Geminiviruses and RNA silencing

Geminiviruses are single-stranded circular DNA viruses that cause economically

signicant diseases in a wide range of crop plants worldwide. In plants, post-
transcriptional gene silencing (PTGS) acts as a natural anti-viral defense
system and plays a role in genome maintenance and development. During the
past decade there has been considerable evidence of PTGS suppression by
viruses, which is often required to establish infection in plants. In particular,
nuclear-replicating geminiviruses, which have no double-stranded RNA phase in
their replication cycle, can induce and suppress the PTGS and become targets
for PTGS. Here, we summarize recent developments in determining how these
viruses trigger PTGS and how they suppress the induced PTGS, as well as how
we can use the system to control these viruses in plants better and manipulate
the system to study functional genomics in crop plants.

Gene silencing in plants

RNA silencing involves suppression of gene expression by sequence-specic
interaction with RNA at the posttranscriptional level in diverse eukaryotes. The
RNA silencing phenomenon was rst discovered and termed post-
transcriptional gene silencing (PTGS) in plants [1], quelling in fungi [2], and
RNA interference (RNAi) in animals [3]. There are at least three different
pathways in the gene silencing mechanism: cytoplasmic short interfering
(siRNA) silencing, silencing of endogenous mRNAs by microRNAs (miRNAs), and
DNA methylation and suppression of transcription [4]. A unifying feature of RNA
silencing is the cleavage of long double-stranded (ds) RNA into short-interfering
(2126 nt) RNAs [5] by a Ribonuclease III-like enzyme termed DICER [6]. In
plants, four Dicer-like (DCL) enzymes have been identied in Arabidopsis
thaliana [7]; however, distinct functions are dened for DCL1, which is involved
in miRNA biognesis [8]; DCL3 is required for retroelement and transposon
siRNA production and chromatin silencing, and DCL2 has been implicated in
viral siRNA production [9]. The function of DCL4 is not known. MicroRNAs are
endogenous, non-coding RNAs (1825 nt) existing both in plants and in animals
[10,11]. These small-RNA fragments serve as the specicity determinant by
being incorporated into the RNA-induced silencing complex (RISC)
endonuclease [12], which degrades mRNAs in a sequence-specic manner or
inhibits protein translation [13].
In virus-infected plants, cytoplasmic siRNA silencing, which is an anti-viral
defense response, is initiated by Corresponding author: Fauquet, C.M.
(ILTAB@danforthcenter.org). dsRNA that could be viral replication intermediates
or aberrant RNAs, or single-stranded (ss) RNAs that become dsRNA by host-
encoded RNA-dependent RNA polymerase (RdRP) [14,15]. Plant viruses are
important pathogens that are poorly controlled and whose molecular basis for
disease development is still under investigation. The majority of plant-infecting
viruses have RNA genomes, except caulimoviruses, nanoviruses and gemini-
viruses. Caulimoviruses have a dsDNA genome that replicates through an RNA
intermediate using reverse transcription [16].
Geminiviruses and nanoviruses have ssDNA genomes and replicate through a
dsDNA intermediate by a rolling circle mechanism [17]. This review covers the
recent discoveries relating to how gemini-viruses are able to trigger, become
targets and suppress the induced PTGS to be replicated and induce disease
symptoms in infected plants. In addition, we describe the efcacy of PTGS-
based strategies to counter ssDNA virus infection in plants.
Geminiviruses can both induce and become targets of gene silencing
Geminiviruses are characterized by small geminate particles (18!20 nm)
containing either one or two single-stranded circular DNA molecules of w2.7 kb
[18]. Based on genome organization, host-range and vector specicity, the
members of the family Geminiviridae are classied into four genera:
Begomovirus, Mastrevirus, Curtovirus and Topocuvirus [19]. The majority of
bego-moviruses have two components, referred to as DNA-A and DNA-B, both
of which are essential for infectivity.
