[Show abstract][Hide abstract] ABSTRACT: Summary Using 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.