide, RB right border, 35S cauliflower mosaic virus 35S promoter, GVG GAL4-binding domain, VP16 activation domain–glucocorticoid receptor fusion, E9 poly (A) addition sequence of the ribulosebisphosphate carboxylase small subunit (rbcS-E9), NOS-P nopaline synthase promoter, NOS-T nopaline synthase terminator; 6×UAS six copies of the GAL4 upstream activating sequence and the −46 to +1 region of the 35S promoter, 3A poly (A) addition sequence of the pea rbcS-3A, LB left border.


简介

诱导型基因表达系统为研究人员提供了在特定发育阶段,特别是组织中同步靶基因表达的机会。糖皮质激素受体(GR),一种脊椎动物类固醇受体,已经在植物中被很好地用于此目的。为了产生类固醇诱导的植物,已经开发了在上游激活序列(UAS)控制下具有靶基因的GAL4结合结构域-VP16激活结构域-GR融合(GVG)的构建体,并广泛用于植物研究中。
免疫受体感知由病原体分泌的保守分子模式,并启动强烈的免疫应答。水稻免疫受体,XA21 识别在所有测序的黄单胞菌属的基因组中高度保守的分子模式,并且赋予对X的强抗性。 oryzae pv。 oryzae ( Xoo )。然而,鉴定XA21 下游的基因受到限制性损伤的阻碍,因此在表达Xa21 的植物中限制了防御反应区域。诱导型表达允许跨大量水稻组织的同步免疫应答,非常适合通过全基因组方法(例如转录组学和蛋白质组学)研究XA21 介导的免疫。在该协议中,我们描述了使用该GVG系统来同步 Xa21 表达。

关键字:地塞米松, Xa21介导的免疫, Xanthomonas oryzae pv。米曲霉, 糖皮质激素受体

材料和试剂

  1. 野生型水稻种子(<北>水稻 ssp。japonica cv。Kitaake)
  2. 含有pTA7002 :: Myc :: Xa21(P ,2012)的转基因水稻种子
  3. 含有Ubi :: Myc :: Xa21(2010)的转基因水稻种子</em>
  4. Xanthomonas oryzae pv。 ( Xoo ;菲律宾第六赛季,PXO99Az系列)
  5. 地塞米松(Sigma-Aldrich,目录号:D1756)
  6. 二甲基亚砜(DMSO)(Thermo Fisher Scientific,目录号:D128-1)
  7. Tween-20(Bio-Rad Laboratories,目录号:170-6531)
  8. 无菌H 2 O(Milli-Q)
  9. TRIzol(Life Technologies,Invitrogen TM,目录号:15596-026)
  10. M-MLV逆转录酶(Life Technologies,Invitrogen TM,目录号:28025-013)
  11. SsoFastEvaGreenSupermix(Bio-Rad Laboratories,目录号:172-5203)
  12. 含有20μg/ml头孢氨苄(MP Biomedicals,目录号:02150585)的蛋白胨蔗糖琼脂(PSA)固体培养基(参见配方)
  13. 地塞米松(见配方)
  14. 温室水稻生长条件(见配方)
  15. 步入式生长室水稻生长条件(见配方)

设备

  1. 喷雾瓶(550 ml)(任何供应商)地塞米松叶面喷雾器
  2. 1.5 ml Eppendorf管(任何供应商)
  3. 外科剪刀(锋利/锋利,直,5½英寸或类似),用于 Xoo 剪裁接种
  4. 5½英寸方形一次性壶
  5. Supertub(24英寸×36英寸×8英寸)(Mac Court Products,型号:ST3608或类似物)
  6. 适用于低至0.0001 g(任何制造商)的测量
  7. 适用于在600 nm(任何制造商)进行光密度测量的分光光度计
  8. 生长室(14小时光照和10小时黑暗光周期,28℃温度)(任何制造商)水稻种子发芽
  9. 用于Xoo 制备的孵育室(28℃)(任何制造商)
  10. 温室能够控制生长水稻的温度和湿度
  11. 用于Xoo 接种和地塞米松治疗的步入式生长室(环境或等同物)
  12. qPCR机(Bio-Rad Laboratories,型号:CFX96实时PCR)

