Cross-Kingdom Communication via Plant-Derived Extracellular Vesicle Nucleic Acids in Genetically Engineered Nicotiana tabacum

Abstract

Background/objectives: Plants release extracellularly lipid bilayer-enclosed vesicles of nanometric size that can be retrieved in their fluids. Plant-derived extracellular vesicles (PDEVs) have mostly been involved in modulating host-pathogen interaction, making them a tool for cross-kingdom communication with a key role in plant immunity. In addition, PDEVs have demonstrated promising therapeutic features, not only in terms of intrinsic nutraceutical properties but also of active molecules' delivery. Transgenic plants have been developed for a variety of purposes, i.e., to improve their functional properties like crops, but also to produce therapeutic molecules. However, it is unclear whether transgenes can end up in PDEVs, thus making them a vehicle for their cross-kingdom diffusion into the environment.

Methods: Here, we investigated the association of transgenic DNA and RNA with PDEVs secreted by tobacco (Nicotiana tabacum) engineered to express the neomycine phosphotransferase II (Npt-II) gene. PDEVs were isolated from leaf apoplastic fluid by ultracentrifugation and characterized for their morphology and size. The association of DNA and RNA was assessed by qRT-PCR and their immunomodulatory properties by assaying PDEVs-induced IL1β and IL10 on THP1 monocytes.

Results: Npt-II RNA, but not DNA, could be amplified from PDEVs, whereas no differences were observed between wt and transgenic tobacco PDEVs in terms of immunomodulatory properties.

Conclusions: Although a different behaviour by other types of RNAs or DNAs could still be possible, our findings indicate that in this model, PDEVs are not associated with transgenic DNA, but they can protect RNA, including transgenic RNA, from degradation, contributing to their cross-kingdom spreading.

Keywords: Nicotiana tabacum; cross-kingdom communication; extracellular RNA; plant-derived extracellular vesicles.

Figure 1

Analysis of the NPT-II gene in tobacco leaves. DNA was extracted from leaves, amplified by PCR with NPT-II.for and NPT-II.rev primers, and run on 1.5% agarose gel. Lanes 1 is a negative control, loaded with the amplification product from wt tobacco, while lane 2 is loaded with the amplification product of Npt-II tobacco. Lane M, 100 bp DNA ladder.

Figure 2

Characterization of PDEVs released from Npt-II tobacco. (A,B) Scanning electron microscopy images of PDEVs. Samples were fixed using 2.5% glutaraldehyde and then allowed to dry at room temperature onto glass coverslips. (A) PDEVs 40 K fraction; (B) PDEVs 100 K fraction. (C) Nanoparticle tracking analysis of PDEVs of the 100 K fraction. Samples were resuspended in 0.22 μm filtered PBS and then loaded into a NS300 Malvern instrument. Data are reported as particles/mL, with respect to size.

Figure 3

Analysis of the ITS, Npt-II, and rps amplicons in DNA and RNA isolated from leaf and 100 K fraction PDEVs of Npt-II tobacco. DNA was extracted from leaf (indicated with A) or from 100 K fraction PDEVs (indicated with B) isolated from the apoplastic fluid obtained from 15 g of leaves, amplified by PCR, and run on 1.5% agarose gel. cDNA was reverse-transcribed from RNA extracted from leaf (indicated with C) or from 100 K fraction PDEVs (indicated with D) isolated from the apoplastic fluid (~15 g of leaves), amplified by PCR, and run on 1.5% agarose gel. The ITS amplicon is indicated with 1, the Npt-II amplicon with B, and the rps amplicon with 3. Lane M, 100 bp DNA ladder.

Figure 4

Gene expression of IL-10 and IL-1β in response to administration of PDEVs isolated either from wild-type (WT) or NPT-II transgenic (TG) tobacco. THP-1 cells were stimulated for 24 h with approximately 0.8 µg (WT 1 and TG 1) or 2.4 µg of PDEVs (WT2 and TG 2), diluted in 1 mL of cell culture medium. LPS (100 ng/mL) was used as control. Relative gene expressions with respect to untreated THP-1 cells (CTRL) are displayed. The analysis was repeated four times in triplicate. The mean ± SE is reported (* p < 0.05; ** p < 0.01).