Pseudomonas syringae pv. tomato DC3000 polymutants deploying coronatine and two type III effectors produce quantifiable chlorotic spots from individual bacterial colonies in Nicotiana benthamiana leaves

Abstract

Primary virulence factors of Pseudomonas syringae pv. tomato DC3000 include the phytotoxin coronatine (COR) and a repertoire of 29 effector proteins injected into plant cells by the type III secretion system (T3SS). DC3000 derivatives differentially producing COR, the T3SS machinery and subsets of key effectors were constructed and assayed in leaves of Nicotiana benthamiana. Bacteria were inoculated by the dipping of whole plants and assayed for population growth and the production of chlorotic spots on leaves. The strains fell into three classes. Class I strains are T3SS⁺ but functionally effectorless, grow poorly in planta and produce faint chlorotic spots only if COR⁺ . Class II strains are T3SS^- or, if T3SS⁺ , also produce effectors AvrPtoB and HopM1. Class II strains grow better than class I strains in planta and, if COR⁺ , produce robust chlorotic spots. Class III strains are T3SS⁺ and minimally produce AvrPtoB, HopM1 and three other effectors encoded in the P. syringae conserved effector locus. These strains differ from class II strains in growing better in planta, and produce chlorotic spots without COR if the precursor coronafacic acid is produced. Assays for chlorotic spot formation, in conjunction with pressure infiltration of low-level inoculum and confocal microscopy of fluorescent protein-labelled bacteria, revealed that single bacteria in the apoplast are capable of producing colonies and associated leaf spots in a 1 : 1 : 1 manner. However, COR makes no significant contribution to the bacterial colonization of the apoplast, but, instead, enables a gratuitous, semi-quantitative, surface indicator of bacterial growth, which is determined by the strain's effector composition.

Keywords: bacterial plant colonization; bacterial polymutants; coronafacic acid; coronatine; effectors; type III secretion system; virulence assays.

Figure 1

Pseudomonas syringae pv. tomato (Pto) DC3000 and D28E derivatives with and without cmaL reveal the relative contributions of effectors and coronatine (COR) to the production of chlorotic spots in dip‐inoculated Nicotiana benthamiana leaves. (A) Plants were dip inoculated at 10⁵ colony‐forming units (CFU)/mL with the indicated strains and photographed at 6 days post‐inoculation (dpi). Representative leaves from a single experiment are shown. Three plants were dipped per strain in each experiment and the experiment was repeated three times with similar results. (B) Quantification of the intensity of chlorotic spots corroborates the enhancement of symptom production by cmaL. Plants were dip inoculated with the indicated D28E derivatives as described above, and images of 40 leaf samples from two independent experiments were collected at 6 dpi and analysed using ImageJ software. The results represent least‐squares means with standard error (SE) of total intensity/cm². (C) Pto DC3000 and D28E derivatives with and without cmaL and a functional type III secretion system (T3SS) reveal that COR is able to promote the production of chlorotic spots in the absence of the T3SS or effectors. Plants were dipped in inoculum of the indicated strains at 10⁵ CFU/mL and leaves were photographed at 6 dpi. Three plants were dipped per strain, and representative leaves are shown. The experiment was repeated twice with similar results.

Figure 2

The discrete chlorotic spots produced by D28E+8 in dip‐inoculated Nicotiana benthamiana are dependent on coronafacic acid (CFA). D28E+2 and D28E+8 derivatives with and without the cfa operon were dip inoculated into N. benthamiana leaves at 10⁵ CFU/mL, which were then photographed at 6 days post‐inoculation (dpi). Three plants were inoculated per strain, and representative leaves are shown. The experiment was repeated twice with similar results.

Figure 3

Coronafacic acid (CFA), coronamic acid (CMA) and coronatine (COR) do not contribute significantly to the growth of D28E+2 or D28E+8 in dip‐ or syringe‐inoculated Nicotiana benthamiana leaves. (A) Plants were dipped with the indicated strains at 10⁵ colony‐forming units (CFU)/mL and bacterial populations in leaf tissue at 6 days post‐inoculation (dpi) were determined by plate counting. The chart shows the combined data from five independent experiments, with each experiment containing different subsets of the strains shown. Each strain was evaluated in at least two independent experiments, with three plants per strain in every experiment. (B) Leaves were inoculated by blunt syringe pressure infiltration with the indicated strains at 10⁴ CFU/mL, and bacterial populations were measured at 6 dpi. The chart shows combined data from seven independent experiments, with each experiment containing different subsets of the strains shown. Each strain was evaluated in at least three independent experiments, with three plants per strain in every experiment. Growth is depicted as the least‐squares means with standard error (SE) of log(CFU/cm²). Means marked with the same letter are not significantly different using the Tukey honestly significant difference (HSD) method of multiple sample comparison (α = 0.05).

