Molecular characterization of tomato brown rugose fruit virus-host interactions and development of biocontrol strategies
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.
ToBRFV, infectious cDNA clone, SGT1-HSP90-SCFFBS1 module, Tm-22, antiviral immunity, cross-protection
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