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Sep 2019

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Quantification of Protein Enrichment at Plasmodesmata
胞间连丝富集蛋白的量化分析   

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Abstract

Intercellular communication plays a crucial role in the establishment of multicellular organisms by organizing and coordinating growth, development and defence responses. In plants, cell-to-cell communication takes place through nanometric membrane channels called plasmodesmata (PD). Understanding how PD dictate cellular connectivity greatly depends on a comprehensive knowledge of the molecular composition and the functional characterization of PD components. While proteomic and genetic approaches have been crucial to identify PD-associated proteins, in vivo fluorescence microscopy combined with fluorescent protein tagging is equally crucial to visualise the subcellular localisation of a protein of interest and gain knowledge about their dynamic behaviour. In this protocol we describe in detail a robust method for quantifying the degree of association of a given protein with PD, through ratiometric fluorescent intensity using confocal microscopy. Although developed for N. benthamiana and Arabidopsis, this protocol can be adapted to other plant species.

Keywords: Plasmodesmata (胞间连丝), PD Index (PD指数), Protein enrichment (蛋白质富集), Confocal data analysis (激光共聚焦数据分析), Confocal microscopy (激光共聚焦显微镜)

Background

Currently, confirmation of protein localization to PD by confocal imaging is based primarily on two different approaches. On the one hand, the molecular composition of the cell wall surrounding PD differs. While the cell wall is highly enriched in cellulose, the environment near the PD is enriched in callose, a beta (1-3) glucan polymer that can easily and specifically be stained with fluorophore Aniline Blue. This staining presents the considerable advantage of being used as a PD marker without crossed lines. On the other hand, proteomic studies have identified specific PD proteins and led to their characterization (Faulkner et al., 2005; Fernandez-Calvino et al., 2011; Grison et al., 2015; Brault et al., 2019). These proteins can be used as PD markers in subsequent studies when tagged with fluorescent proteins in transient or stable expression in plants (Thomas et al., 2008; Simpson et al., 2009). Note that PD proteins can be exclusively associated with PD but can also present a dual localization within different cellular compartments as for Synaptotagmin1 (SYT1), Multiple C2 domains and Transmembrane regions Protein 4 (MCTP4) which associate with both the Endoplasmic Reticulum and PD (Levy et al., 2015; Brault et al., 2019). Since PD are dynamic structures responding to developmental and environmental cues, their molecular constituents may vary conditionally (Benitez-Alfonso et al., 2013; Stahl et al., 2013; Han et al., 2014; Sager and Lee, 2014; Otero et al., 2016; Stahl and Faulkner, 2016). Thus, Receptor Like Kinases (RLKs), such as the Leucine-Rich-Repeat RLKs Qian Shou Kinase 1 (QSK1) and Inflorescence Meristem Kinase 2 (IMK2) or the Cystein-Rich Receptor Kinase 2 (CRK2), are able to dynamically relocate to PD upon osmotic stress conditions (Grison et al., 2019; Hunter et al., 2019). The study of the dynamic localization of protein in vivo requires the development of quantification methodologies. Using confocal microscopy, we developed a ratiometric calculations to evaluate the PD enrichment of a given protein using PD markers, hereinafter referred as “PD Index”. The PD Index can be used for co-localization experiments but also as reference points for characterizing mutants, drugs or growing conditions that could modify the degree of proteins PD association (Perraki et al., 2018; Brault et al., 2019; Grison et al., 2019).

Materials and Reagents

  1. Syringe without needle (1 ml, Dutscher, catalog number: 8SS01H1 )
  2. Razorblade (19 x 38 x 0.27 mm, Dutscher, catalog number: 320529 )
  3. Slides (76 x 26 mm, Dutscher, catalog number: 0 6962 )
  4. Coverslips (22 x 32 mm, Dutscher, catalog number: 100034M )
  5. Tweezer (Pince à écharde forme pointue, Dutscher, catalog number: 711197 )
  6. Plant material (see Procedure A)
  7. Aniline Blue (Biosupplies Australia, catalog number: 100.1 ), storage at 4 °C
    Note: The Aniline Blue stock solution is 1 mg/ml in water. The Aniline Blue working solution is 0.025 mg/ml. Do not use higher concentration of aniline bleu when the protein of interest is GFP tagged otherwise the Aniline Blue signal will crosstalk with the GFP signal.
  8. Distilled water
  9. Luria and Bertani medium (LB broth Miller, Sigma-Aldrich, catalog number: L3152 )
  10. Sucrose (Sigma-Aldrich, catalog number: S7903 )

