The immune NIK1/RPL10/LIMYB signaling module regulates photosynthesis and translation under biotic and abiotic stresses

Abstract

Photosynthesis and translation are targets of metabolic control and development in plants, yet how stress signals coordinately regulate these opposing energy-producing and consuming processes remains enigmatic. Here, we unravel a growth control circuit that ties photosynthesis to translational control in response to biotic and abiotic signals. Our findings reveal that the L10-INTERACTING MYB DOMAIN-CONTAINING PROTEIN (LIMYB), a key player of the NUCLEAR SHUTTLE PROTEIN-INTERACTING KINASE 1 (NIK1)/ RIBOSOMAL PROTEIN L10 (RPL10) antiviral signaling pathway, not only downregulates translation genes, but also represses photosynthesis-related genes and photosynthesis itself. LIMYB repressor activity, regulated by phosphorylation, is crucial for the decline in photosynthesis under stress. NIK1 activation by PAMPs or the phosphomimetic NIK1-T474D represses photosynthesis-related genes and photosynthesis in control but not in limyb lines. Furthermore, heat and osmotic stress also activate the NIK1/RPL10/LIMYB signaling circuit in wild type. These stresses induce NIK1 phosphorylation, but not marker gene repression, in limyb, indicating that LIMYB connects NIK1 activation to stress-mediated downregulation of translation- and photosynthesis-related genes. This coordinated repression via the NIK1/RPL10/LIMYB module may help plants adapt to changing environments.

Fig. 1. LIMYB represses the expression of photosynthetic apparatus-related genes and inhibits photosynthesis.

a Top enriched motifs in the promoter region of LIMYB-regulated genes. The p-value was calculated using a statistical model based on log-likelihood ratios (LLRs) and adjusted for multiple testing using the Benjamini-Hochberg (BH) method. b LIMYB DNA-binding motifs are enriched in the promoter region of LIMYB downregulated genes. In the schematic representation, upstream promoter sequences are green, and coding regions are red. ChIP-seq peaks map to the promoter region, whereas the RNA-seq hits lay in the coding region. Abundance of RNA-sequencing hits in blue (LIMYB) and red (Col-0) shows the relative gene expression in Col-0 and LIMYB-overexpressing lines. c LIMYB represses the PsbP promoter. N. benthamiana leaves were agroinfiltrated with promoter fusions as shown and the 35S: LIMYB construct. After 48 h, luciferase activity was measured from total protein extracts from transformed leaves. An unrelated Ubiquitin promoter (UBQ) was used as a negative control. Results are mean values ± SE (n = 6). Asterisks indicate significant differences from the control line (two-tailed unpaired Student’s t-test, p < 0.05). d LIMYB binds to the CAAAAC DNA binding motif on the RPL18 promoter by EMSA. A 26-bp-biotinylated CAAAAC-containing fragment was incubated with purified His-LIMYB, and the DNA-protein complex was resolved by SDS-PAGE. A 100-fold molar excess of unlabeled RPL18 26-bp dsDNA was used as a specific competitor, and a mutated 26-bp dsDNA containing ATCGTG as a nonspecific competitor. e, f The PsbP (subunit of the PHSII-OEC) and FD1 (Ferredoxin) genes are repressed by LIMYB. The transcript levels were quantified by RT-qPCR in LIMYB-overexpressing (limyb-32-L1 and limyb-32-L3) lines and Col-0. Data are presented as mean ± SE from three independent replicates. Statistical significance was assessed using one-way analysis of variance (ANOVA), followed by Dunnett’s post hoc test for multiple comparisons with the control group. Asterisks indicate significant differences from the control line (p < 0.05). g Electron transport rate (ETR) in LIMYB-overexpressing lines (limyb-32-L1, L3, L4), and limyb knockout line. PAR denotes Photosynthetically Active Radiation. h CO₂ assimilation rate (A) is reduced in LIMYB-overexpressing lines. For g, h data are mean ± SE (n = 6). Asterisks indicate significant differences from the control line (two-tailed unpaired Student’s t-test, p < 0.05). Exact p-values in the Source Data file.

