Study of viral suppressors of RNA silencing is a powerful tool to unravel the mechanism of RNAi

1. VIRAL SUPPRESSORS OF
RNA SILENCING
J.Shamyuktha
2018631508
Dept. of Plant pathology
TNAU

2. Contents
Introduction
RNA Silencing and its mechanism
Viral Suppressors
Functional assays
Mechanism of suppression
Future directions

3. Introduction
• Virus infection usually starts with transmission by vector.
• The virus replicates in the initially infected cell and then
moves into adjacent cells, spreading from cell to cell until it
enters the vascular system, which allows rapid movement to
distant parts of the plant.

4. RNA SILENCING
• RNA interference (RNAi) is a mechanism that inhibits
gene expression at the stage of translation or by
hindering the transcription of specific genes.
• RNAi targets include RNA from viruses and
transposons.
(Mlotshwa et al., 2002)

5. MECHANISM OF RNA SILENCING
1.Sensing and processing viral
RNAs to viral siRNAs
2.Amplifying vsiRNAs
3.Assembling antiviral RISC and
targeting viral mRNAs.

6. The process is initially triggered by dsRNA, which can be
introduced experimentally or arise from
Endogenous transposons
Replicating RNA viruses
Transcription of transgenes.
STEP:1

7. The dsRNA trigger is cleaved by a ribonuclease III (RNAse III)-like
enzyme termed Dicer into 21–24 nucleotide duplexes termed
short-interfering RNAs (siRNAs).
ATP-dependent
step
(Voinnet et al., 2014)
It also involves interaction with
other proteins ,including
i. An argounaute like protein
ii. A dsRNA binding protein and
iii.RNA helicase
STEP:2

8. siRNAs have distinctive characteristics:
They represent both polarities and have two nucleotide 3’ overhangs
with 5’ phosphate and 3 hydroxyl groups
In another ATP-dependent step ,
the siRNAs are denatured and incorporated
into a multi-subunit endonuclease silencing
complex called RNA-induced silencing
complex(RISC)
STEP:3

9. Within the activated RISC, single-stranded siRNAs act as
guides to bring the complex into contact with
complementary mRNAs and thereby cause their
degradation.
(Bologna et al., 2014)
STEP:4

10. miRNA
• Originate from capped & polyadenylated full length
precursors(pri-miRNA)
• Hairpin precursor ~70 nt (pre-miRNA) Mature miRNA ~22
nt(miRNA)

11. Viral Suppressors
• RNA silencing is an innate antiviral immunity response of plants
and animals.
• To counteract this host immune response, viruses have evolved an
effective strategy to protect themselves by the expression of viral
suppressors of RNA silencing(VSRs).
(Csorba et al., 2016)

12. The various VSRs are able to target all effectors of the
silencing pathway, such as
• Viral RNA recognition,
• Dicing,
• RISC assembly,
• RNA targeting and amplification

13. VSRs are multifunctional
Coat proteins,
Replicases,
Movement proteins,
Helper components for viral transmission,
Proteases or
Transcriptional regulators.

15. VSRs suppress RNA silencing pathways mainly:
• Through dsRNA binding.
• Sequestering siRNA duplexes prevents small RNA loading into the
AGO effector proteins to assemble the RNA-induced silencing
complex (RISC).
• Sequestering long dsRNA duplexes inhibits siRNA biogenesis
• By interacting with and inhibiting components of the host RISC
machinery.
• By inhibiting enzymes necessary for host cell-mediated DNA
methylation.

16. Functional assays
• Three major approaches have been widely used to identify
plant viral suppressors of RNA silencing:
Transient
expression
assays,
The reversal
of silencing
assay,
Stable
expression
assays.

17. Transient expression assays
Agrobacterium co-infiltration
• Rapid and easy test of suppressor activity and is currently the technique
most commonly used to identify viral suppressors
• The method makes use of a commonly used bacterial pathogen of plants,
Agrobacterium tumefaciens
The Agrobacterium serves two purposes:
• One strain is used to induce RNA silencing of a reporter gene (usually green
fluorescent protein (GFP))
• Another strain is used to express the candidate suppressor.
(Llave et al., 2000)

18. The overall strategy is to co-infiltrate mixtures of the two bacterial strains
(one acting to induce silencing, the other to suppress it) into a plant leaf and
then examine the infiltrated patch over time for silencing of the reporter.
(A) Assay for suppressors of local silencing.

