In plants, RNA interference constitutes an important defense mechanism against viruses, transposo... more In plants, RNA interference constitutes an important defense mechanism against viruses, transposons, and transgenes. Viruses, on the other hand, use suppressors to counteract silencing. In contrast to mammalian systems, silencing in plants spreads systemically through the whole organism. Since also viruses spread and consequently produce suppressors systemically, a race between silencing and virus replication occurs. Apparently, successful viruses win the race,
ABSTRACT In plants and some animals, viral infection triggers production of virus-derived short i... more ABSTRACT In plants and some animals, viral infection triggers production of virus-derived short interfering (si)RNAs (vsRNAs) by the host gene-silencing machinery, which is thought to restrict virus replication and spread. To counter the silencing-based host defense and thereby establish successful infection, viruses encode suppressor proteins that block different steps of siRNA biogenesis or action. Plants infected with DNA viruses accumulate three major size classes of vsRNAs that are processed from double-stranded RNA precursors by Dicer-like (DCL) proteins. In a model plant Arabidopsis thaliana possessing four DCLs, DCL4 and DCL1 generate 21-nt vsRNAs, DCL2 generates 22-nt vsRNAs, and DCL3 generates 24-nt vsRNAs. In contrast, RNA virus infections are associated with production of DCL4-dependent 21-nt vsRNAs and DCL2-dependent 22-nt vsRNAs. This reflects the difference in life cycles of plant DNA and RNA viruses: the former are transcribed in the nucleus where DCL1 and DCL3 normally generate endogenous miRNAs and heterochromatic siRNAs, respectively, whereas the latter are generally restricted to the cytoplasm. To function in silencing, like endogenous miRNAs and siRNAs, vsRNAs must get associated with Argonaute (AGO) family proteins and guide the resulting RNA-induced silencing complexes to complementary RNA or DNA targets. The nuclear-localized AGO proteins act in transcriptional gene silencing and heterochromatin formation through siRNA-directed DNA methylation, whereas the cytoplasmic AGOs act in posttranscriptional gene silencing through miRNA/siRNA-directed mRNA cleavage and/or translational repression. The plant silencing machinery has a remarkable ability to mediate siRNA amplification and systemic spread; these processes involve RNA-dependent RNA polymerases and plant-specific DNA-dependent RNA polymerases Pol IV and Pol V. Thus, amplification and spread of vsRNAs may also play a role in plant antiviral defense. Here we review the accumulating evidence on the role of nuclear and cytoplasmic components of the plant silencing machinery in the biogenesis and action of vsRNAs. We also describe silencing suppression and evasion strategies evolved by plant viruses and illustrate how viruses and their suppressor proteins could be used as a tool to discover novel features of the plant silencing system.
Biotechnology and Sustainable Agriculture 2006 and Beyond, 2007
ABSTRACT Cassava mosaic disease (CMD) is one of the major problems encountered in cassava fields ... more ABSTRACT Cassava mosaic disease (CMD) is one of the major problems encountered in cassava fields in Africa where the disease can trigger overall yield losses up to 25%. CMD is caused by whitefly-transmitted geminiviruses. Synergism, recombination and pseudo-recombination between different cassava geminivirus species have led to the recent pandemics of severe CMD in Africa (Legg and Fauquet, 2004). Our research aims at developing different CMD prevention strategies in transgenic cassava and therefore providing local farmers with geminivirus resistant lines to secure cassava production in the subsistence faming system.
Small interfering RNA (siRNA)-directed gene silencing plays a major role in antiviral defense. Vi... more Small interfering RNA (siRNA)-directed gene silencing plays a major role in antiviral defense. Virus-derived siRNAs inhibit viral replication in infected cells and potentially move to neighboring cells, immunizing them from incoming virus. Viruses have evolved various ways to evade and suppress siRNA production or action. Here, we show that 21-, 22-, and 24-nucleotide (nt) viral siRNAs together constitute up to 19% of total small RNA population of Oryza sativa plants infected with Rice tungro bacilliform virus (RTBV) and cover both strands of the RTBV DNA genome. However, viral siRNA hotspots are restricted to a short noncoding region between transcription and reverse-transcription start sites. This region generates double-stranded RNA (dsRNA) precursors of siRNAs and, in pregenomic RNA, forms a stable secondary structure likely inaccessible to siRNA-directed cleavage. In transient assays, RTBV protein P4 suppressed cell-to-cell spread of silencing but enhanced cell-autonomous silen...
