CRISPR/Cas9-Mediated Generation of Pathogen-Resistant Tomato against Tomato Yellow Leaf Curl Virus and Powdery Mildew

Abstract

Tomato is one of the major vegetable crops consumed worldwide. Tomato yellow leaf curl virus (TYLCV) and fungal Oidium sp. are devastating pathogens causing yellow leaf curl disease and powdery mildew. Such viral and fungal pathogens reduce tomato crop yields and cause substantial economic losses every year. Several commercial tomato varieties include Ty-5 (SlPelo) and Mildew resistance locus o 1 (SlMlo1) locus that carries the susceptibility (S-gene) factors for TYLCV and powdery mildew, respectively. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) is a valuable genome editing tool to develop disease-resistant crop varieties. In this regard, targeting susceptibility factors encoded by the host plant genome instead of the viral genome is a promising approach to achieve pathogen resistance without the need for stable inheritance of CRISPR components. In this study, the CRISPR/Cas9 system was employed to target the SlPelo and SlMlo1 for trait introgression in elite tomato cultivar BN-86 to confer host-mediated immunity against pathogens. SlPelo-knockout lines were successfully generated, carrying the biallelic indel mutations. The pathogen resistance assays in SlPelo mutant lines confirmed the suppressed accumulation of TYLCV and restricted the spread to non-inoculated plant parts. Generated knockout lines for the SlMlo1 showed complete resistance to powdery mildew fungus. Overall, our results demonstrate the efficiency of the CRISPR/Cas9 system to introduce targeted mutagenesis for the rapid development of pathogen-resistant varieties in tomato.

Conserved Opposite Functions in Plant Resistance to Biotrophic and Necrotrophic Pathogens of the Immune Regulator SRFR1

“…Golden Gate cloning strategy [ 23 ] was used to assemble four independent expression modules into a single plant binary T-DNA vector ( pSlSRFR1-GE ) that included a plant selection marker ( neomycin phosphotransferase ( nptII ), kanamycin resistance), functional SpCas9 expression cassette, and two sgRNA expression cassettes ( Figure 3 c). Finally, pSlSRFR1-GE was transformed into tomato cultivar M82 using Agrobacterium-mediated transformation to produce genome-edited plants by optimized tissue culture conditions [ 24 , 25 ].…”

“…SRFR1 is well-conserved in different organisms [ 4 ] and exists as a single-copy gene in many crop plants, including tomato. Tomato is a major horticultural crop plant, and tissue culture-based genetic engineering protocols are well adopted for precise genome manipulations [ 24 , 25 ]. Despite intensive research in host–microbe interactions, molecular interactions are not yet entirely known in crop plants.…”

“…Indel generation patterns at targeted sites indicated a higher editing rate at Target 1 (SRFR1-sgRNA1) than Target 2 (SRFR1-sgRNA2). A difference in editing activity resulted from a combination of factors reported recently in SlPelo1 , for example, unstable Cas9-sgRNA complex formation, target accessibility, and Cas9 availability while using multiple sgRNAs [ 25 ]. Although the genome editing efficiency was lower in the G0 stage generating biallelic and chimeric editing patterns, screening of segregating populations produced homozygous slsrfr1 mutant lines in G1 generation.…”

“…Agrobacterium tumefacien s GV3101 (MP90)-mediated transformation was conducted to deliver T-DNA containing a plant selection marker, SpCas9, and two guide RNA assemble into tomato. The transformation was followed as described [ 24 , 25 ]. Tomato cultivar M82 was grown on 1/2 MSO media at 25 °C in a 16 h light/8 h dark photoperiod for 7 days.…”

Control of Plant Viral Diseases by CRISPR/Cas9: Resistance Mechanisms, Strategies and Challenges in Food Crops

“…Deleting the chromosomal fragments adjacent to S gene clusters may produce durable virus resistance in different hosts. In addition to virus resistance, CRISPR-mediated gene insertion opens an avenue to study important S genes [ 27 ]. The functional analysis of susceptibility genes enables us to understand the regulation of gene expression.…”