Succinate transport is not essential for symbiotic nitrogen fixation by Sinorhizobium meliloti nor Rhizobium leguminosarum

Symbiotic nitrogen fixation (SNF) is an energetically expensive process performed by bacteria known as rhizobia during endosymbiotic relationships with leguminous plants. The bacteria require the plant to provide a carbon source for generation of the reductant to power SNF. Although it is well known that C4-dicarboxylates (succinate, fumarate, malate) function as the primary, if not sole, carbon source provided to the rhizobia, the relative contribution of each C4-dicarboxylate is not known. Here, we employ genetic and systems-level analyses to address this issue. Expression of a malate specific transporter (MaeP) in Sinorhizobium meliloti Rm1021 dct mutants unable to transport C4-dicarboxylates resulted in malate import rates up to ~ 30% that of wild type S. meliloti. This was sufficient to support SNF with Medicago sativa, with acetylene reduction rates up to ~ 50% those of plants inoculated with wild type S. meliloti. Rhizobium leguminosarum bv. viciae 3841 dct mutants unable to transport C4-dicarboxylates but expressing the maeP transporter had strong symbiotic properties, with Pisum sativum plants inoculated with these strains appearing similar to plants inoculated with wild type R. leguminosarum. This was despite malate transport rates by the mutant bacteroids being < 10% those of the wild type. A transcriptomics analysis of the combined plant/bacterium nodule transcriptome was performed using RNA-sequencing to identify any systems-level adaptations in response to the inability of the bacteria to import succinate or fumarate. Few transcriptional changes, with no obvious pattern, were revealed by this analysis. Overall, these data illustrated that succinate and fumarate are not essential for SNF, and that at least in specific symbioses, L-malate is likely to naturally serve as the primary C4-dicarboxylate provided to the bacterium. Overall design: There were a total of 6 samples for the RNA-sequencing analysis: two conditions performed in biological triplicates. Total plant and bacterial RNA was isolated from Pisum sativum cv. Homesteader nodule tissue containing one of two bacterial strains. The first was wild type Rhizobium leguminosarum bv. viciae 3841. The second was a R. leguminosarum bv. viciae 3841 dctA mutant expressing the maeP gene of Streptococcus gallolyticus in trans. All plants were grown in nitrogen free sand-vermiculate set-ups. Sequencing was performed using Illumina HiSeq 1500 with 51 nt single reads.

共生固氮（Symbiotic nitrogen fixation, SNF）是一类耗能极高的生物学过程，由根瘤菌（rhizobia）在与豆科植物形成内共生关系时执行。根瘤菌需要宿主植物提供碳源以合成还原力，为共生固氮供能。尽管学界已明确C4二羧酸（C4-dicarboxylates，包括琥珀酸succinate、延胡索酸fumarate、苹果酸malate）即使不是唯一也是根瘤菌获取的主要碳源，但各类C4二羧酸的相对贡献仍不明晰。本研究通过遗传学与系统水平分析对此展开探究。
在无法转运C4二羧酸的苜蓿中华根瘤菌（Sinorhizobium meliloti）Rm1021 dct突变体中表达苹果酸特异性转运蛋白（MaeP）后，其苹果酸摄入速率可达野生型苜蓿中华根瘤菌的约30%。该水平足以支持与紫花苜蓿（Medicago sativa）的共生固氮，乙炔还原固氮活性速率可达野生型菌株接种植株的约50%。对于豌豆根瘤菌豌豆生物型（Rhizobium leguminosarum bv. viciae）3841，其无法转运C4二羧酸但表达maeP转运蛋白的dct突变体同样表现出良好的共生特性，接种该菌株的豌豆（Pisum sativum）植株表型与野生型根瘤菌接种植株无显著差异；尽管此类突变类菌体的苹果酸转运速率仅为野生型的10%以下。
本研究通过RNA测序（RNA-sequencing）对植物-类菌体联合结节转录组开展转录组学分析，以探究细菌无法摄取琥珀酸与延胡索酸时的系统水平适应性变化。分析结果显示仅存在少量转录水平变化，且无明显规律。
综上，本研究数据表明琥珀酸与延胡索酸并非共生固氮的必需底物，至少在特定共生体系中，天然状态下L-苹果酸可能是供给类菌体的主要C4二羧酸碳源。

### 总体实验设计
本RNA测序分析共设置6个样本，包含2个实验组，每组设置3次生物学重复。实验材料取自豌豆（Pisum sativum cv. Homesteader）的结节组织，从中分离植物与细菌的总RNA，所对应菌株分为两类：第一类为野生型豌豆根瘤菌豌豆生物型3841；第二类为反式表达解链球菌（Streptococcus gallolyticus）maeP基因的豌豆根瘤菌豌豆生物型3841 dctA突变体。所有植株均种植于无氮砂-蛭石基质培养体系中。测序采用Illumina HiSeq 1500平台，进行51碱基单端读取测序。