Fig. 2. LIMYB-mediated transcriptional repression of target genes causes an earlier reduction in protein levels that precedes the NIK1-mediated suppression of global translation.

a The constitutively activated NIK1 mutant T474D represses the RPL18 promoter activity. Arabidopsis protoplasts were transformed with the proL18:luciferase construct alone or combined with the 35S:T474D construct. Time zero was considered 12 h after transfection. Transcript levels were determined by RT-qPCR (a) and luciferase activity was determined at different time points (b). c, d Time course of LIMYB-controlled mRNA accumulation (c) and luciferase activity under the control of RPL18 promoter (d). Protoplasts from limyb knockout lines co-transformed with 35S:LIMYB-GFP + proL18(2):Luciferase (proL18) or proUBQ:Luciferase (proUBQ) were treated with a viral PAMP (IR-vDNA; intergenic region from the component B of a begomovirus), and mRNA accumulation and luciferase activity were measured in the intervals of 3, 6, 8, and 16 h after the viral PAMP treatment. Time zero was considered 12 h after transfection. e, f NIK1-T474D does not affect the activity of the LIMYB non-target UBQ promoter. Arabidopsis protoplasts were transformed with the proUBQ:luciferase construct alone or with the 35S:T474D construct. Transcript levels were determined by RT-qPCR (e), and luciferase activity was determined at different time points (f). Time zero was considered 12 h after transfection. g, h LIMYB does not target the UBQ promoter but decreases luciferase activity (protein level) as a late response. Progression time of mRNA accumulation and luciferase activity under the control of the UBQ promoter after LIMYB expression. For a, b, e, f, the results are presented as mean values ± SD (n = 6) and for c, d, g, h, data are mean ± SD (n = 3). Asterisks indicate significant differences from the control line (two-tailed unpaired Student’s t-test, p < 0.05). The experiments were repeated at least twice with similar results.

Fig. 3. NIK1 activation by biotic signals represses the expression of photosynthetic apparatus-related genes.

a NIK1 is rapidly phosphorylated by begomovirus-derived nuclei acids. NIK1-HA-expressing Arabidopsis seedlings were treated with RNA or DNA prepared from mock-inoculated (UnRNA and UnDNA) or begomovirus-infected (InRNA and InDNA) plants for the time indicated in the figure. NIK1 was immunoprecipitated with an α-NIK1 antibody and probed with an α-phospho-threonine antibody. b Viral PAMPs require NIK1 and/or NIK2 to repress the expression of the PsbP gene. Leaf discs of the indicated genotypes were treated with InRNA and InDNA, and PsbP transcript levels were quantified by RT-qPCR. NIK1-T474D-6 is a transgenic line that expresses the NIK1-T474D mutant. c Constitutive activation of NIK1 in T474D-expressing (T474D-4 and T474D-6) lines downregulates PsbP and FD1. Total RNA from the indicated genotypes and the transcript levels of the indicated genes were quantified by RT-qPCR. For (b and c) data are presented as mean ± SE from three independent replicates. Statistical significance was assessed using one-way analysis of variance (ANOVA), followed by Dunnett’s post hoc test for multiple comparisons with the control group. Asterisks denote statistically significant differences from the control line at p < 0.05.

Fig. 4. The LIMYB-mediated inhibition of photosynthesis depends on NIK1 activation.

Leaf gas exchange parameters and photochemical processes were measured in expanded leaves from 30-days-old plants from the indicated genotypes. a electron transport rate (ETR), b net CO₂ assimilation rate (A), c stomatal conductance (gs), d transpiration rate (E), e the internal concentration of CO₂ (Ci), f quantum efficiency (ΦPSII), g water-use efficiency (WUE) The experiments were repeated at least twice with similar results. Bars ( ± SD) with an asterisk differ from each other by the two-tailed unpaired Student’s t‐test (p < 0.05), n = 5. h T474D requires the function of LIMYB to mediate photosynthesis-related gene repression. RNA were extracted from three limyb lines, ectopically expressing 35S::T474D, and the expression of photosynthesis-related genes was monitored by RT-qPCR. The experiments were repeated three times with similar results. Data are mean ± SE (n = 6). Asterisks indicate significant differences from the control line (two-tailed unpaired Student’s t-test, p < 0.05). The exact p-values can be found in the Source Data file.

