Transgenic expression of artificial microRNA targeting soybean mosaic virus P1 gene confers virus resistance in plant.

RNA silencing is an innate immune mechanism of plants against invasion by viral pathogens. Artificial microRNA (amiRNA) can be engineered to specifically induce RNA silencing against viruses in transgenic plants and has great potential for disease control. Here, we describe the development and application of amiRNA-based technology to induce resistance to soybean mosaic virus (SMV), a plant virus with a positive-sense single-stranded RNA genome. We have shown that the amiRNA targeting the SMV P1 coding region has the highest antiviral activity than those targeting other SMV genes in a transient amiRNA expression assay. We transformed the gene encoding the P1-targeting amiRNA and obtained stable transgenic Nicotiana benthamiana lines (amiR-P1-3-1-2-1 and amiR-P1-4-1-2-1). Our results have demonstrated the efficient suppression of SMV infection in the P1-targeting amiRNA transgenic plants in an expression level-dependent manner. In particular, the amiR-P1-3-1-2-1 transgenic plant showed high expression of amiR-P1 and low SMV accumulation after being challenged with SMV. Thus, a transgenic approach utilizing the amiRNA technology appears to be effective in generating resistance to SMV.

Robust Response to Plum pox virus Infection via Plant Biotechnology.

Our goal was to target silencing of the Plum pox virus coat protein (PPV CP) gene independently expressed in plants. Clone C-2 is a transgenic plum expressing CP. We introduced and verified, in planta, the effects of the inverse repeat of CP sequence split by a hairpin (IRSH) that was characterized in the HoneySweet plum. The IRSH construct was driven by two CaMV35S promoter sequences flanking the CP sequence and had been introduced into C1738 plum. To determine if this structure was enough to induce silencing, cross-hybridization was made with the C1738 clone and the CP expressing but PPV-susceptible C2 clone. In total, 4 out of 63 clones were silenced. While introduction of the IRSH is reduced due to the heterozygous character in C1738 plum, the silencing induced by the IRSH PPV CP is robust. Extensive studies, in greenhouse containment, demonstrated that the genetic resource of C1738 clone can silence the CP production. In addition, these were verified through the virus transgene pyramiding in the BO70146 BlueByrd cv. plum that successfully produced resistant BlueByrd BO70146 × C1738 (HybC1738) hybrid plums.

Validation of reference genes as internal control for studying viral infections in cereals by quantitative real-time RT-PCR.

BACKGROUND: Reference genes are commonly used as the endogenous normalisation measure for the relative quantification of target genes. The appropriate application of quantitative real-time PCR (RT-qPCR), however, requires the use of reference genes whose level of expression is not affected by the test, by general physiological conditions or by inter-individual variability. For this purpose, seven reference genes were investigated in tissues of the most important cereals (wheat, barley and oats). Titre of Barley yellow dwarf virus (BYDV) was determined in oats using relative quantification with different reference genes and absolute quantification, and the results were compared. RESULTS: The expression of seven potential reference genes was evaluated in tissues of 180 healthy, physiologically stressed and virus-infected cereal plants. These genes were tested by RT-qPCR and ranked according to the stability of their expression using three different methods (two-way ANOVA, GeNorm and NormFinder tools). In most cases, the expression of all genes did not depend on abiotic stress conditions or virus infections. All the genes showed significant differences in expression among plant species. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-tubulin (TUBB) and 18S ribosomal RNA (18S rRNA) always ranked as the three most stable genes. On the other hand, elongation factor-1 alpha (EF1A), eukaryotic initiation factor 4a (EIF4A), and 28S ribosomal RNA (28S rRNA) for barley and oat samples; and alpha-tubulin (TUBA) for wheat samples were consistently ranked as the less reliable controls.The BYDV titre was determined in two oat varieties by RT-qPCR using three different quantification approaches. There were no significant differences between the absolute and relative quantifications, or between quantification using GAPDH + TUBB + TUBA +18S rRNA and EF1A + EIF4A + 28S rRNA. However, there were discrepancies between the results of individual assays. CONCLUSIONS: The geometric average of GAPDH, 18S rRNA and TUBB is suitable for normalisation of BYDV quantification in barley tissues. For wheat and oat samples, a combination of four genes is necessary: GAPDH, 18S rRNA, TUBB and EIF4A for wheat; and GAPDH, 18S rRNA, TUBB and TUBA for oat is recommended.

An Amino Acid Deletion in Wheat streak mosaic virus Capsid Protein Distinguishes a Homogeneous Group of European Isolates and Facilitates Their Specific Detection.

The tritimovirus Wheat streak mosaic virus (WSMV) is widespread throughout the world and represents a severe threat to cereal crop production. To increase knowledge of genetic diversity of WSMV in Europe, until now scarce, capsid protein (CP) sequences of several Czech, French, Italian, Slovak, and Turkish isolates have been determined. A multiple alignment of CP nucleotide sequences using available WSMV sequences revealed only limited sequence variation among 3 previously sequenced European isolates and the 14 European isolates sequenced in this study. Moreover, these isolates were characterized by an identical 3-nucleotide deletion, resulting in the lack of the Gly2761 codon within the CP region of the polyprotein. The results indicate that this monophyletic group of isolates (designated as WSMV-ΔE) is common and widely dispersed throughout the European continent. The close relationship of WSMV-ΔE isolates implies a single common ancestor and, presumably, subsequent dispersal throughout Europe from a single focus. We developed two simple assays for specific and accurate detection of WSMV-ΔE isolates. First, a conserved ClaI restriction site in the core CP gene sequence unique to WSMV-ΔE isolates was used for restriction fragment length polymorphism analysis of amplified polymerase chain reaction (PCR) products. Second, the conserved and specific codon gap in WSMV-ΔE sequences was used as a target to design specific primers functional in one-step reverse-transcription PCR detection of WSMV-ΔE isolates.