Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, OK 73401, USA.
Plant Methods. 2011 Oct 6;7:32. doi: 10.1186/1746-4811-7-32.
The Arabidopsis thaliana-Pseudomonas syringae model pathosystem is one of the most widely used systems to understand the mechanisms of microbial pathogenesis and plant innate immunity. Several inoculation methods have been used to study plant-pathogen interactions in this model system. However, none of the methods reported to date are similar to those occurring in nature and amicable to large-scale mutant screens.
In this study, we developed a rapid and reliable seedling flood-inoculation method based on young Arabidopsis seedlings grown on MS medium. This method has several advantages over conventional soil-grown plant inoculation assays, including a shorter growth and incubation period, ease of inoculation and handling, uniform infection and disease development, requires less growth chamber space and is suitable for high-throughput screens. In this study we demonstrated the efficacy of the Arabidopsis seedling assay to study 1) the virulence factors of P. syringae pv. tomato DC3000, including type III protein secretion system (TTSS) and phytotoxin coronatine (COR); 2) the effector-triggered immunity; and 3) Arabidopsis mutants affected in salicylic acid (SA)- and pathogen-associated molecular pattern (PAMPs)-mediated pathways. Furthermore, we applied this technique to study nonhost resistance (NHR) responses in Arabidopsis using nonhost pathogens, such as P. syringae pv. tabaci, pv. glycinea and pv. tomato T1, and confirmed the functional role of FLAGELLIN-SENSING 2 (FLS2) in NHR.
The Arabidopsis seedling flood-inoculation assay provides a rapid, efficient and economical method for studying Arabidopsis-Pseudomonas interactions with minimal growth chamber space and time. This assay could also provide an excellent system for investigating the virulence mechanisms of P. syringae. Using this method, we demonstrated that FLS2 plays a critical role in conferring NHR against nonhost pathovars of P. syringae, but not to Xanthomonas campestris pv. vesicatoria. This method is potentially ideal for high-throughput screening of both Arabidopsis and pathogen mutants.
拟南芥-丁香假单胞菌模式病理系统是用于了解微生物发病机制和植物先天免疫机制的最广泛应用系统之一。已经使用了几种接种方法来研究该模型系统中的植物-病原体相互作用。然而，迄今为止报道的方法都没有类似于自然发生的方法，也不利于大规模突变体筛选。
在本研究中，我们基于在 MS 培养基上生长的年轻拟南芥幼苗，开发了一种快速可靠的幼苗浸种接种方法。与传统的土培植物接种测定相比，该方法具有多个优势，包括较短的生长和孵育时间、易于接种和处理、均匀的感染和疾病发展、需要更少的生长室空间，并且适合高通量筛选。在本研究中，我们证明了拟南芥幼苗测定法在研究 1）丁香假单胞菌 pv.番茄 DC3000 的毒力因子，包括 III 型蛋白分泌系统（TTSS）和植物毒素冠菌素（COR）；2）效应子触发的免疫；和 3）水杨酸（SA）和病原体相关分子模式（PAMPs）介导途径中受影响的拟南芥突变体的功效。此外，我们使用非宿主病原体，如丁香假单胞菌 pv.番茄，pv.菜豆和 pv.番茄 T1，将该技术应用于研究拟南芥的非宿主抗性（NHR）反应，并证实了 FLAGELLIN-SENSING 2（FLS2）在 NHR 中的功能作用。
拟南芥幼苗浸种接种测定法为研究拟南芥-丁香假单胞菌相互作用提供了一种快速、高效和经济的方法，所需生长室空间和时间最小。该测定法也可提供用于研究丁香假单胞菌毒力机制的优良系统。使用该方法，我们证明 FLS2 在赋予拟南芥对非宿主丁香假单胞菌菌株的 NHR 中起关键作用，但对野油菜黄单胞菌 pv.vesicatoria 不起作用。该方法对于拟南芥和病原体突变体的高通量筛选可能是理想的。