Figure 4

D28E derivatives carrying small sets of effectors, with and without cmaL, reveal relative contributions of effectors and coronatine (COR) to plant cell death and electrolyte leakage in pressure‐infiltrated zones in Nicotiana benthamiana leaves. (A) D28E+avrPtoB+CEL and D28E+8 produce greater cell death in inoculated leaf zones of N. benthamiana if cmaL is restored to their genome. Leaves were infiltrated with the indicated strains at 10⁶ colony‐forming units (CFU)/mL and photographed at 4 days post‐inoculation (dpi). (B) Restoration of cmaL enhances electrolyte leakage from leaf tissue, as assayed by conductivity (µS/cm) of a solution containing leaf discs sampled 3 days after inoculation with D28E+avrPtoB+CEL and D28E+8 derivatives at 1.5 × 10⁶ CFU/mL. Each value represents the mean and standard deviation (SD) of four replicates. Pairwise comparisons with asterisks are significantly different (P < 0.05); ND, not different. These experiments were repeated twice with similar results.

Figure 5

The method of inoculation and level of inoculum strongly influence the nature of the symptoms produced by D28E+2+cmaL and D28E+8+cmaL in Nicotiana benthamiana. (A, B) Plants were dip inoculated (A) or vacuum infiltrated (B) with D28E+2+cmaL at the indicated levels of inoculum, and the plants were photographed at 7 days post‐inoculation (dpi) (dip) or 6 dpi (vacuum). Representative leaves from three plants per treatment are shown. It should be noted that all leaves were of equivalent size when inoculated, and similar results were observed in an independent experiment. (C) Zones in N. benthamiana leaves were inoculated by syringe infiltration with c. 50 µL of bacterial suspension at the indicated colony‐forming units (CFU)/mL and photographed at 7 dpi. Black marks on the leaves were used to delineate the perimeter of the infiltration zones, which are further outlined here in red and yellow for inoculations at 10³ and 10² CFU/mL, respectively. Inoculations were similarly performed in three leaves, with a representative leaf shown, and the experiment was repeated twice with similar results.

Figure 6

Measurements of bacterial populations of D28E+2+cmaL in chlorotic spots relative to symptomless tissue following dip inoculation, and confocal microscopy of D28E+2 derivatives labelled with fluorescent proteins, indicate that a single coronatine‐positive (COR⁺) bacterial colony can produce a chlorotic spot, but COR has no role in establishing colonies in Nicotiana benthamiana leaves. (A) Leaves were dip inoculated with D28E+2+cmaL at 10⁵ colony‐forming units (CFU)/mL, and growth was evaluated in chlorotic leaf spots or green leaf tissue at 6 days post‐inoculation (dpi). Growth is depicted as means with standard deviation of log(CFU/cm²) of 12 leaf discs from three plants per sample. Student's t‐test was used to evaluate the significance of the difference (*P ≤ 0.001). The experiment was repeated three times with similar results. The size of the leaf discs used for bacterial enumeration was 0.13 cm². (B) D28E+2+cmaL labelled with mCherry was used to dip inoculate N. benthamiana plants at 10⁵ CFU/mL, and confocal microscopy was used to image colony formation in leaves at 5 dpi. Colonies were found to be localized in chlorotic spots, whose perimeters were delineated with a black marker before microscopy (interior border outlined in green). Shown here is a representative image with a single colony (arrow) associated with a chlorotic spot in a confocal cross‐section overlay of chlorophyll fluorescence (cyan) and mCherry fluorescence (red). (C) D28E+2+cmaL labelled with mCherry was mixed 1 : 1 with D28E+2 labelled with yellow fluorescent protein (YFP), and the mixture was dip inoculated at 10⁸ CFU/mL into leaves. Leaf areas were imaged using confocal microscopy at 5 dpi. Both D28E+2+cmaL (red colonies) and D28E+2 (yellow colonies) were observed in approximately equal numbers. The representative image shown here is a three‐dimensional stack compressed to one plane, and chlorophyll fluorescence is shown in grey. Additional images from four independent experiments are shown in Fig. S4 (see Supporting Information).