细菌病原体向植物细胞运送水和溶质通道

　　使用KOD热启动聚合酶（Millipore Sigma）和以下引物集，从E. amylovora菌株EA273的基因组DNA中进行了DSPE基因：dspe_forward_primer：5'-at-AtggaattaAtaAtaAtaataAtcactgggaCactgeGaactgaActgaAcacacacacacacacAag -3'';DSPE_REVERSE_PRIMER：5'-GCTCTTCATTTCCCCCTTCCTTCTC-3'。将PCR产物克隆到修改后的PET28A载体（Millipore Sigma）中，作为与含有His8 TAG的麦芽糖结合蛋白（MBP）的C末端融合，预缩蛋白酶裂解位点（PPX）和以MBP – HIS8 – PPPX – Flag-Flag-flag-flag-flag-flag（MBP）的形式（ppx）（ppx）（ppx）。将质粒转化的BL21（DE3）大肠杆菌细胞在37°C的Luria – Bertani培养基中生长，直到600 nm（OD600 nm）的光密度达到0.4-0.6，然后以0.1 mM IPTG诱导，并在18°C下以18°C的生长。将收集的细胞颗粒重悬于含有20 mM HEPE（pH 7.5），300 mM NaCl和2.5％甘油的裂解缓冲液中，并补充了完全无EDTA的无EDTA蛋白酶抑制剂片（ROCHE）和DNase I，并由法国媒体裂解。在20,000 r.p.m.离心之后在4°C下进行30分钟以去除细胞碎片，使用爪龙钴树脂（Takara Bio）纯化融合蛋白。在含有20 mM HEPE（pH 7.5），300 mM KCl，2.5％甘油，1 mm ATP，5 mM MGCL2和10 mM咪唑的缓冲液中进行了大量洗涤后，将融合蛋白在含有20 mm HEPES（pH 7.5），300 mm Nacl，2.5％Glycerol和250 glycerol和250的缓冲液中洗脱。Superose 6增加了10/300 GL柱（Cytiva Life Science），该平衡液在4°C下用含有20 mM HEPE（pH 7.5），150 mM NaCl和1 mM Dithiothreitol（DTT）的缓冲液进行了平衡。将峰处的蛋白质分数等分为等分，并在-80°C下进行闪光冻干以进行存储。 　　DSPE（Δβ-桶）（Δ1278–1566+Δ1649–1813）的构建体是从上述通过融合内克隆所描述的WT MBP – DSPE构建体生成的。通过与WT蛋白相同的程序纯化突变蛋白。 　　在扩展数据中显示了代表性的大小 - 排斥色谱谱和SDS-聚丙烯酰胺凝胶电泳（PAGE）凝胶（PAGE）凝胶，在扩展数据中显示了图4G，h。 　　Constructs of MBP–DspE and MBP–DspE(Δβ-barrel) were made by the in-fusion cloning methods using the NEBuilder HiFi DNA assembly kit (New England Biolabs) with the following primer sets—WT DspE, dspE_forward_primer: 5′-GATGGAATTAAAATCACTGGGAACTGAACACAAG-3′;DSPE_REVERSE_PRIMER：5'-GAAGGAAGGGCTGGAAATGAAGAGCTAATTGATTAA-3';vector_forward_primer：5'-gagctaattgattaAtaAtaAtaAtaCctgctgctaaAcaaag-3';vector_reverse_primer：5'-TTCTGTTCCAGGGGCCCGCGCGCGCGATGGAATTAAAATC-3';DSPE（Δβ-桶）（Δ1278–1566+Δ1649–1813），DSPE forward_primer：5'-cctggacagtgcggcggcggcgcgcggcggtgaccagcagcagcaaa-3';DSPE reverse_primer：5'-aggctgcgcgcgcagcagcagcggaatagct-3';vector_forward_primer：5'-cacagcggaatagctcaggctaatccgcag-3';vector_reverse_primer：5'-gaatacgctgttgttgtcctggacagtgcgga-3'。 　　使用Leica EM GP2自动凹槽冷冻室在湿度控制的腔室中以10°C和85–90％的相对湿度进行制备冷冻EM网格。使用Pelco Easiglow Glow排放清洁系统（TED Pella），在施加样品之前，使用Pelco Easiglow Glow放电系统（TED PELLA）对自制的金量化R1.2/1.3 300英寸网格进行了发光。在样品冷冻过程中，将3μl的DSPE样品（约1.1 mg ML-1）应用于新鲜发光的电网，并在网格上孵育60秒钟，然后用Whatman＃1滤纸将2.8 s吸毒。然后立即在液态乙烷中直接漏气，并在数据采集之前存储在液氮中。 　　在装有K3 Direct Electron检测器（GATAN）的FEI Titan Krios Electron显微镜（Thermo Fisher）上，总共记录了7,810个显微照片堆栈。使用Latitude S（使用3.51.3719.0，GATAN）自动化图像采集套件，使用像素的像素大小为1.08Å至-2.4μm至-0.8μm，以×81,000的名义放大倍数（×81,000）收集显微照片堆栈。每个堆栈暴露于2.8 s，暴露时间为每帧0.047 s，每堆栈60帧。每个堆栈的总剂量约为56.3 E -Å2。 　　通过斑块运动校正模型和CryoSparc40中的斑块运动校正模型和斑块CTF估计模块进行运动校正和对比度传递函数（CTF）估计。根据CTF拟合分辨率，使用4.0Å的截止值从总共7,810张图像中选择了7,141个显微照片。使用预训练的TOPAZ41型号选择了约240万个颗粒，其中约有167,000个与具有高分辨率特征的全长蛋白相对应的167,000个颗粒以生成二维类平均水平。