High-throughput quantitative luminescence assay of the growth in planta of Pseudomonas syringae chromosomally tagged with Photorhabdus luminescens luxCDABE
High-throughput quantitative luminescence assay of the growth in planta of Pseudomonas syringae chromosomally tagged with Photorhabdus luminescens luxCDABE
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DOI：
10.1111/j.1365-313x.2007.03303.x
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发表时间：
2008-01-01
期刊：
影响因子：
7.2
通讯作者：
Lamb, Chris
中科院分区：
文献类型：
--
作者：
Fan, Jun;Crooks, Casey;Lamb, Chris
拟南芥病原菌假单胞菌致病变种（Pseudomonaslingae pathovar（pv.）番茄和pv.通过在组成型启动子的控制下将来自发光光杆菌的luxCDABE操纵子插入到P. luxCDABE的稳定整合不影响细菌适应性、植物生长或疾病结果。发光准确和可靠地报告了在感染的拟南芥叶片中的细菌生长，其中固定的接种物随时间推移而变化，并且在单个时间点测定不同的接种物。此外，生物发光测定法可以检测不同植物基因型之间细菌生长的小（1.3倍）差异，其精度与标准平板测定法相当。luxCDABE-标记的P. lucingae的发光允许快速和方便地定量细菌生长，而不需要组织提取、系列稀释、平板接种和手动评分，所述组织提取、系列稀释、平板接种和手动评分涉及通过来自感染组织的样品的平板培养物中的菌落形成的细菌生长的标准测定。生物发光测定的实用性通过调查普遍毒性的P.在100多个拟南芥生态型中，鉴定出了两个数量性状基因座，分别占拟南芥对斑枯病的基础抗性变异的48%和16%。番茄DC 3000在Col-0 x Fl-1 F-2群体中的生长。用luxCDABE标记的细菌染色体的发光测定应极大地促进基因对基因、基础和获得性抗病性以及植物与细菌病原体相互作用的其他方面的定量差异的遗传解剖，这些方面需要高通量测定或大规模定量筛选。
Bioluminescent strains of the Arabidopsis thaliana pathogens Pseudomonas syringae pathovar (pv.) tomato and pv. maculicola were made by insertion of the luxCDABE operon from Photorhabdus luminescens into the P. syringae chromosome under the control of a constitutive promoter. Stable integration of luxCDABE did not affect bacterial fitness, growth in planta or disease outcome. Luminescence accurately and reliably reported bacterial growth in infected Arabidopsis leaves both with a fixed inoculum followed over time and with varying inocula assayed at a single time point. Furthermore, the bioluminescence assay could detect a small (1.3-fold) difference in bacterial growth between different plant genotypes with a precision comparable to that of the standard plate assay. Luminescence of luxCDABE-tagged P. syringae allows rapid and convenient quantification of bacterial growth without the tissue extraction, serial dilution, plating and manual scoring involved in standard assays of bacterial growth by colony formation in plate culture of samples from infected tissue. The utility of the bioluminescence assay was illustrated by surveying the 500-fold variation in growth of the universally virulent P. syringae pv. maculicola ES4326 among more than 100 Arabidopsis ecotypes and identification of two quantitative trait loci accounting for 48% and 16%, respectively, of the variance of basal resistance to P. syringae pv. tomato DC3000 in the Col-0 x Fl-1 F-2 population. Luminescence assay of bacteria chromosomally tagged with luxCDABE should greatly facilitate the genetic dissection of quantitative differences in gene-for-gene, basal and acquired disease resistance and other aspects of plant interactions with bacterial pathogens requiring high-throughput assays or large-scale quantitative screens.