Development of a rapid screen to identify formulations that enhance plant resistance to viral infection

Abstract

A screening method was developed to identify formulations that enhance plant resistance to viral infection. A modified Tobacco rattle virus (TRV) genome with green fluorescent protein (TRV-GFP) was delivered into Nicotiana benthamiana cells using Agrobacterium tumefaciens. Subjective scales based on TRV-GFP fluorescence in inoculated and systemic leaves were created and validated by examining TRV coat protein expression using RT-PCR. This rapid 7-day Agrobacterium-TRV-GFP/N. benthamiana screen was used to test many formulations containing sodium magnesium chlorophyllin (Mg-chl) with various surfactants. Treatment with Mg-chl formulation 1 and 2 resulted in significant reductions in TRV-GFP levels in N. benthamiana inoculated and systemic leaves compared to mock-treated plants, without causing phytotoxic effects. Given that Mg-chl is activated by light to produce reactive oxygen species (ROS), H₂O₂ levels were examined and shown to increase in Mg-chl-treated leaves. Elevated H₂O₂ levels may have initiated Salicylic acid (SA)- and Jasmonic acid (JA)-mediated resistance pathways as supported by expression of N.benthamiana Pathogenesis-Related1 (NbPR1) and N.benthamiana Myelocytomatosis transcription factor (NbMYC2) in formulation-treated leaves. Treatment with formulations 1 and 2 also provided resistance in Nicotiana tabacum leaves to Tobacco mosaic virus (TMV). Given that the JA pathway gene NbMYC2 was expressed in response to Mg-chl treatment, resistance to necrotrophic pathogens or insects may also be enhanced. Treatment with Mg-chl formulation 1 and 2 conferred resistance to TRV-GFP in N. benthamiana and TMV in N. tabacum without causing phytotoxic effects, indicating these formulations are ideal candidates for commercialization as plant immunity stimulators.