Endocytosis is a crucial mechanism by which eukaryotic cells internalize extracellular and plasma membrane material, and it is required for a multitude of cellular and developmental processes in unicellular and multicellular organisms. In animals and yeast, the best characterized pathway for endocytosis depends on the function of the vesicle coat protein clathrin. Clathrin-mediated endocytosis has recently been demonstrated also in plant cells, but its physiological and developmental roles remain unclear. Here, we assessed the roles of the clathrin-mediated mechanism of endocytosis in plants by genetic means. We interfered with clathrin heavy chain (CHC) function through mutants and dominant-negative approaches in Arabidopsis thaliana and established tools to manipulate clathrin function in a cell type–specific manner. The chc2 single mutants and dominant-negative CHC1 (HUB) transgenic lines were defective in bulk endocytosis as well as in internalization of prominent plasma membrane proteins. Interference with clathrin-mediated endocytosis led to defects in constitutive endocytic recycling of PIN auxin transporters and their polar distribution in embryos and roots. Consistent with this, these lines had altered auxin distribution patterns and associated auxin transport-related phenotypes, such as aberrant embryo patterning, imperfect cotyledon specification, agravitropic growth, and impaired lateral root organogenesis. Together, these data demonstrate a fundamental role for clathrin function in cell polarity, growth, patterning, and organogenesis in plants.
SignificanceMultiple effectors of bacterial pathogens target immune kinases such as BAK1 and BIK1, but it is unclear whether this strategy is employed by fungal pathogens. We reveal here that a fungal effector named NIS1 is broadly conserved in filamentous fungi in the Ascomycota and Basidiomycota, thus being regarded as a core effector, and has the ability to suppress PAMP-triggered immunity. Importantly, NIS1 targets BAK1 and BIK1, interfering with their essential functions for immune activation upon pathogen recognition. Multifaceted analyses including the knockout of NIS1 revealed that it plays a critical role in fungal infection. These findings demonstrate that to infect host plants, filamentous fungi deploy a core effector that attacks conserved immune kinases critical for the ancestral defense system.
Endocytic vesicle trafficking is crucial for regulating activity and localization of plasma membrane components, but the process is still poorly genetically defined in plants. Membrane proteins of the PIN-FORMED (PIN) family exhibit polar localization in plant cells and facilitate cellular efflux of the plant hormone auxin, thereby regulating multiple developmental processes. PIN proteins undergo constitutive endocytosis and GNOM ARF GEF-dependent recycling, and their localization is under extensive regulation by developmental and environmental cues. We designed a fluorescence imaging-based screen to identify Arabidopsis thaliana mutants defective in internalization of proteins including PINs from the plasma membrane. We identified three mutant loci, BFA-visualized endocytic trafficking defective1 (ben1) through ben3 that do not efficiently accumulate PIN1-GFP in intracellular compartments after inhibition of recycling and secretion by fungal toxin brefeldin A (BFA). Fine mapping revealed that BEN1 encodes an ARF GEF vesicle trafficking regulator from the functionally uncharacterized BIG class. ben1 mutant has been previously implicated in pathogen response and shows cell polarity, BFA sensitivity, and growth defects. BEN1 is involved in endocytosis of plasma membrane proteins and localizes to early endocytic compartments distinct from GNOM-positive endosomes. Our results identify BEN1 as the ARF GEF mediating early endosomal traffic.
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