DSPE的冷冻EM样品显示出颗粒的严重取向偏置，这阻止了冷冻EM密度图的高分辨率重建。 　　AVRE或DSPE开放式阅读框（ORF）通过以下底漆集进行扩增 - avre正向引物：5'-TTGCCCGGCGCGCGCCACCATGCAGCAGTCACCACCATCATCGATCCACCGGA-3'（Kozak序列序列下划线）；AVRE反向引物：5'-CCTCTAGATTAGCTTCAGTTCGAACCCCTCTCT-3';DSPE正向引物：5'-TTGCCCGGCGCGCCACCATGGAATTAAATAATCACTGGGAACTG-3'（下划线的Kozak序列）；DSPE反向引物：5'-CCTCTAGATTAGCTTTCATTTCATCCCCTTCC-3'。 　　将PCR放大的AVRE或DSPE ORF（SRFI – XBAI片段）克隆到PGH19（参考42）（用XMAI和XBAI消化）中，以创建PGH-AVRE或PGH-DSPE。为了制备卵母细胞注射的CRNA，将PGH-AVRE或PGH-DSPE与NHEI线性化，然后用T7聚合酶MMESSAGE MMACHINE KIT（AMBION）在体外转录。 　　为了对DSPE进行突变分析，使用Q5站点定向的诱变试剂盒（New England Biolabs）获得了PGH-DSPE的点或缺失突变，并具有以下引物集-pgh-dspeΔβ-barrel（即，即，Δ1278–1566+∆1278-1566+∆1649-1649–1813）5'-GCGGAGCCGGTGACCAGCAACGATA-3';∆1278–1566反向引物：5'-actgtccagggaCaAcagcgtattc-3';∆1649–1813正向引物：5'-GGAATAGCTCAGGCTAATCCGCAGGG-3';∆1649–1813反向引物：5'-GCTGTGGTGCTGTCCGCAGCCTGTTGA-3';PGH-DSPEK1399E/K1401E，K1399E+K1401E正向引物：5'-CTGGAGTTTGAGTTTGAGCTGAGAGAGAGGATGAG-3'（下划线表示突变点）；K1399E+K1401E反向引物：5'-GCTGTTTTTGTGTGTTCCTTGCAGGGGT-3';PGH-DSPEL1776E/L1777E/L1778E，L1776E+L1777E+L1778E正向引物：5'-GAGGAAGAGAGGAGGGGGGAGGAGGAGGAAGCAACAGCCTG-3'（下划线表示突变点）；L1776E+L1777E+L1778E反向引物：5'-CGCTGGGGGTGGTGGTATTGAAGCCTTCGCTTTTTTT-3'。 　　卵母细胞从Xenopus1购买为卵巢。用0.55 mg ml-1胶原酶B（0.191 U mg-1）在无钙的ND96盐盐中处理卵巢43（96 mM NaCl，2 mM KCl，1 mM MMGCL，1 mM MGCL2，HEPES 5 mm HEPES和2.5 mm Na Pyruvate，pH 7.5），pH 7.5），在21 cutanting Mixer上，在21分钟上为20分钟。治疗后立即将酶溶液用ND96浴盐水从卵巢中冲洗掉（96毫米NaCl，2 mM KCl，1 mm MMGCL2，1.8 mm CaCl2，5 mm HEPES，5 mm HEPES，2.5 mm Na Pyruvate和0.5 mm Theopherline，ph 7.5 phylline，pH 7.5）塑料90次塑料材料，均为70次塑料材料。在18°C孵化器中临时存储。同一天，用细镊子手动将覆盖成熟卵母细胞（IV和V阶段）的卵泡细胞和卵泡膜用细镊子剥离，并在18°C下将卵母细胞保存在ND96浴盐水中，直到注射CRNA。在注入27.6 nl的体积时，将CRNA与二乙基氯苯甲酸盐处理的水与所需量（范围从0.01 ng至20 ng）的确定浓度混合在一起。按照制造商的指示，使用纳米注射器（纳米对象II，Drummond Scientific）进行注射。对照卵母细胞注入了二乙基碳酸盐处理的水。将卵母细胞保存在18°C的6孔塑料培养板中，以表达蛋白质。孵育溶液是对照（ND96浴盐水），ND96与抑制剂（PAMAM G0或G1，Niflumic Acid或Fipronil），ND96，具有0.0005％荧光素或0.1％GFP蛋白的ND96。 　　将卵母细胞注入2 ng的WT或突变的DSPE CRNA，并在浴ND96盐水中孵育15小时。如先前所述，使用Pierce细胞表面蛋白生物素化和隔离试剂素（Thermo Fisher）纯化表面暴露的蛋白质，并根据制造商的方案进行了一些修饰，如前所述44。每次治疗使用五个卵母细胞（用生物素，无生物素或总细胞提取物）。简而言之，将细胞在OR2 Buffer44中冲洗了3次，然后在2.5 ml OR2缓冲液中孵育10分钟，在台式轨道振动板中，在6孔培养板中使用或不使用Sulfo-NHS-SS-Biotin，均在85 R.P.M.在室温下。立即将全细胞提取物处理的卵母细胞储存在-80°C下，而生物素化的卵母细胞在Tris Buffer44中进行三次冲洗三次，然后将其置于1.5毫升管中，含有500 µL的裂解缓冲液，其中含有10 µL Halt蛋白酶蛋白酶蛋白酶蛋白质COCKETOL（TORMERETOCORCOCTTAIL）。通过穿过20尺（G）针的裂解混合物匀浆10次，然后在4°C下在蛋黄酱混合器上孵育。其余步骤遵循套件制造商的协议。将最终样品用200 µL洗脱缓冲液洗脱，并与50 µL的5×SDS样品缓冲液混合。同时，将存储用于总细胞提取物处理的五个卵母细胞在250 µL的2×SDS样品缓冲液中匀浆。为了等同的负载，将25 µL的总提取物或抗生物素 - 粉状生物素化蛋白样品添加到每个车道上以进行SDS-PAGE。 　　向卵母细胞注入0.01 ng的WT或突变体DSPE CRNA或0.1 ng的Avre CRNA。AVRE在卵母细胞中的功能较低，而不是DSPE。在沐浴ND96盐水中孵育约15小时后（96毫米NaCl，2 mM KCl，1 mm MgCl2，1.8 mm CaCl，10 mm HEPES，pH 7.5），每个卵母​​细胞都被一个伏特式摩洛西酯和一个电静脉微电极和一个电流的贝尔塞脉络脉冲0.MOHM，而在1毫升的电接地ND96记录盐水中（96毫米NaCl，2 mM KCl，1 mM MGCL2，1.8 mM CaCl，10 mm HEPES，pH 7.5）。