The Knockout of Enterobactin-Related Gene in Pectobacterium atrosepticum Results in Reduced Stress Resistance and Virulence towards the Primed Plants

Abstract

Siderophores produced by microorganisms to scavenge iron from the environment have been shown to contribute to virulence and/or stress resistance of some plant pathogenic bacteria. Phytopathogenic bacteria of Pectobacterium genus possess genes for the synthesis of siderophore enterobactin, which role in plant-pathogen interactions has not been elucidated. In the present study we characterized the phenotype of the mutant strain of Pba deficient for the enterobactin-biosynthetic gene entA. We showed that enterobactin may be considered as a conditionally beneficial virulence factor of Pba. The entA knockout did not reduce Pba virulence on non-primed plants; however, salicylic acid-primed plants were more resistant to ΔentA mutant than to the wild type Pba. The reduced virulence of ΔentA mutant towards the primed plants is likely explained by its compromised resistance to oxidative stress.

Keywords: Pectobacterium; enterobactin; oxidative stress; priming; salicylic acid; siderophores; virulence factors.

Figure 1

Growth curves (A) and activities of extracellular enzymes (B) of the wild type (dark grey) and ΔentA mutant (light grey) of Pectobacterium atrosepticum SCRI1043. PL—pectate lyase, PG—polygalacturonase, Cel—cellulase, Prt—protease. The enzymatic activities were determined in the culture supernatants after one day of bacteria cultivation. The presented values are means ± SD of three biological replicates. The activity levels of the wild type were equated to one. No significant differences in the assessed parameters were revealed between the wild type and ΔentA mutant (Mann-Whitney two-sided test, p < 0.05).

Figure 2

Virulence of ΔentA mutant of Pectobacterium atrosepticum SCRI1043. (A) Number of tobacco plants displaying maceration symptoms after infection with the wild type (dark grey) or ΔentA mutant (light grey) of P. atrosepticum. (B) Bacterial colony forming unit (CFU) titer in the tobacco plants infected with the wild type (dark grey) or ΔentA mutant (light grey) of P. atrosepticum. (C) Expression levels of genes LOX (lipoxygenase) and AOC (allen oxide cyclase) in non-infected plants (hashed column) and infected with the wild type (dark grey) or ΔentA mutant (light grey) of P. atrosepticum. The expression levels and CFU titers were determined one day post inoculation. The presented values in B and C are means ± SD of five biological replicates. Asterisks (*) show the significance of difference (Mann–Whitney two-sided test, p < 0.05).

Figure 3

Resistance of the wild type (dark grey), ΔentA mutant (light grey) and ΔentA complementation mutant (hashed column) of Pectobacterium atrosepticum SCRI1043 to iron depletion (20 µM Na-EDTA) (A), oxidative stress (H₂O₂) (B) and heavy metal (CuSO₄) (C). Cells were cultured 24 h before plating for CFU titer analysis. The presented values are means ± SD of three biological replicates of one of three representative experiments. Inoc—inoculation titer. Asterisks (*) show the significance of difference (Mann–Whitney two-sided test, p < 0.05).

Figure 4

The disease incidents caused by the wild type Pectobacterium atrosepticum SCRI1043 (dark gray) or its ΔentA mutant (light grey) on mock-treated (solid columns) or 0.2 mM salicylic acid-treated (hashed columns) tobacco plants. The number of the diseased plants in mock-treated group infected by the wild type was equated to 100% in each time point. The presented values are means ±SD of four independent experiments performed at 15–25 biological replicates each. Asterisks (*) show the significance of difference (Mann–Whitney two-sided test, p < 0.05).

Figure 5

Expression level of LOX2 (lipoxygenase) gene in non-primed (light grey) and salicylic acid-primed (dark grey) tobacco plants non-infected (control) or infected with the wild type (WT) or ΔentA mutant (ΔentA) of Pectobacterium atrosepticum SCRI1043. The expression levels were determined one day post inoculation. The presented values are means ± SD of four biological replicates. Asterisk (*) shows the significance of difference (Mann–Whitney two-sided test, p < 0.05).

Figure 6

The percent of non-primed (light grey) and salicylic acid-primed (dark grey) tobacco plants showing disease symptoms after infection with the wild type (WT), or ΔentA mutant (ΔentA), or ΔentA complementation mutant (Comp) of Pectobacterium atrosepticum SCRI1043. The priming was performed one day before the inoculation. The symptoms were analyzed three days post infection.

Figure 7

The level of hydrogen peroxide in non-primed (light grey) and salicylic acid-primed (dark grey) tobacco plants non-infected control (C) or infected with the wild type (WT), or ΔentA mutant (ΔentA), or ΔentA complementation mutant (Comp) of Pectobacterium atrosepticum SCRI1043. Plants with symptoms (S) and with no symptoms (N) were analyzed differentially within all experimental groups (WT, ΔentA, Comp) of infected plants. The priming was performed one day before the inoculation. Hydrogen peroxide was determined one day post inoculation. The presented values are means ± SD of three biological replicates. Asterisks (*) show the significance of difference (Mann–Whitney two-sided test, p < 0.05) between non-primed and salicylic acid (SA)-primed plants within a single variant (C, WT, ΔentA, Comp). The table located under the diagram shows significant differences between all experimental groups; (SA)-primed variants are marked by grey in the table. NS—non-significant. FW—fresh weight.