Equipment

  1. Confocal Microscope: plant imaging was performed using a ZEISS microscope (ZEISS, model: LSM880 )
  2. 28 °C Shaking Incubator (Dutscher, MaxQ 4000, catalog number: 0 78381 )
  3. Centrifuge (Dutscher, Spectrafuge 6 C for 10 ml tubes, catalog number: 0 96610 )

Software

  1. FIJI (https://imagej.net/Fiji) (Schindelin et al., 2012)
  2. R (https://www.r-project.org) (R Core Development Team, 2015)

Procedure

  1. Plant material preparation
    In Arabidopsis seedlings
    1. Aniline Blue staining
      This method allows PD staining in the cotyledon and the hypocotyl, the aniline blue staining in roots gives a resolution and staining efficiency that we find difficult to combine with PD Index calculation.
      Note: For roots, we advise the users to do immunolocalization in whole mount roots using monoclonal antibody: Biosupplies Australia, (1-3)-beta-glucan-directed monoclonal, catalogue number 400-2 and the protocol described in Boutté and Grebe, 2014.
      1. Grow Arabidopsis seedlings during 4 to 6 days on ½ MS 1% sucrose agar plate under 16/8 h day/night photoperiodic condition (150 μE/m2/s, 22 °C).
      2. Take 0.2 ml of Aniline blue solution at a concentration of 0.025 mg/ml in water with the 1 ml syringe.
        Note: Abiotic or biotic stress conditions can be tested to compare the PD Index of the protein of interest, in that case the aniline blue can be diluted in water supplemented with different molecules (such as NaCl, Mannitol, …) or can be apply before the aniline blue staining (such as viral infection where the plant is infected few days before aniline blue infiltration). Note that leaves or seedlings cannot be infiltrated twice.
      3. Remove the air from the syringe.
      4. Carefully take the seedling with tweezers.
      5. Gently push the seedling into the syringe, so that the aerial parts of the seedling (i.e., the cotyledons and hypocotyl) are immersed in the solution (Figures 1A-1D). The seedling’s root should protrude from the syringe in order to be able to extract the seedling from the syringe after infiltration.
      6. Position your finger at the extremity of the syringe and slowly pull the piston out of the syringe and reach the 0.21 graduation, bubbles should appear at the surface of the cotyledons (Figures 1E-1H).
      7. Count to 5 and then release the piston very slowly (Figures 1I).
        Note: We recommended the users to be extremely delicate and gentle at both steps (f and g) otherwise the cells will explode under the pressure and it will lead to a general blurry blue coloration.
      8. Carefully remove the seedling from the syringe by grasping the root of it with tweezers (Figures 1J-1K).
      9. Place the seedling on a slide in a drop of water, and gently mount the coverslip.
      10. Immediately proceed to the acquisition under the confocal microscope.
        Note: Aniline Blue bleaches rapidly. Direct observation should be done with a low power of the mercury lamp to avoid the bleaching of the sample. In confocal mode, please use a 405 nm laser power as low as possible for the same reason. With the ZEISS 880 confocal device, the laser power is generally set around 0.2 to 5%, but of course this may vary depending on the microscope used. Aniline blue staining can be visualised immediately and is stable during 10 to 15 min.


        Figure 1. Arabidopsis seedling infiltration. Illustration of the different steps of an Arabidopsis seedling infiltration using a 1 ml syringe.

    2. Using PD proteins as PD marker
      In addition to aniline blue, fluorescently tagged PD proteins such as Plasmodesmata Located Protein 1 (PDLP1) or Plasmodesmata Callose Binding Protein 1 (PDCB1) can be used as PD markers for the PD Index calculation both in transient and stable expression (Thomas et al., 2008; Simpson et al., 2009). However, we highly recommend crossing the Arabidosis lines expressing the protein of interest with the available Arabidopsis lines expressing a fluorescent tagged PD marker protein.
      Notes:
      1. Over expression of PD associated proteins can lead to callose deposition at PD.
      2. Transient expression of fluorescent tagged PD protein markers in Arabidopsis seedlings may be used but the efficiency rate of transformation is low. We do not recommend this method.

