In plants, plasmodesmata establish cytoplasmic continuity between cells to allow for communication and resource exchange across the cell wall. While plant pathogens use plasmodesmata as a pathway for both molecular and physical invasion, the benefits of molecular invasion (cell-to-cell movement of pathogen effectors) are poorly understood. To establish a methodology for identification and characterization of the cell-to-cell mobility of effectors, we performed a quantitative live imaging-based screen of candidate effectors of the fungal pathogen Colletotrichum higginsianum. We predicted C. higginsianum effectors by their expression profiles, the presence of a secretion signal, and their predicted and in planta localization when fused to green fluorescent protein. We assayed for cell-to-cell mobility of nucleocytosolic effectors and identified 14 that are cell-to-cell mobile. We identified that three of these effectors are “hypermobile,” showing cell-to-cell mobility greater than expected for a protein of that size. To explore the mechanism of hypermobility, we chose two hypermobile effectors and measured their impact on plasmodesmata function and found that even though they show no direct association with plasmodesmata, each increases the transport capacity of plasmodesmata. Thus, our methods for quantitative analysis of cell-to-cell mobility of candidate microbe-derived effectors, or any suite of host proteins, can identify cell-to-cell hypermobility and offer greater understanding of how proteins affect plasmodesmal function and intercellular connectivity.

Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

在植物中，胞间连丝在细胞之间建立细胞质连续性，以允许跨细胞壁的通讯和资源交换。虽然植物病原体利用胞间连丝作为分子和物理入侵的途径，但人们对分子入侵（病原体效应子的细胞间运动）的好处知之甚少。为了建立一种识别和表征效应子细胞间迁移性的方法，我们对真菌病原体希金斯炭疽病菌的候选效应子进行了基于实时成像的定量筛选。我们通过其表达谱、分泌信号的存在以及与绿色荧光蛋白融合时的预测和植物定位来预测C. higginsianum效应子。我们分析了核胞浆效应子的细胞间移动性，并鉴定出 14 个具有细胞间移动性的效应子。我们发现其中三个效应器是“超机动性”的，显示出细胞间的机动性大于该大小的蛋白质的预期。为了探索过度活动的机制，我们选择了两种过度活动效应器并测量了它们对胞间连丝功能的影响，发现尽管它们与胞间连丝没有直接关联，但每一个都增加了胞间连丝的运输能力。因此，我们对候选微生物衍生效应子或任何宿主蛋白套件的细胞间流动性进行定量分析的方法可以识别细胞间过度流动性，并更好地了解蛋白质如何影响胞间连丝功能和细胞间连接。

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