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Double-stranded RNA (dsRNA)-specific endonucleases belonging to RNase III classes 3 and 2 process dsRNA precursors to small interfering RNA (siRNA) or microRNA, respectively, thereby initiating and amplifying RNA silencing-based antiviral defense and gene regulation in eukaryotic cells. However, we now provide evidence that a class 1 RNase III is involved in suppression of RNA silencing. The single-stranded RNA genome of sweet potato chlorotic stunt virus (SPCSV) encodes an RNase III (RNase3) homologous to putative class 1 RNase IIIs of unknown function in rice and Arabidopsis. We show that RNase3 has dsRNA-specific endonuclease activity that enhances the RNA-silencing suppression activity of another protein (p22) encoded by SPCSV. RNase3 and p22 coexpression reduced siRNA accumulation more efficiently than p22 alone in Nicotiana benthamiana leaves expressing a strong silencing inducer (i.e., dsRNA). RNase3 did not cause intracellular silencing suppression or reduce accumulation of siRNA in the absence of p22 or enhance silencing suppression activity of a protein encoded by a heterologous virus. No other known RNA virus encodes an RNase III or uses two independent proteins cooperatively for RNA silencing suppression.

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SUMMARYAccumulation of viruses in vegetatively propagated plants causes heavy yield losses. Therefore, supply of virus-free planting materials is pivotal to sustainable crop production. In previous studies, Raspberry bushy dwarf virus (RBDV) was difficult to eradicate from raspberry ( Rubus idaeus ) using the conventional means of meristem tip culture. As shown in the present study, it was probably because this pollen-transmitted virus efficiently invades leaf primordia and all meristematic tissues except the least differentiated cells of the apical dome. Subjecting plants to thermotherapy prior to meristem tip culture heavily reduced viral RNA2, RNA3 and the coat protein in the shoot tips, but no virus-free plants were obtained. Therefore, a novel method including thermotherapy followed by cryotherapy was developed for efficient virus eradication. Heat treatment caused subcellular alterations such as enlargement of vacuoles in the more developed, virusinfected cells, which were largely eliminated following subsequent cryotherapy. Using this protocol, 20-36% of the treated shoot tips survived, 30-40% regenerated and up to 35% of the regenerated plants were virus-free, as tested by ELISA and reverse transcription loop-mediated isothermal amplification. Novel cellular and molecular insights into RBDV-host interactions and the factors influencing virus eradication were obtained, including invasion of shoot tips and meristematic tissues by RBDV, enhanced viral RNA degradation and increased sensitivity to freezing caused by thermotherapy, and subcellular changes and subsequent death of cells caused by cryotherapy. This novel procedure should be helpful with many virus-host combinations in which virus eradication by conventional means has proven difficult.

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The importance of cassava as a food security crop in Africa and the world Cassava, originally from South America, is the fourth most important source of calories in the developing world after the cereal crops wheat, maize, and rice. Worldwide, it feeds an estimated 700 million people directly or indirectly. Cassava production has increased steadily for the last 50 years, with 242 MT harvested in 2012. The increase is likely to continue as farmers in more than 105 countries come to recognize the crop's advantages. A semi-perennial root crop, cassava can stay in the ground for up to 3 years. This makes it an excellent food security crop: when all other crops have been exhausted, cassava roots can still be harvested. It is naturally drought resistant and resilient to climatic changes, high temperatures, and poor soils, and in addition, cassava responds extremely well to high CO 2 concentrations, making it a very important crop for the 21st century. Africa alone accounts for more than 55 % of the world's production, and cassava is the first food crop in fresh tonnage before maize and plantain in sub-Saharan Africa. Cassava is also an important source of income, especially for women in sub-Saharan Africa (SSA). Furthermore, cassava is the second most important source of starch in the world. Cassava is thus a highly valuable crop for the world today and in the future. It is critical that it should not be compromised by viral diseases.