    In Nicotiana benthamiana
    1. Aniline Blue staining
      1. Three days before imaging, infiltrate Nicotiana benthamiana leaves only with agrobacteria previously electroporated with the relevant binary plasmid of the protein of interest (as described in Step A1a of In Arabidopsis seedlings).
      2. Take a small volume, around 0.2 ml, of aniline blue at a concentration of 0.025 mg/ml in water in a 1 ml syringe.
      3. Gently return the leaf to show its abaxial side.
      4. Apply the syringe on the leaf and position your finger at the same location on the other side in order to block the syringe (Figures 2A-2B).
        Note: Avoid infiltration in veins area of the leaf, the area to infiltrate should be as flat as possible. Also avoid the puncture site from the previous Agrobacterium infiltration.
      5. Apply a small pressure on the leaf with the syringe and push slowly the piston in order to infiltrate (Figures 2C-2F).
        Note: When the liquid penetrates and migrates in the leaf, a darker area should appear and expend around the syringe. If the pressure applied is correct, the infiltration should be smooth, without resistance nor loss of liquid, and without wounding the surface of the leaf.
      6. Do not infiltrate the whole leaf, a small area of 1 cm2 is sufficient.
      7. With a sharp razor blade, cut the infiltrated area of the leaf.
      8. Place the sample on a mounting slide, abaxial side of the leaf facing up.
      9. Add a drop of water on the sample.
      10. Cover it with the coverslip.
      11. Immediately proceed to the acquisition under the confocal microscope.


        Figure 2. Nicotiana benthamiana leave infiltration. Illustration of the different steps (A-F) of Nicotiana benthamiana leaf infiltration using a 1 ml syringe.

    2. Using PD proteins as PD marker
      1. Grow Agrobacterium previously electroporated with the relevant binary plasmids of the protein of interest and of the PD marker (Table 1) in liquid Luria and Bertani medium with appropriate antibiotics, at 28 °C and 250 rpm, for 1 day.
      2. Perform a 1/10 dilution of each culture and grow again at 28 °C and 250 rpm until the culture reach an OD600 of about 0.8
      3. Centrifuge the culture at 3,500 x g, discard carefully the supernatant and resuspend in water for a final OD600 of 0.3.
      4. Mix 1:1 volume of both the agrobacteria cultures transformed with the protein of interest and with the PD marker.
      5. Use 5 to 6 leaves stage plant and make a very small puncture on the abaxial side of each selected leaf. Avoid the leaf veins.
      6. Apply a 1 ml syringe containing the agrobacteria mix in water against the leaf at the puncture site
      7. Position your finger on the other side to block the syringe and gently push the piston while applying a small pressure on the leaf so the liquid can infiltrate. Here, it is advantageous to infiltrate a large portion of the leaf.
      8. Place the plant in the appropriate culture room for another 3 days for protein expression.
      9. Using a razor blade, cut a square of approximately 1 cm2 in the infiltrated area of the leaf.
      10. Place the sample on a mounting slide, abaxial side of the leaf facing up.
      11. Add a drop of water on the sample.
      12. Cover it with the coverslip.
      13. Immediately proceed to the acquisition under the confocal microscope.

  2. Confocal acquisition
    1. The use of a water immersion objective 63x (NA ≥1.4) for observation is recommended to have the same refraction index between immersion and mounting. If water immersion objective is not available, the use of an oil-immersion objective is still possible.
    2. The pinhole value needs to be kept at airy 1 to ensure a focal plane as accurate as possible.
    3. The excitation wavelength and the spectral acquisition windows should be adjusted according to the fluorescent proteins chosen in your experiment (Table 1).
    4. Laser power, photomultiplier (PMT) and photomultiplier offset should be set such that the acquired signals are not saturated. Between experiments and in a same experiment the setting parameters of the PD marker can be modified to obtain the better signal as possible without bleaching. In a same experiment the fluorescent tagged protein of interest channel setting must be kept identical while comparing different mutants or conditions. Between experiments we also recommend keeping the same settings for the fluorescently-tagged protein of interest. However, the PD Index is a ratiometric calculation between Region Of Interest (ROI) in a same picture so changes on the fluorescent protein acquisition setting may be acceptable if really needed.
    5. Line average can be applied during image acquisition, the same line average should be kept in all experiments.
    6. During image acquisition, the sequential scanning is preferable compared to simultaneous scanning. It is important to control that the excitation wavelength used for one fluorescent protein does not excite the other one, i.e., ensure there is no crosstalk between the PD marker/Aniline blue and the tag of the protein of interest (Table 1).
      Notes:
      1. The Aniline Blue stock solution is 1 mg/ml in water. The Aniline Blue working solution is 0.025 mg/ml. Do not use higher concentration of aniline bleu when the protein of interest is GFP tagged otherwise the Aniline Blue signal will crosstalk with the GFP signal.
      2. To avoid crosstalk of fluorescence between channels, it is necessary to perform independent and combined excitation and detection at the excitation and emission wavelength of the individual fluorochrome or fluorescent protein, respectively. Control samples labeled only with a single fluorescent protein/PD marker should be prepared in order to verify that the laser excitation wavelength used to excite one fluorochrome does not excite the other.

