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

Enhanced Transgene Expression in Sugarcane by Co-Expression of Virus-Encoded RNA Silencing Suppressors

Post-transcriptional gene silencing is commonly observed in polyploid species and often poses a major limitation to plant improvement via biotechnology. Five plant viral suppressors of RNA silencing were evaluated for their ability to counteract gene silencing and enhance the expression of the Enhanced Yellow Fluorescent Protein (EYFP) or the β-glucuronidase (GUS) reporter gene in sugarcane, a major sugar and biomass producing polyploid. Functionality of these suppressors was first verified in Nicotiana benthamiana and onion epidermal cells, and later tested by transient expression in sugarcane young leaf segments and protoplasts. In young leaf segments co-expressing a suppressor, EYFP reached its maximum expression at 48-96 h post-DNA introduction and maintained its peak expression for a longer time compared with that in the absence of a suppressor. Among the five suppressors, Tomato bushy stunt virus-encoded P19 and Barley stripe mosaic virus-encoded γb were the most efficient. Co-expression with P19 and γb enhanced EYFP expression 4.6-fold and 3.6-fold in young leaf segments, and GUS activity 2.3-fold and 2.4-fold in protoplasts compared with those in the absence of a suppressor, respectively. In transgenic sugarcane, co-expression of GUS and P19 suppressor showed the highest accumulation of GUS levels with an average of 2.7-fold more than when GUS was expressed alone, with no detrimental phenotypic effects. The two established transient expression assays, based on young leaf segments and protoplasts, and confirmed by stable transgene expression, offer a rapid versatile system to verify the efficiency of RNA silencing suppressors that proved to be valuable in enhancing and stabilizing transgene expression in sugarcane

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

Temporal characterization of psyllid colonies.

<p>Percentage of psyllids positive for the presence of <i>Candidatus</i> Liberibacter <i>solanacearum</i> (CLs) were determined by conventional PCR of a 16S rDNA specific region at irregular time points (T1 to T8). The colonies evaluated were C1, a psyllid colony with low titer of CLs, and C2 and C3, which were known to contain higher titers of CLs. The average (Avg) and the standard deviation (STDEV) for each colony were calculated and are shown above each group of time points. Y axis, percentage of positive psyllids; X axis, time points. Sample dates separated by one or more days.</p

Protein content of healthy and ZC affected plants.

*<p>Bradford assays were used to determine the protein concentration of healthy and ZC affected stem tissue. The plant samples were measured in three replicates.</p>**<p>A statistical analysis one-way ANOVA using SPSS 14.0 and Tukey mean comparison with P = 0.01 showed that the ZC diseased plants have a significant increase of protein content per gram of tissue compared to healthy potato plants. The potato plants were sampled in the field and were of different varieties (FL1867, Norkotah or Norgold).</p

Lugol staining of potato stems.

<p>Potato stem - tops were sliced and reacted with lugol solution to determine presence of starch. Positive (+) indicates that tissue reacted with the lugol solution displaying a dark blue color, and negative (−) indicates that the tissue remained the same color.</p

Detection of starch accumulation by lugol staining.

<p><b>A</b>. Typical starch accumulation pattern in different plant tissues of healthy potato varieties. Upper portion of the stem (US), lower portion of the stem (LS). <b>B</b>. Lugol staining of the upper stem (US) of potato FL1867 chipping variety affected by ZC. H1 and H2 are the healthy controls. The results of frying test performed on tuber slices performed are shown in the lower panel.</p

Detection of patatin proteins in healthy and ZC potato plants.

<p><b>I.</b> Alkaline phosphatase western blot using a patatin-specfic antibody crossreacting with two bands; a class I patatin of 40 kDa (arrow) and a class II patatin (*), size markers in kDa. <b>II.</b> Coomasie loading control. HS represents healthy plant stem; HT, healthy tuber and ZS, ZC plant stem.</p

Cyclophylin detection.

<p>A <i>Solanum sogarandinum</i> cyclophilin antibody was used for detection of cyclophylin in ZC and healthy potato plants. <b>A.</b> Western blot analysis showing cyclophilin (17.5 kDa, indicated by arrow) detection in both stem (S) and tubers (T) of healthy (H) tissues, but no detection in tubers of Zebra complex (Z) diseased plants. <b>B.</b> Coomassie brilliant blue stained gel of the protein samples used for the western blot in A, the protein concentration was calculated by Bradford assay and equal amounts of proteins (7.5 ug) were loaded. Size markers are indicated.</p

SDS-PAGE of proteins stained with Coomasie brilliant blue.

<p><b>A</b>. Profile comparison of upper stem (US) of ZC diseased plants with tubers (T) of healthy or ZC plants. <b>B</b>. Profile comparison of the root (R) and stem (S) tissue of ZC diseased (Z) and healthy (H) plants, a and b depict the differentially expressed proteins; (a) contains two proteins, cyclophilin and a putative transcription factor BTF-3 and (b) represents glycoprotein-like product. Approximately 7.5 ug of protein per lane were loaded and run in a 15% polyacryalmide gel, size markers in kDa are shown on the edges.</p