Effects of Calreticulin on Viral Cell-to-Cell Movement

Abstract

Cell-to-cell tobacco mosaic virus movement protein (TMV MP) mediates viral spread between the host cells through plasmodesmata. Although several host factors have been shown to interact with TMV MP, none of them coresides with TMV MP within plasmodesmata. We used affinity purification to isolate a tobacco protein that binds TMV MP and identified it as calreticulin. The interaction between TMV MP and calreticulin was confirmed in vivo and in vitro, and both proteins were shown to share a similar pattern of subcellular localization to plasmodesmata. Elevation of the intracellular levels of calreticulin severely interfered with plasmodesmal targeting of TMV MP, which, instead, was redirected to the microtubular network. Furthermore, in TMV-infected plant tissues overexpressing calreticulin, the inability of TMV MP to reach plasmodesmata substantially impaired cell-to-cell movement of the virus. Collectively, these observations suggest a functional relationship between calreticulin, TMV MP, and viral cell-to-cell movement.Following initial infection (usually by mechanical inoculation), many plant viruses, such as tobacco mosaic virus (TMV), spread from cell to cell through plasmodesmata (for review, see Heinlein, 2002;Waigmann et al., 2004), presumably utilizing the existing cellular pathways of plasmodesmal transport. In the case of TMV, this virus encodes a specific cell-to-cell movement protein (MP), which mediates the transport of the viral genomic RNA through plasmodesmata (Deom et al., 1987). To date, MP has been shown to bind TMV RNA (Citovsky et al., 1990(Citovsky et al., , 1992, associate with the cytoskeleton (Heinlein et al., 1995;McLean et al., 1995;Boyko et al., 2000) and endoplasmic reticulum (ER; Heinlein et al., 1998;Reichel and Beachy, 1999), target to plasmodesmata within plant cell walls (Tomenius et al., 1987;Ding et al., 1992a), increase plasmodesmal permeability (Wolf et al., 1989;Waigmann et al., 1994), and undergo negative regulation by phosphorylation (Citovsky et al., 1993;Waigmann et al., 2000;Trutnyeva et al., 2005). To perform many of these functions, TMV MP most likely cooperates with different cellular factors, some of which, such as cell wall pectin methylesterases (PME; Dorokhov et al., 1999;Chen et al., 2000), microtubules (MTs) and actin filaments (Heinlein et al., 1995;McLean et al., 1995;Boyko et al., 2000), and a RIO protein kinase (Yoshioka et al., 2004), are known, while others remain to be discovered. It would be especially interesting to determine whether cellular proteins exist that not only recognize TMV MP but also coreside with it within plasmodesmata.Using TMV MP as specific ligand in a biochemical purification protocol, we showed that it interacts with plant calreticulin. Calreticulin is a Ca 21 -sequestering protein that is highly conserved between different species, including plants ( Nelson et al., 1997; Borisjuk et al., 1998, and refs. therein;Michalak et al., 1998). Functionally, calreticulin is involved in Ca 21 storage and signaling, chaperone activity, cell ad...

Molecular cloning of a new wheat calreticulin gene TaCRT1 and expression analysis in plant defense responses and abiotic stress resistance

“…In addition, several calreticulins were found to play important roles in plant resistance to virus. For examples, papaya (Carica Papaya) CpCRT gene increased plant resistance to papaya ringspot virus (Shen et al, 2010), and the intra-cellular transportation of tobacco mosaic virus was suppressed in tobacco plants overexpressing a corn gene ZmCRT1 (Chen et al, 2005). These results indicated that calreticulins played key roles in plant defense responses and resistance to environmental stresses (Jia et al, 2009).…”

“…and corn ZmCRT1 in defense responses (Chen et al, 2005), but rice Q9SP22, OsCRO1 and BAG89479 are associated with cold stress resistance (Arun et al, 2004;Komastu et al, 2009). In Group II, tobacco ACH72686 and papaya CpCRT participate in plant defense responses (Lu et al, 2010;Shen et al, 2010), while tobacco NtRPL60 and caster bean AAB71420 play roles in plant growth and development (Denecke et al, 1995;Coughlan et al, 1997).…”

Cell-to-cell movement of three genera (+) ss RNA plant viruses

“…Chen et al (2005) suggested calreticulin engagement in TMV translocation between ER and PD. Calreticulin is a protein, which was localized in PD due to N-terminal signal peptide (Baluska et al 1999;Michalak et al 1999).…”

“…Calreticulin is a protein, which was localized in PD due to N-terminal signal peptide (Baluska et al 1999;Michalak et al 1999). Chen et al (2005) showed that calreticulin interacts with TMV MP and over-expression of this protein in transgenic plants determines TMV relocation from PD to microtubules and thus cell-to-cell transport of the virus. Potential role of calreticulin in PD capacity was discussed also by Boevink and Oparka (2005), who underlined the ability of PD to modify thanks to MP TMV-CRT cooperation by changes in cell wall structure.…”

Modulation of host plant immunity by Tobamovirus proteins

“…Plasmodesmata-associated kinase (PAPK) specifically phosphorylates TMV MP at its C-terminus in vitro (Lee et al, 2005). Calreticulin, a calcium-sequestering ER-resident protein, binds MP in vitro and interferes with TMV movement in overexpressing transgenic plants (Chen et al, 2005). Actin elements (Hofmann et al, 2007) and myosins (Amari et al, 2014) are required for MP-plasmodesmata interaction.…”