Establishment And Validation Of A Real-time Quantitative PCR System For Wheat Dwarf Virus In Wheat And Leafhopper

Wheat dwarf virus (WDV), transmitted by leafhopper (Psammotettix striatus L.) in a persistent circulative manner, belongs to the genus Mastrevirus, family Geminiviridae. WDV and leafhopper or wheat interacted gene expression plays an important role for spread of wheat dwarf disease. It has been proved that Geminiviridae coat protein act as an auxiliary for MP, either actively or by protecting the genome when the virus moves cell to cell through plasmodesmata. The CP protein of WDV has been interacted with leafhopper. The purpose of this study is to analysis WDV gene expression pattern in wheat and leafhopper using real-time quantitative PCR (RT-qPCR).(1) We built the RT-qPCR protocol in regard to study gene expression in the wheat, using 5 housekeeping genes TEF-1-α, GAPDH,18SrRNA, UBC and 28SrRNA as the candidate reference genes. Genes expression stability was assessed in the stem and leaf tissue of wheat using Genorm, NormFinder and Bestkeeper softwares. In the stem tissue, TEF-1-α ranked as the most stable and the 28SrRNA is the least stable one. Whereas, in leaf tissue gene stabilty was varing in all the three software used for analysis gene expression. TEF-1-α followed by 18SrRNA, GAPDH,28SrRNA and UBC showed more stability in Genorm analytical software. GAPDH was proved more stable as compare to 18SrRNA, TEF-1-α, UBC and 28SrRNA in NormFinder software. On the other hand, Bestkeeper software showed 18SrRNA as more stable in leaves tissues as compare to other four genes. However, TEF-1-α and GAPDH were more satble in both tissues and the optimal number of reference genes for normalization was at least five.(2) This study analyzed the relative expression of WDV-CP gene in the stem and leaf of wheat by the RT-qPCR system we built. The relative quantitative of WDV-CP gene in the stem was higher than the leaf. This might be as the result of the entering of WDV in the wheat phloem by the leafhopper and then move in the phloem up to down.(3) The RT-qPCR system was built in the leafhopper using 6 housekeeping genes (RPS23e, UBC, Beta-TUB, GAPDH,18SrRNA and ACT2) which were the first time sequenced from the leafhopper. Our results showed that RPS23e was most stable and ACT2 was less stable as compare to UBC, Beta-TUB, GAPDH and 18SrRNA in leafhopper using either analytical software. However, the most stable combination was Beta-TUB and UBC. The optimal number of reference genes for normalization was also at least five.(4)urthermore, we analyzed the WDV-CP gene expression in leafhopper after 12 h to 156 h with interval of 12 h feeding on virus infected plant. We found the titer of WDV decreased from12 h to 48 h, and then increase from 48 h to 96 h to the highest point, with the reduction again between 96 h to 120 h and increase from 120 h to 156 h. This result suggests the feeding behavior or mouthpart of leafhopper was involed to regulate virus titer in leafhoppers.Taken all together, we successfully quantifed WDV titers in different part of wheat plant and entire leafhopper body. Such quantitative insight of WDV titers in wheat and leafhopper will be useful to understand WDV-Wheat-Leafhopper interaction.