LAMP Detection And Identification Of Gene Silencing Suppressor Of Barley Yellow Dwarf Viruses (BYDVs)

Barley yellow dwarf viruses (BYDVs) are economically important viruses that infect cereal crops worldwide, which can make the plant yellow and dwarf. BYDV shows great importance in economy, while the mechanism of gene express, evolution and the interaction between transmission vector and plant host are also research focuses.Accurate and efficient detection of the pathogens is the backbone for the forecasting and control of wheat/barley yellow dwarf disease. Several methodologies have been used to diagnose BYDVs. These methods include biological techniques, enzyme-linked immunosorbent assay (ELISA), and nucleic acid-based assays such as polymerase chain reaction and dot blot hybridization. A reverse transcription loop-mediated isothermal amplification of DNA (RT-LAMP) for detection of Barley yellow dwarf viruses (BYDVs) was developed. In this procedure, three sets of four primers matching a total of six sequences of the coat protein (CP) or readthrough (RTP) genes of BYDVs-each of three species, namely BYDV-GAV, GPV, and PAV were synthesized to develope a specific and sensitive RT-LAMP assay from total RNA extracts of field-infected wheat plants in such a way that a loop could be formed and elongated during DNA amplification. RT-LAMP was carried out using total RNA isolated from the infected tissues. In a total volume of 25μl, the reaction contained 2μl of RNA, Avian myeloblastosis virus (AMV) reverse transcriptase 1μl, added to provide a final concentration of primers F3 and B3 0.2μM,primers FIP and BIP 1.6μM. dNTP 0.4 mM, betaine 0.8 mM, 20 mM Tris-HCl (pH 8.8 ), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, Bst DNA polymerase 1μl, MgCl2 0.45μl(25 mM), DTT 0.45μl (100 mM), Rnasin 0.1μl (40U/μl) also were included. The mixture was incubated at 65°C for 80 min followed by 5 min at 80°C. To determine the specificity of RT-LAMP, total RNA from fresh leaves infected by the laboratory isolates of BYDV-GPV, GAV, and PAV-CN respectively was separately applied to the reaction system and then using each of four oligonucleotide primers corresponding to three species of BYDV respectively. It suggested that the RT-LAMP system in this study was very specific for each of three species of BYDV in China.Sensitivity comparisons were performed between the RT-LAMP amplification and RT-PCR assay developed for BYDV-GPV detection. Results showed positive RT-LAMP amplifications were reliably observed with weak and inconsistent positives for some replicates at dilutions of 1:10-5. RT-PCR gave positive results up to a 1:10-2 dilutions.RNA silencing is an important natural mechanism of defence against virus. On the other side, in order to infect sucuessfully hosts, viruses also evolved an antidefence mechanism. Most plant viruses can code a kind of protein factor to suppress RNA silencing. Identificating these suppressors and clearing their function is not only good for the mechanism of suppressing RNA silencing, but also significant for the mechanism of RNA silencing.Perfection of the theory of gene silencing and the mature technology provide a new approach to the study of BYDV genome function. In this study, we constructed ten plasmids (GAV-ORF6-pBin,GAV-CP-pBin,GAV-ORF1-pBin,GPV-P0-pBin,GPV-UP0-pBin,GPV-P0-pGD,GPV-UP0-pGD,GPV-MP-pGD,GPV-CP-pGD,161-pBin ) to express 7 kinds of proteins. Each of the plasmid was co-introduced with a plasmid (pBin-mGFP5-ER) carrying 35S-GFP segment into leaves of 16C transgenic N.benthamiana plant and N.benthamiana plant through an Agrobacterium (C58CI) -mediated infiltration procedure. The agroinfiltcated plants were examined for GFP silencing under a long wavelength UV illuminator, and the leaves of N.benthamiana from the 5th days were analysed by western blotting. The results indicated the proteins coded by GAV-ORF6, GPV-P0 were the suppressors of RNA silencing.