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Page 2 Cai et al. Extracell Vesicles Circ Nucleic Acids 2023;4:262-82 https://dx.doi.org/10.20517/evcna.2023.10

Keywords: Extracellular vesicles, small RNAs, cross-kingdom RNA interference, plant-microbial interaction, spray-
induced gene silencing
INTRODUCTION
Extracellular vesicles (EVs) were first observed in plants and mammals in the mid-1900s, half a century
before it would be known that these vesicles are a universally conserved and impactful form of inter-cellular
and inter-organismal communication. The first mammalian observation of extracellular vesicles was made
[1]
in 1946 when platelet-derived particles were detected in human plasma . In the following decades, there
were numerous scattered observations of mammalian EVs in vivo and in cell culture . However,
[2]
throughout the 1900s, these extracellular particles were often considered to be cellular debris, a term that
historically suggested that these particles represented cellular waste . This "debris" designation spurred a
[3]
decades-long delay in the development of EV research, but since the turn of the century, this field has
quickly expanded to show the diversity and influential functions of EVs on cellular development and
interactions.

Research on plant- and microbial-derived EVs has undergone the same slow development as the study of
mammalian EVs, although in recent years, there has been a surge of new studies characterizing EVs from
plant and microbial cells, showing the same scope of diversity and biological significance as mammalian
EVs. In cotton plants, intraluminal vesicles in multivesicular bodies were first observed in 1965 . This was
[4]
followed by the observation in carrots that multivesicular bodies could fuse with the plasma membrane and
release vesicles into the extracellular space, leading to the discovery of plant extracellular vesicles . In the
[5]
early 2,000s, studies of plant and microbial EVs using transmission electron microscopy revealed the
accumulation of vesicles at plant-bacteria and plant-fungus interaction sites . This inspired a new field of
[6-8]
research into the role of plant and microbial EVs in the trafficking of materials not only between cells within
an organism, but also between different organisms and kingdoms of life.

Numerous classifications and diverse roles of EVs have been characterized in mammalian systems. EVs can
be isolated from plasma, serum, urine, and various other physiological fluids [9,10] . They are small lipid-
encapsulated nanoparticles that carry cargoes, such as RNAs [e.g., small RNAs (sRNAs), messenger RNAs
(mRNAs), transfer RNA (tRNA), and long non-coding RNAs], DNA fragments, lipids, and proteins [9,11-13] .
EVs transport these biomolecules between cells and play an important role in cell-to-cell
communication [9,14] . In addition, EVs have emerged as important disease biomarkers and have potential
applications in therapeutics and diagnostics [15-17] . Among all cellular origins, including mammalian, plant,
and microbial cells, EVs can be broadly classified into either exosomes or microvesicles based on their
biogenesis [3,10,18] . Exosomes are small EVs (30-150 nm in diameter) originating from the endosomal organelle
multivesicular bodies (MVBs). Microvesicles, also denoted as ectosomes, are heterogeneous and larger EVs
(100-1,000 nm in diameter) generated by direct budding from the plasma membrane [19,20] . In contrast to
exosomes and microvesicles, mammalian cells also produce several types of vesicle bodies associated with
different forms of cell death . These include necroptotic vesicles released from necroptotic cells [21,22] ,
[3]
pyroptotic inflammasomes released during pyroptosis of cells , and apoptotic bodies (1-5 µm in diameter)
[23]
produced during apoptotic cell disassembly [24-26] . Research on EVs from plants and microbes has progressed
less than research in mammalian systems due to the limitations in EV isolation and detection methods .
[27]
Mammalian EVs are frequently isolated from cell cultures or body fluids such as blood, breast milk, urine,
lung fluid, and semen [2,28] . Since these mammalian samples are fluids, extraction is relatively straightforward.
In contrast, extraction of EVs from plants is more challenging due to the small amount of extracellular fluid
and the extensive procedures required for EV isolation in plants.