Of note, in initial preliminary experiments, when oocytes were injected with a high amount of dspE or avrE cRNA (for example, 1 ng of dspE or 20 ng avrE cRNA per oocyte), membrane potentials at 24 h dropped close to 0 mV (Extended Data Table 1) and current conductance was very large (>50 µA), a condition at which the TEVC equipment no longer works properly.因此，我们将CRNA输入降低至每个卵母细胞0.01 ng dspe，每卵母细胞和评估时间为0.1 ng AVRE，并在注入后15小时，这产生了类似于注入水控制的卵母细胞（扩展数据表1）的静止电位，而Tevc可以记录了TEVC的最低率。 　　对于离子替换实验，通过用96 mM LICL，KCL，RBCL，CSCL，CSCL，CSCL，NDMG-CL，NDMG-CL（N-Methyl----甲基---葡萄糖苷），NAI，NAI，NABLO，NABLO3或NABRO3，MES46或NABRO3，NABRO3，MES 46，NABR，NABRO3，NABRO3，NABRO3，MES HABR，NABLO3，通过96 mM LICL，KCL，CSCL，NDMG-CL，NABR，NABR，NABLO3。电流首先记录在ND96记录缓冲区中。为了用新的阳离子或阴离子代替ND96，使用10 ml的塑料注射器用带有18克针头的10 mL塑料注射器缓慢地添加10 ml新的ND96溶液，而原始ND96则使用Vacuum Full Flof Floff Flow Flofl From Flofflow从腔室的另一端洗净了原始的ND96。玻璃微电极用1.5％的琼脂半填充，其中包含3 M KCl。电极通过氯化银线连接到卵母细胞夹具放大器（OC-725C，Warner仪器）。浴室夹具的末尾通过两条氯化银线连接到沐浴盐，在一次性聚氟乙烯18克管道中，装有1.5％琼脂，其中含有3 M KCl，用作琼脂桥。卵母细胞夹放大器通过模拟 - 数字界面（Digidata 1440a，分子设备）连接到计算机。命令电压协议和数据采集是在PCLAMP v.10.7软件套件（分子设备）中进行的。使用最大夹具增益，电流增益设置为0.1 V UA -1，将卵母细胞夹紧至所需电势。记录电压和电流的信号。在刺穿卵母细胞和夹紧卵子之前，两个电极都能够测量该卵母细胞的静息电位。将卵母细胞固定在静息电位上，并在10 mV增量中以100 ms的测试脉冲向更正面或更多的负电势。记录并分析结果电流。测试脉冲开始后10至20 ms确定电流振幅， 来自内源性卵母细胞通道的膜电容或CA依赖性Cl-电流的最小或没有重叠的时间47。随着各个卵母细胞之间的静止值变化（可能是由于每个卵母细胞的无法控制的固有差异，其大小以及电极位置和电阻的细胞调节），电压 - 电流关系数据适用于二次多项式回归（Sigmaplot 12.5 Systat软件）的互动式间隔和95％的Intermiped Interpirite Interviere和95％。这也允许从处理卵母细胞中的对照卵母细胞中的测试电位引起的电流从处理卵母细胞中减去，因此所得值仅代表DSPE-或AVRE介导的电流流过整个膜。使用Tukey测试的双向方差分析进行了电流的比较，其显着性设置为P值< 0.05. 　　Oocytes injected with 1 or 2 ng dspE or 20 ng avrE cRNA per oocyte were imaged using Motic Images Plus 3.0 software connected to a Moticam X3 camera (Motic China) on an SHR Plan Apo 1×, working distance 60, magnification lens of a stereoscope (Nikon SMZ18). At 0.01 ng dspE or 0.1 ng avrE cRNA per oocyte that was used for TEVC recordings, no baseline oocyte swelling was observed. Baseline swelling began to be observed at >每卵母细胞0.1 ng dspe或> 10 ng avre crna。对于基线卵母细胞肿胀，每次注射后立即记录起始卵母细胞图像，然后每2小时至4小时记录24小时。在整个时期，定位镜室中有或没有PAMAM抑制剂的200 MOSM的盐水中，将卵母细胞保存在18-19°C的位置。用fiji v.2.3.0软件分析了描绘五个卵母细胞（重复）的每张图片48。数据在给定的评估时间或相对于起点（CRNA注射后立即）的体积变化表示为绝对体积。对于低燃料引起的肿胀，将表达DSPE或AVRE的卵母细胞转移到五倍的ND96浴室盐水（40 MOSM）中，并立即对每20秒钟的基线肿胀进行10-20分钟或直到注射DSPE或DSPE或AVRE的卵母细胞开始爆发。数据以稀释盐水中的第一张图片的相关量显示为变化。图片还按顺序排列，以创建延时视频，显示卵母细胞肿胀和破裂。带有Tukey测试的单向方差分析用于数据集中的多次比较，其意义设置为P值< 0.05. For dataset with repeated measures over time, as in the hypoosmotic-induced swelling assay, a two-way repeated measures ANOVA with Dunnett’s test was used instead, with significance also set to a P value < 0.05. 　　