Plant Virus Interaction - Orelvis

Generalidades sobre las interacciones planta-virus.
Sistema inmune en plantas
Orelvis Portal Villafaña, José Efraín González

Ramírez, Dariel Cabrera Mederos
Año 2022
Mecanismos moleculares de defensa de las plantas a los
virus
⚫ Inmunidad innata
⚫ Silenciamiento de ARN
⚫ Represión de la traducción de proteínas
⚫ Resistencia viral dominante atípica
⚫ Degradación de proteínas mediante ubiquitación y autofagia
⚫ Regulación de la traducción de ARN

Wu et al. (2019)
Plant Innate Immunity
Microbial/pathogen-associated molecular patterns (MAMPs/PAMPs)

“Remember that all models are wrong; the practical question is

what errors must be in order not to be useful” (George E.P. Box)
What are the limitations of the Zig-Zag exhibition model?
1. Molecular scope (pathogenesis concept)

2. Absence of environmental context
3. The ordering of events
4. The time scale
5. Physical scale
6. The model is qualitative
Zvereva and Pooggin (2012)

Cucumber mosaic virus P6

RNA interfering in Plants
Timeline of RNA interfering
Types of RNA
RNA silencing pathway
RNA silencing movement
Overcoming Silencing. RNAi suppressors
Silencing and simptoms
Role of temperature in RNA silencing
Application of gene silencing in plants
Conlcuding remarks
1990 - First description of post-transcriptional gene silencing (PTGS) in plants
They attempted to overexpress chalcone synthase (CHS) in pigmented petunia

petals by introducing a chimeric petunia CHS gene. Unexpectedly, the
introduced gene created a block in anthocyanin biosynthesis. The mechanism
responsible for the reversible co-suppression of homologous genes in trans is
unclear, but the erratic and reversible nature of this phenomenon suggests the
possible involvement of methylation.
Napoli, C., Lemieux, C. and Jorgensen, R. (1990) Introduction of a chimeric

chalcone synthase gene into Petunia results in reversible co-suppression of
homologous genes in trans. Plant Cell 2: 279-289.
Wild type PTGS

Strongly pigmented compounds
1992 – Fungi (Quelling)
Up to 36% of Neurospora crassa transformants showing an albino phenotype

were recovered by transforming a wild-type strain with different portions of the
carotenogenic albino-3 (al-3) and albino-1 (al-1) genes.
Romano, N. and Macino, G. (1992) Quelling: transient inactivation of gene

expression in Neurospora crassa by transformation with homologous
sequences. Molecular Microbiology 6: 3343-3353.
Antisense Technology
- Antisense technology has been used for ~25 years.

- Based on introducing an antisense gene (or antisense RNA) into cells or
organisms to try to block translation of the sense mRNA.
-The “antisense effect” was probably due to RNAi rather than inhibiting
translation
1995 - First noticed that sense RNA was as effective as antisense RNA for
suppressing gene expression in the worm Caenorhabditis elegans
Guo, S. and Kemphues, K.J. (1995) par-1, a gene required for establishing
polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is
asymmetrically distributed. Cell 81: 611-620.
Is a free-living,
transparent nematode
(roundworm), about
1 mm in length, which
lives in temperate soil
environments.
C. elegants
1995 - The phenomenon of PTGS had been observed in all three Kingdoms of
multicellular organisms
Understanding of the cellular processes responsible for PTGS (now known as

RNA interference, or RNAi) exploded in 1998 with the discovery that it was
triggered by double-stranded RNA (dsRNA), a discovery that won Fire and
Mello the 2006 Nobel Prize in Physiology or Medicine.
Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E. and Mello, C.C.
(1998) Potent and specific genetic interference by double-stranded RNA in
Caenorhabditis elegans. Nature 391:806-811.
Andrew Z. Fire Craig C. Mello

1998 - Mick Graham and his colleagues at CSIRO Plant Industry established the
role of double-stranded RNA in triggering PTGS in plants
Waterhouse, P.M., Graham, M.W. and Wang, M.-B. (1998) Virus resistance and
gene silencing in plants can be induced by simultaneous expression of sense
and antisense RNA. Proceeding of National Academic of Science USA 95: 13959-
13964.
1999 - It was found that PTGS in plants was accompanied by short RNA
molecules whose sequence corresponded to parts of the mRNA (messenger
RNA) of the silenced gene
PTGS is a nucleotide sequence-specific defense mechanism that can target both

