De novo Assembly of the Pokeweed Genome Provides Insight Into Pokeweed Antiviral Protein (PAP) Gene Expression

Abstract

Ribosome-inactivating proteins (RIPs) are RNA glycosidases thought to function in defense against pathogens. These enzymes remove purine bases from RNAs, including rRNA; the latter activity decreases protein synthesis in vitro , which is hypothesized to limit pathogen proliferation by causing host cell death. Pokeweed antiviral protein (PAP) is a RIP synthesized by the American pokeweed plant ( Phytolacca americana ). PAP inhibits virus infection when expressed in crop plants, yet little is known about the function of PAP in pokeweed due to a lack of genomic tools for this non-model species. In this work, we de novo assembled the pokeweed genome and annotated protein-coding genes. Sequencing comprised paired-end reads from a short-insert library of 83X coverage, and our draft assembly (N50 = 42.5 Kb) accounted for 74% of the measured pokeweed genome size of 1.3 Gb. We obtained 29,773 genes, 73% of which contained known protein domains, and identified several PAP isoforms. Within the gene models of each PAP isoform, a long 5′ UTR intron was discovered, which was validated by RT-PCR and sequencing. Presence of the intron stimulated reporter gene expression in tobacco. To gain further understanding of PAP regulation, we complemented this genomic resource with expression profiles of pokeweed plants subjected to stress treatments [jasmonic acid (JA), salicylic acid, polyethylene glycol, and wounding]. Cluster analysis of the top differentially expressed genes indicated that some PAP isoforms shared expression patterns with genes involved in terpenoid biosynthesis, JA-mediated signaling, and metabolism of amino acids and carbohydrates. The newly sequenced promoters of all PAP isoforms contained cis -regulatory elements associated with diverse biotic and abiotic stresses. These elements mediated response to JA in tobacco, based on reporter constructs containing promoter truncations of PAP-I, the most abundant isoform. Taken together, this first genomic resource for the Phytolaccaceae plant family provides new insight into the regulation and function of PAP in pokeweed.

Ribosome-Inactivating Proteins of Bougainvillea glabra Uncovered Polymorphism and Active Site Divergence

“…The Bougainvillea RIPs possess transcriptional polymorphism, and all the isoforms exhibit diversified expression in the different seasons. It was previously shown that the expression of each isoform is affected by a cis-acting element that combines with the transcription factor under jasmonic acid stress [ 17 ]. Besides this, the Bougainvillea RIPs have a close genetic relationship with PAP, which alters the splicing of viral RNA and reduces viral infection [ 30 , 31 ].…”

“…These isoforms are differentially expressed during leaf development, wounding, and abscisic acid treatment, which suggests that RIP isoforms may play diversified roles during stress responses [ 16 ]. Some studies have shown that plants express various RIP isoforms to improve their defense systems to cope with the stress caused by environmental changes [ 16 , 17 ].…”

The Structural Characterization and Antipathogenic Activities of Quinoin, a Type 1 Ribosome-Inactivating Protein from Quinoa Seeds

“…Since then, experimental evidence has confirmed the control of the expression of RIPs at the gene level during abiotic and biotic stress in plants [39,40]. The de novo assembly of the pokeweed (P. americana) genome showed that the genes of both the pokeweed antiviral protein (PAP) and the PAP isoforms exhibited a long 5 UTR containing one intron and cis-regulatory elements associated with diverse biotic and abiotic stresses [41].…”

“…This model was similar to the gene models retrieved for the PAP isoforms. Therefore, the gene organization suggested that quinoin protein expression could be subjected to particular gene regulation in biotic and abiotic stresses given the presence of the 5 UTR region of both the intron and the leader intron [41].…”

Full-length transcriptome analysis of Phytolacca americana and its congener P. icosandra and gene expression normalization in three Phytolaccaceae species

“…However, these data were both achieved by second generation sequencing (SGS), which could not produce full-length transcripts. Genomic data of P. americana was available at the SRA under project PRJNA544344, but it's raw reads without coding sequences prediction and functional annotation [7].…”