By John A. Lindbo (auth.), John M. Watson, Ming-Bo Wang (eds.)
Studies on the topic of pathogen-mediated virus resistance in vegetation have been instrumental in offering many of the ancient observations which finally resulted in the important discovery of double-stranded RNA (dsRNA)-induced gene silencing or RNA interference (RNAi), which has due to the fact that revolutionized learn on plant-virus interactions. In Antiviral Resistance in vegetation: tools and Protocols, specialist researchers within the box element the various equipment that are now familiar to review the phenomenon of RNA silencing when it comes to viral infections of crops. those comprise tools and strategies for the isolation and quantitative/qualitative analyses of plant small 21-24 nucleotide RNAs reminiscent of small interfering RNAs (siRNAs) and microRNAs (miRNAs) in addition to the research and manipulation of virus-induced gene silencing (VIGS) in either monocotyledonous and dicotyledenous vegetation and using hairpin RNA (hpRNA) transgenes. Written within the hugely winning Methods in Molecular Biology™ sequence structure, chapters contain introductions to their respective subject matters, lists of the required fabrics and reagents, step by step, comfortably reproducible laboratory protocols, and key tips about troubleshooting and fending off identified pitfalls.
Authoritative and useful, Antiviral Resistance in crops: tools and Protocols seeks to help scientists within the additional learn of this crucially very important botanical trait.
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Additional info for Antiviral Resistance in Plants: Methods and Protocols
Benthamiana were recently shown to depend on RDR6; loss of RDR6 activity results in reduced viroid siRNAs and symptoms (95). More recently, it was confirmed that the yellowing symptoms caused by CMV Y-satellite RNA in tobacco is due to Y-satellite siRNA-directed silencing of the host chlorophyll biosynthetic gene CHLI (128, 129). Thus, viral siRNA-induced host gene silencing may be a general mechanism for subviral RNA pathogenicity. Two recent studies have shown that siRNAs from CaMV and Tobacco mosaic virus have many potential target sequences in the host genomes (23, 94).
These proteins efficiently form complexes with 21-nucleotide sRNA duplexes but fail to bind long dsRNA, so it is likely that they suppress silencing mainly by sequestering siRNAs and preventing their incorporation into RISC (105). AGO1 is the main slicer of the antiviral silencing complex. The 2b protein of Cucumber mosaic cucumovirus interacts directly with AGO1 and inhibits its cleavage activity (108). The 2b protein dramatically reduces the accumulation of all three size classes of viral siRNAs in Arabidopsis (22), possibly because its inhibition on AGO1 activity reduces the amount of viral RNA cleavage product that serves as template for RDRs.
This thermodynamic stability bias is a conserved mechanism and is mirrored in the fact that most miRNAs display high thermodynamic asymmetry (41), presumably assisting in the correct incorporation of the functional guide strand into the catalytic complex. It is suggested that HYL1 and DCL1 are able to assess the symmetry of the sRNA molecule (42). A. thaliana also appears to have developed a 5¢ specificity determinant mechanism, whereby AGOs exhibit a preference for sRNAs with a particular 5¢ nucleotide (40, 43, 44).