      Table 1. Fluorescence excitation and emission maximum of Aniline Blue and commonly used fluorescent proteins (FP: Fluorescent Protein or Dye; Ex: Excitation wavelength in nm; Em: Emission wavelength in nm)

Data analysis

  1. Data collection using Fiji
    1. Open the confocal image with Fiji. Do not split channels.
    2. Open ROI Manager (Analyze > Tools > ROI Manager).
    3. Use the circle selection tool from main panel and draw a circle on the PD marker channel that is slightly smaller than the size of the PD marker signal (Figure 3A).
      Note: Only sharp callose signal should be selected as PD ROI. Do not select ROI either for PD ROI and for PM ROI when the “spot” in the aniline blue channel is blurry.
    4. Click “add” on the ROI Manager once the circle is well positioned and selected. A new line should appear in the ROI Manager.
    5. Move the circle, without modifying it, to another signal and click “add” again.
    6. Repeat Step A4 until you have enough ROI (ROI; best between 5 and 10).
    7. Change the view to the other channel (the protein of interest fluorescence channel).
    8. Using the same circle resume from step A5 and add regions that do not overlap with the AB signals but are at the cell periphery (Figure 3B).
    9. Verify the measurement parameters by going to Analyze > Set parameters. Tick the “Mean gray value” box.
    10. Verify that the channel is still on the protein of interest. Then, on the ROI Manager, click on “Measure”. A new window appears with the values.
    11. Select and copy the values.


      Figure 3. ROI selection for PD Index calculation. A. The PD ROI are selected in the PD marker channel (white arrows). B. The PM ROI are selected in the protein of interest fluorescence channel (red arrows). Verify that the ROI selected in the protein of interest fluorescence channel do not correspond to a PD in the PD marker channel.

  2. PD index calculation using Excel
    1. On an Excel sheet, paste the values copied from Fiji (ImageJ)
    2. Calculate the mean of the values from “at PD” ROIs and the mean of the values from “outside PD” ROIs
    3. Calculate the PD index by doing “At PD ROI mean value/Outside PD ROI mean value”. A PD Index value below or equal to 1 means that there is no specific enrichment nor accumulation of the protein of interest at PD, whereas a PD Index value above 1 is significant from enrichment of a protein at PD.
    Note: For example, a PD resident protein like At-PDCB1, which is also located at the plasma membrane, present a PD Index of 1.45 when transiently expressed in N. benthamiana. QSK1 and IMK2, two PM associated LRR-RLKs which conditionally relocalize to PD, display a PD Index rising from 0.9 to 1 in control condition to 1.5 to 2 upon abiotic stress (Grison et al., 2019). It is also important to note that the system of expression may sorely influence the PD Index value. Illustrating this, At-MCTP4 display a PD Index of 1.85 when transiently expressed in N. benthamiana, whereas the PD Index raised to 5 when stably expressed in Arabidopsis (Brault et al., 2019).
      For statistical analysis the use of R software and the Rcmdr package is recommended. Parametrical tests can be use only when n ≥ 20 and when the sample distribution respect the normal law. Non-parametrical tests are systematically used when n < 20.

Acknowledgments

J.D.P. is funded by a PhD fellowship from the Belgian “Formation à la Recherche dans l'Industrie et l'Agriculture” (FRIA grant no. 1.E.096.18).
  This work was supported by the National Agency for Research (Grant ANR-14-CE19-0006-01 to E.M.B), “Osez l’interdisciplinarité” OSEZ-2017-BBRIDGING CNRS program to E.M.B., the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No 772103-BRIDGING to E.M.B).

