Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Yamazaki, Noda, 2641, 278-8510, Chiba, Japan.
BMC Microbiol. 2023 Jul 5;23(1):175. doi: 10.1186/s12866-023-02920-y.
Microorganisms that activate plant immune responses are useful for application as biocontrol agents in agriculture to minimize crop losses. The present study was conducted to identify and characterize plant immunity-activating microorganisms in Brassicaceae plants.
A total of 25 bacterial strains were isolated from the interior of a Brassicaceae plant, Raphanus sativus var. hortensis. Ten different genera of bacteria were identified: Pseudomonas, Leclercia, Enterobacter, Xanthomonas, Rhizobium, Agrobacterium, Pantoea, Rhodococcus, Microbacterium, and Plantibacter. The isolated strains were analyzed using a method to detect plant immunity-activating microorganisms that involves incubation of the microorganism with tobacco BY-2 cells, followed by treatment with cryptogein, a proteinaceous elicitor of tobacco immune responses. In this method, cryptogein-induced production of reactive oxygen species (ROS) in BY-2 cells serves as a marker of immune activation. Among the 25 strains examined, 6 strains markedly enhanced cryptogein-induced ROS production in BY-2 cells. These 6 strains colonized the interior of Arabidopsis plants, and Pseudomonas sp. RS3R-1 and Rhodococcus sp. RS1R-6 selectively enhanced plant resistance to the bacterial pathogens Pseudomonas syringae pv. tomato DC3000 and Pectobacterium carotovorum subsp. carotovorum NBRC 14082, respectively. In addition, Pseudomonas sp. RS1P-1 effectively enhanced resistance to both pathogens. We also comprehensively investigated the localization (i.e., cellular or extracellular) of the plant immunity-activating components produced by the bacteria derived from R. sativus var. hortensis and the components produced by previously isolated bacteria derived from another Brassicaceae plant species, Brassica rapa var. perviridis. Most gram-negative strains enhanced cryptogein-induced ROS production in BY-2 cells via the presence of cells themselves rather than via extracellular components, whereas many gram-positive strains enhanced ROS production via extracellular components. Comparative genomic analyses supported the hypothesis that the structure of lipopolysaccharides in the outer cell envelope plays an important role in the ROS-enhancing activity of gram-negative Pseudomonas strains.
The assay method described here based on elicitor-induced ROS production in cultured plant cells enabled the discovery of novel plant immunity-activating bacteria from R. sativus var. hortensis. The results in this study also suggest that components involved in the ROS-enhancing activity of the bacteria may differ depending largely on genus and species.
激活植物免疫反应的微生物可用于农业中的生物防治剂，以最大程度地减少作物损失。本研究旨在鉴定和表征芸薹属植物中的植物免疫激活微生物。
从芸薹属植物萝卜（Raphanus sativus var. hortensis）内部共分离出 25 株细菌。鉴定出 10 个不同属的细菌：假单胞菌、勒克氏菌、肠杆菌、黄单胞菌、根瘤菌、农杆菌、泛菌、节杆菌、微杆菌和植物杆菌。采用检测植物免疫激活微生物的方法对分离株进行分析，该方法涉及将微生物与烟草 BY-2 细胞共培养，然后用蛋白激发子 cryptogein 处理，以诱导烟草免疫反应。在该方法中，cryptogein 诱导 BY-2 细胞中活性氧（ROS）的产生可作为免疫激活的标志物。在 25 株受检菌株中，有 6 株明显增强了 BY-2 细胞中 cryptogein 诱导的 ROS 产生。这 6 株菌定植于拟南芥植物内部，假单胞菌 RS3R-1 和节杆菌 RS1R-6 分别选择性地增强了植物对细菌病原体丁香假单胞菌 pv.番茄 DC3000 和胡萝卜软腐欧文氏菌亚种胡萝卜软腐亚种 NBRC 14082 的抗性。此外，假单胞菌 RS1P-1 还能有效增强对这两种病原体的抗性。我们还全面研究了源自萝卜的芸薹属植物和源自另一种芸薹属植物 Brassica rapa var. perviridis 的先前分离的细菌产生的植物免疫激活成分的定位（即细胞内或细胞外）。大多数革兰氏阴性菌株通过自身细胞的存在而不是通过细胞外成分增强 cryptogein 诱导的 BY-2 细胞中的 ROS 产生，而许多革兰氏阳性菌株则通过细胞外成分增强 ROS 产生。比较基因组分析支持了这样的假设，即外细胞包膜中脂多糖的结构在革兰氏阴性假单胞菌菌株增强 ROS 活性中起重要作用。
本文描述的基于培养植物细胞中诱导 ROS 产生的测定方法可从萝卜中发现新型植物免疫激活细菌。本研究结果还表明，参与 ROS 增强活性的细菌成分可能在很大程度上取决于属和种而有所不同。