cellular and viral mRNAs.
Short RNA molecules were of uniform length, estimated at 25 nucleotides, and
their accumulation required either transgene sense transcription or RNA virus
replication. Thus, the 25-nucleotide antisense RNA is likely synthesized from an
RNA template and may represent the specificity determinant of PTGS.
Hamilton, A.J. and Baulcombe, D.C. (1999) A species of small antisense RNA in
posttranscriptional gene silencing in plants. Science 286: 950-952.
2000 – Rnase III (Dicer) processes dsRNA into 21-23-nt
Zamore et al. (2000) RNAi: double-stranded RNA directs the ATP-dependent

cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101: 25-33.
2000 - The proteins comprising the silencing complex began to be teased out
and were found to bear striking resemblances between all organisms
These results raise the possibility that PTGS, or at least some of its elements,
could participate in the regulation of gene expression during development in
plants.
Fagard, M., Boutet, S., Morel, J.-B., Bellini, C. and Vaucheret, H. (2000) AGO1,
QDE-2, and RDE-1 are related proteins required for post-transcriptional gene
silencing in plants, quelling in fungi, and RNA interference in animals.
Proceeding of National Academic of Science USA 97: 11650-11654.
2001 – Dicer is Cloned
The RNase III enzyme that is evolutionarily conserved and contains helicase
and PAZ domains, as well as two dsRNA-binding domains.
Bernstein et al. Role for a bidentate ribonuclease in the initiation step of RNA
interference. Nature 2001 409: 363-366.
2002 - In recognition of the overwhelming importance of RNAi as a biological

process and a universally applicable tool, the leading journal Science
announced it "The breakthrough of the year, 2002."
Meanwhile, work continued on dissecting the cellular complex that catalyses

RNAi…
Many excellent reviews of RNAi are available, among which are the
following:
Sen, G.L. and Blau, H.M. (2006) A brief history of RNAi: the silence of the
genes. FASEB Journal 20: 1293-1299.
The use of the RNAi pathway to eliminate gene products has greatly
facilitated the understanding of gene function. Behind this remarkable
pathway is an intricate network of proteins that ensures the degradation of
the target mRNA. In this review, they explore the history of RNAi as well as
highlighting recent discoveries
Kodama, H. and Komamine, A. (2011) RNAi and plant gene function analysis:
methods and protocols. Humana Press (Springer). 210 pp
Feng, X., Fanga, B. and Qia, Y. (2016) RNAi in Plants: An Argonaute-Centered

View. The Plant Cell 28: 272–285
Rosa, C. et al. (2018) RNA Interference Mechanisms and Applications in Plant

Pathology. Annual Review of Phytopathology 56: 581-610.
Types of RNA
Coding: messenger RNA (mRNA).

Non-coding:
- Ribosomal RNA (rRNA).
- Transfer RNA (tRNA).
- Small nuclear RNA (snRNA): includes spliceosomal RNAs.
- Small nucleolar RNA (snoRNA): most known snoRNAs are involved in rRNA
modification.
- Short interfering RNA (siRNA): active molecules in RNA interference;
degrades mRNA.
- Micro RNA (miRNA): short RNAs that regulate gene expression in eukaryotes
by translational inhibition or cleavage of complementary mRNAs. In plants,
miRNAs are known to target mostly transcription factors and are implicated in
diverse aspects of plant growth and development.
RNA silencing pathways
- dsRNA is cleaved into small

fragments of 20–25 bp pairs
by a ribonuclease called DICER.
- siRNA fragments are separated to give
the guide strand, which is
complementary to the target mRNA and
the helper strand, which is further
degraded.
- The guide strand is then incorporated
into the RNA-induced silencing complex
(RISC), which targets it to the cognate
mRNA, forming a duplex with that RNA.
- The cognate mRNA strand
complementary to the bound siRNA is
cleaved by the slicer activity of RISC to
give siRNA fragments.
Transcribed from endogenous gene as pri-miRNA, which is transcribed

from intergenic and intronic regions of the host genome as stem-loop
structure with imperfect base-pairing .
- Primary miRNA: long with multiple hairpins.
- Imperfect internal sequence complementarity.
- pri-miRNA is identified drosha due to its high affinity to
hairpin loops.
Pre-miRNA is exported to the cytoplasm by Exportin 5
Processed by the RNase III enzyme Drosha into a ~70 nt hairpins → Pre-
miRNA.
-Cleaved by Dicer into the mature miRNA
-Symmetric 2nt 3’ overhangs, 5’ phosphate groups
miRNA
Results of the System
- Target RNA degradation: Processing of the cognate viral RNA or mRNA

targeted by si- or mi-RNA in the RISC.
-Translational repression: Prevention of translation of mRNA due to

binding by siRNA (or miRNA).
-Transcriptional repression : Epigenetic effect caused by DNA methylation

