Grant: Discovery Grants Program - Individual

Plants have evolved several strategies to evade virus attack and subsequent spread. One group of proteins involved in defense is the ribosome inactivating proteins (RIPs). RIPs are enzymes that remove a purine base from a conserved region of ribosomal RNA. Early studies showed that depurination inhibited protein translation, and resulting toxicity was cited to explain the antiviral activity of RIPs; that is, localized host cell death would limit virus spread. Pokeweed antiviral protein (PAP) is a RIP synthesized by the pokeweed plant (Phytolacca americana) that inhibits the replication of plant and animal viruses. We were the first to demonstrate that this activity is due to depurination of viral genomic RNA, which inhibits several steps of the virus life cycle. Much of what we know about PAP and other RIPs comes from their expression in heterologous systems; little is known of their function in native plants. For example, expression of PAP in tobacco produces virus-resistant plants; however, they exhibit undesirable phenotypes including stunted growth and sterility. We attribute this to insufficient understanding of how PAP is regulated in pokeweed. ******The long-term goal of our research program is to understand the mechanisms that allow plants to survive virus infections. We chose pokeweed as our model because it has broad-spectrum virus resistance. ***Our short term objectives are: 1) to determine how PAP targets RNAs for depurination; 2) to understand how PAP gene expression is regulated in pokeweed. Together, these objectives will expand our knowledge of pokeweed antiviral mechanisms and regulation of PAP.******To address the first aim, we will identify the sequences and structures of RNAs that bind PAP. We will also determine what cellular RNAs are depurinated by PAP. Pairing this information with identification of proteins that interact with PAP will reveal whether PAP functions as part of a protein complex, which could influence substrate selection.******To address the second aim, we will characterize the promoter of PAP by testing the activity of its proximal region in expression reporter assays. Based on in silico analysis indicating the presence of cis-acting elements involved in signaling plant stress, we suggest that PAP transcription is regulated by defense related proteins. We will identify transcription factors that bind specific regions of the proximal promoter to support this hypothesis. We will also investigate the function of a 5' UTR intron of PAP to test whether it contributes to transcriptional control of PAP. ******Together these experiments will contribute to understanding how PAP functions as an antiviral protein without causing cytotoxicity, and how its expression is regulated in pokeweed. Though pokeweed is not a model plant, knowledge gleaned can be useful for the agricultural application of PAP, and in understanding the function of RIPs in plant defense. **