DNA-A has six genes: AC1 encodes a replication-associated protein (Rep)
essential for viral DNA replication in association with host DNA polymerase
[20]; AC2 encodes a transcription activator protein (TrAP) [21]; AC3 encodes a
replication enhancer protein (REn) [22]; AV1 and AV2 encode coat protein and
pre-coat protein, respectively [23]; but no function has been attributed to AC4-
encoded protein in relation to virus multiplication. DNA-B has BV1 and BC1
genes that encode a nuclear-shuttle protein (NSP) and movement protein (MP),
respectively [24] (Figure 1). Monopartite begomoviruses with a single genomic
component equivalent to DNA-A have been identied, such as isolates of
Tomato yellow leaf curl Sardinia virus (TYLCSV) [25]. In addition, ssDNA satellite
DNA-b molecules identied in association with monopartite begomoviruses are
involved in symptom enhancement [26].
In these viruses, DNA replication is accomplished through a rolling circle
mechanism with a dsDNA intermediate, and gene transcription is bi-directional
from the common region [20]. Recently, TYLCSV, a monopartite begomovirus
[27], and ve distinct cassava-infecting bipartite begomoviruses, namely
African cassava mosaic virus from Cameroon (ACMV-[CM]), East African cassava
mosaic Cameroon virus (EACMCV), EACMV from Uganda (EACMV-[UG]), Sri-
Lankan cassava mosaic virus (SLCMV) and Indian cassava mosaic virus (ICMV)
were shown to trigger the PTGS system in infected plants with the production
of virus-specic siRNAs [28]. It is puzzling how these viruses that do not have a
dsRNA phase in their replication cycle can trigger PTGS in plants.
The possibility that the bi-directional transcription of these viruses with
transcripts occurring from opposite polarity [29] overlap at their 30-ends was
conrmed using strand-specic probes; the dsRNA formed in this way partly
explains how these viruses induce PTGS in infected plants [28] (Figure 2a).
Alternatively, the early and abundant AC1-transcript of these viruses could
serve as the template for the host RdRP to make dsRNAs to induce the system
[14]. Yet another possibility is that the strong fold-back structure of geminivirus
transcripts could simply become a template for DICERs to cleave at specic
locations and produce siRNAs [28] (Figure 1) In plants, some virushost
interactions naturally lead to host recovery. The natural recovery responses
induced by a nepovirus [30] and a caulimovirus [31] are similar to RNA-
mediated virus resistance. This symptom recovery phenomenon is unusual for
geminiviruses. However, in ACMV-[CM]-infected N. benthamiana and cassava,
and SLCMV-infected cassava, symptom recovery has been observed; in both
cases, mosaic formation and leaf distortion in the systemically infected leaves
were observed for 23 weeks after inoculation, after which the plants recovered
from the infection symptoms. This recovery phenomenon is associated with the
production of virus-derived siRNAs beginning one week post-inoculation and
becoming abundant in the newly developed symptom-less recovered leaves,
both in N. benthamiana and cassava. This increase in virus-derived siRNA
accumulation was accompanied by a reduction in the levels of both viral DNA
and mRNA accumulation [28]. More-detailed analysis of the composition of
siRNAs has revealed that the majority of siRNAs in ACMV-[CM]-infected plants
were derived from the DNA-A component, particularly a region that corresponds
to the C-terminus of AC1 that overlaps with the N-terminus of the AC2 gene
(Figure 2b). Therefore, it is conceivable that in ACMV-[CM]-infected plants, the
presence of virus-specic siRNAs down-regulates the corresponding mRNAs,
which in turn affects the viral replication and its further movement. As a result,
there is a reduction in both virus titer and symptom intensity in the newly
developed leaves [28].
Symptom recovery is not a usual plant response to virus infection. Virus-
derived siRNAs implicating virus-induced PTGS has been shown for RNA viruses
[5] and for geminiviruses such as TYLCSV, EACMCV, EACMV-UG and ICMV, even
though the infected plants do not recover. Although non-recovery type cassava
geminiviruses (EACMCV, EACMV-[UG] and ICMV) are able to trigger the host
PTGS system, the amount of siRNA production was less than 10% compared
with the amount that accumulated with ACMV-[CM], the recovery type virus. A
reverse correlation was found between virus-derived siRNAs and the levels of
viral DNA and mRNA accumulation in EACMCV-infected plants. Moreover, the
EACMCV-induced PTGS mostly targets the DNA-B component (Figure 2c),
suggesting that it has a much weaker effect on virus replication. These ndings
indicate that the ongoing silencing of viral RNAs can occur in successful virus
infections and might not be sufcient to lead to a recovery or that these viruses
encode strong silencing suppressors.
