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

Abstract

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.

A Violaxanthin Deepoxidase Interacts with a Viral Suppressor of RNA Silencing to Inhibit Virus Amplification

“…Previous studies indicated that potyviral HC-Pros were distributed to the cytoplasm (Riedel et al, 1998;Mlotshwa et al, 2002;Zheng et al, 2011;Sahana et al, 2014), nucleus (Riedel et al, 1998;Sahana et al, 2014), cytoplasm filaments, cell membrane, or nuclear envelope (Zheng et al, 2011). In addition, Turnip mosaic virus HC-Pro and Potato virus Y (PVY) HC-Pro could form aggregates in the cytoplasm (Zheng et al, 2011;del Toro et al, 2014), and an amino acid change of Papaya ringspot virus HC-Pro also led to the formation of the aggregates (Sahana et al, 2014). Interestingly, PVY HC-Pro also could be present in the chloroplasts of PVY-infected plants (Gunasinghe and Berger, 1991).…”

High Temperature, High Ambient CO2 Affect the Interactions between Three Positive-Sense RNA Viruses and a Compatible Host Differentially, but not Their Silencing Suppression Efficiencies

“…benthamiana [ 23 ] expressing an inverted repeat fragment of its RNA-dependent RNA polymerase 6 (RDR6 ) gene that reduced the levels of the endogenous transcript mRNA to 4% of that found in non-silenced plants was also used, kindly provided by Prof. D. C. Baulcombe (University of Cambridge, UK). Viruses: we used an aphid transmissible PVY isolate (Scottish ordinary variety PVY-O, Scottish Agricultural Science Agency) from which the P1-6x-HCPro sequence contained in the binary constructs P1-6x-HCPro and PVX-P1-6x-HCPro [ 24 , 25 ], respectively, and described below derive. We used a cloned CMV isolate, strain Fny, obtained by inoculating combined in vitro transcripts of full-length infectious clones of viral RNAs 1, 2, and 3 [ 24 ].…”

The HCPro from thePotyviridaefamily: an enviable multitasking Helper Component that every virus would like to have

“…The most recent and thorough examination of subcellular localization comes from a study performed by del Toro et al . () with the PVY HCPro fused to diverse fluorophores. In addition to a diffuse presence of this viral protein in the cytoplasm, they also observed distinct protein distributions (e.g.…”