or remodelling of the chromatin because of the presence of siRNA. There
is circumstantial evidence for methylation of both geminiviral DNA and of
modification of chromatin structure.
Components of the System
dsRNA
Dicer
Products of Dicer
RISC
Sources of
dsRNA
A. Replicating ssRNA virus; note

that replicative intermediate
and secondary structure in the
ss form can be targets.
B. Replicating dsRNA virus.
C. Retro- and pararetro-virus. The
secondary structure in the
terminal repeats (TR) can be a
target.
D. Replicating ssDNA virus. The
overlapping ends of transcripts
from complementary strands can
be targets.
E. Viroids.
F. Structure of a hairpin construct
for transformation into plant to
give protection against the virus.
The branched model
Vaucheret et al. (2001)

- 200 kDa family of proteins that usually contain an ATPase/RNA Dicer

helicase domain, a PAZ domain, two RNaseIII domains, and a C-
terminal dsRNA binding domain.
- The distance between the two RNase III domains determines the size
of the siRNA that it produces.
- Dicer progressively cleaves dsRNA at 21–25 bp intervals
to generate siRNA with 2-nt 3’ overhangs and phosphorylated 5’
termini.
- Dicer cleaves pri-miRNA to give 20–25 bp fragments with blunt ends.
- There are various dicers that have distinct roles; those from plants
are called dicer-like (DCL).
Products
siRNA: Twenty to 25 nucleotide dsRNA fully base-paired of Dicer
with a 2 nucleotide 3’ overhang. It is complementary only
to the dsRNA from which it arose.
miRNA. Twenty-one to 23 nucleotide dsRNA produced

from priming miRNA.
A. Structure of pri miRNA.
B. Diagram of the structure

of an siRNA showing a 19–21
base-pair RNA duplex with 2
nucleotide 3’ overhangs:
OH, 3’ hydroxyl; P, 5’
phosphate.
RISC
- RNA-induced silencing complex (RISC) includes, besides the si-
or mi-RNA, the members of the Argonaute (AGO) protein
family and various accessory factors.
- AGO proteins are 100 kDa and have a PAZ domain and a PIWI
domain, which is related to RNaseH endonuclease and
functions in slicer activity.
- Various AGO proteins are probably associated with the
variations of the basic silencing pathway.
- Arabidopsis encodes 10 putative AGOs, with AGO1 being
involved in the RISC.
The silencing of a virus can spread systemically throughout the plant

after induction in the initially infected cell.
- Viral primary 21 nt siRNA produced by the dicer/RISC system in the
initially infected cell moves through plasmodesmata to about 10–15
surrounding cells either with or ahead of the infection front .
- The host RNA dependent RNA polymerase amplifies the siRNAs to give
ds forms, which are then processed into secondary siRNAs. These then
move to surrounding cells and are amplified.
- This reiterative process moves the siRNA signal to the vascular tissue
from where it, possibly associated with small-RNA binding proteins, is
distributed to other parts of the plant.
- This systemic movement of the silencing signal ahead of viral movement
primes the defense response prior to the arrival of the virus.
UV phenotypes of RNA silencing of a GFP transgene in N. benthamiana
(A) Not silenced; (B) spontaneous short-range local silencing; (C) induced local
silencing; (D) fully silenced; (E) systemic silencing; (F) systemic (centre and right-
hand leaves) and extensive local silencing (left-hand leaf).
Kalantidis et al. (2008)

Overcoming Silencing
Viruses and viroids have developed two basic strategies to suppress RNA
silencing:
- Suppression of silencing
Protein suppressors
Nucleic acid suppressors
- Avoidance silencing
Protein suppressors
- Viral suppressors often also have other functions and frequently are
identified as “pathogenicity determinants.” However, these other
functions, such as replication enhancement, determining virus movement,
and symptom production, can be attributed to the suppression of
silencing.
- Silencing suppressors bind dsRNA, some binding both long and short
dsRNAs and others just binding ds siRNA like p19 of tombusviruses, p20 of
closteroviruses, p15 of Peanut clump virus, TGB1 of Barley stripe mosaic
virus, and HC-Pro of potyviruses.
- Silencing suppressors inhibit the assembly of RISC by competing more
efficiently for the ds siRNA than the RISC assembly complex.
- HC-Pro, p19, and p21 suppressors bind 21-nt siRNA duplexes more
efficiently than 24-nt siRNA duplexes; however, p21 and HC-Pro require a
2-nt 3’ end overhang, whereas p19 does not.
- Not all suppressors function by binding ds RNA and thereby inhibiting the
assembly of RISC. For instance, 2b of Cucumber mosaic virus interacts with
Argonaute 1 protein in RISC and inhibits its cleavage activity.
Silencing suppressors can inhibit the key components of RNA-silencing

pathways.
Song et al. (2011)