程序

  1. 水稻生长条件
    1. 在培养皿(10cm直径)的滤纸上播种12-20个水稻种子, 用10ml无菌水并将其置于孵育室中 一周。
    2. 将一周龄的幼苗移植到5 1/2英寸的盆中 (每盆三棵幼苗),并在温室中种植 大约再五个星期。 对于灌溉,保持个别盆 在充满受精水的supertubs,直到四个星期,然后继续   用超滤水装满超级桶。
      注意:
      1. 水   水平应保持在土壤表面附近,但不要高于 防止绿藻覆盖盆中的土壤。
      2. 最大值   每个supertub可以容纳24个罐,但是12个罐是最佳的 这些实验允许盆之间的间距。
    3. 当。。。的时候 旗叶完全延伸,但在穗出现前(约 发芽后6周)将植物转移到步入式生长室中 用于 Xoo 接种和地塞米松治疗 注意:
      1. 在接种之前允许植物至少三天使植物平衡到室条件。
      2. 根根经常生长在花盆底部。 小心 在将植物转移到步入式小室时最小化根损害

  2. 用 Xoo
    接种水稻
    1. 在将植物转移到步入式生长室前两天, 将30μl的Xoo 从-80℃转移到含有PSA的固体培养基中 头孢氨苄,并在28℃孵育三天,直到生物膜 形成。
      注意:如果需要,生物膜形成后可以储存Xoo 在4°C直至准备好进行,但不应储存超过 两个星期。
    2. 在接种水稻前两天,传代培养 距离原始PSA固体介质约1cm直径的Xoo 含有头孢氨苄的新型PSA固体培养基  在28℃下孵育另外两天直到生物膜形成。
    3. 在接种当天,从最新的PSA中暂停 Xoo 含有头孢氨苄的固体培养基在无菌H 2 O至OD 600 = 0.5中。
    4. 通过将剪刀尖浸入Xoo悬浮液中接种稻叶 并切割叶片大约2-3厘米远离叶尖 1 st 和2 nd 个叶子,或只是2个 nd 叶子。预期细菌负荷 接种后立即为约1×10 5 cfu/ml(Song et al al。,1995)。
      注意:对于未接种的对照,应该用剪刀将水稻叶片浸入水中。
    5. 允许接种后五至七天,以便从中扩展 Xoo 接种的区域并且在稻上形成更均匀的分布 叶。
      注:选择接种后5至7天 因为基于以前的实验(Song等人,1995),显示了Xoo   在这段时间内完成对数增长阶段。 后 七天,疾病症状开始更明显。

  3. 地塞米松叶面喷雾
    1. 在叶面施用当天,在550ml喷雾瓶中制备30μM地塞米松+ 0.01%吐温-20(400ml)。
    2. 从12-24的距离每个supertub喷射200毫升地塞米松 英寸远离植物,走在浴缸,当你喷雾和移动 上下运动,确保喷雾均匀地施加到所有 植物的部分。
      注意:
      1. 避免碰撞或干扰植物在这一步或地塞米松溶液将滑落叶子。
      2. 如果步入式生长室有一个强大的循环风扇,植物 应放置在生长室内较不风的区域 地塞米松应用作为球迷将引起地塞米松 蒸发太快,地塞米松吸收可能不一样均匀。 植物可以在地塞米松溶液不再可见后返回   在叶子表面(1到2小时后)。
    3. 收获组织 地塞米松治疗后所需的时间点。收获 组织,在接种的叶尖处3至5cm用灭菌切割 剪刀并立即在液氮中冷冻。

代表数据



图1.使用地塞米松诱导系统的实验设计。 用Xoo 接种六周大的稻植物。在地塞米松处理之前,pTA7002 :: Myc :: XA21植物(A)不表达XA21并对易感的Kitaake植物(B)进行phenocopy。接种后6天,将地塞米松应用于水稻植物,并且XA21开始表达,诱导强烈的免疫应答。在施用地塞米松后0小时,12小时和24小时收获叶组织,并在液氮中冷冻用于下游应用。在玉米泛素启动子控制下过表达的Ubi :: Myc :: XA21植物(C)(Park等人, 2010)抗 Xoo 并用作阳性对照。


