CRISPR/Cas9 mutagenesis of tomato genes encoding proteins associated with intracellular pospiviroid movement and pathogenesis

Authors: Hammond, Rosemarie; Kovalskaya, Natalia; WHEATLEY, MATTHEW - Pennsylvania State University; YANG, YINONG - Pennsylvania State University

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 3/29/2023
Publication Date: N/A
Citation: N/A

Interpretive Summary: Viroids are small, single-stranded, circular, non-coding RNAs that cause disease and reduced yield in several crop plants. In commercial tomato greenhouses, infection by potato spindle tuber viroid and tomato planta macho viroid reduce fruit set and fruit size, significantly impacting yield. A greater understanding of the disease process will lead to novel disease control strategies for these pathogens. Genome editing of the tomato host is an approach that we have employed in this study to individually target and knock-out two genes that are proposed to be important in viroid infection and spread. Our results will be of interest to plant pathologists and plant physiologists who study plant-pathogen interactions and plant development.

Technical Abstract: Viroids - small, single-stranded, circular, non-coding RNAs - cause disease and reduced yield in several crop plants. In tomato, the pospiviroids potato spindle tuber viroid (PSTVd) and tomato planta macho viroid (TPMVd) replicate in the nucleus and trigger signaling cascades leading to stunting, leaf epinasty, and reduced fruit size and yield. PSTVd- and TPMVd-derived small RNAs target specific plant mRNAs, resulting in cleavage of the mRNA and corresponding reduced transcript levels. Viroid infection also results in transcriptional up-regulation of plant genes, the mechanism of which is unknown. In our study of viroid-host interactions, we developed CRISPR/Cas9-edited gene knockout lines in tomato (cv. Moneymaker) by individually targeting two single-copy genes encoding ViRP1 (viroid RNA binding protein 1), a bromodomain protein that binds to viroid RNA and may play a role in viroid movement into the nucleus, and PKV (protein kinase viroid induced), an AGCVIIIa protein kinase that is transcriptionally activated in viroid infected plants . T0 plants generated for each edited gene consisted of homozygous and heterozygous lines for the expected deletions, and small indels. Phenotypes of the T0 and T1 plants for the ViRP1 mutation were similar to wild type, while PKV T0 mutants had small fruits and few or no viable seeds. Current work focuses on the heritability of the mutations, phenotypes of stable, homozygous lines, and subsequent effects of the mutations on viroid infectivity and pathogenesis when challenged with PSTVd and TPMVd.