Competing interests

The authors declare no competing financial interests.

References

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简介

[摘要] 细胞间通讯通过组织和协调生长,发育和防御反应,在建立多细胞生物中起着至关重要的作用。在植物中,细胞之间的通信通过称为胞质膜(PD)的纳米膜通道进行。了解PD如何决定细胞连通性很大程度上取决于对PD成分的分子组成和功能表征的全面了解。虽然蛋白质组学和遗传学方法对于鉴定PD相关蛋白至关重要,但在体内 荧光显微镜结合荧光蛋白标记对于可视化目标蛋白的亚细胞定位并获得有关其动态行为的知识同样至关重要。在此协议中,我们详细描述了一种稳健的方法,可通过使用共聚焦显微镜通过比例荧光强度来定量定量给定蛋白质与PD的结合程度。虽然是为N 开发的。 本塔米亚纳和拟南芥,该协议可以适应其他植物物种。

[背景 ] 目前,蛋白质定位到PD通过共焦成像的确认主要基于两种不同的方法。一方面,PD周围细胞壁的分子组成不同。虽然细胞壁高度富含纤维素,但PD附近的环境中富含call质(一种β (1-3)g 卢康聚合物),可以轻松且特异地用荧光团苯胺蓝染色。这种染色显示出用作没有交叉线的PD标记的显着优势。另一方面,蛋白质组学研究已经鉴定出特定的PD蛋白并对其进行了表征(Faulkner 等人,2005; Fernandez-Calvino 等人,2011; Grison 等人,2015; Brault 等人,2019)。当用荧光蛋白标记植物中的瞬时或稳定表达时,这些蛋白可用作后续研究中的PD标记(Thomas 等,2008; Simpson 等,2009 )。请注意,PD蛋白可以与PD排他性地结合在一起,也可以在不同的细胞区中出现双重定位,如突触结合蛋白1(SYT1),多个C2域和跨膜区蛋白4(MCTP4),它们与内质网和PD (Levy 等,2015; Brault 等,2019 )。由于PD是响应发育和环境线索的动态结构,因此其分子组成可能有条件地发生变化(Benitez-Alfonso 等人,2013; Stahl 等人,2013; Han 等人,2014; Sager和Lee,2014; Otero 等人等人,2016年;Stahl和Faulkner,2016年)。因此,类似亮氨酸重复的RLK的千寿激酶1(QSK1)和花序分生组织的激酶2(IMK2)或半胱氨酸富集的受体激酶2(CRK2)等受体样激酶(RLK)能够动态重定位。在渗透胁迫条件下对PD的抑制(Grison 等,2019; Hunter 等,2019)。体内蛋白质动态定位的研究需要定量方法的发展。使用共聚焦显微镜,我们开发了一种比例计算,以使用PD标记物(以下称为“ PD指数”)评估给定蛋白质的PD富集度。该PD指数可用于共定位实验也可作为参考点用于表征突变体,药物或生长,可以修饰蛋白质PD关联程度的条件(Perraki 等人,2018; Brault 等人,2019; 的Grison 等等。,2019)。

关键字:胞间连丝, PD指数, 蛋白质富集, 激光共聚焦数据分析, 激光共聚焦显微镜

材料和试剂


 


小号yringe无针(1毫升,Dutscher ,目录号:8SS01H1)
剃刀(19 x 38 x 0.27 mm,Dutscher ,目录号:320529)
滑轨(76 x 26 mm,Dutscher ,目录号:06962 )
盖玻片(22 x 32 mm,Dutscher ,目录号:100034M)
镊子(P inceàéchardeforme pointue,Dutscher,目录号:711197)
植物材料(请参阅步骤A)
苯胺蓝(澳大利亚生物材料公司,目录号:100.1),在4°C下储存
注意:苯胺蓝储备溶液在水中为1 mg / ml。苯胺蓝工作溶液为0.025 mg / ml。当目标蛋白被GFP标记时,请勿使用更高浓度的苯胺蓝,否则苯胺蓝信号将与GFP信号串扰。


蒸馏水
Luria和Bertani培养基(LB brot h Miller,Sigma -Aldrich ,目录号:L3152)
蔗糖(Sigma -Aldrich ,目录号:S7903)
 


