Potato virus Y HCPro localization at distinct, dynamically-related and environment-influenced structures in the cell cytoplasm

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: 1-a small number of large, amorphous cytoplasm inclusions that contained α-tubulin; 2-a pattern of numerous small, similarly-sized, dot-like inclusions distributing regularly throughout the cytoplasm and associated or anchored to the cortical endoplasmic reticulum (ER) and the microtubule (MT) cytoskeleton; 3-a pattern that coated smoothly the MTs. Furthermore, mixed and intermediate forms from the last two patterns were observed, suggesting dynamic transports between them. HCPro did not co-localize with actin filaments or the Golgi apparatus. Despite its association to MTs, this network integrity was required neither for HCPro suppression of silencing in agropatch assays nor for its mediation of virus transmission by aphids.