Simultaneously induced mutations in eIF4E genes by CRISPR/Cas9 enhance PVY resistance in tobacco

Abstract

Tobacco is an important commercial crop and a rich source of alkaloids for pharmaceutical and agricultural applications. However, its yield can be reduced by up to 70% due to virus infections, especially by a potyvirus Potato virus Y (PVY). The replication of PVY relies on host factors, and eukaryotic translation initiation factor 4Es (eIF4Es) have already been identified as recessive resistance genes against potyviruses in many plant species. To investigate the molecular basis of PVY resistance in the widely cultivated allotetraploid tobacco variety K326, we developed a dual guide RNA CRISPR/Cas9 system for combinatorial gene editing of two clades, eIF4E1 (eIF4E1-S and eIF4E1-T) and eIF4E2 (eIF4E2-S and eIF4E2-T) in the eIF4E gene family comprising six members in tobacco. We screened for CRISPR/Cas9-induced mutations by heteroduplex analysis and Sanger sequencing, and monitored PVYO accumulation in virus challenged regenerated plants by DAS-ELISA both in T0 and T1 generations. We found that all T0 lines carrying targeted mutations in the eIF4E1-S gene displayed enhanced resistance to PVYO confirming previous reports. More importantly, our combinatorial approach revealed that eIF4E1-S is necessary but not sufficient for complete PVY resistance. Only the quadruple mutants harboring loss-of-function mutations in eIF4E1-S, eIF4E1-T, eIF4E2-S and eIF4E2-T showed heritable high-level resistance to PVYO in tobacco. Our work highlights the importance of understanding host factor redundancy in virus replication and provides a roadmap to generate virus resistance by combinatorial CRISPR/Cas9-mediated editing in non-model crop plants with complex genomes.

Plant eIF4E isoforms as factors of susceptibility and resistance to potyviruses

“…In tomato, resistance to multiple viruses was achieved by inactivating both eIF4E1 and eIF4E2 (Gauffier et al, 2016). In tobacco, the knockout of a single eIF4E gene was overcome by PVY (Julio et al, 2015), whereas the inactivation of two or more eIF4E factors from multiple ones in this plant rendered tobacco almost immune to PVY (Udagawa et al, 2021;Le et al, 2022). Therefore, potyviruses overcome resistance based on the absence of a particular eIF4E isoform primarily, if not exclusively, via the use of another eIF4E isoform.…”

“…To improve resistance durability, this backup isoform needs to be modified. In tobacco, PVY resistance durability mediated by eIF4E-1 inactivation could be markedly improved by inactivating the backup susceptibility isoform (Udagawa et al, 2021;Le et al, 2022). In B. rapa, durable broad-spectrum resistance to TuMV besides eIF(iso)4E.a inactivation also required a certain resistant allele (named ConTR01) of eIF(iso) 4E.c, which is apparently used by TuMV as backup susceptibility factor (Rusholme et al, 2007;Nellist et al, 2014).…”

AGRICULTURAL DEVELOPMENT BASED ON CRISPR-CAS9 AND RETRONs TECHNIQUES: A PERSPECTIVE APPLICATION ON TOMATO

“…These two reports are the first to apply CRISPR-Cas9 gene-editing technology to generate mutation in the eIF4E1 gene for broad-spectrum resistance to potyvirus. Aside from that, CRISPR/Cas9 application successfully modified eIF4E genes in other crops (Le et al, 2022;Pechar et al, 2022).…”

Knockout of elF4E using CRISPR/Cas9 for large-scale production of resistant cucumber cultivar against WMV, ZYMV, and PRSV

“…For example, deletion mutations in Arabidopsis thaliana elF4E and elF (iso)4E provide complete resistance to turnip mosaic virus (TuMV) without affecting plant vigor (Pyott et al, 2016). Induced deletion mutations in tobacco elF4E genes (elF4E1-S, elF4E1-T, elF4E2-S, and elF4E2-T) also conferred higher level of resistance to potato virus Y (PVY), another member of the potyvirus family (Le et al, 2022). Silencing of elF4E has shown broad-spectrum resistance against RNA viruses in tomato (Mazier et al, 2011), besides determining resistance to potyvirus in naturally occurring mutant plants for elF4E and eIF(iso)4E (Goḿez et al, 2009).…”