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Genome editing technology is important for plant science and crop breeding. Genome-edited plants prepared using general CRISPR-Cas9 methods usually contain foreign DNA, which is problematic for the production of genome-edited transgene-free plants for vegetative propagation or highly heterozygous hybrid cultivars. Here, we describe a method for highly efficient targeted mutagenesis in Nicotiana benthamiana through the expression of Cas9 and single-guide (sg)RNA using a potato virus X (PVX) vector. Following Agrobacterium-mediated introduction of virus vector cDNA, >60% of shoots regenerated without antibiotic selection carried targeted mutations, while ≤18% of shoots contained T-DNA. The PVX vector was also used to express a base editor consisting of modified Cas9 fused with cytidine deaminase to introduce targeted nucleotide substitution in regenerated shoots. We also report exogenous DNA-free genome editing by mechanical inoculation of virions comprising the PVX vector expressing Cas9. This simple and efficient virus vector-mediated delivery of CRISPR-Cas9 could facilitate transgene-free gene editing in plants.

Pattern recognition receptors (PRRs) bind microbe- and damage-associated molecular patterns (MAMPs/DAMPs, respectively) to enhance host immunity in animals and plants. Here, we report that PRRs also confer salt tolerance in the model plant Arabidopsis thaliana following recognition of cognate ligands, such as bacterial flagellin and EF-Tu and the endogenous Pep peptides. Pattern-triggered salt tolerance (PTST) requires the PRR-associated kinases BAK1 and BIK1, and the NADPH oxidase RBOHD. Transcriptome profiling reveals an inventory of PTST target genes, which increase or acquire salt responsiveness following an exposure to immunogenic patterns. In their regulatory DNA sequences, specific binding sites for a subset of WRKY transcription factors are over-represented. Accordingly, PTST requires WRKY40 and WRKY18, which activate salt tolerance-related genes but attenuate pathogen defense-related genes, including the EDS1 immunity activator. PRR signaling leads to sustained WRKY40/WRKY18 accumulation under salt stress and utilizes both WRKYs for salt tolerance. The PRR-WRKY40/WRKY18 module also confers salt tolerance after challenge with non-pathogenic bacteria. Our findings give molecular insight into signaling plasticity underlying biotic-abiotic stress cross-tolerance in plants conferred by PRRs.

Plant genome editing is achieved by the expression of sequence-specific nucleases (SSNs). RNA virus vector-mediated expression of SSNs is a promising approach for transgene integration-free targeted mutagenesis in plants. However, the removal of virus vectors from infected plants is challenging because no antiviral drugs are available against plant viruses. Here, we developed a removable RNA virus vector that carries the target site of tobacco microRNA398 (miR398) whose expression is induced during shoot regeneration. In the inoculated leaves in which expression of miR398 is not induced, insertion of the miR398 target site did not affect the practicability of the virus vector. When shoots were regenerated from the infected leaves, miR398 was expressed and viral RNA was eliminated. The virus vector successfully expressed SSNs in inoculated leaves, from which virus-free genome-edited plants were regenerated via tissue culture.

Eutrema salsugineum (also known as Thellungiella salsuginea and formerly Thellungiella halophila), a species closely related to Arabidopsis thaliana, shows tolerance not only to salt stress, but also to chilling, freezing, and high temperatures. To identify genes responsible for stress tolerance, we conducted Full-length cDNA Over-eXpressing gene (FOX) hunting among a collection of E. salsugineum cDNAs that were stress-induced according to gene ontology analysis or over-expressed in E. salsugineum compared with A. thaliana. We identified E. salsugineum CSP41b (chloroplast stem-loop-binding protein of 41 kDa; also known as CRB, chloroplast RNA binding; named here as EsCSP41b) as a gene that can confer heat and salinity stress tolerance on A. thaliana. A. thaliana CSP41b is reported to play an important role in the proper functioning of the chloroplast: the atcsp41b mutant is smaller and paler than wild-type plants and shows altered chloroplast morphology and photosynthetic performance. We observed that AtCSP41b-overexpressing transgenic A. thaliana lines also exhibited marked heat tolerance and significant salinity stress tolerance. The EsCSP41b-overexpressing transgenic A. thaliana lines showed significantly higher photosynthesis activity than wild-type plants not only under normal growth conditions but also under heat stress. In wild-type plants, the expression levels of both EsCSP41b and AtCSP41b were significantly reduced under heat or salinity stress. We conclude that maintenance of CSP41b expression under abiotic stresses may alleviate photoinhibition and improve survival under such stresses.

Osmotic stress caused by drought, salt or cold decreases plant fitness. Acquired stress tolerance defines the ability of plants to withstand stress following an initial exposure1. We found previously that acquired osmotolerance after salt stress is widespread among Arabidopsis thaliana accessions2. Here, we identify ACQOS as the locus responsible for ACQUIRED OSMOTOLERANCE. Of its five haplotypes, only plants carrying group 1 ACQOS are impaired in acquired osmotolerance. ACQOS is identical to VICTR, encoding a nucleotide-binding leucine-rich repeat (NLR) protein3. In the absence of osmotic stress, group 1 ACQOS contributes to bacterial resistance. In its presence, ACQOS causes detrimental autoimmunity, thereby reducing osmotolerance. Analysis of natural variation at the ACQOS locus suggests that functional and non-functional ACQOS alleles are being maintained due to a trade-off between biotic and abiotic stress adaptation. Thus, polymorphism in certain plant NLR genes might be influenced by competing environmental stresses.