Isolation of a putative tobacco host factor interacting with cucumber mosaic virus-encoded 2b protein by yeast two-hybrid screening

The cucumber mosaic virus (CMV)-encoded 2b protein has been implicated to p lay a role in long distance movement of the virus through the plant's trans port system. It is unknown, however, how it mediates virus movement and whe ther any intrinsic components of plant cells also participate in this proce ss. To isolate a host factor that interacts with 2b, the yeast two-hybrid s ystem was used. First, it was found that the 2b protein per se could functi on as a transcriptional activator in yeast, However, its two carboxyl termi nal deletion mutants, 2b Delta 98 and 2b Delta 95, which lacked 12 and 15 a mino acids from the carboxyl terminus respectively, showed complete absence of transcriptional activation in yeast. A tobacco cDNA library expressing the GAL4 activation domain fusion proteins was screened using 2b Delta 98 a s a bait. A clone named 2bip (2b-interacting protein) was isolated whose tr anslation product apparently interacted with 2b, Consistent with this obser vation, bacterially expressed GST-2bip fusion protein bound tightly to 2b D elta 95 and 2b Delta 98 polypeptides in vitro, as well as to the unmodified 2b protein. Nucleotide sequencing and database searches revealed that the amino acid sequence deduced from it was similar to a prokaryotic LytB prote in and an unknown protein of Ambidopsis. DNA and RNA gel blot analyses show ed that 2bip-related sequences were present in the tobacco genome and that transcripts corresponding to 2bip were expressed constitutively in various plant organs and in response to CMV infection. These results suggest 2bip a s a novel host factor that is capable of interacting with CMV2b.