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Potato virus Y HCPro Localization at Distinct, Dynamically Related and Environment-Influenced Structures in the Cell Cytoplasm

¹Environmental Biology Department, Centro de Investigaciones Biológicas, CIB-CSIC, Ramiro de Maeztu 9, 28040 Madrid; ²The James Hutton Institute, Cell and Molecular Sciences, Invergowrie, Dundee, DD2 5DA, U.K.; ³Biomedical Sciences Research Complex, University of St Andrews, Fife KY16 9ST, Scotland, U.K.; ⁴National Institute of Horticultural & Herbal Science, Agricultural Research Center for Climate Change, 281, Ayeon-ro, Jeju, 690-150, Jeju Island, Republic of Korea; ⁵Division of Plant Pathology, Indian Agricultural Research Institute, 110 012 New Delhi, India

Potyvirus HCPro is a multifunctional protein that, among other functions, interferes with antiviral defenses in plants and mediates viral transmission by aphid vectors. We have visualized in vivo the subcellular distribution and dynamics of HCPro from Potato virus Y and its homodimers, using green, yellow, and red fluorescent protein tags or their split parts, while assessing their biological activities. Confocal microscopy revealed a pattern of even distribution of fluorescence throughout the cytoplasm, common to all these modified HCPros, when transiently expressed in Nicotiana benthamiana epidermal cells in virus-free systems. However, in some cells, distinct additional patterns, specific to some constructs and influenced by environmental conditions, were observed: i) a small number of large, amorphous cytoplasm inclusions that contained α-tubulin; ii) a pattern of numerous small, similarly sized, dot-like inclusions distributing regularly throughout the cytoplasm and associated or anchored to the cortical endoplasmic reticulum and the microtubule (MT) cytoskeleton; and iii) a pattern that smoothly coated the MT. Furthermore, mixed and intermediate forms from the last two patterns were observed, suggesting dynamic transports between them. HCPro did not colocalize with actin filaments or the Golgi apparatus. Despite its association with MT, this network integrity was required neither for HCPro suppression of silencing in agropatch assays nor for its mediation of virus transmission by aphids.