Endophytic commensal bacteria capitalize on the AvrPto-FER pathway to enhance proliferation during early stages of pathogen invasion

Abstract

Leaves typically harbor a community of pre-existing beneficial and commensal bacteria that contribute to plant health. When pathogens invade, plants employ a series of strategies to response to the invasion, including the modulation of the microbial community structure. However, it remains unclear how commensal bacteria respond to pathogen at the early stage, and whether this response is specifically regulated. Here, we show that infection of Arabidopsis thaliana leaves by the pathogen Pseudomonas syringae pv. tomato DC3000 leads to a significant increase in the population of commensal bacteria, characterized by enrichment of Gammaproteobacteria and Alphaproteobacteria, alongside a reduction in Firmicutes and Betaproteobacteria. This cascade of events specifically occurs because AvrPto, an effector secreted by Pst DC3000, targets and inhibits the host receptor kinase FER, resulting in suppression of FER-mediated pattern-triggered immunity via the previously identified RIPK-RBOHD module. This specific suppression via FER pathway creates a condition that facilitates rapid proliferation of pre-existing commensal bacteria during early pathogen invasion. Our work provides a paradigm for the study of the interaction and ecological generality between commensal bacteria and pathogens with spatiotemporal patterns.

Keywords: commensal bacteria; endosphere; immunity; microbial community; receptor kinase.

Figure 1

The commensal bacterial population in Col-0 leaves increases during early pathogen invasion. (A) Schematic diagram showing how the effect of pathogens on the commensal community in leaves was evaluated. Two-week-old plants were preincubated with the commensal community, and pathogens (10⁶ CFU mL⁻¹) were sprayed onto the plants on the third day of culture. The leaves were surface-sterilized and the bacterial population was counted; the population of commensal bacteria was determined by subtracting the number of pathogenic bacteria on selective medium from the total bacterial count; (B) population dynamics of the commensal community in Col-0 leaves sprayed with 10⁶ CFU mL⁻¹ of commensal bacteria; (C) representative images of Col-0 plants sprayed with bacterial suspension at a concentration of 10⁶ CFU mL⁻¹. The plants were pre-sprayed with commensal community, and pathogens were spayed after 3 days. Photographs were taken 5 days after spaying with pathogens. Bar, 0.5 cm; (D) Changes in the commensal bacterial population in Col-0 following invasion by the pathogen Pst DC3000 or Psm ES4326. The leaves were incubated with commensal community for 3 days, and pathogens (10⁶ CFU mL⁻¹) were added on the third day. Relative commensal bacteria levels were calculated by dividing the population of the commensal community incubated with pathogens by that of the community not incubated with pathogens; (E) the effects of the non-pathogens DC3000^Δ28E and Nitrobacter sp. C2 (10⁶ CFU mL⁻¹) on the population of endophytic bacteria; plants were treated with DC3000^Δ28E of Nitrobacter sp. C2 after 3 days of culture; (F) the population dynamics of Pst DC3000 in Col-0 leaves in the presence and absence of commensal bacteria. Experiments were performed three times. Data were analyzed by a two-sided Student’s t test, *** indicates a significant difference (P < 0.001) at the indicated time point. Dpi, days post-inoculation.

Figure 2

Commensal bacteria in fer-4 leaves do not respond to invasion by Pst DC3000. (A) Representative images of fer-4 plants sprayed with bacterial suspension at a concentration of 10⁶ CFU mL⁻¹. The plants were pre-sprayed with commensal community, and pathogens were spayed after 3 days. Photographs were taken 5 days after spaying with pathogens. Bar, 0.5 cm; (B) the population of the commensal community in fer-4 leaves sprayed with bacteria at a density of 10⁶ CFU mL⁻¹, the data for Col-0 are the same as in Fig. 1B; (C) changes in the commensal bacterial population in fer-4 leaves following invasion by the pathogen Pst DC3000 or Psm ES4326. The leaves were incubated with the commensal community for the first 3 days, and pathogens (10⁶ CFU mL⁻¹) were added on the third day. Relative commensal bacteria levels were calculated by dividing the population of the commensal community incubated with pathogens by that of the community not incubated with pathogens; (D) the population of Pst DC3000 in fer-4 leaves with or without commensal bacteria; (E) changes in the commensal bacterial population in fls2 and bak1–4 leaves following invasion by the pathogen Pst DC3000; (F) relative abundance of endophytic leaf bacteria at the phylum level and class level for Proteobacteria. Experiments were repeated three times. Dpi, days post-inoculation.