19. (B) Assay for suppressors of systemic silencing.
(Ruiz et al., 1998)

20. (Johansen and Carrington., 2001).

21. Reversal of silencing assay
• Used to identify candidate viruses that may suppress
silencing.
Overall strategy
• To infect a silenced plant with the candidate virus and
determine whether the silenced phenotype is reversed
• The most common version of this technique uses the GFP-
expressing transgenic N. benthamiana line .

22. (Voinnet et al., 1999)

23. Stable expression assays
• A stable transgenic line expressing a candidate suppressor of
silencing (usually initially identified in one of the previous two
assays) is crossed to a series of well-characterized transgenic
lines silenced for a reporter gene
Advantage:
• Examine the effect of the suppressor on different well-defined
types of transgene-induced RNA silencing.
• Providing information about the mechanism of suppression.
(Anandalakshmi et al., 1998)

24. (A) Genetic crosses with silenced transgenic lines.

25. • The stable expression assays are also well suited to
investigate the role of suppressors in systemic silencing using
grafting
• In these assays, the ability of a plant to send a mobile
silencing signal is assayed by grafting an expressing line onto
the top of it (the bottom plant is called the rootstock and the
expressing plant grafted onto the top is called the scion)
(Palauqui et al., 2001)
Contd.,

26. Role of suppressors in systemic silencing using
grafting
(Mallory et al., 2001)

27. GENUS VIRUS SUPPRESSOR EVIDENCE REFERENCE
Carmovirus Turnip
crinkle virus
(TCV)
CP TCV infection does not reverse silencing. In
agro-co infiltration assay, CP blocks sense
and antisense induced local silencing and
prevents systemic silencing.
Qu et al., (2003)
Closterovirus Beet yellows
virus (BYV)
p21 Suppresses inverted repeat (IR) induced
local silencing in agro-coinfiltration assay
Reed et al., (2003)
Cucumovirus Cucumber
mosaic virus
(CMV)
2b Infection with CMV or with PVX-2b vector
blocks silencing.
Li et al., (2002)
Geminivirus Tomato
yellow leaf
curl virus-
China
(TYLCV-C)
AC2 Blocks sense induced silencing in agro-
coinfiltration assay.
Dong et al., (2003)
Tombusvirus Tomato
bushy stunt
virus (TBSV
P19 Limited activity in reversal of silencing;
strong activity in agro-coinfiltration
Voinnet et al.,
(2003)

28. GENUS VIRUS SUPPRESSOR EVIDENCE REFERENCE
Potexvirus Potato virus X (PVX) p25 PVX infection does not
suppress silencing. In
agro-co infiltration, p25
blocks systemic but not
always local silencing
Hamilton et al.,2002
Potyvirus Potato virus Y (PVY) HC-Pro Does not block systemic
silencing in stable
expression grafting
assay, but does in agro-
coinfiltration assay
Mallory et al.,2001
Tombusvirus Tomato bushy stunt
virus (TBSV
P19 Limited activity in
reversal of silencing;
strong activity in agro-
coinfiltration
Voinnet et al., (2003)

29. Mechanism of suppression
• Conflicting results from different assays have made it
difficult to draw firm conclusions for many suppressors.
• The currently known suppressors share no obvious
similarities at either
 The nucleic acid or
 The protein level
perhaps reflecting differences at the mechanistic level as well.

31. Two major classes of suppressor
action
SUPPRESSOR
Affects
Small RNA metabolism Systemic Silencing
1. P19
2. HC-Pro
1. HC-Pro
2. CMV-2b

32. Suppressors that affect small RNA metabolism
• Many suppressors reduce the accumulation of siRNAs,
raising the possibility that silencing is blocked at the step at
which Dicer processes the dsRNA that triggers silencing.
• Thus, it may be that many suppressors prevent silencing by
blocking production of the siRNAs that provide the
sequence specificity of the process.

