RNA virus-mediated gene editing for tomato trait breeding

Abstract

Virus-induced genome editing (VIGE) leverages viral vectors to deliver CRISPR-Cas components into plants for robust and flexible trait engineering. We describe here a VIGE approach applying an RNA viral vector based on potato virus X (PVX) for genome editing of tomato, a mayor horticultural crop. Viral delivery of single-guide RNA into Cas9-expressing lines resulted in efficient somatic editing with indel frequencies up to 58%. By proof-of-concept VIGE of PHYTOENE DESATURASE (PDS) and plant regeneration from edited somatic tissue, we recovered loss-of-function pds mutant progeny displaying an albino phenotype. VIGE of STAYGREEN 1 (SGR1), a gene involved in fruit color variation, generated sgr1 mutant lines with recolored red-brown fruits and high chlorophyll levels. The obtained editing events were heritable, overall confirming the successful breeding of fruit color. Altogether, our VIGE approach offers great potential for accelerated functional genomics of tomato variation, as well as for precision breeding of novel tomato traits.

Figure 1

RNA virus/CRISPR-Cas9 editing in tomato. (A) Scheme of pLX-PVX, a mini T-DNA vector for agroinoculation of recombinant potato virus X (PVX) constructs for sgRNA delivery. The derivatives pLX-PVX::sgPDS and pLX-PVX::sgSGR include single-guide RNAs (sgRNAs) targeting the tomato PHYTOENE DESATURASE (PDS) and STAYGREEN 1 (SGR1), respectively. (B) Agrobacterium-mediated inoculation of pLX-PVX in tomato. RT–PCR detection of a PVX genomic fragment (CP) in a Cas9-expressing tomato cv. Micro-Tom (MT-Cas9) plant agroinoculated with pLX-PVX::sgPDS; samples were collected from upper uninoculated leaves; MW, DNA size standards; N, negative control. (C) Virus-induced genome editing (VIGE) of PDS in tomato somatic cells. MT-Cas9 plants were agroinoculated with pLX-PVX::sgPDS; after 6 weeks, VIGE was assessed in upper uninoculated leaf samples by PCR of a PDS genomic fragment and Sanger trace deconvolution. Indel frequency percentages are shown; CTRL, uninoculated MT-Cas9 plants. (D) Experimental workflow for tomato trait breeding using the PVX/CRISPR-Cas9 system. sgRNAs are delivered into MT-Cas9 by pLX-PVX agroinoculation, whole plants are regenerated in vitro from upper uninoculated leaf tissue, and progeny is screened for mutant line identification. (E) VIGE of PDS in regenerated tomato plants. Indel frequency percentages and genotypic classification of 10 regenerated plants are shown. (F) Rescue of homozygous PDS mutant progeny. Phenotype and Sanger trace of the progeny line PDS02.3 with a homozygous loss-of-function PDS allele (pds +1/+1); CTRL, control condition. (G) Rescue of homozygous SGR1 mutant progeny. Sanger traces and mature fruit phenotypes of progeny lines with homozygous loss-of-function SGR1 alleles; CTRL, unedited MT-Cas9. (H) Chlorophyll quantification in fruits of SGR1 mutants. Normalized fluorometric amounts of chlorophyll a in mature fruit samples are plotted (mean ± SD, n = 4); significance levels versus the control unedited MT-Cas9 (CTRL) as per Student’s t-test are shown; ^*, p ≤ 0.05; ^**, p ≤ 0.01; ^***, p ≤ 0.001.