Two hours after injection with 1 or 2 ng of dspE or 20 ng of avrE cRNA, oocytes were placed in ND96 bath saline with or without 5 µg ml−1 fluorescein, 1 mg ml−1 GFP and/or 5 mM PAMAM G1 inhibitor and incubated until evaluation time, as indicated in the figure legends. Oocytes were rinsed twice in ND96 bath saline and imaged as described above for oocyte swelling assay, with a few exceptions: they were imaged at a ×2 magnification with either a bright-field or GFP-B filter. In the Motic Images Plus software, the green channel gain was increased to improve green fluorescence detection. Although specific values of the green channel gain value varied across different independent assays, all configurations were kept the same across all treatments within the same experiment. Bright-field and fluorescence images of each oocyte were stacked using Fiji software and the integrated density of fluorescence was measured within oocyte boundaries and subtracted from the fluorescence values observed on the image background, so data are presented as corrected total cell fluorescence (for short: cell fluorescence). Two-way ANOVA, with Tukey’s test, was used for multiple comparisons within a dataset, with significance set to a P value < 0.05. 　　For eGFP purification, pET28-eGFP49 was transformed into E. coli Rosetta(DE3). eGFP production was induced by adding 0.25 mM IPTG to bacterial culture for 4 h at 28 °C. eGFP was purified from total cell lysate using Ni-NTA agarose beads in the extraction buffer (50 mM Tris-Cl, pH 8.0, 250 mM NaCl, 5% glycerol, 0.1 mM phenylmethylsulfonyl fluoride). Before the oocyte uptake test, the buffer was exchanged to ND96 bath saline using Amicon Ultra-4 centrifugal filter units (MilliporeSigma). 　　Five oocytes (15 mg) or 10 mg fresh plant leaf tissue was homogenized in 100 µl of 2× SDS sample buffer. After 10 min boiling, cell lysates were briefly centrifuged and 10 µl was loaded to each lane of an SDS–PAGE gel. After separation, proteins were blotted onto a PVDF membrane. AvrE, β-actin, DspE or PR1 was detected by anti-AvrE20 (1:5,000 dilution), anti-β-actin [HRP] (GenScript; 1:5,000), anti-DspE50 (1:5,000), anti-PR1 antibody (a gift from Xinnian Dong; 1:5,000), respectively, on an Invitrogen iBright 1500 system. The secondary antibodies anti-rabbit IgG (whole molecule)–alkaline phosphatase (Sigma) or anti-rabbit IgG (whole molecule)–HRP antibody (Sigma) were used with 1:10,000 or 1:5,000 dilution, respectively. 　　