设备


 


共聚焦显微镜:使用蔡司显微镜(蔡司,型号:LSM880)进行植物成像
28°C摇动培养箱(Dutscher ,MaxQ 4000,货号:078381)
离心机(d utscher ,Spectrafuge 6下10毫升管,目录号:096610)
 


软件


 


斐济(https://imagej.net/Fiji)(Schindelin 等人。,20 12 )
R(https://www.r-project.org)(R核心开发团队,2015 )




程序


 


植物材料准备
在拟南芥幼苗中


苯胺蓝染色
这种方法可以使子叶和下胚轴上的PD染色,而根中的苯胺蓝染色则提供了分辨率和染色效率,我们发现很难与PD Index计算结合使用。


注意:对于根,我们建议用户使用单克隆抗体在整个根上进行免疫定位:澳大利亚生物材料公司(1-3)-β- 葡聚糖定向单克隆抗体,目录号400-2,以及Boutté 和Grebe中所述的方案, 2014。


在16/8小时的日/夜光周期条件下,在½MS 1%蔗糖琼脂平板上生长拟南芥幼苗4至6天(150  μ ë /米2 /秒,22 ℃)。
采取0.2毫升的苯胺蓝溶液以0.025的浓度毫克/毫升在水中与1ml注射器。
注意:可以测试非生物或生物应激条件以比较目标蛋白质的PD指数,在这种情况下,可以将苯胺蓝稀释在补充有不同分子(例如NaCl,甘露醇等)的水中,也可以在应用之前苯胺蓝染色(例如病毒感染,即在苯胺蓝渗透前几天被植物感染)。请注意,叶片或幼苗不能渗透两次。


从注射器中排出空气。
用镊子小心地取苗。
将幼苗轻轻推入注射器中,使幼苗的空中部分(即子叶和下胚轴)浸入溶液中(图1A-1D)。幼苗的根部应从注射器突出,以便能够在渗透后从注射器中拔出幼苗。
将手指放在注射器的末端,然后慢慢将活塞从注射器中拉出并达到0.21刻度,子叶表面会出现气泡(图1E-1H)。
数到5,然后非常缓慢地释放活塞(图1I)。
无德:我们推荐用户是在极其细腻与温柔两个步骤S(f和g) ,否则细胞就会发生爆炸的压力下,它会导致一般的模糊蓝色着色。


小心通过抓住袋鼠除去从注射器幼苗吨的它与镊子(图1J-1K)。
将幼苗放在一滴水滴的幻灯片上,然后轻轻地盖上盖玻片。
立即在共聚焦显微镜下进行采集。
注意:苯胺蓝快速漂白。应使用低功率的水银灯进行直接观察,以避免样品漂白。在共焦模式下,出于相同的原因,请使用尽可能低的405 nm激光功率。使用ZEISS 880共聚焦设备,激光功率通常设置为0.2%到5%,但是当然这可能会因所使用的显微镜而异。苯胺蓝染色可以立即显现,并且在10到15分钟内稳定。


 






图1。拟南芥幼苗浸润。使用1 ml注射器进行拟南芥幼苗浸润的不同步骤的图示。


 


使用PD蛋白作为PD标记
除苯胺蓝外,荧光标记的PD蛋白(如Plasmodesmata定位蛋白1(PDLP1)或Plasmodesmata ose 糖结合蛋白1(PDCB1))可用作PD指数的PD标记,可瞬时和稳定表达(Thomas 等。 ,2008 ;辛普森等,2009 )。然而,我们强烈建议过路的Arabidosis 表达与可用感兴趣的蛋白线拟南芥系中表达荧光标记的PD标记蛋白。


笔记:


PD相关蛋白的过度表达可导致PD处的call质沉积。
可以使用拟南芥幼苗中荧光标记的PD蛋白标记的瞬时表达,但转化效率较低。我们不建议使用此方法。
 


在烟草本塞姆氏


苯胺蓝染色
三成像前几天,渗入烟草本塞姆氏叶只用农杆菌预先用感兴趣的蛋白质的有关二元质粒电穿孔(如在步骤中描述阿1 一个的在拟南芥幼苗)。
在1毫升注射器中以0.025毫克/毫升的浓度取少量约0.2 毫升的苯胺蓝。
轻轻地将叶子退回以显示其背面。
将注射器放在叶子上,然后将手指放在另一侧的同一位置,以阻塞注射器(图2A-2B)。
注意:避免在叶片的叶脉区域渗透,要渗透的区域应尽可能平坦。还应避免先前农杆菌浸润的穿刺部位。