33. TOMBUSVIRUS P19
• RNA silencing suppressor p19 (also known as Tombusvirus P19
core protein and 19 kDa symptom severity modulator) is a
protein expressed from the ORF4 gene in the genome of
tombusviruses
• Tomato bushy stunt virus and
• Carnation Italian ringspot virus
The protein dimer
formed by two p19
molecules
• P19 is a star, blocking both local and systemic
silencing and apparently eliminating all small
RNAs.
• Following the elucidation of its role as a
suppressor of RNA silencing, p19 has also been
used as a tool in molecular biology research on
RNA silencing, RNA interference, and related
processes (Silhavy et al., 2002)

34. P19 binds siRNAs and that binding depends on characteristics of
RNase III products (dsRNAs with two nucleotide 3’ overhangs)
P19 suppresses silencing by sequestering siRNAs, thereby
preventing their incorporation into the RISC complex to serve as
guides
FUNCTIONS-Local silencing

35. This is a novel mechanism among suppressors, and because it
theoretically stems from an intrinsic property of the protein to
bind functional siRNAs, it is possible that P19 could interfere with
silencing in a broad range of different plants
“Molecular caliper"
(Silhavy et al., 2002)

36. Suppressors that affect systemic silencing
• Systemic silencing can be assayed in either transient expression
experiments or in experiments with stably transformed
transgenic lines.
• Many suppressors have been demonstrated to block systemic
silencing in at least one of these assays. CMV 2b primarily
targets systemic silencing because it blocks movement of the
signal in many assays, but has a lesser effect on local silencing

37. Cucumber mosaic virus (CMV) 2b-Systemic
Silencing
• Stable expression assays and grafting experiments provided an
elegant demonstration that 2b blocks the movement of the
systemic silencing signal
• The stable expression assays made use of a well-characterized
silenced tobacco line called 6b5
(Guo and Ding, 2002)

39. How does CMV 2b protein block the movement of
the mobile silencing signal?
• Either sequesters or inactivates the signal in the phloem
stream
• 2b localizes to the nucleus suggests that the suppressor acts
indirectly, perhaps by activating one or more processes that
subsequently affect the signal.
(Lucy et al., 2008)

40. • The virus infection triggers the enhanced expression of AGO1
mRNA and, consequently, the AGO1 proteins as a part of the
host defence respond.
• The enhanced AGO1 level facilitates the formation of vsiRNA-
loaded RISCs.
• Recent work has revealed a new strategy for antiviral silencing
suppression through the specific induction of miR168, which is
ubiquitous in plant-virus infections
• Eg. Cymbidium ringspot virus
TOMBUSVIRUS P19-Systemic infection
(Varallyay et al., 2010)

41. • However, when the P19 VSR is expressed by the virus it
induces the overexpression of miR168, which presumably
loads into AGO10 and arrests the translation AGO1 mRNA.
• Thus, P19 VSR, in addition to its siRNA sequestering ability,
ensures that the loading of viral siRNAs onto AGO1 is
efficiently inhibited, thereby allowing successful systemic
infection.
Cont.,

42. Repressing AGO1 through the specific induction
of miR168 by the tombusvirus P19 protein
(Varallyay et al., 2010)

43. HC Pro - Poty viral helper-component
protease
• HC-Pro was the first identified suppressor of RNA silencing.
• The original reports demonstrated that it suppresses both
transgene- and virus-induced silencing
• In contrast to CMV 2b protein, it is able to reverse
established silencing in the reversal of silencing assay
• Although HC-Pro alone has suppressor activity, it is
frequently expressed as the proteinase 1 (P1)/HC-Pro
polyprotein in suppression of silencing studies.
(Anandalakshmi et al., 2000)

44. HC-Pro and small RNAs
• HC-Pro has been reported to alter the
accumulation of several classes of
small RNAs.
• In stable expression assays in tobacco,
HC-Pro has been reported to suppress
three classes of transgene-induced
RNA silencing, in each case interfering
with the accumulation of siRNAs
(Xie et al., 2004)

