Hak, H. | Search bioRxiv

bioRxiv2021

Differential detection of Tomato mosaic virus (ToMV) and Tomato brown rugose fruit virus (ToBRFV) using CRISPR-Cas12

CRISPR/Cas12-based detection is a novel approach for efficient, sequence-specific identification of viruses. Here we adopt the use of CRISPR/Cas12a to identify the Tomato brown rugose fruit virus (ToBRFV), a new and emerging Tobamovirus causing substantial damage to the global tomato industry. Specific guide RNAs (gRNAs) were designed to detect either ToBRFV or the closely related Tomato mosaic virus (ToMV). This technology enabled the differential detection of ToBRFV and ToMV. Sensitivity assays revealed that viruses can be detected from 15-30 ng of RT-PCR product, and that specific detection could be achieved from a mix of ToMV and ToBRFV. In addition, we show that this method enabled the identification of ToBRFV in samples collected from commercial greenhouses. These results demonstrate a new method for species-specific detection of plant viruses. This could provide a platform for the development of efficient and user-friendly ways to distinguish between closely related strains and resistance-breaking pathogens.

molecular biology↗

bioRxiv2020

The Tomato brown rugose fruit virus movement protein overcomes Tm-22 resistance while attenuating viral transport

Tomato brown rugose fruit virus (ToBRFV) is a new virus of the Tobamovirus genus, causing substantial damage to tomato crops in the Middle East. Reports of recent ToBRFV outbreaks from around the world indicate an emerging global epidemic. ToBRFV overcomes all tobamovirus resistances in tomato, including the durable Tm-22 resistance gene. Here, we show that the ToBRFV movement protein (MPToBRFV) is the cause for overcoming Tm-22 resistance. Transient expression of MPToBRFV failed to activate the Tm-22 resistance response. Replacement of the original MP sequences of Tomato mosaic virus (ToMV) with MPToBRFV enabled this recombinant virus to overcome Tm-22 resistance. Hybrid protein analysis revealed that the resistance-breaking elements are located between MPToBRFV amino acids 1 and 216, and not the C terminus as previously assumed. Interestingly, replacement of Tobacco mosaic virus (TMV) and ToMV MPs with MPToBRFV caused an attenuation of systemic infection of both viruses. Cell-to-cell movement analysis revealed that MPToBRFV moves less effectively compared to the TMV MP (MPTMV). These findings suggest that overcoming Tm-22 is associated with attenuated MP function. This viral fitness cost may explain the high durability of Tm-22 resistance, which had remained unbroken for over 60 years.

plant biology↗