The revolutionary CRISPR/Cas9 genome-editing technology has emerged as a powerful tool for plant improvement, offering unprecedented precision and efficiency in making targeted gene modifications. This powerful and practical approach to genome editing offers tremendous opportunities for crop improvement, surpassing the capabilities of conventional breeding techniques. This article provides an overview of recent advancements and challenges associated with the application of CRISPR/Cas9 in plant improvement. The potential of CRISPR/Cas9 in terms of developing crops with enhanced resistance to biotic and abiotic stresses is highlighted, with examples of genes edited to confer disease resistance, drought tolerance, salt tolerance, and cold tolerance. Here, we also discuss the importance of off-target effects and the efforts made to mitigate them, including the use of shorter single-guide RNAs and dual Cas9 nickases. Furthermore, alternative delivery methods, such as protein- and RNA-based approaches, are explored, and they could potentially avoid the integration of foreign DNA into the plant genome, thus alleviating concerns related to genetically modified organisms (GMOs). We emphasize the significance of CRISPR/Cas9 in accelerating crop breeding processes, reducing editing time and costs, and enabling the introduction of desired traits at the nucleotide level. As the field of genome editing continues to evolve, it is anticipated that CRISPR/Cas9 will remain a prominent tool for crop improvement, disease resistance, and adaptation to challenging environmental conditions.

Gene silencing is a natural antiviral defense mechanism in plants. For effective infection, plant viruses encode viral silencing suppressors to counter this plant antiviral response. The geminivirus-encoded C4 protein has been identified as a gene silencing suppressor, but the underlying mechanism of action has not been characterized. Here, we report that Cotton Leaf Curl Multan virus (CLCuMuV) C4 protein interacts with S-adenosyl methionine synthetase (SAMS), a core enzyme in the methyl cycle, and inhibits SAMS enzymatic activity. By contrast, an R13A mutation in C4 abolished its capacity to interact with SAMS and to suppress SAMS enzymatic activity. Overexpression of wild-type C4, but not mutant C4^R13A, suppresses both transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS). Plants infected with CLCuMuV carrying C4^R13A show decreased levels of symptoms and viral DNA accumulation associated with enhanced viral DNA methylation. Furthermore, silencing of NbSAMS2 reduces both TGS and PTGS, but enhanced plant susceptibility to two geminiviruses CLCuMuV and Tomato yellow leaf curl China virus. These data suggest that CLCuMuV C4 suppresses both TGS and PTGS by inhibiting SAMS activity to enhance CLCuMuV infection in plants. Geminiviruses are single-stranded DNA (ssDNA) viruses that infect a wide range of plant species and are responsible for substantial crop damage worldwide. However, how geminiviruses inhibit plant antiviral gene silencing defense is unclear. Here, we report that a single geminiviral protein CLCuMuV C4 inhibits both plant transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS) to promote an effective viral infection. We show that CLCuMuV C4 protein interacts with SAMS, a core enzyme in methyl cycle, and inhibits SAMS activity. Overexpression of CLCuMuV C4 reduces the DNA methylation levels of both a transgene and an endogenous locus. Further, silencing of SAMS reduced both TGS and PTGS, and enhanced viral infection while CLCuMuV virus carrying a mutation in C4 that fails to interact with SAMS showed decreased infection. These findings reveal a novel mechanism by which the CLCuMuV C4 protein suppress SAMS mediated TGS and PTGS, leading to enhanced viral infection in plant.

The Arabidopsis flowering locus T ( FT ) gene encodes the mobile florigen essential for floral induction. While movement of the FT protein has been shown to occur within plants, systemic spread of FT mRNA remains to be unequivocally demonstrated. Utilizing novel RNA mobility assay vectors based on two distinct movement-defective viruses, Potato virus X and Turnip crinkle virus , and an agroinfiltration assay, we demonstrate that nontranslatable FT mRNA, independent of the FT protein, moves throughout Nicotiana benthamiana and mutant Arabidopsis plants and promotes systemic trafficking of viral and green fluorescence protein RNAs. Viral ectopic expression of FT induced flowering in the short-day N. tabacum Maryland Mammoth tobacco under long-day conditions. Recombinant Potato virus X bearing FT RNA spread and established systemic infection more quickly than the parental virus. The cis -acting element essential for RNA movement was mapped to the nucleotides 1 to 102 of the FT mRNA coding sequence. These data demonstrate that a plant self-mobile RNA molecule can mediate long-distance trafficking of heterologous RNAs and raise the possibility that FT RNA, along with the FT protein, may be involved in the spread of the floral stimulus throughout the plant.

Autophagy plays an important role in plant–pathogen interactions. Several pathogens including viruses induce autophagy in plants, but the underpinning mechanism remains largely unclear. Furthermore, in virus–plant interactions, viral factor(s) that induce autophagy have yet to be identified. Here, we report that the βC1 protein of Cotton leaf curl Multan betasatellite (CLCuMuB) interacts with cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC), a negative autophagic regulator, to induce autophagy in Nicotiana benthamiana. CLCuMuB βC1 bound to GAPCs and disrupted the interaction between GAPCs and autophagy-related protein 3 (ATG3). A mutant βC1 protein (βC13A) in which I45, Y48, and I53 were all substituted with Ala (A), had a dramatically reduced binding capacity with GAPCs, failed to disrupt the GAPCs-ATG3 interactions and failed to induce autophagy. Furthermore, mutant virus carrying βC13A showed increased symptoms and viral DNA accumulation associated with decreased autophagy in plants. These results suggest that CLCuMuB βC1 activates autophagy by disrupting GAPCs–ATG3 interactions.