Table_1_A complex regulatory network governs the expression of symbiotic genes in Sinorhizobium fredii HH103.docx

IntroductionThe establishment of the rhizobium-legume nitrogen-fixing symbiosis relies on the interchange of molecular signals between the two symbionts. We have previously studied by RNA-seq the effect of the symbiotic regulators NodD1, SyrM, and TtsI on the expression of the symbiotic genes (the nod regulon) of Sinorhizobium fredii HH103 upon treatment with the isoflavone genistein. In this work we have further investigated this regulatory network by incorporating new RNA-seq data of HH103 mutants in two other regulatory genes, nodD2 and nolR. Both genes code for global regulators with a predominant repressor effect on the nod regulon, although NodD2 acts as an activator of a small number of HH103 symbiotic genes.MethodsBy combining RNA-seq data, qPCR experiments, and b-galactosidase assays of HH103 mutants harbouring a lacZ gene inserted into a regulatory gene, we have analysed the regulatory relations between the nodD1, nodD2, nolR, syrM, and ttsI genes, confirming previous data and discovering previously unknown relations.Results and discussionPreviously we showed that HH103 mutants in the nodD2, nolR, syrM, or ttsI genes gain effective nodulation with Lotus japonicus, a model legume, although with different symbiotic performances. Here we show that the combinations of mutations in these genes led, in most cases, to a decrease in symbiotic effectiveness, although all of them retained the ability to induce the formation of nitrogen-fixing nodules. In fact, the nodD2, nolR, and syrM single and double mutants share a set of Nod factors, either overproduced by them or not generated by the wild-type strain, that might be responsible for gaining effective nodulation with L. japonicus.

引言：根瘤菌-豆科植物固氮共生关系的建立依赖于两种共生体之间的分子信号的相互交换。我们先前通过RNA测序技术研究了共生调节因子NodD1、SyrM和TtsI在用异黄酮类化合物金雀异黄素处理苏云金根瘤菌HH103后，对其共生基因（nod调控子）表达的影响。在本研究中，我们进一步通过整合HH103突变体在另外两个调节基因nodD2和nolR的新RNA测序数据，对这一调节网络进行了深入研究。这两个基因编码的均为全局调节因子，对nod调控子具有显著的抑制效应，尽管NodD2在少数HH103共生基因的激活中发挥重要作用。
方法：通过结合RNA测序数据、qPCR实验以及携带插入至调节基因中的lacZ基因的HH103突变体的β-半乳糖苷酶分析，我们分析了nodD1、nodD2、nolR、syrM和ttsI基因之间的调节关系，验证了先前数据并发现了先前未知的关联。
结果与讨论：先前研究表明，nodD2、nolR、syrM或ttsI基因的HH103突变体与模式豆科植物大豆形成有效根瘤，尽管共生性能有所不同。本研究表明，这些基因突变组合在大多数情况下导致共生效率降低，尽管所有突变体均保留了诱导形成固氮根瘤的能力。实际上，nodD2、nolR和syrM的单突变体和双突变体共享一组Nod因子，这些因子可能由它们过量产生或野生型菌株未产生，这可能是它们与大豆形成有效根瘤的原因。