Mikhail Pooggin

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.

Plant viruses have evolved several unconventional translational strategies that allow efficient expression of more than one protein from their compact, multifunctional RNAs, as well as regulation of polycistronic translation in the infected plant cell. Here, we review recent advances in our understanding of these unconventional mechanisms, which include leaky scanning, ribosome shunting, internal initiation, reinitiation, stop codon suppression and frameshifting,

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

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 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.

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. 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...

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...

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 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 ...

Due to the compactness of their genomes, viruses are well suited to the study of basic expression mechanisms, including details of transcription, RNA processing, transport, and translation. In fact, most basic principles of these processes were first described in viral systems. Furthermore, viruses seem not to respect basic rules, and cases of "abnormal" expression strategies are quiet common, although such strategies are usually also finally observed in rare cases of cellular gene expression. Concerning translation, viruses most often violate Kozak's original rule that eukaryotic translation starts from a capped monocistronic mRNA and involves linear scanning to find the first suitable start codon. Thus, many viral cases have been described where translation is initiated from noncapped RNA, using an internal ribosome entry site. This review centers on other viral translation strategies, namely shunting and virus-controlled reinitiation as first described in plant pararetroviruses (Caulimoviridae). In shunting, major parts of a complex leader are bypassed and not melted by scanning ribosomes. In the Caulimoviridae, this process is coupled to reinitiation after translation of a small open reading frame; in other cases, it is possibly initiated upon pausing of the scanning ribosome. Most of the Caulimoviridae produce polycistronic mRNAs. Two basic mechanisms are used for their translation. Alternative translation of the downstream open reading frames in the bacilliform Caulimoviridae occurs by a leaky scanning mechanism, and reinitiation of polycistronic translation in many of the icosahedral Caulimoviridae is enabled by the action of a viral transactivator. Both of these processes are discussed here in detail and compared to related processes in other viruses and cells.

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 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...

The 5' untranslated leader of potato virus X (PVX) RNA is shown when contiguous to the coding sequence, to enhance the expression of the neomycin phosphotransferase II gene (NPTII) in Nicotiana tabacum protoplasts in vivo. The level of transient expression of the NPTII gene in protoplasts provided by a plasmid containing the coding sequence of the NPTII gene under the control of 35S cauliflower mosaic virus (CaMV) promoter and terminator elements served as the baseline control. Insertion of the viral 5' untranslated leader sequence upstream of the NPTII ATG codon increased the level of expression 4-fold. An 83 nucleotide (nt) leader sequence (lacking only one nucleotide of the complete PVX leader) and a truncated version with a 28 deletion at the 3' end both had similar enhancing abilities. The 28 nt CA region of the PVX leader alone had no enhancement properties.

... 1). Recent reviews of the function of eukaryotic translation initiation factors include Jackson; Hershey; Gallie and Pestova. ... View Record in Scopus | Cited By in Scopus (34). Fütterer, J., Gordon, K., Bonneville, JM, Sanfaçon, H., Pisan, B., Penswick, J. and Hohn, T., 1988. ...

... ing ribosomes are deficient in factors responsible for cor-rect AUG start codon recognition. A similar behavior was observed in certain cases of internal ribosome entry (Jackson 2000). ... Virology 167: 125. Bonneville JM, Sanfaçon H., Fütterer J., and Hohn T. 1989. ...