AGO2: A New Argonaute Compromising Plant Virus Accumulation

Plant viruses use several strategies to transport their nucleic acid genomes throughout the plants. Regardless of the movement mechanism, a universal major block to uninterrupted viral trafficking is the induction of antiviral silencing that degrades viral RNA. To counteract this defense, viruses encode suppressors that block certain steps in the RNA silencing pathway, and consequently these proteins allow viral spread to proceed. There is a constant battle between plants and viruses and sometimes viruses will succeed and invade the plants and in other cases the RNA silencing mechanism will override the virus. A key role in the silencing versus suppression conflict between plants and viruses is played by one or more members of the Argonaute protein (AGO) family encoded by plants. Here we review the mechanisms and effects of antiviral silencing with an emphasis on the contribution of AGOs, especially the recently discovered role of AGO2

Development of Molecular Markers Tightly Linked to Pvr4 Gene in Pepper Using Next-Generation Sequencing

It is imperative to identify highly polymorphic and tightly linked markers of a known trait for molecular marker-assisted selection. Potyvirus resistance 4 (Pvr4) locus in pepper confers resistance to three pathotypes of potato virus Y and to pepper mottle virus. We describe the use of next-generation sequencing technology to generate molecular markers tightly linked to Pvr4. Initially, comparative genomics was carried out, and a syntenic region of tomato on chromosome ten was used to generate PCR-based markers and map Pvr4. Subsequently, the genomic sequence of pepper was used, and more than 5000 single-nucleotide variants (SNVs) were identified within the interval. In addition, we identified nucleotide binding site–leucine-rich repeat-type disease resistance genes within the interval. Several of these SNVs were converted to molecular markers desirable for large-scale molecular breeding programmes

Identification of Pns6, a putative movement protein of RRSV, as a silencing suppressor

RNA silencing is a potent antiviral response in plants. As a counterdefense, most plant and some animal viruses encode RNA silencing suppressors. In this study, we showed that Pns6, a putative movement protein of Rice ragged stunt virus (RRSV), exhibited silencing suppressor activity in coinfiltration assays with the reporter green fluorescent protein (GFP) in transgenic Nicotiana benthamiana line 16c. Pns6 of RRSV suppressed local silencing induced by sense RNA but had no effect on that induced by dsRNA. Deletion of a region involved in RNA binding abolished the silencing suppressor activity of Pns6. Further, expression of Pns6 enhanced Potato virus × pathogenicity in N. benthamiana. Collectively, these results suggested that RRSV Pns6 functions as a virus suppressor of RNA silencing that targets an upstream step of the dsRNA formation in the RNA silencing pathway. This is the first silencing suppressor to be identified from the genus Oryzavirus

Subcellular localization and expression of bamboo mosaic virus satellite RNA-encoded protein

The satellite RNA of bamboo mosaic virus (satBaMV) has a single open reading frame encoding a non-structural protein, P20, which facilitates long-distance movement of satBaMV in BaMV and satBaMV co-infected plants. Immunohistochemistry and immunoelectron microscopy revealed that the P20 protein accumulated in the cytoplasm and nuclei in co-infected cells. P20 and the helper virus coat protein (CP) were highly similar in their subcellular localization, except that aggregates of BaMV virions were not labelled with anti-P20 serum. The BaMV CP protein was fairly abundant in mesophyll cells, whilst P20 was more frequently detected in mesophyll cells and vascular tissues. The expression kinetics of the P20 protein was similar to but slightly earlier than that of CP in co-infected Bambusa oldhamii protoplasts and Nicotiana benthamiana leaves. However, satBaMV-encoded protein levels declined rapidly in the late phase of co-infection. During co-infection, in addition to the intact P20, a low-molecular-mass polypeptide of 16 kDa was identified as a P20 C-terminally truncated product; the possible method of generation of the truncated protein is discussed