用注射器在叶片上施加较小的压力,并缓慢推动活塞以使其渗入(图2C-2F)。
注意:当液体渗透并在叶片中迁移时,应该出现较暗的区域,并在注射器周围扩展。如果施加的压力正确,则渗透应该是平滑的,没有阻力,也不会损失液体,也不会伤及叶片表面。


不要渗入整片叶子,只要1 cm 2的小面积即可。
用锋利的剃刀刀片切开叶子的浸润区域。
将样品放在安装滑片上,叶片的背面朝上。
在样品上加一滴水。
用盖玻片盖上它。
立即在共聚焦显微镜下进行采集。
 






图2. 烟草本塞姆氏假浸润。的不同步骤说明(AF)的烟草本塞姆氏使用1ml注射器叶的渗透。


 


使用PD蛋白作为PD标记
生长的农杆菌事先在28°C和250 rpm的液体Luria和Bertani培养基中与合适的抗生素一起电穿孔了感兴趣的蛋白质和PD标记物的相关二元质粒(表1),持续1天。
对每种培养物进行1/10稀释,然后在28°C和250 rpm下再次生长,直到培养物的OD 600 达到约0.8
以3500 xg 离心培养物,小心丢弃上清液,重悬于水中,最终OD 600 为0.3。
将用目标蛋白和PD标记物转化的两种农杆菌培养物按1:1体积混合。
使用5到6片叶期植物,并在每片选定叶片的背面刺破一个很小的孔。避免叶脉。
在穿刺部位将1 ml装有农杆菌混合物的注射器用水冲在叶子上
将手指放在另一侧以阻塞注射器,并在叶子上施加较小压力的同时轻轻推动活塞,以使液体可以渗透。在此,有利的是渗透大部分的叶子。
再将植物放在适当的培养室中3天以表达蛋白质。
用剃须刀在叶子的浸润区域切一个大约1 c m 2 的正方形。
将样品放在安装滑片上,叶片的背面朝上。
在样品上加一滴水。
用盖玻片盖上它。
立即在共聚焦显微镜下进行采集。
 


共聚焦采集
建议使用63 x (NA≥1.4)的水浸物镜进行观察,以使浸入和安装之间的折射率相同。如果水浸泡的目的是不可用,在使用油浸物镜的仍然是可能的。
针孔值需要保持为通风1,以确保焦平面尽可能准确。
激发波长和光谱采集窗口应根据实验中选择的荧光蛋白进行调整(表1)。
应设置激光功率,光电倍增管(PMT)和光电倍增管偏移,以使采集的信号不饱和。在实验之间和同一实验中,可以修改PD标记的设置参数以获得尽可能好的信号而不会漂白。在同一实验中,比较不同的突变体或条件时,必须将荧光标记的目标通道标记的蛋白质保持相同。在实验之间,我们还建议对感兴趣的荧光标记蛋白质保持相同的设置。然而,PD指数是一个比例区之间的计算中在相同的画面(ROI)利息等荧光蛋白获取设定变更是可以接受的,如果确实需要。
线平均可以在图像采集期间应用,所有实验中都应保持相同的线平均。
在图像获取期间,与同时扫描相比,顺序扫描更为可取。重要的是要控制用于一种荧光蛋白的激发波长不会激发另一种荧光蛋白,即。e。确保PD标记物/苯胺蓝与目标蛋白的标记物之间没有串扰(表1)。
笔记:


苯胺蓝储备液在水中为1 mg / ml。苯胺蓝工作溶液为0.025 mg / ml。当目标蛋白被GFP标记时,请勿使用更高浓度的苯胺蓝,否则苯胺蓝信号将与GFP信号串扰。
为了避免通道之间的荧光串扰,有必要分别在单个荧光染料或荧光蛋白的激发和发射波长下进行独立和组合的激发和检测。应该准备仅用单个荧光蛋白/ PD标记物标记的对照样品,以验证用于激发一种荧光染料的激光激发波长不会激发另一种荧光染料。




表1.苯胺蓝和常用荧光蛋白的荧光激发和发射最大值(FP:荧光蛋白或染料; Ex:激发波长(nm);Em :发射波长(nm ))