Vegetatively propagated crop plants often suffer from infections with persistent RNA and DNA viru... more Vegetatively propagated crop plants often suffer from infections with persistent RNA and DNA viruses. Such viruses appear to evade the plant defenses that normally restrict viral replication and spread. The major antiviral defense mechanism is based on RNA silencing generating viral short interfering RNAs (siRNAs) that can potentially repress viral genes posttranscriptionally through RNA cleavage and transcriptionally through DNA cytosine methylation. Here we examined the RNA silencing machinery of banana plants persistently infected with six pararetroviruses after many years of vegetative propagation. Using deep sequencing, we reconstructed consensus master genomes of the viruses and characterized virus-derived and endogenous small RNAs. Consistent with the presence of endogenous siRNAs that can potentially establish and maintain DNA methylation, the banana genomic DNA was extensively methylated in both healthy and virus-infected plants. A novel class of abundant 20-nucleotide (nt)...
Plants infected with DNA viruses produce massive quantities of virus-derived, 24-nucleotide short... more Plants infected with DNA viruses produce massive quantities of virus-derived, 24-nucleotide short interfering RNAs (siRNAs), which can potentially direct viral DNA methylation and transcriptional silencing. However, growing evidence indicates that the circular double-stranded DNA accumulating in the nucleus for Pol II-mediated transcription of viral genes is not methylated. Hence, DNA viruses most likely evade or suppress RNA-directed DNA methylation. This review describes the specialized mechanisms of replication and silencing evasion evolved by geminiviruses and pararetoviruses, which rescue viral DNA from repressive methylation and interfere with transcriptional and post-transcriptional silencing of viral genes.
The frontline of plant defense against non-viral pathogens such as bacteria, fungi and oomycetes ... more The frontline of plant defense against non-viral pathogens such as bacteria, fungi and oomycetes is provided by transmembrane pattern recognition receptors that detect conserved pathogen-associated molecular patterns (PAMPs), leading to pattern-triggered immunity (PTI). To counteract this innate defense, pathogens deploy effector proteins with a primary function to suppress PTI. In specific cases, plants have evolved intracellular resistance (R) proteins detecting isolate-specific pathogen effectors, leading to effector-triggered immunity (ETI), an amplified version of PTI, often associated with hypersensitive response (HR) and programmed cell death (PCD). In the case of plant viruses, no conserved PAMP was identified so far and the primary plant defense is thought to be based mainly on RNA silencing, an evolutionary conserved, sequence-specific mechanism that regulates gene expression and chromatin states and represses invasive nucleic acids such as transposons. Endogenous silencin...
Proceedings of the National Academy of Sciences of the United States of America, Jan 30, 2001
Cauliflower mosaic virus (CaMV) is a DNA-containing pararetrovirus replicating by means of revers... more Cauliflower mosaic virus (CaMV) is a DNA-containing pararetrovirus replicating by means of reverse transcription of a terminally redundant pregenomic 35S RNA that is also used as a polycistronic mRNA. The leader of 35S RNA is long, highly structured, and contains multiple short ORFs (sORFs), which strongly interfere with the ribosome scanning process. Translation of this RNA is initiated by a ribosome shunt mechanism, in which ribosomes translate the most 5'-proximal short ORF (sORF A), then skip a large region of the leader containing a putative RNA encapsidation signal and reinitiate translation at the first long viral ORF. Here, we demonstrate that the efficiency of the sORF A-mediated ribosome shunt is an important determinant of viral infectivity. Point mutations in sORF A, which reduced the basal level of shunt-dependent expression and the degree of shunt enhancement by a CaMV-encoded translation transactivator (TAV), consequently reduced infectivity of the virus in turnip...
In plant pararetroviruses, pregenomic RNA (pgRNA) directs synthesis of circular double-stranded v... more In plant pararetroviruses, pregenomic RNA (pgRNA) directs synthesis of circular double-stranded viral DNA and serves as a polycistronic mRNA. By computer-aided analysis, the 14 plant pararetroviruses sequenced so far were compared with respect to structural organization of their pgRNA 5'-leader. The results revealed that the pgRNA of all these viruses carries a long leader sequence containing several short ORFs and having the potential to form a large stem-loop structure; both features are known to be inhibitory for downstream translation. Formation of the structure brings the first long ORF into the close spatial vicinity of a 5'-proximal short ORF that terminates 5 to 10 nt upstream of the stable structural element. The first long ORF on the pgRNA is translated by a ribosome shunt mechanism discovered in cauliflower mosaic (CaMV) and rice tungro bacilliform viruses, representing the two major groups of plant pararetroviruses. Both the short ORF and the structure have been ...