Intracellular Transport of Plant Viruses: Finding the Door out of the Cell

Plant viruses are a class of plant pathogens that specialize in movement from cell to cell. As part of their arsenal for infection of plants, every virus encodes a movement protein (MP), a protein dedicated to enlarging the pore size of plasmodesmata (PD) and actively transporting the viral nucleic acid into the adjacent cell. As our knowledge of intercellular transport has increased, it has become apparent that viruses must also use an active mechanism to target the virus from their site of replication within the cell to the PD. Just as viruses are too large to fit through an unmodified plasmodesma, they are also too large to be freely diffused through the cytoplasm of the cell. Evidence has accumulated now for the involvement of other categories of viral proteins in intracellular movement in addition to the MP, including viral proteins originally associated with replication or gene expression. In this review, we will discuss the strategies that viruses use for intracellular movement from the replication site to the PD, in particular focusing on the role of host membranes for intracellular transport and the coordinated interactions between virus proteins within cells that are necessary for successful virus spread

Molecular and Physiological Properties Associated with Zebra Complex Disease in Potatoes and Its Relation with Candidatus Liberibacter Contents in Psyllid Vectors

Zebra complex (ZC) disease on potatoes is associated with Candidatus Liberibacter solanacearum (CLs), an α-proteobacterium that resides in the plant phloem and is transmitted by the potato psyllid Bactericera cockerelli (Šulc). The name ZC originates from the brown striping in fried chips of infected tubers, but the whole plants also exhibit a variety of morphological features and symptoms for which the physiological or molecular basis are not understood. We determined that compared to healthy plants, stems of ZC-plants accumulate starch and more than three-fold total protein, including gene expression regulatory factors (e.g. cyclophilin) and tuber storage proteins (e.g., patatins), indicating that ZC-affected stems are reprogrammed to exhibit tuber-like physiological properties. Furthermore, the total phenolic content in ZC potato stems was elevated two-fold, and amounts of polyphenol oxidase enzyme were also high, both serving to explain the ZC-hallmark rapid brown discoloration of air-exposed damaged tissue. Newly developed quantitative and/or conventional PCR demonstrated that the percentage of psyllids in laboratory colonies containing detectable levels of CLs and its titer could fluctuate over time with effects on colony prolificacy, but presumed reproduction-associated primary endosymbiont levels remained stable. Potato plants exposed in the laboratory to psyllid populations with relatively low-CLs content survived while exposure of plants to high-CLs psyllids rapidly culminated in a lethal collapse. In conclusion, we identified plant physiological biomarkers associated with the presence of ZC and/or CLs in the vegetative potato plant tissue and determined that the titer of CLs in the psyllid population directly affects the rate of disease development in plants

RNA Interference Is Responsible for Reduction of Transgene Expression after Sleeping Beauty Transposase Mediated Somatic Integration

Integrating non-viral vectors based on transposable elements are widely used for genetically engineering mammalian cells in functional genomics and therapeutic gene transfer. For the Sleeping Beauty (SB) transposase system it was demonstrated that convergent transcription driven by the SB transposase inverted repeats (IRs) in eukaryotic cells occurs after somatic integration. This could lead to formation of double-stranded RNAs potentially presenting targets for the RNA interference (RNAi) machinery and subsequently resulting into silencing of the transgene. Therefore, we aimed at investigating transgene expression upon transposition under RNA interference knockdown conditions. To establish RNAi knockdown cell lines we took advantage of the P19 protein, which is derived from the tomato bushy stunt virus. P19 binds and inhibits 21 nucleotides long, small-interfering RNAs and was shown to sufficiently suppress RNAi. We found that transgene expression upon SB mediated transposition was enhanced, resulting into a 3.2-fold increased amount of colony forming units (CFU) after transposition. In contrast, if the transgene cassette is insulated from the influence of chromosomal position effects by the chicken-derived cHS4 insulating sequences or when applying the Forg Prince transposon system, that displays only negligible transcriptional activity, similar numbers of CFUs were obtained. In summary, we provide evidence for the first time that after somatic integration transposon derived transgene expression is regulated by the endogenous RNAi machinery. In the future this finding will help to further improve the molecular design of the SB transposase vector system