In addition, virushost interactions are strongly modied by environmental
factors, particularly by temperature. Molecular analysis of Cymbidium ring spot
virus (CymRSV) has revealed that low temperature (158C) inhibits RNA
silencing-mediated defense by controlling siRNA generation. By contrast, RNA
silencing was activated with an increase in siRNA accumulation with rising
temperature (278C) [32]. In ssDNA viruses, irrespective of the recovery and
non-recovery type viruses, there is a general trend for symptom decrease with
high temperature, indicating that ssDNA viruses follow the same trend as RNA
viruses (P. Chellappan et al., unpublished).
Geminiviruses can suppress the induced RNA silencing
In recent years, 29 RNA silencing-inhibiting proteins that counter the antiviral
RNA silencing have been identied in several plant and animal viruses [3335].
Three distinct phases have been identied in the RNA silencing process:
initiation, maintenance and systemic signaling or the effector step [36]. These
suppressor proteins do not share homology at either sequence or viral
functional levels; however, these identied suppressor proteins might target
similar or different steps of the RNA silencing pathway.
Recently, our group conducted a comprehensive analysis of representatives of
four distinct species of cassava-infecting geminiviruses that belong to recovery
(ACMV-[CM] and SLCMV) and non-recovery (EACMCV and ICMV) type viruses.
An Agrobacterium leaf inltration assay using GFP transgenic N. benthamiana
revealed that AC4 of ACMV-[CM] and SLCMV have the capacity to suppress the
induced-PTGS [37]. A previous study had indicated that AC2 of ACMV-[KE] was
a suppressor of silencing [38]. However, in our study, AC2 of ACMV-[CM]
showed little suppression activity (20%) compared with AC4, which showed
strong activity (72%). By contrast, the AC2 of EACMCV and ICMV, the non-
recovery type viruses, were identied as suppressors of induced RNA silencing
[37] (Figure 3). In monopartite Tomato yellow leaf curl China virus (TYLCCNV),
the C2 protein, a positional homolog of AC2, was found to possess silencing
suppression activity [39]. In addition, TYLCCNV-C2 requires the nuclear
localization signal, DNA binding and zinc-nger motif for anti-PTGS activity
[40,41]. AC2 of bipartite geminiviruses shares a considerable sequence
homology (5688%), and they cluster according to a geographical distribution
of 92 begomoviruses in a similar manner to the rest of the genome (C.M.
Fauquet, unpublished). Therefore, the AC2 PTGS-suppressors (AC2C) are closely
related to the AC2 non-PTGS-suppressors (AC2K).
By contrast, the AC4 is highly variable among bipartite geminiviruses and, to
date, no denitive and universal function has been attributed to this gene
product, which is expressed from an embedded open reading frame (ORF) into
the Rep ORF. There is much less sequence homology (2558%) between the
PTGS-suppressors (AC4C) and non-suppressors (AC4K). In monopartite
begomoviruses and curtoviruses, C4, a positional homolog of AC4, is a major
determinant of pathogenesis [42,43]. The identication of two types of
geminiviral PTGS suppressor proteins, AC2 and AC4, (Figure 3), implies that
these proteins play different roles and as a consequence might target different
steps in the silencing pathway, or might interact with different host proteins,
which all contribute to the suppression of RNA silencing.
Synergism and PTGS suppression
Mixed viral infections occur frequently with biological and epidemiological
implications in nature. Synergistic viral diseases in plants have been known for
a long time; recently it has been suggested that synergism can occur A PTGS
strategy to control geminiviruses Initially it was thought that engineering
transgenic plants that express viral proteins would confer protection against
the virus. In some studies, the expression of proteins has been responsible for
virus resistance but in several cases the resistance was demonstrated to occur
at the RNA level [51]. A natural recovery from ACMV-[CM] infection and its
correlation with high levels of virus-derived siRNA accumulation support PTGS
as a strategy to control geminiviruses effectively [28]. In plants, transcriptional
silencing of a promoter is evident because of the intracellular signaling
between the cytoplasm and the nucleus in which RNA degradation is typically
correlated with methylation of cognate DNA in the nucleus [52]. In
geminiviruses, a transgene (GUS) driven by the TLCV promoter was silenced
upon homologous virus infection but not with heterologous virus infection; this
phenomenon occurs because of the hypermethylation of the TLCV-derived
promoter sequences [53]. However, transient expression of long dsRNA
targeting the promoter region of Mungbean yellow mosaic virus-Vigna showed
recovery from virus infection [54]. Furthermore, a PTGS-based strategy to
control virus replication was demonstrated when plant cells simultaneously
transfected with ACMV-[CM] and with a synthetic siRNA designed to target the
AC1 gene of the virus showed a reduction in the accumulation levels of AC1
mRNA by more than 90% and viral DNA by 70% compared with controls [55].