Cauliflower mosaic virus pregenomic 35S RNA begins with a long leader sequence containing an exte... more Cauliflower mosaic virus pregenomic 35S RNA begins with a long leader sequence containing an extensive secondary structure and up to nine short open reading frames (sORFs), 2 to 35 codons in length. To test whether any of these sORFs are required for virus viability, their start codons were mutated either individually or in various combinations. The resulting viral mutants were tested for infectivity on mechanically inoculated turnip plants. Viable mutants were passaged several times, and the stability of the introduced mutations was analyzed by PCR amplification and sequencing. Mutations at the 5'-proximal sORF A and in the center of the leader resulted in delayed symptom development and in the appearance of revertants. In the central leader region, the predicted secondary structure, rather than the sORF organization, was restored, while true reversions or second-site substitutions in response to mutations of sORF A restored this sORF. Involvement of sORF A and secondary struct...
ABSTRACT In the past 7-8 years considerable effort has been expended to produce transgenic plants... more ABSTRACT In the past 7-8 years considerable effort has been expended to produce transgenic plants that resist virus infection, insects, herbicides and disease development. The crop improvement and the desired phenotype is conferred by transformation, usually with a single foreign gene. Selection of transformants demands the use of selectable markers, which usually encode enzymes which inactivate either a herbicide or an antibiotic. Also we use certain foreign genes known as reporter genes (for review see [7]).
Virus-infected plants accumulate abundant, 21-24 nucleotide viral siRNAs which are generated by t... more Virus-infected plants accumulate abundant, 21-24 nucleotide viral siRNAs which are generated by the evolutionary conserved RNA interference (RNAi) machinery that regulates gene expression and defends against invasive nucleic acids. Here we show that, similar to RNA viruses, the entire genome sequences of DNA viruses are densely covered with siRNAs in both sense and antisense orientations. This implies pervasive transcription of both coding and non-coding viral DNA in the nucleus, which generates double-stranded RNA precursors of viral siRNAs. Consistent with our finding and hypothesis, we demonstrate that the complete genomes of DNA viruses from Caulimoviridae and Geminiviridae families can be reconstructed by deep sequencing and de novo assembly of viral siRNAs using bioinformatics tools. Furthermore, we prove that this 'siRNA omics' approach can be used for reliable identification of the consensus master genome and its microvariants in viral quasispecies. Finally, we utili...
In plants, RNA interference constitutes an important defense mechanism against viruses, transposo... more In plants, RNA interference constitutes an important defense mechanism against viruses, transposons, and transgenes. Viruses, on the other hand, use suppressors to counteract silencing. In contrast to mammalian systems, silencing in plants spreads systemically through the whole organism. Since also viruses spread and consequently produce suppressors systemically, a race between silencing and virus replication occurs. Apparently, successful viruses win the race,
ABSTRACT In plants and some animals, viral infection triggers production of virus-derived short i... more ABSTRACT In plants and some animals, viral infection triggers production of virus-derived short interfering (si)RNAs (vsRNAs) by the host gene-silencing machinery, which is thought to restrict virus replication and spread. To counter the silencing-based host defense and thereby establish successful infection, viruses encode suppressor proteins that block different steps of siRNA biogenesis or action. Plants infected with DNA viruses accumulate three major size classes of vsRNAs that are processed from double-stranded RNA precursors by Dicer-like (DCL) proteins. In a model plant Arabidopsis thaliana possessing four DCLs, DCL4 and DCL1 generate 21-nt vsRNAs, DCL2 generates 22-nt vsRNAs, and DCL3 generates 24-nt vsRNAs. In contrast, RNA virus infections are associated with production of DCL4-dependent 21-nt vsRNAs and DCL2-dependent 22-nt vsRNAs. This reflects the difference in life cycles of plant DNA and RNA viruses: the former are transcribed in the nucleus where DCL1 and DCL3 normally generate endogenous miRNAs and heterochromatic siRNAs, respectively, whereas the latter are generally restricted to the cytoplasm. To function in silencing, like endogenous miRNAs and siRNAs, vsRNAs must get associated with Argonaute (AGO) family proteins and guide the resulting RNA-induced silencing complexes to complementary RNA or DNA targets. The nuclear-localized AGO proteins act in transcriptional gene silencing and heterochromatin formation through siRNA-directed DNA methylation, whereas the cytoplasmic AGOs act in posttranscriptional gene silencing through miRNA/siRNA-directed mRNA cleavage and/or translational repression. The plant silencing machinery has a remarkable ability to mediate siRNA amplification and systemic spread; these processes involve RNA-dependent RNA polymerases and plant-specific DNA-dependent RNA polymerases Pol IV and Pol V. Thus, amplification and spread of vsRNAs may also play a role in plant antiviral defense. Here we review the accumulating evidence on the role of nuclear and cytoplasmic components of the plant silencing machinery in the biogenesis and action of vsRNAs. We also describe silencing suppression and evasion strategies evolved by plant viruses and illustrate how viruses and their suppressor proteins could be used as a tool to discover novel features of the plant silencing system.