图2.在地塞米松应用后 Xa21 的表达。在施用后在指定的时间点收获pTA7002 :: Myc :: XA21 <地塞米松。使用TRIzol使用标准方案提取RNA;用于定量PCR的cDNA用M-MLV逆转录酶转录;并在PCR仪上用SsoFastEvaGreenSupermix进行定量PCR。使用水稻泛素基因(LOC_Os06g46770)作为内部对照来标准化Xa21的基因表达,并且在Ubi :: Myc :: XA21 植物中表达水平作为 XA21 )设置为1.0

图3.地塞米松处理抑制pTA7002 :: Myc :: XA21植物中损伤长度的延长。六周大的水稻植株用Xoo >。在接种后6天(6DAI),在将地塞米松(DEX)施用于植物之前测量疾病进展(红线)。直到6个DAI,pTA7002 :: Myc :: XA21 植物不表达Xa21,并且观察到的DEX和没有DEX处理的植物之间的疾病发展变化不具有统计学显着性。使疾病进展持续到接种后12天(12DAI,DEX处理后6天),并如所示测量病变长度(蓝线)。与非地塞米松处理的植物相比,用地塞米松处理的pTA7002 :: Myc :: XA21 植物显示出显着更少的疾病进展,表明DEX处理成功诱导Xa21的表达, XA21 - 介导的免疫被触发。

笔记

  1. 在没有激素的情况下的"泄漏"表达先前已经在化学诱导系统中报道(Padidam,2003; Zuo和Chua,2000)。在我们的系统中,约一半的独立转化的水稻品系表现出弱或中度渗漏表达(Park等人,2012)。因此,确定显示最低泄漏表达同时保持高度诱导表达的转基因株系是至关重要的。为了确认 Xa21 的非渗漏表达,在没有DEX的情况下,所选择的行应该完全易受 Xoo 的影响,并且在DEX之后对 Xoo 应用。另外,在DEX处理之前,通过定量RT-PCR和western印迹分析,Xa21基因表达和蛋白质丰度应当低或不可检测,并且应该通过DEX应用诱导(Park等人, em>,2012)。
  2. 为了确保地塞米松治疗成功,接种另外的植物并保持而不收获叶子以在接种后12至14天测量损伤。
  3. 在我们的经验中,当我们使用分开的步入式生长室用于地塞米松和非地塞米松处理时,我们观察到Kitaake(地塞米松处理的)和地塞米松诱导的XA21细胞之间的 Xoo 即使当相同的设置应用于两个步入式生长室(数据未显示)时,植物(非地塞米松诱导的)因此,我们建议将实验限制于单一生长室,其中地塞米松应用于所有植物,并且Kitaake野生型稻用作对照。
  4. 根据大学生物安全协议,将植物转移到步入式生长室中以接种Xoo。作者怀疑,接种和地塞米松的应用在温室中也应该是成功的,尽管这些条件没有被测试。
  5. 在我们的预实验中,我们将30μM地塞米松叶面施用与10μM地塞米松灌溉组合。但是,与单独的叶面应用相比,我们没有看到更多的 Xa21 表达式(数据不是 显示)。

食谱

  1. 含有头孢氨苄(1L)的蛋白胨蔗糖琼脂(PSA)固体培养基 头孢氨苄储备液的制备:将头孢氨苄水合物溶解在50%乙醇中的10mg/ml,并将2ml等分试样储存在-20℃,直到准备好准备培养基。
    将10克蛋白胨,10克蔗糖和1克L-谷氨酸溶于900毫升无菌H 2 O中。
    使用1N NaOH将培养基的pH调节至7.0,并使体积达到1L
    加入16g琼脂和高压灭菌器灭菌
    使溶液冷却至55℃,并加入2ml原料头孢氨苄至终浓度为20μg/ml。
  2. 地塞米松,含有0.01%(w/v)Tween-20(400ml)的30μM溶液
    对于两个supertubs,将0.0048g地塞米松溶解在400ml DMSO中的1.5ml eppendorf管中
    溶解后,将其加入到含有40μltween-20的400ml无菌H 2 O中。
  3. 温室水稻生长条件
    温室(26-28℃光,18-20℃黑暗,40-60%湿度) 从4月到9月,自然光线
    从10月到3月,从上午6点到晚上10点(14小时光照和10小时黑暗光周期),补充了1000W金属卤化物灯泡的光
  4. 步入式生长室水稻生长条件
    步入式生长室(14小时光照,28℃,80%湿度; 10小时黑暗,24℃,85%湿度)
  5. 受精水成分
    受精水保持在EC:0.8-1
    当以1:200:
    注射时PPM的理论分解
    N
    173.58
    P
    45.39
    K
    165.64
    Ca
    60.05
    Mg
    38.88
    S
    146.42
    Fe
    5.23
    Cu
    0.18
    B
    0.4
    Mn
    1.20
    Mo
    0.05
    Zn
    0.37

