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.

大豆与重氮根瘤菌（Bradyrhizobium diazoefficiens） USDA110的共生关系一直与该领域的其他根瘤菌竞争，在农业和环境方面具有重要意义。在本文中，利用双重萤光素酶报告基因测定法来监测两个主要的大豆根瘤菌感染大豆根的动态。更明确地说，设计了萤光素酶标记的重氮芽孢杆菌USDA110（USDA110-FLuc）和Bradyrhizobium elkanii USDA 94（USDA94-RLuc），共接种到大豆种子中，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，但随着植物的生长，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）的竞争力不及野生型。我构建了siRNA，在不同靶位点敲低nodD1并将其转化为细菌。出乎意料的是，尽管具有3'末端靶位点的siRNA能够抑制nodD1表达的65％，但使用生物分析仪对总RNA进行分析后发现，siRNA转化的和野生型USDA110菌株的23S / 16S-rRNA比率为相似，但低于nodD1突变体。

总之，目前的工作-而报告的竞争力B. diazoefficiens大豆根瘤和基因的早期入住USDA110 nodD1因为这种感染的关键因素-提供了对微生物的siRNA沉默的洞察力，并演示了高效的成像方法这可能为许多生物学研究领域带来许多新途径。