Global translational induction during NLR-mediated immunity in plants is dynamically regulated by CDC123, an ATP-sensitive protein

Abstract

The recognition of pathogen effectors by their cognate nucleotide-binding leucine-rich repeat (NLR) receptors activates effector-triggered immunity (ETI) in plants. ETI is associated with correlated transcriptional and translational reprogramming and subsequent death of infected cells. Whether ETI-associated translation is actively regulated or passively driven by transcriptional dynamics remains unknown. In a genetic screen using a translational reporter, we identified CDC123, an ATP-grasp protein, as a key activator of ETI-associated translation and defense. During ETI, an increase in ATP concentration facilitates CDC123-mediated assembly of the eukaryotic translation initiation factor 2 (eIF2) complex. Because ATP is required for the activation of NLRs as well as the CDC123 function, we uncovered a possible mechanism by which the defense translatome is coordinately induced during NLR-mediated immunity. The conservation of the CDC123-mediated eIF2 assembly suggests its possible role in NLR-mediated immunity beyond plants.

Keywords: ATP; CDC123; ETI; NLR; eIF2 complex assembly; effector-triggered immunity; nucleotide-binding leucine-rich repeat; plant immune response; translational reprogramming.

Figure 1.. Global translational activity is elevated during ETI

(A and B) Translational induction of the LS_TBF1-FLUC reporter during RPS2-mediated ETI. WT or rps2 plants with the reporter were inoculated with MgCl₂ (Mock) or Psm/AvrRpt2 (AvrRpt2). FLUC activity (A) was normalized to 1 hpi for each genotype and presented as mean ± SEM (n = 12). FLUC mRNA level at 7 hpi (B) was normalized to Mock for each genotype and presented as mean ± SD (n = 3). Two-tailed Student’s t test; ns, not significant. (C) Polysome profiling of lysates from WT at 7 hpi with Mock or Psm/AvrRpt2. Polysome/monosome (P/M) ratios are presented as mean ± SD (n = 3). Two-tailed Student’s t test; **, p < 0.01. (D) Translational dynamics of the LS_TBF1-FLUC reporter in WT or rps2 plants carrying DEX:AvrRpt2. Data are presented as mean ± SEM (n = 6) after normalizing to 1 hpi of DEX for each genotype. (E) Conductivity assay measuring cell death upon DEX-induced expression of AvrRpt2. Data are presented as mean ± SEM (n = 3). (F–H) New protein synthesis during ETI was detected using the SUnSET assay. Leaf discs were treated with or without DEX (F). Plants in the Col-0 ecotype were infiltrated with Psm or Psm/AvrRpm1 (G) and in the Ws-2 ecotype were treated with Pf Pf0–1/AvrRps4 or Pf Pf0–1/AvrRps4^mut (H). RuBisCo large subunit (RbcL) was stained by Ponceau S as a loading control and was probably unlabeled in the SUnSET assay explaining the blank around 55 kDa in the immunoblot. See also Figure S1.

Figure 2.. The dst7 mutant is deficient in ETI-induced translation and defense

(A) ETI-induced translational changes in dst7 and the independent complementation lines, C7 and C8. Data are presented as mean ± SEM (n = 12) after normalizing to 1 hpi for each genotype. (B) Polysome profiling of WT and dst7 at 7 hpi with Mock or Psm/AvrRpt2 (Avr). Polysome/monosome (P/M) ratios are presented as mean ± SD (n = 3). Two-tailed Student’s t test; **, p < 0.01; ns, not significant. (C) SUnSET analysis of WT and dst7 upon DEX:AvrRpt2-induced ETI. Ponceau S-stained RbcL was used as a loading control. (D) Conductivity assay measuring cell death induced by Psm/AvrRpt2 in dst7 and the complementation lines. Data are presented as mean ± SEM (n = 4). (E) Bacterial growth in dst7 and the complementation lines. Data are presented as a box-and-whisker plot (n = 8). Different letters indicate significant differences, one-way ANOVA, p < 0.05. See also Figure S2.

Figure 3.. eIF2γ, an interactor of CDC123, is a positive regulator of ETI

(A) Interactions between CDC123 and eIF2 subunits. Tagged proteins were transiently expressed in N. benthamiana. Numbers below the blot show relative band intensity normalized to the IP of CDC123-YFP. nd, not detected. (B) Effects of CDC123 on the eIF2 complex assembly. eIF2 subunits synthesized in vitro were incubated together with CDC123 (WT), CDC123-D251N (D251N), or control (−). Numbers below the blot show relative band intensity normalized to IP of eIF2γ-YFP. (C–E) ETI phenotypes of the inducible eIF2γ-silencing plants. FLUC activity (C) was normalized to 1 hpi of Psm/AvrRpt2 for each DEX:RNAi-eIF2γ line (#1 and #5, n = 12). Cell death rate (D) was assessed by conductivity measurement (n = 3). Data in (C) and (D) are presented as mean ± SEM. The bacterial population (E) is presented as a box-and-whisker plot (n = 8). Two-tailed Student’s t test; ***, p < 0.001; ns, not significant. See also Figure S3 and Table S1.

Figure 4.. Elevated ATP level during ETI enhances CDC123-mediated eIF2 complex assembly to induce translation

(A and B) Interaction dynamics of CDC123 and eIF2 subunits during DEX:AvrRpt2-induced ETI. Relative band intensity of the immunoblots (A) was normalized to IP of eIF2γ-myc (numbers below the blot) and their relative ratios (B) are presented as mean ± SD. (C) ATP level changes during ETI induced by DEX:AvrRpt2 in WT or rps2. Data are presented as mean ± SD (n = 3). One-way ANOVA; **, p < 0.01; ***, p < 0.001. (D) The effect of oligomycin A on ETI-induced eIF2 assembly. Numbers below the blot show relative band intensity normalized to IP of eIF2γ-myc. (E) eIF2 complex assembly in WT or dst7 plants upon ETI induction. Numbers below the blot show relative band intensity normalized to IP of eIF2α. nd, not detected. (F) ATP concentration in WT or dst7 plants in response to DEX:AvrRpt2 induction. Data are presented as mean ± SD (n = 3). Two-tailed Student’s t test; ***, p < 0.001; ****, p < 0.0001. (G) The effect of oligomycin A treatment on ETI cell death measured by the conductivity assay. Conductivity was measured at 16 hpi of DEX. Data are presented as a box-and-whisker plot (n = 6). Two-way ANOVA; ***, p < 0.0001. (H) Proposed model for the CDC123 function in regulating translation during ETI. See also Figure S4.