45. HC-Pro and systemic silencing

46. Potato virus X (PVX) p25
• PVX is the only potexvirus for which a suppressor of RNA
silencing has subsequently been characterized.
• In grafting experiments or with movement defective forms
of PVX, could not detect systemic silencing unless the 25
kDa viral movement protein (p25) was made non-functional.
(Voinnet et al., 2000)

48. RNA silencing suppression by satellite viruses
• The CP of TCV suppresses RNA silencing in N. benthamiana.
• TCV CP is a weak suppressor of RNA silencing when assembled
into the intact virions but a strong suppressor when expressed
freely in the cytosol.
• The satC associated with TCV can reduce the accumulation of
virions, thereby increasing the level of free CP, which leads to
the suppression of RNA silencing and subsequent exacerbation
in symptom severity.
(Manfre et al., 2008)

49. Suppresses RNA silencing :
The CP of TCV suppresses RNA
silencing in N. benthamiana by
obstructing the Dicer-like protein
DCL2/DCL4 silencing pathway
(Manfre et al.,2008)

50. • Beet curly top virus (BCTV), the model species for the Curtovirus
genus, is able to infect the widest range of plants among Gemini
viruses
• BCTV V2 acts as a potent PTGS suppressor, possibly by impairing
the RDR6(RNA-dependent RNA polymerase 6)/suppressor of gene
silencing 3 (SGS3) pathway.
(Luna et al.,2017)

53. RESULTS:
Taking into account all the results obtained in this work, we
conclude that inspite of limited sequence homology, BCTV V2, as
with its begomo virus counterpart:
(i) is required for a systemic infection,
(ii) accumulates in the nucleus and the ER, and
(iii) is a strong suppressor of intracellular PTGS by impairing the
RDR6/SGS3 pathway without impacting on local cell-to-cell
silencing movement.
(Luna et al., 2017)

54. Do all plant viruses have suppressors of
silencing?
• Although many different viral suppressors have been
identified, which suppress the RNA silencing.
• Some viruses may have evolved other ways to try to avoid
silencing, such as by replicating within spherules in the ER
• where the dsRNA is hidden,
• or by replicating and
• moving rapidly enough to outrun the mobile silencing signal.
(Schwartz et al., 2002)

55. FUTURE DIRECTIONS
• Viral suppressors often target RNA-silencing pathways such as
siRNAs or effectors such as AGO and DCL proteins, in some cases a
single VSR can target more than one element in the silencing
pathway
• It is possible that many other VSRs interact in multiple ways with
RNA-silencing pathways, which remain to be discovered.

56. • There are still several gaps in our knowledge regarding the
effectors of plant silencing machinery
• The replication, subcellular localization and regulation of the
expressions of viral genes including VSRs, which are still not
known for many plant viruses.

57. Conclusion

58. REFERENCE
• Braden M. Roth, Gail J. Pruss , Vicki B. Vance,2004. Plant viral
suppressors of RNA silencing. Science Direct. Virus Research 102.
97–108
• Baulcombe,2004. RNA silencing in plants. Nature 431, 356–363
• Jozsef Burgya´n and Zolta´n Havelda, 2011. Viral suppressors of
RNA silencing. Trends in Plant Science , Vol. 16, No. 5 268
• Varallyay, et al., (2010) Plant virus-mediated induction of miR168 is
associated with repression of ARGONAUTE1 accumulation. EMBOJ.
29, 3507–3519

59. • Luna et al., 2017. V2 from a curtovirus is a suppressor of post-
transcriptional gene silencing. Journal of General Virology.
98:2607–2614
• Csorba, T. et al., 2010. Polerovirus protein P0 prevents the
assembly of small RNA-containing RISC complexes and leads to
degradation of ARGONAUTE1. Plant J. 62, 463–472
• Guo, H.S. and Ding, S.W. 2002. A viral protein inhibits the long
range signaling activity of the gene silencing signal. EMBO J. 21,
398–407