Biotechnology and Sustainable Agriculture 2006 and Beyond, 2007
ABSTRACT Cassava mosaic disease (CMD) is one of the major problems encountered in cassava fields ... more ABSTRACT Cassava mosaic disease (CMD) is one of the major problems encountered in cassava fields in Africa where the disease can trigger overall yield losses up to 25%. CMD is caused by whitefly-transmitted geminiviruses. Synergism, recombination and pseudo-recombination between different cassava geminivirus species have led to the recent pandemics of severe CMD in Africa (Legg and Fauquet, 2004). Our research aims at developing different CMD prevention strategies in transgenic cassava and therefore providing local farmers with geminivirus resistant lines to secure cassava production in the subsistence faming system.
Small interfering RNA (siRNA)-directed gene silencing plays a major role in antiviral defense. Vi... more Small interfering RNA (siRNA)-directed gene silencing plays a major role in antiviral defense. Virus-derived siRNAs inhibit viral replication in infected cells and potentially move to neighboring cells, immunizing them from incoming virus. Viruses have evolved various ways to evade and suppress siRNA production or action. Here, we show that 21-, 22-, and 24-nucleotide (nt) viral siRNAs together constitute up to 19% of total small RNA population of Oryza sativa plants infected with Rice tungro bacilliform virus (RTBV) and cover both strands of the RTBV DNA genome. However, viral siRNA hotspots are restricted to a short noncoding region between transcription and reverse-transcription start sites. This region generates double-stranded RNA (dsRNA) precursors of siRNAs and, in pregenomic RNA, forms a stable secondary structure likely inaccessible to siRNA-directed cleavage. In transient assays, RTBV protein P4 suppressed cell-to-cell spread of silencing but enhanced cell-autonomous silen...
Vegetatively propagated crop plants often suffer from infections with persistent RNA and DNA viru... more Vegetatively propagated crop plants often suffer from infections with persistent RNA and DNA viruses. Such viruses appear to evade the plant defenses that normally restrict viral replication and spread. The major antiviral defense mechanism is based on RNA silencing generating viral short interfering RNAs (siRNAs) that can potentially repress viral genes posttranscriptionally through RNA cleavage and transcriptionally through DNA cytosine methylation. Here we examined the RNA silencing machinery of banana plants persistently infected with six pararetroviruses after many years of vegetative propagation. Using deep sequencing, we reconstructed consensus master genomes of the viruses and characterized virus-derived and endogenous small RNAs. Consistent with the presence of endogenous siRNAs that can potentially establish and maintain DNA methylation, the banana genomic DNA was extensively methylated in both healthy and virus-infected plants. A novel class of abundant 20-nucleotide (nt)...
Plants infected with DNA viruses produce massive quantities of virus-derived, 24-nucleotide short... more Plants infected with DNA viruses produce massive quantities of virus-derived, 24-nucleotide short interfering RNAs (siRNAs), which can potentially direct viral DNA methylation and transcriptional silencing. However, growing evidence indicates that the circular double-stranded DNA accumulating in the nucleus for Pol II-mediated transcription of viral genes is not methylated. Hence, DNA viruses most likely evade or suppress RNA-directed DNA methylation. This review describes the specialized mechanisms of replication and silencing evasion evolved by geminiviruses and pararetoviruses, which rescue viral DNA from repressive methylation and interfere with transcriptional and post-transcriptional silencing of viral genes.
The frontline of plant defense against non-viral pathogens such as bacteria, fungi and oomycetes ... more The frontline of plant defense against non-viral pathogens such as bacteria, fungi and oomycetes is provided by transmembrane pattern recognition receptors that detect conserved pathogen-associated molecular patterns (PAMPs), leading to pattern-triggered immunity (PTI). To counteract this innate defense, pathogens deploy effector proteins with a primary function to suppress PTI. In specific cases, plants have evolved intracellular resistance (R) proteins detecting isolate-specific pathogen effectors, leading to effector-triggered immunity (ETI), an amplified version of PTI, often associated with hypersensitive response (HR) and programmed cell death (PCD). In the case of plant viruses, no conserved PAMP was identified so far and the primary plant defense is thought to be based mainly on RNA silencing, an evolutionary conserved, sequence-specific mechanism that regulates gene expression and chromatin states and represses invasive nucleic acids such as transposons. Endogenous silencin...