致谢

该协议由Park等人修改((2012)。
这项研究由国家卫生研究所(NIH,GM55962),国家科学基金会(NSF,IOS-0817738)和孟山都的Beachell-Borlaug国际学者计划支持。

参考文献

  1. Aoyama,T。和Chua,N.H。(1997)。 转基因植物中糖皮质激素介导的转录诱导系统。植物J/em> 11(3):605-612。
  2. Padidam,M。(2003)。 植物中的化学调节基因表达。Curr Opin Plant Biol 6(2):169-177。
  3. Park,C.J.,Canlas,P.E.and Ronald,P.C。(2012)。 建立糖皮质激素介导的水稻转录诱导XA21 模式识别受体。 55(1):43-49
  4. Park,C.J.,Lee,S.W.,Chern,M.,Sharma,R.,Canlas,P.E.,Song,M.Y.,Jeon,J.S.and Ronald, 稻的异位表达 Xa21 克服了发育上受控的对黄单胞菌的抗性米曲霉oryzae 。 Plant Sci 179(5):466-471。
  5. Song,WY,Wang,GL,Chen,LL,Kim,HS,Pi,LY,Holsten,T.,Gardner,J.,Wang,B.,Zhai,WX,Zhu,LH,Fauquet, P.(1995)。 由水稻抗病基因编码的受体激酶样蛋白, Xa21 。 Science 270(5243):1804-1806。
  6. Zuo,J。和Chua,N.H。(2000)。 化学诱导系统,用于调控植物基因的表达。 Curr Opin Biotechnol 11(2):146-151。

