Aug 2026· Plant Protection· Vol 10, pp. 753-763· 0 citations· 27 references
TL;DR
The results demonstrate the cutting-edge potential of CRISPR/Cas9 multiplex approaches as next-generation methods for designing sustainable resistance to multifaceted plant virus diseases and contribute to understanding of CRISPR-based antiviral response mechanisms.
Abstract
Cotton leaf curl disease (CLCuD), caused by begomoviruses and their betasatellites, is a major threat to cotton production, especially in South Asia, where periodic viral outbreaks continue to affect cotton yields, quality, and livelihoods. The advent of CRISPR/Cas9 gene-editing technology has transformed plant biotechnology, offering efficient, accurate, and programmable methods for combating viral pathogens at the genetic level. Here, the antiviral efficacy of two most widely used CRISPR/Cas9 binary plant expression vectors, pKSE401 and pHSE401, was tested in Nicotiana benthamiana against Cotton leaf curl Kokhran virus (CLCuKoV) and Cotton leaf curl Multan betasatellite (CLCuMuB). The guide-RNAs (gRNAs) were designed to target viral genes that play significant roles in pathogenicity and replication, with pHSE401 encoding a single gRNA and pKSE401 a multiplex of two gRNAs. Agrobacterium-mediated transient transformation and viral inoculation experiments revealed that both CRISPR/Cas9 vectors effectively delayed symptom onset and reduced virus titers relative to infected controls. Remarkably, the multiplex pKSE401 system was more effective at suppressing viral infection, achieving about a 90% reduction in viral accumulation compared with a 75% reduction by the single gRNA pHSE401 construct. pKSE401-treated plants showed delayed symptom development, reduced severity, and partial recovery, demonstrating the improved efficiency of multiplex genome editing. The results demonstrate the cutting-edge potential of CRISPR/Cas9 multiplex approaches as next-generation methods for designing sustainable resistance to multifaceted plant virus diseases. This research not only contributes to our understanding of CRISPR-based antiviral response mechanisms but also provides a promising avenue for designing broad-spectrum, sustainable resistance against viral epidemics in cotton and other commercially valuable crops.
The results demonstrate the successful deployment of CRISPR/Cas9 for targeted genome engineering in sugarbeet and establish a reliable platform for future gene-editing efforts aimed at enhancing resistance to a wide range of pathogens and diseases affecting the crop.
Z. Khan, Tinley Hathaway, C. Chu et al.· Frontiers in Genome Editing· 0 citations
Huanglongbing (HLB), caused by Candidatus Liberibacter spp., remains the most destructive disease affecting citrus worldwide. To support host-directed genome-editing strategies aimed at reducing susceptibility, we optimized key regeneration steps in Citrus sinensis and validated a dual-gRNA CRISPR/Cas9 approach targeting the susceptibility gene CsDMR6. Juvenile explants of ‘Valencia’ and hybrid genotypes (CsH1–CsH3) were successfully established in vitro, and shoot elongation was markedly improved by supplementing Citrus Shoot Multiplication (CiSM) medium with 1 mg L−1 GA3. Callus induction was most efficient in Citrus Callus Induction (CiCM) medium under dark conditions, while a 48 h NAA pulse (100 µM) significantly enhanced rooting, increasing efficiencies to 37.1% in ‘Valencia’ and 52.9% in CsH1. Two guide RNAs targeting conserved regions of CsDMR6 were designed and shown to be identical across all evaluated genotypes. The dual-gRNA cassette was assembled into a CRISPR/Cas9 geminivirus-based vector and transiently delivered into sweet orange leaf tissue via Agrobacterium. GFP fluorescence verified construct expression, and PCR amplification across the target region produced a diagnostic ~447 bp fragment corresponding to the expected ~5.8 kb deletion. Sanger sequencing confirmed precise junction formation between the two cut sites. These results demonstrate efficient large-fragment deletion of CsDMR6 in sweet orange and establish an experimentally validated, genotype-compatible regeneration and editing platform. This study provides a transient validation of the dual-gRNA system and establishes the technical foundation required for future stable, non-transgenic edited lines. Together, these advances support the downstream functional evaluation of CsDMR6 loss-of-function alleles under HLB pressure.
It is demonstrated that Avr4 does not explain the resistance of Calcutta 4, suggesting that resistance is instead triggered by the recognition of other hitherto unknown effectors.
Maikel B. F. Steentjes, Gregory Ashe, Patricia Schöppl et al.· bioRxiv· 0 citations
This study demonstrates that expression of tomato leaf curl New Delhi virus CP confers effective resistance against tomato yellow leaf curl China virus (TYLCCNV), another prominent geminivirus, and uncovers a novel mechanism by which a viral effector antagonizes viral CP-mediated host resistance during mixed viral infections.
Shi Wu, Yuzhen Mei, Yuan Chen et al.· Phytopathology Research· 0 citations
BACKGROUND
Tomato mottle mosaic virus (ToMMV) is an emerging tobamovirus that posing a significant threat to the yield and quality of solanaceous crops globally. To elucidate the regulatory role of small RNAs in the host response to ToMMV, we used Nicotiana tabacum var. Xanthi nc as a model system and performed small RNA sequencing across three distinct disease stages: mosaic symptom onset, linear leaf symptom development, and the recovery phase. By integrating qRT-PCR validation with functional assays, including miRNA overexpression, virus-induced gene silencing, and biological inoculation, several miRNAs specifically associated with ToMMV resistance were identified.
RESULTS
Functional characterization revealed that overexpression of miR171c, miR172a, and a novel miRNA (novel-miR363) significantly suppressed ToMMV accumulation and thereby enhancing plant resistance, whereas silencing of miR171c resulted in an approximately threefold increase in viral accumulation. In contrast, overexpression of another novel miRNA, novel-miR1022, promoted viral accumulation and facilitated viral infection. Further analysis through target prediction and experimental validation confirmed that miR171c and miR172a target transcripts encoding Scarecrow-like (SCL) and APETALA2 (AP2) class transcription factors, respectively. Specifically, miR171c appears to confer resistance by repressing NtSCL6 and NtSCL22, whereas miR172a likely mediates this effect through the downregulation of NtRAP2-7.
CONCLUSION
These findings elucidate key miRNA-mediated regulatory modules governing ToMMV resistance, advancing the understanding of host defense mechanisms against tobamoviruses.
Yue-Yue Li, Yu-Lian Cao, Guanlin Tan et al.· BMC Plant Biology· 1 citation
The first successful application of CRISPR/Cas9 genome editing in G. boninense is reported, establishing a robust platform for functional genetic analysis and dissecting pathogenicity in G. boninense, ultimately advancing strategies to mitigate basal stem rot disease in oil palm.
Anis Farhan Fatimi Ab Wahab, Mohd Azinuddin Ahmad Mokhtar, S. Vetaryan et al.· Journal of Fungi· 0 citations
AI is making software generation faster, but speed does not remove the need for expertise. As more work is delegated to AI, tacit knowledge may become one of the most important human advantages in software engineering. The post Beyond Prompt Engineering: The Role of Tacit Knowledge in Software Engineering appeared first on GPT-Lab.
MIT News · Artificial Intelligence· news.mit.eduSep 14, 2026
The “HardFlow” algorithm could help generative AI models produce high-quality outputs that obey strict requirements when “pretty close” doesn’t cut it.
MIT News · Artificial Intelligence· news.mit.eduSep 11, 2026
The handheld catheterization device AI-GUIDE, created by Lincoln Laboratory and Massachusetts General Hospital, promises improved health outcomes for injured service members and civilians.
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