双荧光素酶检测和 siRNA 干扰 nodD1 研究大豆根瘤中德氏固氮菌 USDA110 的竞争力。

Dual-luciferase assay and siRNA silencing for nodD1 to study the competitiveness of Bradyrhizobium diazoefficiens USDA110 in soybean nodulation.

机构信息

Department of Life Science Frontiers, Center for iPS Cell Research and Application, Graduate School of Medicine, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan; Department of Agronomy and Horticultural Science, Graduate School of Agriculture, Kyoto University, Oiwake, Kitashirakawa, Sakyo-ku, Kyoto, 606-8502, Japan.

出版信息

Microbiol Res. 2020 Aug;237:126488. doi: 10.1016/j.micres.2020.126488. Epub 2020 May 3.

Abstract

The symbiosis of soybean with Bradyrhizobium diazoefficiens USDA110, which always competes with other rhizobia in the field, is of great agronomic and environmental importance. Herein, a dual-luciferase reporter assay was utilized to monitor the dynamics of two dominant bradyrhizobia infecting roots of soybean. More explicitly, luciferase-tagged B. diazoefficiens USDA110 (USDA110-FLuc) and Bradyrhizobium elkanii USDA 94 (USDA94-RLuc) were designed, co-inoculated into soybean seeds, and observed for their colonization in root nodules by bioluminescence imaging. The results showed that USDA110-FLuc initiated infection earlier than USDA94-RLuc, but its occupancy in the nodules decreased as the plant grew. A nodulation test showed that nodD1 mutant USDA110 strains, including CRISPR engineered mutants, were less competitive than wild type. I constructed siRNAs to knockdown nodD1 at different target sites and transformed them into the bacteria. Surprisingly, although siRNAs - with 3' end target sites - were able to repress up to 65% of nodD1 expression, the profiling of total RNAs with a bioanalyzer revealed that 23S/16S-rRNA ratios of siRNA-transformed and wild type USDA110 strains were similar, but lower than that of nodD1 mutant. In short, the current work - while reporting the competitiveness of B. diazoefficiens USDA110 in early occupancy of soybean nodules and the gene nodD1 as a key determinant of this infection - gives an insight on siRNA silencing in microbes, and demonstrates a highly efficient imaging approach that could entail many new avenues for many biological research fields.

摘要

大豆与慢生根瘤菌(Bradyrhizobium diazoefficiens USDA110)的共生关系非常重要,因为它在农业和环境方面都具有巨大的意义。慢生根瘤菌 USDA110 总是与田间的其他根瘤菌竞争,而本研究利用双荧光素酶报告基因检测方法监测了两种优势根瘤菌感染大豆根系的动态。具体而言,本研究设计了携带荧光素酶的慢生根瘤菌 USDA110(USDA110-FLuc)和布氏杆菌 USDA94(USDA94-RLuc),将它们共同接种到大豆种子中,通过生物发光成像观察它们在根瘤中的定植情况。结果表明,USDA110-FLuc 比 USDA94-RLuc 更早开始感染,但随着植物的生长,其在根瘤中的占有率下降。结瘤试验表明,包括 CRISPR 工程突变体在内的 nodD1 突变 USDA110 菌株比野生型菌株的竞争力更低。本研究构建了针对不同靶位点的 nodD1 siRNA,并将其转化到细菌中。令人惊讶的是,尽管 3'端靶位点的 siRNA 能够抑制多达 65%的 nodD1 表达,但生物分析仪分析总 RNA 的结果显示,siRNA 转化和野生型 USDA110 菌株的 23S/16S-rRNA 比值相似,但低于 nodD1 突变体。总之,本研究报告了慢生根瘤菌 USDA110 在早期占领大豆根瘤中的竞争力以及基因 nodD1 作为这种感染的关键决定因素,同时也深入了解了微生物中的 siRNA 沉默,并展示了一种高效的成像方法,可为许多生物学研究领域开辟新的途径。

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