In a new publication, VIRTIGATION partner JKI built a novel ToBRFV infectious clone to explore how the virus spreads and infects plants.
Article by Marianna Granatier from the VIRTIGATION project
Background and aims of the study
The study, which JKI published recently in the prestigious Plant Disease journal, presents a new use of a full-length infectious ToBRFV clone to confirm its host range and assess the effectiveness of Agrobacterium-based delivery methods across various plant species. The JKI researchers successfully constructed such a clone, which can now serve as a standardised tool for infectivity testing in resistance screening, host range studies, and investigations into molecular interactions between ToBRFV and its hosts.
From resistance genes to viral vectors: Tools for tackling ToBRFV
Tobamoviruses like ToBRFV have a broad host range, mainly affecting plants in the Solanaceae and Cucurbitaceae families, but some also infect Apocynaceae, Brassicaceae, Cactaceae, and Malvaceae. Although resistance genes like the N-gene, L-gene, and Tm-1/Tm-2 have been identified and introduced into commercial tomato varieties, many of these genes offer only partial or limited protection against ToBRFV. To address challenges associated with virus spread, the JKI scientists note that effective strategies must be developed, including identifying new resistance genes, genetically modifying crops to prevent infection, and exploring the use of mild virus strains for cross-protection.
Infectious clones of plant viruses are valuable tools for addressing challenges in virus research and control. Researchers used tobamoviruses as one of the first plant viruses to develop infectious clones, initially using cDNA to produce infectious RNA transcripts, and later employing methods like the 35S promoter for in vivo transcription, particle bombardment, and Agrobacterium-mediated delivery of viral sequences. For ToBRFV, scientists created infectious clones based on Chinese isolates to infect tomato and test plants, with some tobacco varieties showing resistance. They also used these clones to study how ToBRFV overcomes resistance genes.
Building and testing a ToBRFV clone for host range studies
In their study, VIRTIGATION JKI researchers successfully constructed a full-length infectious clone of ToBRFV, named pJL89-ToBRFV, based on an Israeli isolate. The clone differs from the original sequence by a single nucleotide in the 3′ UTR, which does not affect the encoded proteins. With Gibson Assembly, the JKI researchers inserted the viral genome into a binary plasmid suitable for propagation in both E. coli and Agrobacterium tumefaciens, which allowed for stable storage and delivery.
JKI scientists successfully delivered the clone to Nicotiana benthamian, N. tabacum, and Solanum lycopersicum, inducing symptoms like those caused by wild-type ToBRFV. They found these particles also in plants infected with wild-type virus, confirming the clone’s ability to produce coat protein and form virions. Also, they observed that extracted virions and infected leaf tissue were infectious and induced typical symptoms in new host plants.
To evaluate the clone as a screening tool, JKI project researchers compared symptoms caused by the clone and wild-type virus across known and previously untested hosts. They found that mechanical inoculation led to local lesions in several species, confirming their susceptibility and the clone’s ability to trigger hypersensitive responses similar to the wild-type virus. The project scientists also observed systemic infection in some plants depending on the inoculation method and temperature, highlighting the clone’s utility for host range studies and resistance screening.
First report on C. perfoliate being a susceptible host to ToBRFV
Moreover, VIRTIGATION JKI researchers investigated the susceptibility of various crop-related plants to ToBRFV using both mechanical and agro-inoculation methods. They found that C. perfoliata, P. floridana, S. melongena, and S. lycopersicum could be systemically infected through mechanical inoculation, while P. floridana and S. lycopersicum showed systemic infection via agro-inoculation. Notably, they observed that C. annuum showed no signs of infection, likely due to its L1 resistance gene.
The JKI study is the first to report C. perfoliata as a susceptible host under experimental conditions, supported by findings from wild plants near infected tomato fields. They found that the virus accumulated at low levels in C. perfoliata without visible symptoms. This suggests that it could act as a silent reservoir, and therefore relevant to consider in control strategies. The susceptibility of S. melongena appears to vary by cultivar, with some previously considered non-hosts now showing infection under certain conditions. The JKI scientists observed that symptom development was influenced by plant growth stage, environmental conditions, and inoculation method.
To test whether differences in infection were due to incompatibility with Agrobacterium, JKI experts used three strains and a GFP-expressing control plasmid. GFP fluorescence confirmed successful agro-inoculation in several hosts, while others showed no expression, indicating potential host-specific limitations. These results suggest that both the method of virus delivery and host compatibility with Agrobacterium play crucial roles in ToBRFV infection outcomes. In cases where agro-inoculation fails, alternative methods like in vitro transcript delivery may be necessary, according to the JKI authors of the publication.
Unlocking new insights into plant-virus interactions with ToBRFV infectious clones
In summary, infectious viral clones are valuable tools for achieving consistent and controlled infections in plants, which is essential for resistance screening and comparing infection outcomes across different conditions. They also enable the identification of mild virus strains that may protect crops from more severe infections. The recent JKI publication exemplifies these findings through their novel ToBRFV infectious clone, which can be used not only for these purposes, but also as a viral vector for protein production and gene function studies, including overexpression and gene silencing.
Their clone has shown high expression levels in host plants like Nicotiana tabacum and Solanum lycopersicum, especially when delivered via agro-infiltration. It also proved useful for host range screening, identifying C. perfoliata as a new ToBRFV host. Its design allows for site-directed mutagenesis, enabling studies on viral replication, symptom development, and resistance mechanisms. Finally, having infectious clones from different genetic backgrounds is important for testing the durability of resistance and understanding how ToBRFV overcomes plant defenses.
More info about JKI's new publication in Plant Disease
The full version of the study in Plant Disease titled ‘A novel ToBRFV cDNA full-length infectious clone provides insights on virus host range and inoculation strategies’, authored by JKI, is available online here since 20 March 2025. JKI’s principal investigators in VIRTIGATION wrote this paper: Mareike Rohde, Heiko Ziebell and Annette Niehl. They deposited the sequence of the infectious clone in the NCBI database (GenBankaccession number PQ653969), and have made the datasets underlying their peer-reviewed, open access scientific publication directly available in the publication. Find out more about VIRTIGATION’s scientific publications on our website here.
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