Development Of CDNA Clones For RNAs In Vitro Transcription Of Wheat Yellow Mosaic Virus And Their Infections Of Cell Culture Systems

Wheat yellow mosaic disease caused by Wheat yellow mosaic virus (WYMV) has done severe damage to wheat production in China. However, due to the complex genetic background of the host, the narrow host range of the virus, the difficultness of mechanical transmission, and the instability of the purified virus and its RNAs, it is difficult to conduct WYMV research. To understand the molecular and cellular mechanisms of WYMV infection and replication, we have attempted to establish an experimental system using cDNA clones suitable for production of infectious RNA transcripts in vitro, and to inoculate these to cultured wheat cell lines and tobacco protoplasts. This system will also provide basic tools for research on other viruses.First, the 5' termini of genomic RNA1 and RNA2 of the WYMV-HC, were determined by 5' RACE to be 5' -AAAATAAAACCACCA-3' and 5' -AAAATAAAACCA-3', respectively. Then, on the basis of the primers designed according to the terminal sequences, full-length cDNA clones were constructed the under the control of an upstream T7 promoter to permit in vitro transcription of RNAs 1 and 2. WYMV RNAs transcribed in vitro were inoculated by electroporation onto wheat cell calli (Triticum aestivwn L EEaNo.l) and tobacco protoplasts (Nicotina tobacum Lev. Bright Yellow 2. cell line BY-2), attempted to establish a cellular infectious system suitable for the infectivity of WYMV particle and transcripts in vitro to the plant cell.Evaluation of these systems by Western and northern blots revealed that both systems were capable of supporting virus infection. Moreover, neither an extra G between the T7 promoter and the 5' terminal sequence of WYMV-HC and Japan isolation RNA1, nor a 3' terminal poly(A) tail of 4 or 26 A residues affected the infectivity of the in vitro transcripts.In addition, primary research on the function of the P1 protein-encoded by WYMV RNA2 was carried out in this infectious system. Some transient express vectors of P1 protein was created by fusing GFP to both the N and C termini of the P1 protein, and were inoculated into BY-2 protoplasts and wheat cells by electroporation.Green fluorescence was detected by fluorescence microscopy in the nuclei of BY-2 protoplasts, and in the cytoplasm, cell membrane and cell walls of wheat cells. These differences may be accounted for by the fact that wheat is a host of WYMV and that the P1 protein behaves as it normally would in this host system. However, tobacco is not a normal host of WYMV, and may lack some of the factors needed for interactions with the P1 protein, so this results in an abnormal localization. In conclusion, these results suggest that normal function of P1 protein is dependent on the specificity of the natural WYMV host. In additional studies, some transient expression vectors containing WYMV RNA2 P1 protein fused to GFP were cloned into pBIN35S-nos. After infiltration of an Agrobacterium GFP derivative into 16C transgenic tobacco, the normal fluorescence of the plant disappeared, suggesting posttranscriptional gene silencing (PTGS). In contrast the P1 :GFP derivative fluoresced intensely under a long wave ultra violet lamp suggesting that P1 interferes with PTGS.