Phylogeny and domain architecture of plant ribosome inactivating proteins.

Ribosome inactivating proteins (RIPs) are rRNA N-glycosylases (EC 3.2.2.22) best known for hydrolyzing an adenine base from the conserved sarcin/ricin loop of ribosomal RNA. Protein translation is inhibited by ribosome depurination; therefore, RIPs are generally considered toxic to cells. The expression of some RIPs is upregulated by biotic and abiotic stress, though the connection between RNA depurination and defense response is not well understood. Despite their prevalence in approximately one-third of flowering plant orders, our knowledge of RIPs stems primarily from biochemical analyses of individuals or genomics-scale analyses of small datasets from a limited number of species. Here, we performed an unbiased search for proteins with RIP domains and identified several-fold more RIPs than previously known - more than 800 from 120 species, many with novel associated domains and physicochemical characteristics. Based on protein domain configuration, we established 15 distinct groups, suggesting diverse functionality. Surprisingly, most of these RIPs lacked a signal peptide, indicating they may be localized to the nucleocytoplasm of cells, raising questions regarding their toxicity against conspecific ribosomes. Our phylogenetic analysis significantly extends previous models for RIP evolution in plants, predicting an original single-domain RIP that later evolved to acquire a signal peptide and different protein domains. We show that RIPs are distributed throughout 21 plant orders with many species maintaining genes for more than one RIP group. Our analyses provide the foundation for further characterization of these new RIP types, to understand how these enzymes function in plants.

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