Subsequently, transgenic expression of AC1-dsRNA revealed reliable resistance
to Cotton leaf curl virus in tobacco [56] and TYLCV-[Is] in tomato [57].
By contrast, for the monopartite TYLCSV, N-Rep expressing lines conferred
resistance to homologous and heterologous TYLCV and it seems that it did not
use the PTGS system [27]. Cassava plants stably integrated with multiple
copies of a mutated version of ACMV-AC1 gene induced transgene silencing
whereas transgene-derived siRNAs expressing plant-lines imparted high levels
of resistance to homologous ACMV as well as to two heterologous
cassava-infecting geminiviruses (EACMCV and SLCMV), particularly to
the synergistic mixture ACMV-[CM] and EACMCV (Figure 4) [58]. Although
sequence similarities are low across the genomes of the three geminiviruses
species studied here, there is greater similarity within the AC1 coding sequence
itself (6677%). Therefore, the strong cross-protection imparted by integrated
AC1 sequences could be because of a population of siRNAs produced from the
silenced transgene that have high homology across representatives of several
cassava geminivirus species. In addition, computer analysis over a length of
22-nt revealed that 12 siRNAs and 11 siRNAs generated by the ACMV
transgene would have 100% identity with SLCMV and EACMCV, respectively. If
a tolerance of one mis-matched nucleotide is considered, the predicted number
of siRNAs with homology to SLCMV is 117 and 68 for EACMCV [58]. Hence, it is
concluded that AC1 gene-based RNAi appears to be a highly promising strategy
for developing virus resistance.

Virus-induced gene silencing and endogenous gene inactivation Virus-induced

gene silencing (VIGS) is a type of RNA silencing that is initiated by viral vectors
carrying portions of host genes that can suppress gene expression in plants by
degrading the homologous transcripts [59]. Although geminiviruses have
genome size constraints for movement, the dispensability of CP (for some
viruses) for viral infection has allowed them to be used as silencing vectors.
Initially, Dominique Robertsons group elegantly demonstrated ssDNA-based
VIGS. The CP gene (AV1) slot of the TGMV [60,61] and the Cabbage leaf curl
virus [62] have been used to carry inserts ranging from 100800 bp
representing either phytoene desaturase (PDS), an enzyme required for
carotenoid pigment production, or a gene encoding a component of the
magnesium chelatase complex (ChlI), which is required for chlorophyll
production, to generate silencing vectors. More-detailed analysis has revealed
the requirement of cellular RdRP for DNA-VIGS, but not for RNA-VIGS, in
Arabidopsis [63]. Similarly, the ACMV-[CM] has been shown to carry PDS, ChlI
and CYP79D1 and CYP79D2 (involved in linamarin synthesis), and induce
silencing in infected N. benthamiana and cassava [64]. In addition, a satellite
ssDNA-b associated with the TYLCCNV-isolate Y10 is a silencing vector [65]. In
conclusion, DNA-VIGS is equally effective as RNA-VIGS and hence can be used
efciently as silencing vectors. In general, use of viral-vectors to silence host
genes is more advantageous than making transgenic plants because this
requires an enormous amount of time and energy to study the functional
genomics.
Conclusions and future prospects
RNA silencing is an ancient cellular defense mechanism conserved across
kingdoms. It is understandable that viruses using dsRNA, during replication
would have developed mechanisms to counteract this plant defense system.
Geminiviruses, in spite of being ssDNA viruses with no dsRNA phase in their
replication cycle, have been shown to be involved in the gene silencing
pathway. They are able to trigger the host PTGS system with the production of
specic virus-derived siRNAs and have evolved an active mechanism to
counteract silencing. The uniqueness of geminiviruses is that they encode at
least two types of PTGS suppressor proteins such as AC2 and AC4, indicating
that these viruses evolved differently with regard to interaction with the host.