Silencing suppressors can recruit endogenous negative regulators of RNA

silencing.
Song et al. (2011)

Some silencing suppressors act by interacting with molecules from host

cells.
Song et al. (2011)

Song et al. (2011)

LIiet al. (2001)
Nucleic acid suppressors
Viroids do not encode any proteins. Yet, the finding of viroid-specific siRNAs
shows that their highly structured RNAs are processed by the silencing
pathway. That viroids successfully infect plants indicates that they must be
able to suppress the silencing. It is suggested that the secondary structure
may also have the property of suppressing silencing of the replication of, at
least, some viroids.
Viroids are plants pathogens that consist of a short stretch of highly complementary,
circular, single-stranded RNA without the typical viral coat protein.
Avoidance of Silencing
For some viruses no suppressor of silencing has yet been identified. The
susceptible stage in the viral replication cycle to the silencing
defense system is when dsRNA is exposed at stages such as RNA
replication or translation.
Some viruses avoid exposure to the defense system by replicating in

inaccessible sites, such as vesicles. Furthermore, if a virus replicates and
expresses rapidly and then safely encapsidated its genome, it may
outcompete the defense system.
Silencing and symptoms
Although some symptoms are attributable to silencing and suppression not

all are. Replication, movement, and accumulation of viruses in plants can
cause upsets to the physiology and metabolism of a plant.
- Recovery
- Dark Green Islands and Mosaics
- miRNA
- siRNA effects
- Synergistic effects
Recovery
A virus-infected plant, especially with Nepoviruses, may show severe

viral symptoms on the inoculated and first systemically infected leaves;
however, new growth appears in which symptoms are milder or
absent.
For other viruses, such as Alfalfa mosaic virus, the virus content in the plant
can increase and decrease with concomitant changes in symptoms
sometimes in a cyclical manner. These recovery phenomena likely reflect
changes in the balance between silencing and suppression.
The threshold model
Waterhouse et al. (1999)

Recovery
Recovery in tobacco
plants infected with
Tobacco ringspot virus
Dark green islands and mosaics
One of the most common symptoms of virus

infection is a mosaic of light and dark green
areas on the leaves. The mosaic pattern
develops in leaves that are undergoing cell
division and leaf expansionand can include
well-defined dark green areas in which there
is no detectable virus and they are
resistant to subsequent virus infection.
It would appear that they result from one or a

small group of cells in which there is silencing but
no suppression and that divide to form a discrete
tissue zone.
miRNA
As well as suppressing siRNA silencing, protein suppressors can

affect the miRNA pathway.
Not all suppressors impact upon this pathway, and not all miRNAs
are affected.
However, it is likely that the perturbation of the miRNA pathway
leads to some symptom production. There is evidence for this in
that some transgenic plants expressing suppressors of silencing
have the phenotype of virus infected plants even though there is
no virus present.
siRNA effects
Transgenic tomato plants expressing portions of Potato spindle tuber

viroid contained viroid-specific siRNAs and had a phenotype
similar to the symptoms of viroid infection.
This suggests that the siRNAs might be responsible for the symptom
production possibly by targeting plant genes for RNA silencing.
Synergistic Effects
- The effects of the combined infection by two viruses are more severe than that
of either of the constituent viruses alone. It can be caused by the infection with
two viruses that belong to the same genus or by two viruses from different
genera. The silencing suppressor of one virus can affect the replication of
another virus in a joint infection leading to synergistic effects.
- African cassava mosaic virus (UgACMV) is caused by synergism between two
begomoviruses: African cassava mosaic virus (ACMV) and East African cassava
mosaic virus (EACMV). The synergistic effect is mediated by the differential and
complementary suppression of silencing by the AC4 gene of ACMV and the AC2
gene of EACMV.
- The joint infection of tobacco plants with PVX and PVY is characterized by
severe veinal necrosis in the first systemically infected leaves. Leaves showing
this synergistic reaction contain up to 10 times as much PVX as with single
infections but only the same amount of PVY. The HC-Pro silencing suppressor
from PVY is responsible for the increase in the amount of PVX.
Szittya et al. (2003)

Applications
To protect the genome from viruses (or other invading DNAs or RNAs)
- Antiviral defense
- Suppress transposon activity
- Response to aberrant RNAs
Genetic tool: Probing gene function
Advantages
- Highly gene specific
- High gene silencing efficiency
- Screening targeted plants takes less time
- Highly inducible (heat shock gene promoter)
- Fast
Disadvantages
- It does not knockout a gene for 100%
- siRNA tends to activate unwanted pathways
- Expensive
Balcoumbe et al. (1999)

Summary of features of VIGS vectors
Burch-Smith et al. (2004)

Comparison of VIGS with other functional genomics approaches

Wang et al. 2001; Welse et al. (2001)

• The RNA-silencing defence system is found in eukaryotes.

