R.solanacearum spot inoculations and quantification of bacterial populations
R.solanacearum spot inoculation assay and statistics of R.solanacearum populations were
performed as previously reported.R.solanacearum was grown in phi medium,washed and
resuspended in sterile water.OD600 was adjusted to 0.02 for inoculation.M.truncatula plants
were cultivated in BNM medium for 4-5 days.Sterile needles were used to pierce the root
segments(2cm from the root tip)of each plant for co-inoculating Nod factor with Ralstonia or H2O with Ralstonia viafilter paper.To determine R.solanacearum populations 5 dpi in the inoculation    area of roots,root segments were individually placed in tubes and ground.The material was
resuspended in sterile waterand serial dilutions were plated on Phi medium.Colonies were counted after incubation at 28℃ for 48h,with eGFP colonies counted under a  fluorescent protein lamp 
操作方法
如先前报道所述，进行了R. solanacearum斑点接种实验及细菌种群定量分析，并统计了R. solanacearum种群数量。R. solanacearum在phi培养基中培养后，经洗涤并重悬于无菌水中，将OD600值调整至0.02用于接种。Truncatula植株在 BNM 培养基中培养4-5天。使用无菌针头刺穿每株植物根段（距根尖2厘米处），通过滤纸将结瘤因子与Ralstonia菌或水与Ralstonia菌共同接种。为测定接种后第5天根部接种区域的R. solanacearum种群数量，将各根段单独置于试管中研磨，将材料重悬于无菌水中，进行系列稀释后接种于Phi培养基。于28℃培养48小时后计数菌落，其中eGFP阳性菌落使用荧光蛋白灯进行筛选。
采用BL21(DE3)菌株在大肠杆菌中表达GST或His标签融合蛋白，而GFP标签融合蛋白则在本氏烟草中表达。对于BL21菌株的蛋白表达实验：将携带相应表达载体的BL21菌株过夜培养物稀释于200毫升LB培养基中，于37℃培养至OD600值达0.6-0.8（添加1 mM IPTG），继续振荡培养3小时；随后在4℃、4000 g条件下离心15分钟收集细胞。对于烟草中的蛋白表达实验：将携带相应表达载体的GV3101菌株按前述方法转染至烟草叶片；培养1-3天后，使用便携式 GFP 激发光源（LUYOR ，3415RG）检测荧光区域，并按指定方法进行处理。