附录

  1. Myc :: Xa21 的核苷酸序列。下划线区域定义N末端Myc标签。如先前所述,将Myc标签插入推定的信号肽(结构域A)之后的Xa21基因的结构域B中(Park等, ,2010)。
    atgatatcactcccattattgctcttcgtcctgttgttctctgcgctgctgctctgcccttcaagcagtgacgacgatggtgatgctgccggcgacgaactcgcgctgctctctttcaagtcatccctgctataccaggggggccagtcgctggcatcttggaacacgtccggccacggccagcactgcacatgggtgggtgttgtgtgcggccgccgccgccgccggcacccacacagggtg GAGCAAAAGCTGAACCCACAACACACGCCGGCGGCGGCGGCGGCCGTGGGTGTGTCCCACCTCGTTTTCGACTTTTCTGAGGAGGATCTGCTGGAGAAGCACAGGCTG gtgaagctgctgctgcgctcctccaacctgtccgggatcatctcgccgtcgctcggcaacctgtccttcctcagggagctggacctcggcgacaactacctctccggcgagataccaccggagctcagccgtctcagcaggcttcagctgctggagctgagcgataactccatccaagggagcatccccgcggccattggagcatgcaccaagttgacatcgctagacctcagccacaaccaactgcgaggtatgatcccacgtgagattggtgccagcttgaaacatctctcgaatttgtacctttacaaaaatggtttgtcaggagagattccatccgctttgggcaatctcactagcctccaggagtttgatttgagcttcaacagattatcaggagctataccttcatcactggggcagctcagcagtctattgactatgaatttgggacagaacaatctaagtgggatgatccccaattctatctggaacctttcgtctctaagagcgtttagtgtcagagaaaacaagctaggtggtatgatccctacaaatgcattcaaaacccttcacctcctcgaggtgatagatatgggcactaaccgtttccatggcaaaatccctgcctcagttgctaatgcttctcatttgacagtgattcagatttatggcaacttgttcagtggaattatcacctcggggtttggaaggttaagaaatctcacagaactgtatctctggagaaatttgtttcaaactagagaacaagatgattgggggttcatttctgacctaacaaattgctccaaattacaaacattgaacttgggagaaaataacctggggggagttcttcctaattcgttttccaatctttccacttcgcttagttttcttgcacttgaattgaataagatcacaggaagcattccgaaggatattggcaatcttattggcttacaacatctctatctctgcaacaacaatttcagagggtctcttccatcatcgttgggcaggcttaaaaacttaggcattctactcgcctacgaaaacaacttgagcggttcgatcccgttggccataggaaatcttactgaacttaatatcttactgctcggcaccaacaaattcagtggttggataccatacacactctcaaacctcacaaacttgttgtcattaggcctttcaactaataaccttagtggtccaatacccagtgaattattcaatattcaaacactatcaataatgatcaatgtatcaaaaaataacttggagggatcaataccacaagaaatagggcatctcaaaaatctagtagaatttcatgcagaatcgaatagattatcaggtaaaatccctaacacgcttggtgattgccagctcttacggtatctttatctgcaaaataatttgttatctggtagcatcccatcagccttgggtcagctgaaaggtctcgaaactcttgatctctcaagcaacaatttgtcaggccagatacccacatccttagcagatattactatgcttcattccttgaacctttctttcaacagctttgtgggggaagtgccaaccattggtgctttcgcagctgcatccgggatctcaatccaaggcaatgccaaactctgtggtggaatacctgatctacatctgcctcgatgttgtccattactagagaacagaaaacatttcccagttctacctatttctgtttctctggccgcagcactggccatcctctcatcactctacttgcttataacctggcacaagagaactaaaaagggagccccttcaagaacttccatgaaaggccacccattggtctcttattcgcagttggtaaaagcaacagatggtttcgcgccgaccaatttgttgggttctggatcatttggctcagtatacaaaggaaagcttaatatccaagatcatgttgcagtgaaggtactaaagcttgaaaatcctaaggcgctcaagagtttcactgccgaatgtgaagcactacgaaatatgcgacatcgaaatcttgtcaagatagttacaatttgctcgagcattgataacagagggaacgatttcaaagcaattgtgtatgacttcatgcccaacggcagtctggaagattggatacaccctgaaacaaatgatcaagcagaccagaggcacttgaatctgcatcgaagagtgaccatactacttgatgttgcctgcgcactggactatcttcaccgccatggccctgaacctgttgtacactgtgatattaaatcaagcaatgtgctgttagattctgatatggtagcccatgttggagattttgggcttgcaagaatacttgttgatgggacctcattgatacaacagtcaacaagctcgatgggatttatagggacaattggctatgcagcaccagagtatggcgttgggctcattgcatcaacgcatggagatatttacagctatgg aattctagtgctggaaatagtaaccgggaagcggccaactgacagtacattcagacccgatttgggcctccgtcagtacgttgaactgggcctacatggcagagtgacggatgttgttgacacgaagctcattttggattctgagaactggctgaacagtacaaataattctccatgtagaagaatcactgaatgcattgtttggctgcttagacttgggttgtcttgctctcaggaattgccatcgagtagaacgccaaccggagatatcatcgacgaactgaatgccatcaaacagaatctctccggattgtttccagtgtgtgaaggtgggagccttgaattctga
  2. pTA7002 :: Myc :: Xa21 构建地图。
    SP信号肽,RB右边界,35花椰菜花叶病毒35S启动子,GVG GAL4结合结构域,VP16激活结构域 - 糖皮质激素受体融合物,E9聚(A)核糖核酸双磷酸羧化酶小亚基(rbcS-E9),NOS-P胭脂碱合酶启动子,NOS-T胭脂碱合酶终止子的加成序列; 6×UAS 6个拷贝的GAL4上游激活序列和 35 S启动子的-46至+1区,豌豆rbcS-3A,LB左边界的3A聚(A)添加序列。


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引用:Caddell, D. F., Wei, T., Park, C. and Ronald, P. C. (2015). The Use of a Dexamethasone-inducible System to Synchronize Xa21 Expression to Study Rice Immunity. Bio-protocol 5(9): e1468. DOI: 10.21769/BioProtoc.1468.
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