Page 68 - Read Online

Page 99 Rutter et al. Extracell Vesicles Circ Nucleic Acids 2023;4:90-106 https://dx.doi.org/10.20517/evcna.2023.04

RNA CONTENT
Much of the excitement surrounding EVs is related to their ability to shuttle RNA molecules between cells.
Multiple studies have reported the presence of both coding and non-coding RNAs associated with
mammalian EVs . The majority of EV RNAs are small (< 200 nt), including microRNAs (miRNAs),
[91]
structural RNAs and fragments of ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), messenger RNAs
[92]
(mRNAs) and long non-coding RNAs (lncRNAs) . While much attention has been paid to the presence of
miRNAs inside mammalian EVs as disease biomarkers and potential therapeutic molecules, miRNAs are
often underrepresented in EVs compared to other species of non-coding RNAs [91,92] and are enriched at
higher levels in non-vesicular, extracellular particles, such as the recently defined exomeres and
supermeres [93,94] . Other larger species of RNAs have also been detected in EV samples, including full-length
mRNAs, lncRNAs and circular RNAs (circRNAs) .
[95]

Fungal EVs are also associated with a rich assortment of RNAs. While some of these RNAs are long,
including full-length mRNAs and lnRNAs, the majority are small (< 250 nt) and non-coding, including
microRNA-like sequences, small RNAs (sRNAs), small nucleolar RNAs (snoRNAs), small nuclear RNAs
(snRNAs), tRNAs and fragmented rRNAs . The presence of so many non-coding RNAs (ncRNAs)
[96]
suggests that fungal EVs may regulate intercellular protein synthesis and could be utilized by pathogenic
fungi to interfere with host cell processes .
[96]

Some of the most abundant species of small RNAs in fungal EVs are milRNAs . Similar to miRNAs in
[96]
plants and animals, milRNAs are typically generated from the cleavage of a precursor stem-loop RNA by
Dicer-like (DCL) proteins and loaded onto an Argonaute (AGO) protein to negatively regulate the
[97]
expression of mRNAs . While miRNAs and milRNAs are similar in their biogenesis, there are at least four
distinct pathways for the formation of fungal milRNAs compared to the single, canonical miRNA pathway
[97]
found in plants or animals . Additionally, milRNA precursors can be transcribed by RNA Polymerase III
instead of by RNA Polymerase II, as in plants and animals .
[98]
Small RNAs such as milRNAs are thought to promote the virulence of fungal phytopathogens. milRNAs
have been predicted in multiple fungal phytopathogens, including Sclerotinia sclerotiorum, Fusarium
oxysporum, Puccinia striiformis f. sp. tritici, Z. tritici and Verticillium dahliae [99-104] . These milRNAs can
regulate virulence-related genes within the fungus. For example, in V. dahliae, the milRNA VdMILR1
downregulates the essential fungal virulence factor VdHy1 through histone methylation . Other milRNAs
[102]
are predicted to enhance host susceptibility through trans-kingdom gene silencing. For example, in
Fusarium oxysporum f. sp. lycopersici (Fol), Fol-milR is exported into tomato cells, where it enhances
susceptibility to the fungus by downregulating the expression of the tomato CBL-interacting protein kinase
[104]
SlyFRG4 . Similarly, in Puccinia striiformis f. sp. tritici, Pst-milR1 regulates the expression of predicted
wheat pathogenesis-related 2 (PR2) protein and positively contributes to the virulence of the fungus .
[101]
Only one study so far has examined the RNA contents of EVs from a phytopathogenic fungus [Table 1].
Kwon et al. isolated EV RNA from the biotrophic maize pathogen U. maydis. The study generated poly-A
enriched RNA libraries from both mock- and RNase-treated EVs to rule out the presence of extra-vesicular
RNAs . The majority of EV RNAs were < 200 nt in length and likely comprised tRNAs and fragmented
[57]
mRNAs and rRNAs. Due to the way in which the libraries were generated, the study did not comment on
the presence of small ncRNAs, but it was able to identify several EV-enriched, full-length coding sequences.
These transcripts appear to be selectively packaged into EVs and were enriched for biological gene ontology
(GO) terms related to metabolic processes, proteaosomal degradation, vesicle-mediated transport,
cytokinesis and iron uptake . The study also identified transcripts for nine bona fide effectors as well as 161
[57]