Fig. 2.

DC3000D28E (CUCPB5585) is functionally effectorless but otherwise WT in diagnostic assays. (A) Equivalent growth of DC3000D28E and DC3000ΔhopQ1-1 in liquid mannitol glutamate minimal medium supplemented with 50 μM iron citrate (means ± SD of the absorbance of triplicate cultures). (B) Equivalent translocation of an AvrPto-Cya fusion by DC3000D28E and DC3000. Plasmid pCPP5702 encoding the reporter gene under transcriptional control of the avrPto promoter was introduced into DC3000D28E, DC3000, and the ΔhrcQ-U T3SS⁻ mutant. The resulting strains were infiltrated into N. benthamiana leaves at two different densities (10⁷ and 10⁸ cfu/mL) to control for assay saturation. cAMP concentrations were determined from tissues sampled from three independent leaves per treatment 7 h postinoculation. Means ± SD from one of two replicate experiments are shown. (C) Reduced ability of DC3000D28E to elicit ETI-like rapid plant cell death in N. benthamiana and N. tabacum. DC3000D28E and controls DC3000 (incompatible on both Nicotiana spp.), ΔhopQ1-1 (incompatible on N. tabacum), and ΔhrcQ-U cell suspensions in MgCl₂ buffer, adjusted to three densities covering the dynamic range of the assay, were infiltrated into leaves, and the plant response was photographed 48 h later. Cell death response: +, positive; −, null; ±, partial. Each experiment was repeated at least three times with similar results. (D) Ability of DC3000D28E to be transcomplemented in mixed infections with virulent DC3000ΔhopQ1-1. Equal volumes of DC3000D28E and ΔhopQ1-1 strains (Left) or ΔhrcQ-U and ΔhopQ1-1 strains (Right) standardized at 3 × 10⁶ cfu/mL (an inoculum level high enough for DC3000ΔhopQ1-1 to produce conditions favoring bacterial growth throughout the inoculated tissue) were mixed and infiltrated into leaves of N. benthamiana. Values (means ± SD of three samples) for the query strains and DC3000ΔhopQ1-1 were calculated by subtracting colony-forming unit counts on nonselective media (all strains) from colony-forming units on selective media (spectinomycin-resistant query strains). The experiment was repeated three times with similar results.