Plant Extracellular Vesicles

Contain Diverse Small RNA Species

and are Enriched in 10-17
Nucleotide“Tiny” RNAs
Presented by,
Athira P Anil
P V Shivaprasad’s lab
(20/12/2019)
Introduction:
siRNAs and miRNAs are two major classes small RNAs, differ in
their biogenesis and mode of action.

(Molesini et.al.,2012)

Fig.1.(a) siRNA biogenesis pathway. (b) miRNA biogenesis pathway

sRNAs are often mobile and function in non-autonomous silencing:
• Local silencing
• Systemic silencing
Local silencing involves the transport of sRNAs through
plasmodesmata.
Systemic silencing requires the sRNAs into and through the phloem.

(Dunoyer et.al.,2019)

Fig.2. Local silencing(Left) Systemic silencing(Right)

• Host-Induced Gene Silencing (HIGS)

Through this mechanism sRNAs produced in a plant cell can regulate

expression in an invading pathogen or parasite. Eg. Transfer of siRNAs
from Arabidopsis into Botrytis cinerea. But how this translocation
happens is not underdstood.

In mammals Extracellular Vesicles are known to mediate long

distance transport of RNAs.
Aim :
To determine whether plant EVs contain small RNA
molecules, and if so, their origin and potential
functions.

Plant Extracellular Vesicles:

o Most cells shed small bubbles of membrane called

Extracellular Vesicles.
o Plant Evs were reported long back in 1960.
o Their function and composition remained unknown.
o Rutter and Innes isolated and purified plant EVs for the
first time and they expected role of EV in plant immunity
based on their findings.
o The RNA content of plant Evs has not been carefully
studied yet due to challenges in purifying EVs from plant
tissue.
 (Rutter and Innes,2017)

Fig.3. Negative staining and TEM (left). Cryo-electron microscopy of EV(Right)

 Result & Discussion

Analysis of sRNA contents of Evs:

• sRNA libraries were constructed from 3 biological replicants of
Arabidopsis rosette leaves(total RNA, EVs & apoplastic wash fluids
depleated of EVs)
• Standard read trimming(adapter removal and size selection from 18-
34 nt).
Observation:
• Only 20% of reads from EV samples were retained
• This might be due to very large proportion of 10-17nt long sRNAs in
EV sample.
Size Distribution of sRNAs:

Fig.4.a. Total abundance of the reads. X axis indicates the sRNA

size and y axis indicates its proportion.
 Fig.4.b. Distinct reads. X axis indicates sRNA size and y Axis indicates
proportion.

EV samples continued to show enrichment for tyRNAs. TyRNAs in Evs

are not only abundant but also highly diverse.
Genomic origin of TyRNAs:

Fig.5.Abundances of reads mapping to nine different features of the arabidopsis

genome. (a)Abundance of tyRNAs(10-17nt) (b) Abundance of sRNA reads(18 to 34nt)

Findings:
1. Majority of tyRNAs mapped back to miRNA precursors,Tes,CDSs,
Pol4 precursors and intergenic regions in both total RNA and EV
samples.
2. TyRNAs derived from miRNA precursors was twice in abundance
with that from snRNAs,tRNAs and snoRNAs.
Analysis of relative position of tyRNAs in noncoding RNAs:

Fig.6. sRNAs(18-34nt) and tyRNAs(10-17nt) mapped to different regions of miRNA

precursors. X axis indicates regions of a miRNA precursor and the y axis
represents abundance, in reads per million.

EV tyRNAs are enriched in sequences that map to the loop region

followed by 5’ and 3’ regions.
• miRNA derived tyRNAs are largely products of the reminants after
cleavage by DCL1.

• Terminal regions of miRNA precursors are known to undergo

exonucleolytic decay.

• Degradation of leftover loop is less well examined but apparently

results in the production of tyRNAs.
Fig.7. Abundance of tyRNAs in dcl234, hen1, small DNA degrading
nucleases(sdn1/sdn2) mutants and hen1/sdn1/sdn2 triple mutant.

Evidence TAS derived tyRNAs.

Mapping 1-Hit tyRNAs to genome :
Previous analysis using only reads that map once to the
genome. These 1-hit tyRNAs have a different size
distribution of 13-17nt with majority being 16-17nt.

Fig.8. Size class distribution of 1-hit tyRNAs

Fig.9. Abundance of 1-hit tyRNAs mapping to different features of Arabidopsis
genome.
Fig.10. Abundance of tyRNAs mapping to various regions of gene.
Fig.11. Abundance of 1-hit tyRNAs mapping to different relative positions in
miRNAs. The x axis represents relative position in full length mRNA, expressed in
percentage of the total length and y axis represents abundance.
Do EVs have any preference for
small RNAs
Study on differential accumulation of sRNAs in EVs
relative to apoplast and total leaf samples:

Fig.12. A subset of sRNAs display differential abundance in EVs versus total leaf RNA or
Apoplastic RNA. Samples being compared are indicated below each heatmap.
Observations:
o Possibility of two separate pathways for secretion of sRNAs: EV
dependent & EV independent.
o While Group I & Group II miRNAs may be secreted through
either pathways, group two has slight preference for EV
dependent one.
o Group III strictly depend on EV for secretion while Group IV
miRNAs are primarily secreted via. EV-independent pathway.
o Group I phasi RNA loci had high accumulation in EV and
apoplast samples.
o Group II phasi RNA had low accumulation in EV compared with
the rest.
o None of TAS-derived sRNAs accumulated in Evs instead there is
a preferential accumulation in the apoplast. This may indicate
that phasiRNAs and tasiRNAs are preferentially to EV
independent apoplastic secretion pathway.
o hc-siRNAs had no clear pattern of accumulation in EV samples.

miRNAs have shown specificity in their EV accumulation but

no evidence of specificity in EV localization loci generating
other siRNAs analyzed here.
CONCLUSIONS:

o Evs are highly enriched in sRNAs 10-17 nt in length, named

“tinyRNAs”.

o TyRNAs appear to be degradation products from different sources.

o Some miRNAs are specifically loaded into Evs. This reinforces the
theory that sRNAs use Evs for long-distance movement through
plants and possibly cross kingdom delivery system.

o Discrepancy with Cai et. Al.(2018) regarding transfer of plant

tasiRNAs to B.cinerea involved vesicle trafficking. This might be due
to difference in isolation methods.

o Plants employ multiple mechanisms for the secretion and long

distance transport of sRNAs.
Future Directions:

o Whether tyRNAs have any biological function.

o Do EV associated RNAs play a role in plant- microbe interactions or in

intercellular communication within the plant.

o Do extracellular small RNAs that are not associated with Evs play a
role in these processes.