A comparison of two methods of microinjection for assessing altered plasmodesmal gating in tissues expressing viral movement proteins

Abstract

SummaryUsing two different microinjection methods the effect of transgenic expression of the movement proteins (MPs) of tomato spotted wilt virus (TSWV) and tobacco mosaic virus (TMV) on plasmodesmal size exclusion limit (SEL) was assessed. By pressure mediated injection of fluorescent probes of different molecular mass, in both TSWV and TMV MP-transgenic tissues a similar enlargement of the plasmodesmal SEL was observed, in line with the general conception that alteration of plasmodesmal gating is an intrinsic activity of viral MPs. Strikingly, when iontophoretic injection of the same probes was performed, the plasmodesmal SEL was found to be decreased in transgenic tissues expressing either MP. As iontophoresis is a less invasive technique than pressure injection, these different results may indicate that increase of plasmodesmal SEL in the presence of viral MPs, as measured by the broadly applied pressure injection technique, may not reflect a genuine biochemical activity of these viral proteins. Rather this increase may be the consequence of disturbance of the plasmodesmatal structure due to the application of pneumatic pressure as additional stress on plasmodesmata which have become more prone to disruption by the presence of these viral proteins.

Plasmodesmata targeting and intercellular trafficking of Tomato spotted wilt tospovirus movement protein NSm is independent of its function in HR induction

“…The NSm protein of TSWV has typical characteristics of plant viral movement protein, including localization into plasmodesmata (PD) (Feng et al, 2016;Kormelink et al, 1994), modification of size-exclusion limit of PD (Prins et al, 1997;Storms et al, 1998), tubule formation (Storms et al, 1995), RNA-binding activity and interaction with N protein (Soellick et al, 2000). Using a heterologous Tobacco mosaic virus (TMV)-expression system, domains and amino-acid residues of the TSWV NSm protein necessary for tubule formation, intercellular movement and symptom †These authors contributed equally to this work.…”

An RNA-dependent RNA polymerase gene of a distinct Brazilian tospovirus

“…The M RNA encodes a nonstructural cell-to-cell movement protein in v sense and the envelope glycoproteins (Gn and Gc) precursor in vc sense [6,7]. The L RNA differs from the other two genomic segments since it has a negative polarity and encodes a single ORF (protein L) with motifs found in RNA-dependent RNA polymerases (RdRp) [8].…”

Disruption of Microtubule Organization and Centrosome Function by Expression ofTobacco Mosaic VirusMovement Protein

“…This protein modifies the size exclusion limit of Pd (70,88,95) and binds nucleic acids in vitro, which led to the suggestion that the protein may chaperone vRNA to form a viral ribonucleoprotein (vRNP) complex whose size and structure are compatible with transport through the modified Pd (16,17). Studies to elucidate the pathway by which MP may target viral RNA to Pd have used MP in fusion with the green fluorescent protein (GFP) and shown that, in both plant cells and protoplasts, MP interacts with the cytoskeleton (32,57) and with elements of the endoplasmic reticulum (ER) (33,77).…”