Gene-editing technology, specifically the CRISPR-Cas13a system, has shown promise in breeding plants resistant to RNA viruses. This system targets RNA and, theoretically, can also combat RNA-based viroids. To test this, the CRISPR-Cas13a system was introduced into tomato plants via transient expression and into Nicotiana benthamiana through transgenic methods, using CRISPR RNAs (crRNAs) targeting the conserved regions of both sense and antisense genomes of potato spindle tuber viroid (PSTVd). In tomato plants, the expression of CRISPR-Cas13a and crRNAs substantially reduced PSTVd accumulation and alleviated disease symptoms. In transgenic N. benthamiana plants, the PSTVd levels were lower as compared to wild-type plants. Several effective crRNAs targeting the PSTVd genomic RNA were also identified. These results demonstrate that the CRISPR-Cas13a system can effectively target and combat viroid RNAs, despite their compact structures.

Strawberry mottle virus (SMoV) significantly compromises strawberry fruit quality and yield. The Pro2Glu protein of SMoV, an RNA silencing suppressor, is pivotal for viral infection; however, its interactions with host factors remain underexplored. This study identifies a critical interaction between SMoV Pro2Glu and strawberry NADH dehydrogenase (ubiquinone) 1 β subcomplex subunit 9 (FaNDUFB9). Using yeast two‐hybrid assays and in vivo protein interaction analyses, we confirm that amino acid residues 31–45 of FaNDUFB9 are essential for binding Pro2Glu. Sequence analysis places FaNDUFB9 within the Complex1_LYR_superfamily, and subcellular localisation reveals its presence in both the mitochondria and nucleus. Silencing of NbNDUFB9 in Nicotiana benthamiana elevated Pro2Glu expression, whereas co‐expression of FaNDUFB9 with Pro2Glu suppressed Pro2Glu levels and weakened its silencing activity. Furthermore, overexpression of FaNDUFB9 in transgenic N. benthamiana reduced transient SMoV RNA expression. These findings suggest that FaNDUFB9 inhibits expression of Pro2Glu, affecting its silencing activity, and thereby potentially further influencing viral infection.

SMoV and SVBV are two major viruses that pose significant threats to the global strawberry industry. Both are latent viruses, making early detection difficult due to their uneven distribution and low concentration in host tissues. Traditional RT-PCR techniques are insufficient for precise and quantitative detection. In this study, TaqMan RT-qPCR detection systems for SMoV and SVBV were established for application in practical production settings, enabling accurate, rapid, and efficient detection of strawberry viruses. When viral accumulation in plants is low, the highly sensitive TaqMan RT-qPCR technique allows for accurate quantification, facilitating the early identification of infected plants and preventing large-scale outbreaks in cultivation areas. The development of a duplex TaqMan RT-qPCR assay enables simultaneous quantification of SMoV and SVBV in a single reaction, improving detection efficiency and providing technical support for risk assessment and effective control of strawberry viral diseases.