To establish and maintain a symbiotic relationship, the ectomycorrhizal fungus Laccaria bicolor releases mycorrhiza-induced small secreted proteins (MiSSPs) into host roots. Here, we have functionally characterized the MYCORRHIZA-iNDUCED SMALL SECRETED PROTEIN OF 7.6 kDa (MiSSP7.6) from L. bicolor by assessing its induced expression in ectomycorrhizae, silencing its expression by RNAi, and tracking in planta subcellular localization of its protein product. We also carried out yeast two-hybrid assays and bimolecular fluorescence complementation analysis to identify possible protein targets of the MiSSP7.6 effector in Populus roots. We showed that MiSSP7.6 expression is upregulated in ectomycorrhizal rootlets and associated extramatrical mycelium during the late stage of symbiosis development. RNAi mutants with a decreased MiSSP7.6 expression have a lower mycorrhization rate, suggesting a key role in the establishment of the symbiosis with plants. MiSSP7.6 is secreted, and it localizes both to the nuclei and cytoplasm in plant cells. MiSSP7.6 protein was shown to interact with two Populus Trihelix transcription factors. Furthermore, when coexpressed with one of the Trihelix transcription factors, MiSSP7.6 is localized to plant nuclei only. Our data suggest that MiSSP7.6 is a novel secreted symbiotic effector and is a potential determinant for ectomycorrhiza formation.

中文翻译：

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建立外生菌根共生需要双色的拉卡里亚小分泌效应蛋白MiSSP7.6。

为了建立和维持共生关系，外生菌根真菌双孢子菌将菌根诱导的小分泌蛋白（MiSSPs）释放到宿主根中。在这里，我们通过评估其在外生菌根中的诱导表达，通过RNAi沉默其表达并在植物亚细胞定位其蛋白质产物中进行了跟踪，从功能上鉴定了双色乳杆菌中7.6 kDa（MiSSP7.6）的MYCORRHIZA诱导的小分泌蛋白。我们还进行了酵母双杂交测定和双分子荧光互补分析，以鉴定胡杨根中MiSSP7.6效应子的可能蛋白质靶标。我们显示在共生发展的后期，外生菌根和相关的基质外菌丝体中的MiSSP7.6表达上调。具有降低的MiSSP7的RNAi突变体。6表达具有较低的菌根发生率，提示在建立与植物共生中的关键作用。MiSSP7.6是分泌的，它既定位于植物细胞的核，又定位于细胞质。显示MiSSP7.6蛋白与两个杨Trihelix转录因子相互作用。此外，当与Trihelix转录因子之一共表达时，MiSSP7.6仅定位于植物核。我们的数据表明，MiSSP7.6是一种新型的分泌型共生效应物，是外生菌根形成的潜在决定因素。当与Trihelix转录因子之一共表达时，MiSSP7.6仅定位于植物核。我们的数据表明，MiSSP7.6是一种新型的分泌型共生效应物，是外生菌根形成的潜在决定因素。当与Trihelix转录因子之一共表达时，MiSSP7.6仅定位于植物核。我们的数据表明，MiSSP7.6是一种新型的分泌型共生效应物，是外生菌根形成的潜在决定因素。