Application of phiLOV2.1 as a fluorescent marker for visualization of Agrobacterium effector protein translocation

Abstract

Agrobacterium tumefaciens can genetically transform plants by translocating a piece of oncogenic DNA, called T-DNA, into host cells. Transfer is mediated by a type IV secretion system (T4SS). Besides the T-DNA, which is transferred in a single-stranded form and at its 5' end covalently bound to VirD2, several other effector proteins (VirE2, VirE3, VirD5, and VirF) are translocated into the host cells. The fate and function of the translocated proteins inside the host cell are only partly known. Therefore, several studies were conducted to visualize the translocation of the VirE2 protein. As GFP-tagged effector proteins are unable to pass the T4SS, other approaches like the split GFP system were used, but these require specific transgenic recipient cells expressing the complementary part of GFP. Here, we investigated whether use can be made of the photostable variant of LOV, phiLOV2.1, to visualize effector protein translocation from Agrobacterium to non-transgenic yeast and plant cells. We were able to visualize the translocation of all five effector proteins, both to yeast cells, and to cells in Nicotiana tabacum leaves and Arabidopsis thaliana roots. Clear signals were obtained that are easily distinguishable from the background, even in cases in which by comparison the split GFP system did not generate a signal.

Agrobacterium VirE2 Protein Modulates Plant Gene Expression and Mediates Transformation From Its Location Outside the Nucleus

“…The protoplasts were imaged 16 h later using confocal microscopy (Supplementary Figure 1). Consistent with data from previous studies (Bhattacharjee et al, 2008;Grange et al, 2008;Lee et al, 2008;Li et al, 2014Li et al, , 2020Shi et al, 2014;Li and Pan, 2017;Lapham et al, 2018;Roushan et al, 2018), VirE2-Venus localized to the cytoplasm (Supplementary Figure 1A); however, VirE2-Venus-NLS localized to the nucleus (Supplementary Figure 1B). Although we have not precisely identified where in the cytoplasm VirE2-Venus localizes, it does not localize to the nucleus, and for convenience we shall hereafter refer to the subcellular localization of VirE2-Venus as "cytoplasmic.…”

“…In the plant cell, VirE2-GFP complexes formed filamentous structures mainly in the cytoplasm and with a few that appeared within the nucleus. Roushan et al (2018) used phiLOV2.1 to tag VirE2 internally and showed that, when transferred from Agrobacterium, the protein localized to the cytoplasm of Arabidopsis roots and Nicotiana tabacum leaves. Li et al (2020) further demonstrated that only very small amounts of VirE2 could be detected in the nucleus in the presence of VirD2 and T-strands, solving the conundrum of conflicting results from different laboratories.…”

Agrobacterium expressing a type III secretion system delivers Pseudomonas effectors into plant cells to enhance transformation

“…1 ). Previously, PhiLOV has been used to tag T3Es of animal pathogens to monitor effector secretion and translocation 52 and to visualize translocation and localization of A. tumefaciens virulence proteins 53 . In another study, a split GFP system was used to monitor the secretion of T3Es of P. syringae and R. solanacearum 29 , 30 .…”

Myosin VIII and XI isoforms interact withAgrobacteriumVirE2 protein and help direct transport from the plasma membrane to the perinuclear region during plant transformation

“…However, the subcellular localization of VirE2 remains controversial. Early reports demonstrated nuclear localization of VirE2 (Citovsky et al, 1992), but other studies from several laboratories indicated that VirE2 localizes in the cytoplasm (Grange et al, 2008; Gelvin, 2010; Roushan et al, 2018). A recent report claimed that a small amount of VirE2 can enter the nucleus, but only in the presence of VirD2 and T-strands (Li et al, 2020).…”