Grant: Discovery Grants Program - Individual

Plants have evolved several strategies to survive virus infection. 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 synthesis, supporting the idea that antiviral activity was due to toxicity. Upon pathogen invasion, RIPs would be released from subcellular spaces to kill plant cells and limit virus spread. Our recent search for RIPs within sequenced plant genomes showed that most were localized with ribosomes in cells, raising questions regarding their toxicity. Moreover, we have shown that some RIPs depurinate viral RNAs directly, reducing viral levels without cytotoxicity. How RIPs are antiviral remains an open question as most of our knowledge comes from their over-expression in heterologous systems. Little is known about their function and regulation in the plants that synthesize them. The long-term goal of our research program is to understand how RIPs function in plant defense. We study pokeweed (Phytolacca americana) because the plant is resilient to environmental stress and expresses several RIPs, one of which is pokeweed antiviral protein (PAP). Our short-term objectives are: 1) to determine the effect of PAP on pokeweed infected with Pokeweed mosaic virus (PkMV); 2) to identify what RNAs are targeted for depurination by PAP; and 3) to understand how PAP expression is regulated. Together, these objectives will expand our knowledge of PAP antiviral mechanisms and regulation in pokeweed. To address the first aim, we will infect wild-type and mutant pokeweed not expressing PAP with PkMV. We will identify patterns of transcriptomic change over time and correlate these with microscopy visualization of virus life cycle stages. We will also identify whether PAP binds viral or cellular proteins that may influence its activity. To address the second aim, we will identify the sequences of RNAs that bind PAP. We will also determine what cellular and/or viral RNAs are depurinated by PAP. These results will tell us how PAP selects its substrates and whether specificity exists for its RNA targets. To address the third aim, we will characterize the role of the 5'UTR intron of the PAP gene. We will investigate whether the intron enhances transcription or post-transcription levels of PAP. In addition, some transcripts retain the intron, and we will determine the role of an upstream open reading frame within retained introns of PAP mRNA. Together this work will contribute to understanding how PAP functions in defense and how its expression is regulated in pokeweed. There is merit in studying non-model plants like pokeweed as they provide insight into the defense mechanisms of plants beyond those observed in research models (RIP genes are absent in Arabidopsis). Their study may yield novel approaches to improving the resilience of crops to environmental challenges.