Figure 3

AvrPto interacts with FER and inhibits its kinase activity. (A) Analysis of the effects of Pst DC3000 and DC3000^Δ28E on the phosphorylation of FER in Col-0 leaves; (B) the structure of the AvrPto–FER complex predicted by ColabFold based on the crystal structures of AvrPto (2QKW) and FER-KD (7XDV); (C) AvrPto interacts with FER in a luciferase complementation assay; (D) GST pull-down assay showing that AvrPto interacts with FER-KD. Recombinant AvrPto-GST was co-incubated with his-tag FER-KD (FER-KD-His), and the protein complex was affinity-purified with glutathione-conjugated agarose beads; (E) AvrPto interacts with FER in a co-immunoprecipitation (CoIP) assay. Proteins were extracted from 10-day-old Col-0 and pACTIN2::avrPto transgenic Arabidopsis 2 h post-inoculation for CoIP with myc beads. FER and AvrPto-myc proteins were detected by immunoblotting with anti-FER and anti-myc antibody, respectively; (F) FER-KD autophosphorylation is inhibited by AvrPto in vitro. FER-KD (1 mM) and AvrPto were incubated with 10 mM ATP and 5 mM MgCl₂ at 25°C for 30 min; (G) FER-KD phosphorylation is inhibited by AvrPto in vivo. The phosphorylation of Y648 and S701 of FER was detected in 7-day-old pACTIN2::avrPto seedlings. As a positive control, 200 nM RALF1 was added for inducing phosphorylation of FER. The results are representative of three independent experiments; (H) FER is phosphorylated following treatment with DC3000(ΔavrPto) and D28E(avrPto); (I) the commensal bacterial population in Col-0 leaves changes after Pst DC3000 and DC3000(ΔavrPto) treatment. The leaves were incubated with commensal bacteria for the first 3 days, and Pst DC3000 or DC3000(ΔavrPto) (10⁶ CFU mL⁻¹) was added on the third day. Relative commensal bacteria levels were calculated by dividing the population of the commensal community incubated with Pst DC3000 or DC3000(ΔavrPto) by that of the community not incubated with those two strains. Dpi, days post-inoculation. Experiments were performed three times. Data were analyzed by a two-sided Student’s t test, * indicates significant difference (P < 0.05) at the indicated time point.

Figure 4

The response of commensal bacteria to Pst DC3000 invasion is dependent on the kinase activity of FER. (A) Changes in the commensal bacterial population in Col-0 leaves in response to invasion by Pst DC3000 after treatment with 5 μM FER kinase inhibitors (reversine and lavendustin A). Relative commensal bacteria levels were calculated by dividing the population of the commensal community incubated with pathogens by that of the community not incubated with pathogens; (B) Changes in the commensal bacterial populations in Col-0, fer-4/FER^WT, fer-4/FER^P740A, and fer-4 leaves in response to invasion by Pst DC3000; (C) representative images of pACTIN2::avrPto plants were sprayed with bacterial suspension at a concentration of 10⁶ CFU mL⁻¹. The plants were pre-sprayed with commensal bacteria, and pathogens were sprayed after 3 days. Photographs were taken 5 days after spaying with pathogens. Bar, 0.5 cm; (D) Changes in the commensal bacterial population in Col-0 and pACTIN2::avrPto leaves in response to invasion by Pst DC3000. Experiments were performed three times. Asterisks indicate significant differences at the indicated time points, as analyzed by a two-sided Student’s t test, where **, P < 0.01; ***, P < 0.001. dpi, days post-inoculation.

Figure 5

FER affects the response of commensal bacteria to Pst DC3000 invasion via RIPK–RBOHD and PTI. (A) Representative images of fec and eds1-22 plants sprayed with bacterial suspension at a concentration of 10⁶ CFU mL⁻¹. The plants were pre-sprayed with commensal community, and pathogens were spayed after 3 days. Photographs were taken 5 days after spaying with pathogens. Bar, 0.5 cm; (B) Changes in the commensal bacterial population in fer-4, fec, and eds1-22 leaves in response to invasion by Pst DC3000. Relative commensal bacteria levels were calculated by dividing the population of the commensal community incubated with pathogens by that of the community not incubated with pathogens; (C) Representative images of Ler and ripk/Ler plants sprayed with bacterial suspension at a concentration of 10⁶ CFU mL⁻¹. The plants were pre-sprayed with commensal community, and pathogens were spayed after 3 days. Photographs were taken 5 days after spaying with pathogens. Bar, 0.5 cm; (D) Changes in the commensal bacterial population in ripk/Ler and Ler leaves in response to invasion by Pst DC3000; (E) Representative images of rbohD plants sprayed with bacterial suspension at a concentration of 10⁶ CFU mL⁻¹. The plants were pre-sprayed with commensal community, and pathogens were spayed after 3 days. Photographs were taken 5 days after spaying with pathogens. Bar, 0.5 cm; (F) Changes in the commensal bacterial population in rbohD and Col-0 leaves in response to invasion by Pst DC3000. The plants were sprayed with the commensal bacteria community (10⁶ CFU mL⁻¹) on the first day and treated with Pst DC3000 (10⁶ CFU mL⁻¹) on the third day. Experiments were repeated three times. Asterisks indicate significant differences at the indicated time points, as analyzed by a two-sided Student’s t test, where *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 6

Mechanistic model for the role of FER in regulating commensal bacteria response to pathogens. (A) Under normal conditions, FER plays a vital role in controlling the excessive growth of endophytic bacteria in the leaf and maintaining a balanced microbial population. (B) When pathogens invade, they inhibit the kinase activity of FER by releasing the effector AvrPto, decreasing the participation of FER/RIPK–RBOHD in the regulation of PTI. The increased population of commensal bacteria effect early pathogen proliferation.