Sweet potato chlorotic stunt virus (genus Crinivirus) belongs to the family Closteroviridae, members of which have a conserved overall genomic organization but are variable in gene content. In the bipartite criniviruses, heterogeneity is pronounced in the 39-proximal region of RNA1, which in sweet potato chlorotic stuat virus (SPCSV) encodes two novel proteins, RNase3 (RNase III endonuclease) and p22 (RNA silencing suppressor). This study showed that two Ugandan SPCSV isolates contained the p22 gene, in contrast to three isolates of the East African strain from Tanzania and Peru and an isolate of the West African strain from Israel, which were missing a 767 nt fragment of RNA1 that included the p22 gene. Regardless of the presence of p22, all tested SPCSV isolates acted synergistically with potyvirus sweet potato feathery mottle virus (SPFMV; genus Potyvirus, family Potyviridae) in co-infected sweetpotato plants (Ipomoea batatas), which greatly enhanced SPFMV titres and caused severe sweetpotato virus disease (SPVD). Therefore, the results indicate that any efforts to engineer pathogen-derived RNA silencing-based resistance to SPCSV and SPVD in sweetpotato should not rely on p22 as the transgene. The data from this study demonstrate that isolates of this virus species can vary in the genes encoding RNA silencing suppressor proteins. This study also provides the first example of intraspecific variability in gene content of the family Closteroviridae and may be a new example of the recombination-mediated gene gain that is characteristic of virus evolution in this virus family. INTRODUCTIONViruses belonging to the family Closteroviridae have singlestranded, positive-sense RNA genomes that are the largest among plant viruses . These viruses share a conserved set of 'core' genes, but they also show a high level of variability in gene content at the 39 end of the monopartite genomes in the genera Closterovirus and Ampelovirus and at the 39 end of RNA1 in viruses of the genus Crinivirus, which have bipartite genomes . Gene content and organization of the variable 39-proximal genomic region have evolutionary implications (Karasev et al., 1996;Dolja et al., 2006), which are interesting not least because some of the genes in the 39 region encode RNA silencing suppression (RSS) proteins (Reed et al., 2003;Lu et al., 2004;Kreuze et al., 2005;Chiba et al., 2006). RSS proteins combat the fundamental antiviral resistance that is based on RNA silencing in plants (Lindbo & Dougherty, 2005). The functional similarities of the RSS proteins contrast with their very low or non-significant sequence homology with other viral and host proteins Mérai et al., 2006). It has been suggested that these characteristics might indicate a relatively recent acquisition of the genes for RSS proteins in viral genomes to counteract evolving eukaryotic host defence responses to viral pathogens (Voinnet, 2005).Sweet potato chlorotic stunt virus (SPCSV; genus Crinivirus, family Closteroviridae) is a phloem-limited, whitefly-transmitted, bipartite viru...

Resistance to antibiotics mediated by selectable marker genes remains a powerful selection tool for transgenic event production. However, regulatory agencies and consumer concerns favor these to be eliminated from food crops. Several excision systems exist but none have been optimized or shown to be functional for clonally propagated crops. The excision of the nptII gene conferring resistance to kanamycin has been achieved here using a gene construct based on a heat-inducible cre gene producing a recombinase that eliminates cre and nptII genes flanked by two loxP sites. First-generation regenerants with the Cre-loxP system were obtained by selection on kanamycin media. Following a heat treatment, second generation regenerants were screened for excision by PCR using nptII, cre, and T-DNA borders primers. Excision efficiency appeared to be at 4.7% depending on the heat treatment. The footprint of the excision was shown by sequencing between T-DNA borders to correspond to a perfect recombination event. Selectable marker-free sprouts were also obtained from tubers of transgenic events when submitted to similar heat treatment at 4% frequency. Spontaneous excision was not observed out of 196 regenerants from untreated transgenic explants. Biosafety concerns are minimized because the expression of cre gene driven by the hsp70 promoter of Drosophila melanogaster was remarkably low even under heat activation and no functional loxP site were found in published Solanum sequence database. A new plant transformation vector pCIP54/55 was developed including a multiple cloning site and the self-excision system which should be a useful tool not only for marker genes in potato but for any gene or sequence removal in any plant.