School of Life and Environmental Sciences, University of Sydney, Sydney, NSW, Australia.
Koala Health Hub, University of Sydney, Sydney, NSW, Australia.
Appl Microbiol Biotechnol. 2018 Sep;102(17):7521-7539. doi: 10.1007/s00253-018-9086-2. Epub 2018 Jun 23.
Inoculation of legume seed with rhizobia is an efficient and cost-effective means of distributing elite rhizobial strains to broad-acre crops and pastures. However, necessary drying steps after coating seed expose rhizobia to desiccation stress reducing survival and limiting potential nitrogen fixation by legumes. Rhizobial tolerance to desiccation varies with strain and with growth conditions prior to drying. Cells grown in peat generally survive desiccation better than cells grown in liquid broth. We aimed to identify peat-induced proteomic changes in rhizobia that may be linked to desiccation tolerance. Proteins expressed differentially after growth in peat extract when compared with a minimal defined medium were measured in four rhizobial strains. Proteins showing the greatest increase in abundance were those involved in amino acid and carbohydrate transport and metabolism. Proteins involved in posttranslational modification and cell defence mechanisms were also upregulated. Many of the proteins identified in this study have been previously linked to stress responses. In addition, analysis using nucleic acid stains SYTO9 and propidium iodide indicated that membranes had been compromised after growth in peat extract. We targeted the membrane repair protein PspA (ΔRL3579) which was upregulated in Rhizobium leguminosarum bv. viceae 3841 after growth in peat extract to validate whether the inability to repair membrane damage after growth in peat extract reduced desiccation tolerance. The ΔRL3579 mutant grown in peat extract had significantly lower survival under desiccation stress, whereas no difference in survival between wild-type and mutant strains was observed after growth in tryptone yeast (TY) or minimal medium (JMM) media. Staining mutant and wild-type strains with SYTO9 and propidium iodide indicated that membranes of the mutant were compromised after growth in peat extract and to a lesser extent in TY. This study shows that growth in peat extract causes damage to cell membranes and exposes rhizobia to sub-lethal stress resulting in differential expression of several stress-induced proteins. The induction of these proteins may prime and protect the cells when subjected to subsequent stress such as desiccation. Identifying the key proteins involved in desiccation tolerance and properties of peat that stimulate this response will be important to inform development of new inoculant technology that maximises survival of rhizobia during delivery to legume crops and pastures.
将根瘤菌接种到豆科植物种子上是一种有效且经济高效的方法，可以将优良根瘤菌菌株分布到大面积的作物和牧场上。然而，在涂层种子后进行必要的干燥步骤会使根瘤菌暴露在干燥胁迫下，从而降低其存活率并限制豆类的潜在固氮作用。根瘤菌对干燥的耐受性因菌株和干燥前的生长条件而异。在泥炭中生长的细胞通常比在液体肉汤中生长的细胞更能耐受干燥。我们的目的是确定与耐旱性相关的根瘤菌在泥炭中诱导的蛋白质组变化。在与最小定义培养基相比，在泥炭提取物中生长后表达差异的蛋白质在四个根瘤菌株中进行了测量。表达丰度增加最大的蛋白质是那些参与氨基酸和碳水化合物运输和代谢的蛋白质。参与翻译后修饰和细胞防御机制的蛋白质也被上调。本研究中鉴定的许多蛋白质以前都与应激反应有关。此外，使用核酸染料 SYTO9 和碘化丙啶的分析表明，在用泥炭提取物生长后，细胞膜已经受损。我们针对膜修复蛋白 PspA（ΔRL3579）进行了靶向研究，在 Rhizobium leguminosarum bv. viceae 3841 中，在用泥炭提取物生长后，该蛋白上调，以验证在用泥炭提取物生长后无法修复细胞膜损伤是否会降低耐旱性。在泥炭提取物中生长的ΔRL3579 突变体在干燥胁迫下的存活率明显较低，而在在 tryptone yeast (TY) 或 minimal medium (JMM) 培养基中生长时，野生型和突变型菌株之间的存活率没有差异。用 SYTO9 和碘化丙啶对突变体和野生型菌株进行染色表明，在用泥炭提取物生长后，突变体的细胞膜受损，在 TY 中受损程度较小。本研究表明，在泥炭提取物中生长会导致细胞膜受损，并使根瘤菌暴露在亚致死应激下，从而导致几种应激诱导蛋白的差异表达。这些蛋白质的诱导可能会在随后的应激（如干燥）时使细胞做好准备并得到保护。确定与耐旱性相关的关键蛋白质和刺激这种反应的泥炭特性对于为新的接种剂技术的开发提供信息非常重要，该技术可以最大限度地提高根瘤菌在向豆类作物和牧场输送过程中的存活率。