• The defence system is activated by dsRNA that is cleaved to give small RNA
molecules that target the cognate RNA.
• There are two main sources of dsRNA: perfectly base-paired from viruses,
transposons, transgenes, and aberrations in transcription, which are processed
to give small interfering (si)RNA; and imperfectly base-paired priming-micro
(pri-mi)RNA, which is processed to give micro (mi)RNAs that control host
development.
The dsRNAs are processed through three pathways that have a common basic
section that cleaves the dsRNA and targets the si- or miRNA to its cognate
mRNA.
• The three pathways lead to degradation of mRNA, translational repression of
mRNA, and transcriptional repression. Defense against RNA viruses is usually
by the first pathway and against DNA viruses by the first and third pathways.
The second pathway is frequently used by miRNA.
• In plants the silencing response spreads systemically and primes the defense
ahead of the virus infection front.
• Viruses encode suppressors of silencing that act as a counterdefence against
the silencing system.
Muchas Gracias
Dr. José E. González: jefrain@nauta.cu

Dr. C. Orelvis Portal: orelvispv@uclv.cu

Plant Virus Interaction - Orelvis

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Generalidades sobre las interacciones planta-virus.

Sistema inmune en plantas

Orelvis Portal Villafaña, José Efraín González

⚫ Represión de la traducción de proteínas

⚫ Resistencia viral dominante atípica

⚫ Degradación de proteínas mediante ubiquitación y autofagia

⚫ Regulación de la traducción de ARN

Microbial/pathogen-associated molecular patterns (MAMPs/PAMPs)

“Remember that all models are wrong; the practical question is

What are the limitations of the Zig-Zag exhibition model?

1. Molecular scope (pathogenesis concept)

Zvereva and Pooggin (2012)

Cucumber mosaic virus P6

Timeline of RNA interfering

RNA silencing pathway

RNA silencing movement

Overcoming Silencing. RNAi suppressors

Silencing and simptoms

Role of temperature in RNA silencing

Application of gene silencing in plants

1990 - First description of post-transcriptional gene silencing (PTGS) in plants

They attempted to overexpress chalcone synthase (CHS) in pigmented petunia

Napoli, C., Lemieux, C. and Jorgensen, R. (1990) Introduction of a chimeric

Wild type PTGS

1992 – Fungi (Quelling)

Up to 36% of Neurospora crassa transformants showing an albino phenotype

Romano, N. and Macino, G. (1992) Quelling: transient inactivation of gene

- Antisense technology has been used for ~25 years.

Understanding of the cellular processes responsible for PTGS (now known as

Andrew Z. Fire Craig C. Mello

PTGS is a nucleotide sequence-specific defense mechanism that can target both

2000 – Rnase III (Dicer) processes dsRNA into 21-23-nt

Zamore et al. (2000) RNAi: double-stranded RNA directs the ATP-dependent

2001 – Dicer is Cloned

2002 - In recognition of the overwhelming importance of RNAi as a biological

Meanwhile, work continued on dissecting the cellular complex that catalyses

Feng, X., Fanga, B. and Qia, Y. (2016) RNAi in Plants: An Argonaute-Centered

Rosa, C. et al. (2018) RNA Interference Mechanisms and Applications in Plant

Coding: messenger RNA (mRNA).

RNA silencing pathways

- dsRNA is cleaved into small

Transcribed from endogenous gene as pri-miRNA, which is transcribed

Results of the System

- Target RNA degradation: Processing of the cognate viral RNA or mRNA

-Translational repression: Prevention of translation of mRNA due to

-Transcriptional repression : Epigenetic effect caused by DNA methylation

Components of the System

A. Replicating ssRNA virus; note

The branched model

Vaucheret et al. (2001)

- 200 kDa family of proteins that usually contain an ATPase/RNA Dicer

miRNA. Twenty-one to 23 nucleotide dsRNA produced

A. Structure of pri miRNA.

B. Diagram of the structure

The silencing of a virus can spread systemically throughout the plant

Kalantidis et al. (2008)

Silencing suppressors can inhibit the key components of RNA-silencing

Song et al. (2011)

Silencing suppressors can recruit endogenous negative regulators of RNA

Song et al. (2011)

Some silencing suppressors act by interacting with molecules from host

Song et al. (2011)

Song et al. (2011)