Suppression of plant defense responses by extracellular metabolites from Pseudomonas syringae pv. tabaci in Nicotiana benthamiana

Abstract

Background: Pseudomonas syringae pv. tabaci (Pstab) is the causal agent of wildfire disease in tobacco plants. Several pathovars of Pseudomonas syringae produce a phytotoxic extracellular metabolite called coronatine (COR). COR has been shown to suppress plant defense responses. Interestingly, Pstab does not produce COR but still actively suppresses early plant defense responses. It is not clear if Pstab produces any extracellular metabolites that actively suppress early defense during bacterial pathogenesis.

Results: We found that the Pstab extracellular metabolite extracts (Pstab extracts) remarkably suppressed stomatal closure and nonhost hypersensitive response (HR) cell death induced by a nonhost pathogen, P. syringae pv. tomato T1 (Pst T1), in Nicotiana benthamiana. We also found that the accumulation of nonhost pathogens, Pst T1 and P. syringae pv. glycinea (Psgly), was increased in N. benthamiana plants upon treatment with Pstab extracts . The HR cell death induced by Pathogen-Associated Molecular Pattern (INF1), gene-for-gene interaction (Pto/AvrPto and Cf-9/AvrCf-9) and ethanol was not delayed or suppressed by Pstab extracts. We performed metabolite profiling to investigate the extracellular metabolites from Pstab using UPLC-qTOF-MS and identified 49 extracellular metabolites from the Pstab supernatant culture. The results from gene expression profiling of PR-1, PR-2, PR-5, PDF1.2, ABA1, COI1, and HSR203J suggest that Pstab extracellular metabolites may interfere with SA-mediated defense pathways.

Conclusions: In this study, we found that Pstab extracts suppress plant defense responses such as stomatal closure and nonhost HR cell death induced by the nonhost bacterial pathogen Pst T1 in N. benthamiana.

Figure 1

Suppression of stomatal closure and hypersensitive response (HR) cell death by the host bacterial pathogen Pstab in N. benthamiana. (A) Inoculation of GFPuv-expressing bacterial pathogen of Pstab (6×10³ CFU/ml) in N. benthamiana and Pst T1 (6×10³ CFU/ml) in tomato. Plants were spray inoculated with appropriate bacteria and photographed three days after inoculation. (B) Confocal microscopic images of GFPuv-expressing Pstab and Pst T1 on epidermal peels of N. benthamiana and tomato, respectively. Pstab in N. benthamiana and Pst T1 in tomato were able to reopen stomata 3 hrs after bacterial inoculation, localized around open stomata and in apoplastic space. Epidermal peels were stained with FM4-64 (Invitrogen, Grand Island, N.Y.) to visualize the plasma membrane. Several confocal images were taken from z-series by focusing the stomatal and apoplastic region. (C) Suppression of nonhost HR cell death by Pstab in N. benthamiana. N. benthamiana leaves were syringe-infiltrated with host or nonhost pathogens, Pstab or Pst T1, with a concentration of 2×10⁶ CFU/ml, respectively. Image was taken 16 hours after inoculation.

Figure 2

Suppression of stomatal defense by the metabolite extract from Pstab supernatant in N. benthamiana. (A) Suppression of stomatal closure by Pstab extracts in N. benthamiana epidermal peels. KCl-MES; stomata opening buffer, MG ext/Pst T1; bacterial suspension of Pst T1 with MG media extracts, and Pstab ext/Pst T1; bacterial suspension of Pst T1 with Pstab extracts. (B) Ability of Pst T1 migration through stomata and a number of bacterial cells of Pst T1 in apoplastic space. Detached leaves of N. benthamiana were floated on Pst T1 suspension (1×10⁷ CFU/ml) along with MG media, Pstab supernatant, and Pstab cell pellet extracts, and samples were collected at 1 hr and 4 hrs after incubation. Bars represent the means ± standard deviation (SD) (P<0.05, student’s t-test).

Figure 3

Suppression of nonhost HR cell death and enhancement of bacterial multiplication by Pstab extracts in N. benthamiana. (A) Suppression of nonhost HR cell death by the ethyl acetate extract from Pstab culture supernatant (Pstab ext.). Pst T1 (2.1 x 10⁶ CFU/ml) was infiltrated in N. benthamiana leaves, and photo was taken at 48 hpi. (B) Non-phytotoxin activity of Pstab extracts in N. benthamiana leaf. A high concentration of the Pstab extracts was used, 24 μl/ml instead of 6 μl/ml. Photographs were taken three days after infiltration. (C and D) Bacterial multiplications of nonhost pathogens P. s. pv. tomato T1 (Pst T1) and P. s. pv. glycinea (Psgly) after inoculation with Pstab extracts in N. benthamiana. Pst T1 (C) and Psgly (D) (3×10⁴ CFU/ml) were inoculated with Pstab extracts, and the bacterial growth was measured. The experiment was repeated twice (three replications for each experiment) with similar results. Bars represent the means ± standard deviation (SD) (P<0.05, student’s t-test). (E) Temperature stability of Pstab extracts and suppression of nonhost HR cell death for three nonhost pathogens, P. s. pv. phaseolicola (Psp), P. s. pv. maculicola (Psm), and Pst T1. Pstab extracts were boiled (5 min/95°C) and co-infiltrated with the bacterial suspensions. Photographs were taken at 24 hpi.

Figure 4

HR cell death-induced by Avr-R interactions, PAMP, and ethanol was not altered by Pstab extracts in N. benthamiana. N. benthamiana leaves were infiltrated with either Agrobacterium carrying the binary vector that can express INF1 or Cf-9 + AvrCf-9 or Pto + AvrPto. Photographs were taken three days after agroinfiltration for Pto/AvrPto, five days after inoculation for INF1 and Cf-9/AvrCf-9. To determine chemical induced cell death, 20% EtOH was infiltrated and photographs were taken two days after infiltration.

Figure 5

UHPLC-qTOF-MS total ion current chromatograms (TIC) obtained in the negative electrospray ionization mode of the extracts from the Pstab culture supernatant (A) and the MG medium used for Pstab culture as the control (B). The list of extracellular metabolites from Pstab is shown in Supplementary Table 1.

Figure 6

Expression patterns of PR1, PR2, PR5, PDF1.2, ABA1, COI1 and HSR203J genes after inoculation with Pst T1 + Pstab extracts in N. benthamiana. The expression level of PR1, PR2, PR5, PDF1.2, ABA1, COI1 and HSR203J was determined by qRT-PCR after inoculation of Pst T1 with MG medium extracts or Pstab extracts in N. benthamiana. Gene expression analyses were performed using three biological replications, where each biological replicate consists of three technical replicates. Bars represent the means ± standard deviation (SD).). Same letters above bars indicate no statistically significant difference (P<0.05) among plant genotypes for a given time point using one-way ANOVA followed by LSD test analysis. The qRT-PCR data were normalized to NbActin transcript and shown as relative to that of target gene expressions in 0 hr N. benthamiana leaves without any treatment.