A Breach in Plant Defences: Pseudomonas syringae pv. actinidiae Targets Ethylene Signalling to Overcome Actinidia chinensis Pathogen Responses

Abstract

Ethylene interacts with other plant hormones to modulate many aspects of plant metabolism, including defence and stomata regulation. Therefore, its manipulation may allow plant pathogens to overcome the host's immune responses. This work investigates the role of ethylene as a virulence factor for Pseudomonas syringae pv. actinidiae (Psa), the aetiological agent of the bacterial canker of kiwifruit. The pandemic, highly virulent biovar of this pathogen produces ethylene, whereas the biovars isolated in Japan and Korea do not. Ethylene production is modulated in planta by light/dark cycle. Exogenous ethylene application stimulates bacterial virulence, and restricts or increases host colonisation if performed before or after inoculation, respectively. The deletion of a gene, unrelated to known bacterial biosynthetic pathways and putatively encoding for an oxidoreductase, abolishes ethylene production and reduces the pathogen growth rate in planta. Ethylene production by Psa may be a recently and independently evolved virulence trait in the arms race against the host. Plant- and pathogen-derived ethylene may concur in the activation/suppression of immune responses, in the chemotaxis toward a suitable entry point, or in the endophytic colonisation.

Keywords: bacterial canker of kiwifruit; plant hormones; plant immunity; stomata opening; virulence factors.

Figure 1

Ethylene emissions from bacterial cultures of several Pseudomonas syringae pv. actinidiae strains. Measures were taken 3 days after inoculation of fresh plant extract. Negative control (axenic medium) was taken for reference, and its reading was subtracted to all the values. Strain CFBP7286-Δbep is a defective mutant for the gene Bacterial Ethylene Putative Producer (bep). Different letters indicate statistically different ethylene contents (p ≤ 0.05, n = 3) according to ANOVA and Fisher’s least significant difference test. A strain of P. syringae pv. glycinea (Psg NCPPB1883), used as the positive control, was not considered for statistics.

Figure 2

Comparative kinetics of population growth and ethylene release from Pseudomonas syringae pv. actinidiae. The strain CFBP7286 was grown in liquid culture. Values are presented as the average ± standard error (n = 4).

Figure 3

Ethylene emissions of Pseudomonas syringae pv. actinidiae strain CFBP7286. Measures were taken 72 h after inoculation in modified liquid media containing 1 mM L-methionine (L-Met), 10 mM 2-oxoglutarate (2OG), 2 mM NAD⁺, 2 mM dehydroascorbate (DHA), 1 mM oxidised glutathione (GSSG), 2 mM NADH, 2 mM ascorbic acid (AsA), or 2 mM glutathione (GSH). Different letters indicate significantly different (p ≤ 0.05, n = 3) ethylene emissions, according to ANOVA and Fisher’s least significant difference test.

Figure 4

Expression of genes related to bacterial motility and virulence in liquid cultures of Pseudomonas syringae pv. actinidiae strain CFBP7286 exposed to ethylene. Data are shown as the relative expression compared to housekeeping genes. All data pairs, except hopZ5 and bep, are significantly (p ≤ 0.05, n = 3) different according to Student’s T test.

Figure 5

In planta growth of Pseudomonas syringae pv. actinidiae. (a) Epiphytic and (b) endophytic population of Pseudomonas syringae pv. actinidiae strains CFBP7286 and CFBP7286-Δbep in in vitro Actinidia deliciosa plants was measured 1–7 days post inoculation. Values are presented as the average ± standard error (n = 3). An asterisk indicates a significant difference (determined by Student’s T test with p ≤ 0.05) in the relative data pair.

Figure 6

Stomata colonisation and stomata opening induced by Pseudomonas syringae pv. actinidiae. In vivo stomata colonisation (a,b) and stomatal conductance of Actinidia deliciosa (c) and A. chinensis (d) plants, after inoculation with Pseudomonas syringae pv. actinidiae strains CFBP7286 or Psa-K2. (a) In confocal laser scanning micrographs of Actinidia deliciosa stomata colonised by Pseudomonas syringae pv. actinidiae strains CFBP7286-GFPuv, each green rod is a single pathogen cell; (b) stomata in an uninfected leaf. (c,d) Stomatal conductance measured in real-time by gas exchange analyser (CIRAS-1). For each time point, significant differences (determined by ANOVA followed by Fisher’s least significant difference test with p ≤ 0.05, n = 4) are indicated by different letters.

Figure 7

Interaction of Pseudomonas syringae pv. actinidiae with ABA on stomata. (a) Stomatal conductance of Actinidia deliciosa or (b) A. chinensis plants treated with ABA 1 day before inoculation with Pseudomonas syringae pv. actinidiae strains CFBP7286 or Psa-K2. For each time point, significant differences (determined by ANOVA followed by Fisher’s least significant difference test with p ≤ 0.05, n = 4) are indicated by different letters.

Figure 8

Time-dependent ethylene release from infected plants. (a) Ethylene release from in vitro Actinidia deliciosa ‘Hayward’ plants, inoculated with Pseudomonas syringae pv. actinidiae (Psa strain CFBP7286) or the compatible pathogen Pseudomonas syringae pv. syringae (Pss strain LT23). Data show the typical emission of singular samples. (b) Ethylene release from in vitro Actinidia deliciosa ‘Hayward’ plants, inoculated with several Psa strains belonging to biovars 1, 2 and 3. Values are presented as the average ± standard error (n = 3). In both experiments, the light:dark cycle was 16:8 h.

Figure 9

Ethylene emission from Actinidia deliciosa microcuttings, included in 500-mL jars with AVG-containing medium, 1 week after inoculation with Pseudomonas syringae pv. actinidiae strain CFBP7286. Different lower-case letters indicate significant differences (determined by two-way ANOVA followed by Fisher’s least significant difference test with p ≤ 0.05, n = 3) due to interaction between AVG treatment and pathogen inoculation. Upper-case letters refer to single-factor differences between AVG treatment and water.

Figure 10

Expression of genes related to ethylene signalling in in vitro Actinidia deliciosa and A. chinensis plants. Gene expression was measured 6 h after inoculation with Pseudomonas syringae pv. actinidiae strain CFBP7286, or after treatment with ethylene. Data are expressed as fold change of relative gene expression, calculated with relation to housekeeping genes, compared to untreated plants of the same species. Values are presented as the average ± standard error (n = 3).

Figure 11

Effect of modulators of ethylene metabolim on bacterial colonisation. Endophytic and epiphytic populations of Pseudomonas syringae pv. actinidiae strain CFBP7286-GFPuv were measured in in vitro Actinidia deliciosa plants treated with 1-methylcyclopropene (1-MCP), ethylene (either pre- or post-inoculation) or aminoethoxyvinylglycyne (AVG). Values are presented as the average ± standard error (n = 4). Significant differences, determined by ANOVA and Fisher’s least significant difference test with p ≤ 0.05, are indicated by lower case (for endophytic populations) or upper case (for epiphytic populations) letters.