Soy extract lipids in chloroform were purchased from Avanti Polar Lipids and stored in glass vials24. These solutions were evaporated under a stream of nitrogen until a thin lipid film formed and then dried in a vacuum desiccator chamber overnight. On the second day, the lipid film was dissolved in a suspension buffer (HBS buffer: 20 mM HEPES, 300 mM NaCl, pH 8.0) containing 50 mM 5(6)-carboxyfluorescein (Novabiochem) or 50 mg ml−1 polysucrose 40–fluorescein isothiocyanate conjugate (FITC–polysucrose, molecular mass of 30–50 kDa with an estimated diameter of 80 Å, MilliporeSigma). To solubilize lipids, the solution in the glass vial was sonicated for 15 min and then incubated in a 37 °C water bath for at least 1 h. Then the lipid solution was subjected to eight freeze–thaw cycles, in which lipids were frozen in liquid nitrogen for 5 min and then thawed in a 37 °C water bath for 10 min, to reduce the formation of multilamellar liposomes. To control the liposome size, liposomes were extruded through a polycarbonate filter (200 nm, Whatman) 25 times using a mini extruder (Avanti Polar Lipids) with Hamilton glass syringes. Carboxyfluorescein– or FITC–polysucrose-loaded liposomes were purified by centrifugation at 41,000 r.p.m. for 20 min in a TLA 100.3 rotor incorporating three sequential wash steps. After the final wash, carboxyfluorescein– or FITC–polysucrose-loaded liposomes were resuspended in HBS buffer to give a final carboxyfluorescein-loaded liposome concentration of 1 mg ml−1 and FITC–polysucrose-loaded liposome concentration of 0.5 mg ml−1 (ref. 51). 　　Release of the liposome contents was assessed using the self-quenching property and fluorescence of carboxyfluorescein– and FITC–polysucrose. The HBS buffer composition in and outside the liposome was the same (20 mM HEPES, 300 mM NaCl, pH 8.0). Permeability induced by DspE was evaluated by incubating 10 µl DspE protein solution with 90 µl carboxyfluorescein- or FITC–polysucrose-loaded liposomes (0.25 μg μl−1). The fluorescence intensity was measured every 30 s continuously for 2 h after addition of the purified DspE protein (WT or mutant) to the liposomes in a SpectraMax M3 (Molecular Devices). Then 5 µl of 20% Triton X-100 (Sigma-Aldrich) was added to the 100-µl solution to fully release the dye and its readings were measured for 20 min. The average reading of the last 3 min was used for normalization (100% dye release). In the compound inhibition assays, the buffer, DspE protein and PAMAM G1 inhibitor, at a total volume of 10 µl, were first mixed thoroughly with pipetting, and then 90 µl carboxyfluorescein– or FITC–polysucrose-loaded liposomes was added to a total volume 100 μl. The spectrofluorometric excitation and emission parameters were set at the wavelengths of 485 and 510 nm for carboxyfluorescein– and FITC–polysucrose molecules. 　　