Proceedings of the National Academy of Sciences of the United States of America, Jan 30, 2001
Cauliflower mosaic virus (CaMV) is a DNA-containing pararetrovirus replicating by means of revers... more Cauliflower mosaic virus (CaMV) is a DNA-containing pararetrovirus replicating by means of reverse transcription of a terminally redundant pregenomic 35S RNA that is also used as a polycistronic mRNA. The leader of 35S RNA is long, highly structured, and contains multiple short ORFs (sORFs), which strongly interfere with the ribosome scanning process. Translation of this RNA is initiated by a ribosome shunt mechanism, in which ribosomes translate the most 5'-proximal short ORF (sORF A), then skip a large region of the leader containing a putative RNA encapsidation signal and reinitiate translation at the first long viral ORF. Here, we demonstrate that the efficiency of the sORF A-mediated ribosome shunt is an important determinant of viral infectivity. Point mutations in sORF A, which reduced the basal level of shunt-dependent expression and the degree of shunt enhancement by a CaMV-encoded translation transactivator (TAV), consequently reduced infectivity of the virus in turnip...
In plant pararetroviruses, pregenomic RNA (pgRNA) directs synthesis of circular double-stranded v... more In plant pararetroviruses, pregenomic RNA (pgRNA) directs synthesis of circular double-stranded viral DNA and serves as a polycistronic mRNA. By computer-aided analysis, the 14 plant pararetroviruses sequenced so far were compared with respect to structural organization of their pgRNA 5'-leader. The results revealed that the pgRNA of all these viruses carries a long leader sequence containing several short ORFs and having the potential to form a large stem-loop structure; both features are known to be inhibitory for downstream translation. Formation of the structure brings the first long ORF into the close spatial vicinity of a 5'-proximal short ORF that terminates 5 to 10 nt upstream of the stable structural element. The first long ORF on the pgRNA is translated by a ribosome shunt mechanism discovered in cauliflower mosaic (CaMV) and rice tungro bacilliform viruses, representing the two major groups of plant pararetroviruses. Both the short ORF and the structure have been ...
Cauliflower mosaic virus pregenomic 35S RNA begins with a long leader sequence containing an exte... more Cauliflower mosaic virus pregenomic 35S RNA begins with a long leader sequence containing an extensive secondary structure and up to nine short open reading frames (sORFs), 2 to 35 codons in length. To test whether any of these sORFs are required for virus viability, their start codons were mutated either individually or in various combinations. The resulting viral mutants were tested for infectivity on mechanically inoculated turnip plants. Viable mutants were passaged several times, and the stability of the introduced mutations was analyzed by PCR amplification and sequencing. Mutations at the 5'-proximal sORF A and in the center of the leader resulted in delayed symptom development and in the appearance of revertants. In the central leader region, the predicted secondary structure, rather than the sORF organization, was restored, while true reversions or second-site substitutions in response to mutations of sORF A restored this sORF. Involvement of sORF A and secondary struct...
ABSTRACT In the past 7-8 years considerable effort has been expended to produce transgenic plants... more ABSTRACT In the past 7-8 years considerable effort has been expended to produce transgenic plants that resist virus infection, insects, herbicides and disease development. The crop improvement and the desired phenotype is conferred by transformation, usually with a single foreign gene. Selection of transformants demands the use of selectable markers, which usually encode enzymes which inactivate either a herbicide or an antibiotic. Also we use certain foreign genes known as reporter genes (for review see [7]).
Virus-infected plants accumulate abundant, 21-24 nucleotide viral siRNAs which are generated by t... more Virus-infected plants accumulate abundant, 21-24 nucleotide viral siRNAs which are generated by the evolutionary conserved RNA interference (RNAi) machinery that regulates gene expression and defends against invasive nucleic acids. Here we show that, similar to RNA viruses, the entire genome sequences of DNA viruses are densely covered with siRNAs in both sense and antisense orientations. This implies pervasive transcription of both coding and non-coding viral DNA in the nucleus, which generates double-stranded RNA precursors of viral siRNAs. Consistent with our finding and hypothesis, we demonstrate that the complete genomes of DNA viruses from Caulimoviridae and Geminiviridae families can be reconstructed by deep sequencing and de novo assembly of viral siRNAs using bioinformatics tools. Furthermore, we prove that this 'siRNA omics' approach can be used for reliable identification of the consensus master genome and its microvariants in viral quasispecies. Finally, we utili...
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