Molecular characterization of tomato brown rugose fruit virus-host interactions and development of biocontrol strategies

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Zhang_Shaokang_PhD_2026_thesis.pdf (13.76 MB)

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2026-02-05

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Tomato brown rugose fruit virus (ToBRFV, Tobamovirus fructirugosum) is an emerging RNA virus that threatens global tomato production. The virus can break down durable extreme resistance (ER) to its related tobamoviruses conferred by the NLR gene Tm-22. Currently, no effective control strategies are available. This thesis research was designed to better understand ToBRFV at the molecular level, explore the mechanisms by which ToBRFV overcomes Tm-22-mediated resistance, and develop a disease management approach through biocontrol.

In this dissertation, the genomes of three ToBRFV Canadian isolates were cloned and sequenced, and full-length cDNA infectious clones were constructed. Defective mutation analyses revealed that ToBRFV movement protein (MP) and coat protein (CP) contribute to ToBRFV local accumulation and are critical for systemic infectivity. Two highly conserved CP residues, D89 and R114, are essential for ToBRFV long-distance movement. Alanine substitutions of these two residues disrupted CP self-interaction and virion formation. The 126 kDa replicase was identified as the viral RNA silencing suppressor.

Although ToBRFV has evolved the ability to evade Tm-22-mediated ER, graded inoculation assays indicated that it still confers attenuated, dose-dependent resistance in both Tm-22-carrying Nicotiana benthamiana and tomato plants. This novel discovery ascertains the value of Tm-22 in resistance breeding programs. Mechanistic studies demonstrated that the SGT1 (suppressor of G-two allele of Skp1)/HSP90 (heat shock protein 90) complex, a core complex known to be required for innate immunity in plants, interacts with ToBRFV MP, which is responsible for breakdown of Tm-22 resistance. This interaction boosts ubiquitination and facilitates proteasome degradation via SCFFBS1, a prototypical Cullin-RING E3 ubiquitin ligase complex. Phospho-mimetic assays further revealed that phosphorylated SGT1 and HSP90 negatively regulate MP abundance and ToBRFV accumulation. Application of exogenous salicylic acid (SA) could activate SGT1 and HSP90 through stabilizing the master regulator NPR1 (nonexpressor of pathogenesis-related genes 1). As a counteracting mechanism, ToBRFV CP antagonizes host defenses by specifically targeting the SGT1-HSP90 complex for proteasomal degradation.

To develop a biocontrol agent through cross-protection, a number of ToBRFV mutants were generated and screened. Three and four single amino acid mutants substantially suppressed ToBRFV symptom development and viral accumulation in N. benthamiana and tomato, respectively. Among them, the strong protection efficacy of the S643F mutant was verified in a greenhouse trial under growth conditions that mimic commercial greenhouse production.

Summary for Lay Audience

Like other living organisms, plants are constantly attacked by viral pathogens. Recently, a new virus species named tomato brown rugose fruit virus (ToBRFV), has brought serious concerns to greenhouse vegetable growers worldwide. Infection by this virus not only reduces fruit yield but also causes brown or yellow spots on tomato fruits, significantly impairing marketability. Because there are no resistant tomato cultivars or effective chemicals, ToBRFV is recognized as one of the most serious challenges to tomato production. Although management strategies are urgently needed, we only have limited knowledge about how the virus establishes successful infection in plants and how plants defend themselves against its invasion. This study characterized the viral genomes of Canadian isolates and the ongoing “arms race” between plants and ToBRFV. To initiate its infection in plant cells, ToBRFV requires two key proteins, the movement protein (MP) and coat protein (CP). Without them, ToBRFV cannot move long-distance to reach the newly emerged leaves. In response, two plant proteins called SGT1 (suppressor of the G2 allele of Skp1) and HSP90 (heat shock protein 90) protect plants by targeting and reducing MP accumulation. These two proteins also positively regulate a plant hormone signaling pathway to limit infection. As a counter approach, CP specifically targets and suppresses SGT1 and HSP90 protein accumulation, weakening the plant’s defenses to promote virus infection. This research is also committed to find tools to control ToBRFV. By introducing tiny changes into the virus genome, a number of variants were generated and several of them were found to induce mild symptoms in tomato. Pre-inoculation of these mild variants acted like vaccines, protecting plants against subsequent infection by severe ToBRFV strains. One promising candidate, carrying a single mutation, was tested in a greenhouse under near commercial greenhouse production conditions and shown to protect tomato against ToBRFV infection. Collectively, this study advanced knowledge about ToBRFV, uncovered the fine-tuned interplay between ToBRFV and infected plants, and developed viral vaccines to protect tomato production.

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ToBRFV, infectious cDNA clone, SGT1-HSP90-SCFFBS1 module, Tm-22, antiviral immunity, cross-protection

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https://hdl.handle.net/20.500.14721/39389

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