The DspE protein stock solutions (2.5−25 μM) contained 1 mM DTT. The liposome assays were carried out at 0.05–0.15 μM DspE concentrations with the final DTT concentration less than 0.02 mM. The presence of DTT in the protein buffer did not affect the fluorescence of carboxyfluorescein (Extended Data Fig. 5e). Similarly, the presence of PAMAN G1 at concentrations in the range of 0.3–300 μM did not affect the intrinsic fluorescence of carboxyfluorescein (Extended Data Fig. 7e). 　　Bacterial strains used were WT Pst strain DC3000 and its mutants: the avrE deletion mutant (ΔE)11, the hopM1 deletion mutant (ΔM)11 and the avrE and hopM1 deletion mutant (ΔEM)11 and WT E. amylovora strain Ea273 and its dspE mutant8. Bacteria were grown in low-salt Luria–Bertani medium at 28 °C. The antibiotic ampicillin, gentamicin, kanamycin, rifampicin or spectinomycin was added at 200, 10, 50, 100 or 50 µg ml−1, respectively. Arabidopsis thaliana Col-0 and Col-0/DEX::his-avrE20 plants were grown in Redi-Earth potting soil (Sun Gro Horticulture) in air-circulating growth chambers. Plants were grown at a relative humidity of 60%, temperature of 20 °C, light intensity of 100 µE m−2 s−1 and a photoperiod cycle of 8 h light–16 h dark. Four- to five-week-old plants were used for bacterial disease assay. Immature pear fruits were gifts from George Sundin at Michigan State University. N. benthamiana plants were grown in a growth chamber with 12 h light/12 h dark at 23 °C day and 21 °C night, about 55% humidity and about 100 μmol m−2 s−1 light intensity. Four- to six-week-old plants were used for transient expression assay. 　　Disease assays with immature pear fruits were carried out as previously reported8. Pears were surface-sterilized with 10% bleach for 5 min and rinsed in sterile water twice. Then a small hole was made in the pear using a 200-µl tip. Ten microlitres of a solution containing 103 CFUs per millilitre of Ea273 or the dspE mutant was loaded into the hole. Inoculated pears were placed on a wet paper towel in a sterile box to maintain high humidity at 28 °C for 10 days. Disease assays with Arabidopsis plants were carried out as follows. Arabidopsis plant leaves were infiltrated with Pst DC3000, ΔE, ΔM or ΔEM at 106 CFUs per millilitre with a needle-less syringe. After signs of water soaking were no longer visible (within 1 h), plants were kept under high humidity (about 99%) at 23 °C. The population of bacteria in leaves was determined at day 3 post infiltration. Detached leaves were surface-sterilized in 75% ethanol for 30 s and rinsed in sterile water twice. Then, leaf discs (1 cm2 in diameter) were punched out and ground in 100 µl sterile water. Ten microlitres of each