Moreover, synergism between ACMV and EACMCV is because of the differential
and complementary activity of two PTGS suppressor proteins. Geminiviruses
could be subject to transcriptional gene silencing when their promoters are
integrated in the plant genome. These viruses and their satellites can be used
as effective silencing vectors in a variety of host plants and therefore could be
useful in post-genomic studies. However, there are still many basic questions
that remain unanswered. The rst, most intriguing question relates to how do
geminiviruses trigger the RNA silencing mechanism without having a dsRNA in
their replication cycle? There are some hypotheses, such as overlapping
transcripts, abundance of mRNAs or secondary folding of the viral messengers,
but no direct evidence. How do the different suppressor proteins regulate PTGS
and their spatial and temporal expressions? Do these suppressors interfere
with the miRNA pathway? What kind of host protein interacts with the viral
suppressors? The answers to these questions should give us a better
understanding of virus-induced disease in plants.
An increasing number of geminiviruses, and the satellites associated with
them, are devastating crops and therefore we need control measures to
enhance food production. Manipulating the viral sequences to identify highly
conserved and effective sequences that can target multiple viruses and their
PTGS-suppressors could potentially control many heterologous viruses in a
single attempt. This would pave the way to being able to control geminivirus
disease complexes, which are continuously emerging as a threat to crop
production.
Figure 1. A model depicting geminivirus-induced RNA silencing in plants. The
geminiviral replication cycle is shown in the nucleus of the cell. RNAi is
triggered by dsRNAs generated from overlapping, abundant and folded forms
of mRNAs. An enzyme called DICER cleaves long dsRNA into siRNAs. An RNA-
induced silencing complex (RISC) then distinguishes between the different
strands of the siRNA. The sense-strand (depicted in blue) is degraded. The anti-
sense strand (depicted in red) is used to target genes for silencing. The anti
sensestrand(redcolor),boundtoanRdRPenzyme,canpairwithacomplementarymR
NAandactasastart ointforthesynthesisofanewlongdsRNA.DICERis then required
to generate transitive siRNA (depicted in green), which are specic to different
sequences on the same mRNA. All these processes lead to mRNA degradation.
Figure 2. Identication of transcript overlap and origin of siRNAs-derived from
ACMV-[CM] and EACMCV infected plants. (a) Transcript overlap at the 30-ends
of CP and AC3 genes in ACMV-[CM]. Existing transcript overlap is indicated by
the green box. RdRP-mediated possible transcript extensions are indicated with
broken lines. Genome organization of (b) ACMV-[CM] DNA-A and DNA-B and (c)
EACMCV DNA-A and DNA-B. Arrows indicate the direction of ORFs (arrows
pointing forwards represent virion-sense ORFs and arrows pointing backwards
represent complementary-sense ORFs). The components of the genome were
PCR-amplied into 400-bp fragments (x-axis shows a total of seven fragments
for each component), and probed with puried virus-derived siRNAs that were
(b) ACMV-[CM]-specic and (c) EACMCV-specic.
Figure 3. Identication of cassava mosaic virus-encoded PTGS suppressors.
Symptom severity of cassava plants (from left to right): non-infected healthy
control plant; plant infected with ACMV-[CM]; plant infected with EACMCV; dual
infection with both viruses that leads to synergistic severe disease. The middle
line indicates the presence of AC2 and the bottom line indicates the presence
of AC4-type PTGS suppressor proteins. EACMCV-AC2 (EAC2; middle red panel)
and ACMV-[CM]-AC4 (AAC4; bottom red panel) are positive for PTGS
suppression. Synergism is explained in part by dual suppression of PTGS (red
panels on the right). Green left panels represent frame-shift mutants of ACMV-
AC4 (mAAC4) and EACMCV-AC2 (mEAC2) that lost the capacity to suppress
PTGS. AAC2 is considered negative as PTGS suppression was weak, and EAC4 is
not a PTGS suppressor.
 Figure 4. Geminivirus control via gene silencing-based strategy. (a) Non-
transgenic control plant and (b) AC1-transgenic plant. The plants were
challenged with a dual inoculation of ACMV and EACMV