FP或染料


前最大


Em最大


苯胺蓝


405


450


CFP


433


475


绿松石


433


475


微蓝藻


433


475


绿色荧光蛋白


488


507


眼动计划


514


527


金星


515


528


柠檬黄素


516


529


mRFP


584


607


mCherry


587


610


 


数据分析


 


使用斐济收集数据
使用斐济打开共聚焦图像。不要拆分通道。
打开ROI管理器(分析>工具> ROI管理器)。
使用主面板上的圆圈选择工具,并在PD标记通道上绘制一个稍小于PD标记信号大小的圆圈(图3A)。
注意:仅应选择尖锐的ose 信号作为PD ROI。当苯胺蓝色通道中的“斑点”模糊时,请勿为PD ROI和PM ROI选择ROI。


圆的位置和位置正确后,在ROI Manager上单击“添加”。新行应出现在ROI管理器中。
将圆(不对其进行修改)移动到另一个信号,然后再次单击“添加”。
重复步骤A4,直到有足够的ROI(ROI ;最好在5到10之间)。
将视图更改为另一个通道(感兴趣的蛋白质荧光通道)。
使用相同的圆圈从步骤A5 恢复并添加不与AB信号重叠但是在细胞外围的区域(图3B)。
通过转到分析>设置参数来验证测量参数。勾选“平均灰度值”框。
验证通道仍在目标蛋白上。然后,在ROI管理器上,单击“测量”。将出现一个包含值的新窗口。
选择并复制值。
 






图3.用于PD指数计算的ROI选择。A.在PD标记通道中选择PD ROI(白色箭头)。B.在感兴趣的蛋白质荧光通道(红色箭头)中选择PM ROI。验证在目标蛋白荧光通道中选择的ROI是否不对应于PD标记通道中的PD。


 


使用Excel计算PD指数
在Excel工作表上,粘贴从斐济(ImageJ)复制的值
计算“ PD处” ROI的平均值和“ PD处” ROI的平均值
通过执行“达到PD ROI平均值/ 外部PD ROI平均值”来计算PD指数。小于或等于1的PD指数值表示在PD处没有目标蛋白质的特异性富集或积累,而大于1的PD指数值对PD处的蛋白质富集意义重大。
注意:例如,PD驻留蛋白(如At-PDCB1)也位于质膜,当在本氏烟草中瞬时表达时,其PD指数为1.45 。QSK1和IMK2是两个与PM相关的LRR-RLK,它们有条件地重新定位于PD,在非生物胁迫下,PD指数从对照条件下的0.9升高到1到1.5升高到1.5 (Grison等,2019)。同样重要的是要注意,表达系统可能会严重影响PD指数值。说明这一点的是,At-MCTP4在本氏烟草中瞬时表达时显示出1.85 的PD指数,而在拟南芥中稳定表达时,PD指数上升到5 (Brault等人,2019)。


  为了进行统计分析,建议使用R软件和Rcmdr 软件包。仅当n≥20且样本分布符合正常规律时才能使用参数测试。当n <20时,系统地使用非参数检验。






致谢


 


JDP由来自比利时的“地层点菜的博士奖学金资助RECHERCHE 丹斯欧莱雅工业等L'农业”(FRIA授予没有。1.E.096.18) 。


  这项工作是由美国国家研究机构(批准ANR-14-CE19-0006-01到EMB),“支持Osez L'interdisciplinarité ” OSEZ-2017-CNRS BBRIDGING程序,EMB,根据欧洲研究委员会(ERC)欧盟的Horizon 2020研究与创新计划(授予EMB的授权协议号772103-BRIDGING )。


 


利益争夺


 


作者宣称没有任何竞争的经济利益。


 


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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Grison, M. S., Petit, J. D., Glavier, M. and Bayer, E. M. (2020). Quantification of Protein Enrichment at Plasmodesmata. Bio-protocol 10(5): e3545. DOI: 10.21769/BioProtoc.3545.
  2. Grison, M. S., Kirk, P., Brault, M. L., Wu, X. N., Schulze, W. X., Benitez-Alfonso, Y., Immel, F. and Bayer, E. M. (2019). Plasma membrane-associated receptor-like kinases relocalize to plasmodesmata in response to osmotic stress. Plant Physiol 181(1): 142-160.
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