tenfold serial-diluted leaf extract was plated on Luria–Bertani rifampicin and kept at 28 °C for 24 h. CFUs were counted under a microscope before colonies started to coalesce and were analysed by GraphPad Prism software. Two-way ANOVA with Tukey’s test was used for multiple comparisons within a dataset, with significance set to a P value < 0.05. For inhibition assays, 50 nM PAMAM G1 was added to bacterial suspension and co-inoculated into plants. 　　Sequences of E. amylovora DspE, P. carotovorum DspE, Pst DC3000 AvrE and P. stewartii WtsE were aligned using Clustal Omega52. Sequences were from Uniprot (https://www.uniprot.org) as follows—E. amylovora Ea321 DspE (O54581), P. carotovorum Er18 DspE (D5GSK5), Pst DC3000 AvrE (Q887C9) and P. stewartii subsp. stewartii SS104 WtsE (Q9FCY7). 　　dspE and dspE mutant ORFs were PCR-amplified with dspE ORF forward primer (5′-TTGGGCCCATGGAATTAAAATCACTGGGAACTG-3′, underline indicates ApaI site) and dspE ORF reverse primer (5′-TTTACTAGTTTAGCTCTTCATTTCCAGCCCTTCC-3′, underline indicates SpeI site) and pGH-dspE or pGH-dspE mutant plasmids as a template. PCR-amplified dspE and dspE mutant ORFs (ApaI–SpeI fragment) were cloned into the binary vector pER853 to create pER-dspE and pER-dspE mutant constructs. All constructs were transformed into Agrobacterium tumefaciens GV3101 for the transient expression assay. A total of 1 × 108 CFUs per millilitre of A. tumefasciens GV3101 containing pER8 empty vector, pER-dspE or pER-dspE mutant were syringe-inoculated into leaves of N. benthamiana and kept at 22 °C for 24 h before leaves were painted with 90 µM oestradiol. Eight hours later, leaf samples were collected for western blotting. Water-soaking and necrosis symptoms were recorded at 8 h and 24 h after oestradiol treatment under high humidity (>95％）。 　　从5周龄的拟南芥col-0和转基因col-0/dex :: His-avre20中分离出叶叶片原生质体在胶带三明治方法54之后分离出。为了进行肿胀测试，将分离的原生质体在原生质体分离培养基（甘露醇镁培养基）中孵育，其中含有400 mM（杂种）或320 mm（低溶血）甘露醇1小时。使用ICC50W相机使用Leica DM500显微镜拍摄原生质体图像。使用Image J V.1.53软件分析原生质体积。 　　如先前所述进行13。使用Zeiss Axiophot D-7082显微镜拍摄callo氏图像。使用数量一1-D分析软件v。4.6.6（Bio-Rad）确定callose沉积的数量。 　　根据先前发表的文献选择实验样本量足以进行统计分析。根据单个实验，分析了每次处理的三到四个植物（生物学重复）和/或每个基因型。对所有测定进行了两个或多个独立的实验。使用了以下统计分析：单向方差分析用于具有一个变量的多样本实验，随后是Tukey的多伴侣的诚实显着差异测试；双向方差分析用于多变量分析，然后进行多个多动子的诚实显着差异测试，或者Dunnett的测试与共同控制治疗进行比较；双向重复测量方差分析用于在同一实验单元上进行重复测量，然后进行邓内特的测试，以与共同的对照处理进行比较。学生的t检验用于比较两组数据。如果对残差的正态性和方差平等的测试失败，则使用非参数替代方差方差分析或Mann-Whitney等级总和测试。所有统计检验均在图图和方法中描述。图形图由Sigmaplot 12.5生成，并显示平均值±S.E.M.和各个数据点。 　　图像和动画片是在Coreldraw V.22（加拿大渥太华）和Pymol（1.8.0.4）中创建或组装的。所有图形数据均在Microsoft Excel V.2016中组织，然后在Sigmaplot 12.5（Systat软件）上绘制。将图形进一步编辑为颜色和布置，作为Coreldraw V.22（Corel）中的图形面板。 　　有关研究设计的更多信息可在与本文有关的自然投资组合报告摘要中获得。