A disease resistance assay in Nicotiana benthamiana reveals the immune function of Response to HopBA1

Abstract

A receptor protein variant lacking 2′,3′-cAMP/cGMP synthetase activity but retaining NADase activity does not induce cell death but confers resistance to Potato virus X.

Figure 1.

Disease resistance assay using Potato virus X expressing YFP in N. benthamiana. A) Schematic representation of disease resistance assay using Potato virus X expressing YFP (PVX-YFP). B) The emission spectra for leaf extracts expressing PVX-YFP or PVX. The y axis shows relative fluorescence units. The emission spectra were determined by a confocal microscope. C) A linear correlation between YFP fluorescence and dilution factor in a leaf lysate containing PVX-YFP in the presence and in the absence of 0.1% Triton X-100 in the extraction buffer. The x axis indicates percentage of lysate from leaves infected with PVX-YFP. Leaf lysate from nontransformed plants was used for dilution of lysate from leaves infected with PVX-YFP. The R² values with the linear regression model are >0.99 for both conditions (with or without Triton X-100). D) Representative stereomicroscopic fluorescence images of N. benthamiana leaves infiltrated with the Agrobacteria carrying the pSfinx-mYFP binary vector with OD₆₀₀ values of 0.01, 0.001, 0.0001, or 0 (from top to bottom). Means and standard errors of YFP intensity of N. benthamiana leaf lysate infiltrated with the Agrobacteria carrying the pSfinx-mYFP binary vector measured by plate reader are shown (n = 4 for OD₆₀₀ values of 0.01, 0.001, and 0.0001; n = 3 for OD₆₀₀ value of 0). Samples were examined 4 d after infiltration. Scale bars indicate 5 mm. E) Violin plots showing the degree of dispersion of the quantified YFP intensities from leaves coinfiltrated with the Agrobacteria carrying the pSfinx-mYFP binary vector and the Agrobacteria carrying a GUS gene expression binary vector. The Wilcoxon rank sum test was used to compare the 2 groups (P = 0.67). F) The R protein of the Response to HopBA1 (RBA1) variant (C83A), which lacks 2′,3′-cAMP/cGMP synthetase activity but retains NADase activity (Yu et al. 2022), does not induce cell death but confers resistance to PVX. Bar plots describe the results of 4 independent replicates for wild type and 3 independent replicates for wild type and the eds1 pad4 sag101a sag101b (epss) mutant of N. benthamiana. Red asterisks indicate significant differences from the GUS control and black asterisks depict significant differences between the 2 genotypes as determined by analysis of variance followed by Tukey's honest significant difference test (P < 0.05). Error bars indicate standard error.