Faculty of Agriculture, Ryukoku University, Otsu, Japan.
Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan.
Plant Signal Behav. 2023 Dec 31;18(1):2245995. doi: 10.1080/15592324.2023.2245995.
In addition to rhizobia, many types of co-existent bacteria are found in leguminous root nodules, but their habitats are unclear. To investigate this phenomenon, we labeled USDA122 and sp. SSBR45 with sp. red fluorescent protein (DsRed) or enhanced green fluorescent protein (eGFP). USDA122 enhances soybean growth by forming effective root nodules, but SSBR45 does not form any nodules. Using low-magnification laser scanning confocal microscopy, we found that infected cells in the central zone of soybean nodules appeared to be occupied by USDA122. Notably, high-magnification microscopy after co-inoculation of non-fluorescent USDA122 and fluorescence-labeled SSBR45 also revealed that SSBR45 inhabits the intercellular spaces of healthy nodules. More unexpectedly, co-inoculation of eGFP-labeled USDA122 and DsRed-labeled SSBR45 (and vice versa) revealed the presence of USDA122 bacteria in both the symbiosomes of infected cells and in the apoplasts of healthy nodules. We then next inspected nodules formed after a mixed inoculation of differently-labeled USDA122, without SSBR45, and confirmed the inhabitation of the both populations of USDA122 in the intercellular spaces. In contrast, infected cells were occupied by single-labeled USDA122. We also observed in the intercellular spaces of active wild-type nodules of using transmission electron microscopy. Compatible intercellular rhizobia have been described during nodule formation of several legume species and in some mutants, but our evidence suggests that this type of colonization may occur much more commonly in leguminous root nodules.
除了根瘤菌，豆科植物根瘤中还存在许多共生细菌，但它们的栖息地尚不清楚。为了研究这一现象，我们用 sp. 红色荧光蛋白（DsRed）或增强型绿色荧光蛋白（eGFP）标记了 USDA122 和 sp. SSBR45。USDA122 通过形成有效的根瘤来促进大豆生长，但 SSBR45 不会形成任何根瘤。使用低倍激光扫描共聚焦显微镜，我们发现大豆根瘤中央区的感染细胞似乎被 USDA122 占据。值得注意的是，共接种非荧光 USDA122 和荧光标记 SSBR45 后进行高倍显微镜观察也表明 SSBR45 栖息在健康根瘤的细胞间隙中。更令人意外的是，共接种 eGFP 标记的 USDA122 和 DsRed 标记的 SSBR45（反之亦然）揭示了 USDA122 细菌存在于感染细胞的共生体和健康根瘤的质外体中。然后，我们接下来检查了在没有 SSBR45 的情况下混合接种不同标记的 USDA122 形成的根瘤，证实了两种 USDA122 种群都栖息在细胞间隙中。相比之下，感染细胞被单标记的 USDA122 占据。我们还通过透射电子显微镜观察到活跃的野生型根瘤中存在的。在几种豆科植物的结瘤形成过程中和在一些突变体中已经描述了相容的细胞间根瘤菌，但我们的证据表明，这种定植类型可能在豆科植物根瘤中更为常见。
