Intracellular Trafficking of Potato Leafroll VirusMovement Protein in Transgenic Arabidopsis

Abstract

Intracellular trafficking of viral movement proteins (MPs) in plants has mainly been studied using Tobacco mosaic virus MP30 (TMV MP30) as a model system. Because of the limitations of TMV MP30 expression in Arabidopsis thaliana, these studies have mostly been restricted to tobacco plants. Here we present data on the analysis of transgenic Arabidopsis plants expressing Potato leafroll virus 17-kDa movement protein (MP17) fused to green fluorescent protein. MP17 localizes to secondary branched plasmodesmata (PD) in source but not to simple PD in sink tissues, where MP17 is believed to be degraded by proteolysis. To unravel the intracellular transport path of MP17, we analyzed the relevance of the cytoskeleton and of the secretory pathway on MP17 targeting. To this end, a new incubation system for in vivo analysis of immediate and long-term responses of whole Arabidopsis plants to inhibitor treatments was established. Microscopic and histochemical analysis showed that MP17 is targeted to PD in an actinand endoplasmic reticulum-Golgi-dependent manner. In contrast, degradation of MP17 in sink tissues required intact microtubules and occurred at 26S proteasomes. Interestingly, inhibition of the 26S proteasome led to aggregation of MP17 in aggresome-like structures. Formation of these structures could be inhibited by colchicine, as was shown for aggresomes in mammalian cells.

The Silver Lining of a Viral Agent: Increasing Seed Yield and Harvest Index in Arabidopsis by Ectopic Expression of the Potato Leaf Roll Virus Movement Protein

“…5D and 6) suggested the absence or reduced affinity of putative PD receptor proteins and subsequent proteolytic degradation of nontargeted MP17 in senescent leaves, strongly resembling the situation in sink leaves ( Fig. 1; Vogel et al, 2007). In general, leaf senescence is regarded as a developmental process that is induced to recycle and mobilize nutrients for relocation to other plant organs such as reproducing seeds (Lim et al, 2006).…”

Differential vascularization of nematode-induced feeding sites

“…The results obtained by monitoring the movement of sGFP within the RK suggested that PD with a size exclusion limit (SEL) that allows for trafficking of 27-kDa sGFP are present between nSEs. We investigated number and type of PD in RKs on P 35s :MP17-GFP plants (18). MP17 has been shown to specifically target branched, secondary PD (19).…”

“…Concomitantly, we monitored the formation of secondary PD during feeding site development in both RKN-and CN-infested plants expressing a fusion construct of GFP to the potato leafroll virus movement protein MP17 (18). Around both syncytia and GCs, the newly formed SEs were heavily interconnected by PD.…”

Caracterización molecular y funcional del gen PATHOGEN AND CIRCADIAN CONTROLLED 1 (PCC1) en Arabidopsis thaliana.

“…Para comprobar que la fluorescencia detectada se limita a membranas celulares se procedió a una tinción específica de membranas con FM64 (Bolte et al, 2004) y con yoduro de propidio (Link et al, 2011;Vogel et al, 2007). En ambos casos, la señal del canal verde de GFP coincide con la señal del canal rojo de cada uno de los colorantes, dando lugar a un color mayoritariamente amarillo en la imagen superpuesta de ambos canales (Figura 18).…”

“…Las membranas celulares son impermeables a este compuesto, por lo que se ha utilizado además para la tinción de paredes celulares en células vivas intactas (Link et al, 2011). El protocolo de tinción con yoduro de propidio ha sido descrito previamente (Vogel et al, 2007). Para la tinción de estomas en plántulas de Arabidopsis se incubaron plántulas enteras durante 5 minutos en una solución acuosa de yoduro de propidio 1 µg/ml preparada a partir de una solución stock a 0.1 mg/ml diluída 1:100.…”