Plant Roots Release Small Extracellular Vesicles with Antifungal Activity

Abstract

Extracellular Vesicles (EVs) play pivotal roles in cell-to-cell and inter-kingdom communication. Despite their relevant biological implications, the existence and role of plant EVs released into the environment has been unexplored. Herein, we purified round-shaped small vesicles (EVs) by differential ultracentrifugation of a sampling solution containing root exudates of hydroponically grown tomato plants. Biophysical analyses, by means of dynamic light scattering, microfluidic resistive pulse sensing and scanning electron microscopy, showed that the size of root-released EVs range in the nanometric scale (50-100 nm). Shot-gun proteomics of tomato EVs identified 179 unique proteins, several of which are known to be involved in plant-microbe interactions. In addition, the application of root-released EVs induced a significant inhibition of spore germination and of germination tube development of the plant pathogens Fusarium oxysporum, Botrytis cinerea and Alternaria alternata. Interestingly, these EVs contain several proteins involved in plant defense, suggesting that they could be new components of the plant innate immune system.

Keywords: Alternaria; Botrytis; Fusarium; biocargo; extracellular vesicles; fungal pathogens; plant-pathogen interactions; proteomics; root exudate; tomato.

Figure 1

Experimental design used for collection and characterization of small Extracellular Vesicles (EVs) released by tomato roots. (A) Twenty-day-old tomato plants after removal from the nutrient solution and before transfer to ultrapure water for 2 days for the collection of root exudates (B) Schematization of the experimental design for plant growth, root exudate collection and EVs isolation and characterization.

Figure 2

Physical characteristics of root-derived extracellular vesicles. (A) EVs size distribution curve obtained by dynamic light scattering (DLS). (B) Electron micrograph of three EVs. Scale bar, 100 nm. (C) Large view of density-sensitive backscattered electron imaging showing monodispersed EVs together with some aggregates. Scale bar, 100 nm. (D) Close-up view image of (C) showing the outer lipid layer with lower density with respect to the inner nano-sized structure. Scale bar, 30 nm.

Figure 3

Gene Ontology analysis (Biological Process) of the proteome of tomato root-released EVs.

Figure 4

Tomato root-derived EVs have antifungal activity against plant pathogens. (A) Inverted microscope micrographs (20× magnification, Leica DMi8) of Fusarium oxysporum, Botrytis cinerea and Alternaria alternata spore germination at 24 h post inoculation (hpi) with increasing concentrations of the EVs. Scale bar, 50 μm. (B) Average spore germination (n = 3) (%) for F. oxysporum, B. cinerea and A. alternata at 48 hpi of treatment with increasing concentrations of the EVs. EV resuspension buffer was added to the growth medium of spores of the control sample. Results are representative of three independent experiments. * indicate significance with p < 0.05 by using one way ANOVA Tukey post hoc test.