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.

大豆与重氮慢生根瘤菌USDA110 的共生具有重要的农艺和环境重要性，该种植物始终与田间的其他根瘤菌竞争。在此，利用双荧光素酶报告基因测定来监测感染大豆根部的两种优势缓生根瘤菌的动态。更明确地说，设计了荧光素酶标记的B. diazoefficiens USDA110 (USDA110-FLuc) 和Bradyrhizobium elkanii USDA 94 (USDA94-RLuc)，将其共同接种到大豆种子中，并通过生物发光成像观察它们在根瘤中的定殖。结果表明，USDA110-FLuc 比 USDA94-RLuc 更早开始感染，但随着植物生长，其在根瘤中的占有率下降。结瘤测试表明， nodD1突变体 USDA110 菌株（包括 CRISPR 工程突变体）的竞争力低于野生型。我构建了 siRNA 以在不同的靶位点敲低nodD1 ，并将其转化到细菌中。令人惊讶的是，虽然带有 3' 末端靶位点的 siRNA 能够抑制高达 65% 的nodD1表达，但使用生物分析仪对总 RNA 进行分析显示，siRNA 转化菌株和野生型 USDA110 菌株的 23S/16S-rRNA 比率低于预期。相似，但低于nodD1突变体。

简而言之，当前的工作在报告B. diazoefficiens USDA110 在早期占据大豆根瘤方面的竞争力以及基因nodD1作为这种感染的关键决定因素的同时，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这可能为许多生物研究领域带来许多新途径。