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Summary: CRISPR/Cas‐based genome editing is now extensively used in plant breeding and continues to evolve. Most CRISPR/Cas current applications in plants focus on gene knock‐outs; however, there is a pressing need for new methods to achieve more efficient delivery of CRISPR components and gene knock‐ins to improve agronomic traits of crop cultivars. We report here a genome editing system that combines the advantages of protoplast technologies with recent CRISPR/Cas advances to achieve seamless large fragment insertions in the model Solanaceae plant Nicotiana tabacum. With this system, two resistance‐related regions of the N′ gene were replaced with homologous fragments from the N′alata gene to confer TMV‐U1 resistance in the T0 generation of GMO‐free plants. Our study establishes a reliable genome‐editing tool for efficient gene modifications and provides a detailed description of the optimization process to assist other researchers adapt this system for their needs.

Tomato spotted wilt virus (TSWV) is one of the most destructive viral pathogens of plants. Recently, a single dominant gene conferring complete resistance to TSWV (RTSW) was identified in Nicotina alata and introgressed into cultivated tobacco (N. tabacum). However, whether the TSWV carries an avirulence (Avr) factor directed against RTSW remains obscure. In the present study, we identified the non-structural protein (NSm), the movement protein of TSWV, which is an RTSW-specific Avr factor, by using two different transient expression systems. Using amino acid (aa) substitution mutants, we demonstrated the ability to induce RTSW-mediated hypersensitive response (HR) of NSm is independent of its movement function. Moreover, key substitutions (C118Y and T120N), a 21-aa viral effector epitope, and different truncated versions of NSm, which are responsible for the recognition of the Sw-5b resistance gene of tomato, were tested for their ability to trigger HR to TSWV in tobacco. Together, our results demonstrated that RTSW-mediated resistance is triggered by NSm in the same way as by Sw-5b, however, via different elicitor active sites. Finally, an Avr gene-based diagnostic approach was established and used to determine the presence and effectiveness of resistance genes in tobacco.

The most common response of a host to pathogens is arguably the asymptomatic response. However, the genetic and molecular mechanisms responsible for asymptomatic responses to pathogens are poorly understood. Here we report on the genetic cloning of two genes controlling the asymptomatic response to tobacco mosaic virus (TMV) in cultivated tobacco (Nicotiana tabacum). These two genes are homologous to tobamovirus multiplication 2A (TOM2A) from Arabidopsis, which was shown to be critical for the accumulation of TMV. Expression analysis indicates that the TOM2A genes might play fundamental roles in plant development or in responses to stresses. Consistent with this hypothesis, a null allele of the TOM2A ortholog in tomato (Solanum lycopersicum) led to the development of bent branches and a high tolerance to both TMV and tomato mosaic virus (ToMV). However, the TOM2A ortholog in Nicotiana glauca did not account for the asymptomatic response to TMV in N. glauca. We showed that TOM2A family is plant-specific and originated from Chlorophyte, and the biological functions of TOM2A orthologs to promote TMV accumulation are highly conserved in the plant kingdom—in both TMV host and nonhost species. In addition, we showed that the interaction between tobacco TOM1 and TOM2A orthologs in plant species is conserved, suggesting a conserved nature of TOM1–TOM2A module in promoting TMV multiplication in plants. The tradeoff between host development, the resistance of hosts to pathogens, and their influence on gene evolution are discussed. Our results shed light on mechanisms that contribute to asymptomatic responses to viruses in plants and provide approaches for developing TMV/ToMV-resistant crops.

Linkage drag can hinder the integration of resistance genes from wild crop relatives into breeding programs. We used a chromosome-scale Nicotiana alata genome assembly and a segregating population exceeding 160,000 plants to dissect the complex genetic architecture and overcome the tight linkage between resistance and deleterious loci to produce plants free from linkage drag. We cloned N. alata RTSW , encoding an immune receptor that confers broad-spectrum resistance to orthotospoviruses through the interaction of its carboxyl-terminal domain with an orthotospovirus-encoded protein. Notably, despite recognizing the same avirulence factor, RTSW genes from N. alata and Sw-5b from Solanum peruvianum have evolved independently of adjacent nonorthologous ancestral loci. Our work illustrates the potential of wild relative genomes as resources from which to precisely introduce disease resistance into cultivated crops.