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

Byung Kook Ham, Tai Ho Lee, Jin Sam You, Young Woo Nam, Jeong Kook Kim, Kyung Hee Paek

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

The cucumber mosaic virus (CMV)-encoded 2b protein has been implicated to play a role in long distance movement of the virus through the plant's transport system. It is unknown, however, how it mediates virus movement and whether any intrinsic components of plant cells also participate in this process. To isolate a host factor that interacts with 2b, the yeast two-hybrid system was used. First, it was found that the 2b protein per se could function as a transcriptional activator in yeast. However, its two carboxyl terminal deletion mutants, 2bΔ98 and 2bΔ95, which lacked 12 and 15 amino 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 2bΔ98 as a bait. A clone named 2bip (2b-interacting protein) was isolated whose translation product apparently interacted with 2b. Consistent with this observation, bacterially expressed GST-2bip fusion protein bound tightly to 2bΔ95 and 2bΔ98 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 protein and an unknown protein of Arabidopsis. DNA and RNA gel blot analyses showed 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 as a novel host factor that is capable of interacting with CMV2b.

Original languageEnglish
Pages (from-to)548-555
Number of pages8
JournalMolecules and cells
Volume9
Issue number5
Publication statusPublished - 1999 Oct 31

Keywords

  • 2b Protein
  • Cucumber Mosaic Virus
  • Protein-protein Interaction
  • Tobacco Host Factor
  • Yeast Two-hybrid Screening

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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