Fig. 5. Biotic stimuli induce LIMYB phosphorylation via NIK1 activation.

a The bacterial PAMP, flg22, and viral PAMP, InRNA, induce phosphorylation of endogenous LIMYB. Col-0 seedlings were incubated with RNA prepared from begomovirus-infected leaves (InRNA), mock-inoculated leaves (UnRNA), H₂O, and 100 mM flg22 for 3 h. LIMYB was immunoprecipitated from total protein extracts with an anti-LIMYB polyclonal antibody and immunoblotted with α-phosphoserine (α-pSer) (top) and α-LIMYB (bottom) antibodies. b Viral PAMP-induced LIMYB phosphorylation requires the NIK1/NIK2 function. limyb and nik1/nik2 protoplasts transfected with LIMYB-GFP were treated with InRNA or UnRNA, and the immunoprecipitated LIMYB-GFP was probed with an α-phosphoserine (α-pSer) antibody. c Flg22-induced phosphorylation of LIMYB requires the NIK1/NIK2 function. Col-0 and nik1/nik2 protoplasts transfected with LIMYB-GFP were treated with flg22. Half of the immunoprecipitated LIMYB was fractionated by SDS-PAGE and immunoblotted with an α-phosphoserine (α-pSer) antibody, and the other half with an α-GFP antibody. d LIMYB-S157A does not mediate the repression of the RPL18 promoter in the presence of InRNA. Col-0 protoplasts, LIMYB-S157A-expressing limyb protoplasts, and nik1nik2 protoplasts were transfected with plasmids carrying the RPL13 promoter fused to luciferase and treated with UnRNA or InRNA. After 16 h, luciferase activity was measured from total protein extracts of transfected protoplasts. Data are mean values ± SD (n = 3) (two-tailed unpaired Student’s t-test, *p < 0.01, ns = not significant). e Ser157 is a possible phosphorylation site on LIMYB. The limyb protoplasts transfected with LIMYB-GFP or LIMYB-S157A-GFP were treated with InRNA or UnRNA and protein phosphorylation was assayed by immunoblotting immunoprecipitated GFP fusions with anti-phosphoserine antibody. f UnRNA fails to cause downregulation of NIK1 signaling-associated marker genes in LIMYB-S157A-expressing limyb protoplasts. RNA was extracted from UnRNA and InRNA-treated protoplasts, and transcript accumulation was determined by RT-qPCR. Results are mean values ± SD (n = 3) (two-tailed unpaired Student’s t-test, *p < 0.01, ns = not significant). All the above experiments were repeated at least twice with similar results.

Fig. 6. Viral PAMPs require NIK1/NIK2, but not FLS2 and BAK1, to activate the RPL10/LIMYB signaling module.

a The bacterial PAMP flg22 requires BAK1 and FLS2 to induce RPL10 phosphorylation. Arabidopsis protoplasts prepared from the indicated genotypes were transformed with 35S:RPL10-GFP. After 12-h for RPL10-GFP expression, the protoplasts were treated with PAMPs. Immunoprecipitated RPL10-GFP by an α-GFP antibody was probed with an α-phosphoserine antibody. The experiments were repeated at least three times with similar results. b, c RNA from infected plants requires NIK1/NIK2, but not FLS2 and BAK1, to mediate RPL10 phosphorylation. RNA prepared from mock-inoculated (Un) or begomovirus-infected (In) plants was used to activate NIK1 signaling. NIK1-T474D served as a positive control. Half of the immunoprecipitated RPL10-GFP from total protein extracts was fractionated by SDS-PAGE and immunoblotted with an α-phosphoserine (α-pSer) antibody and the other half was immunoblotted with an α-GFP antibody. d Viral PAMP induces LIMYB phosphorylation independent on BAK1 and FLS2. Seedlings from the indicated genotypes were treated with viral PAMPs for 30 min. LIMYB was immunoprecipitated with α-LIMYB antibody and probed with an α-phosphoserine antibody. e–g Viral PAMP activates NIK1 signaling and mediates repression of the indicated marker genes. Seedlings of the indicated genotypes were treated with viral PAMP InRNA for 1 h and 3 h and transcript accumulation was determined by RT-qPCR, using actin as an endogenous control for normalization. Data are presented as mean ± SE from three independent replicates. Statistical significance was assessed using one-way analysis of variance (ANOVA), followed by Dunnett’s post hoc test for multiple comparisons with the control group. Asterisks indicate significant differences at p < 0.05.

Fig. 7. High temperature activates NIK1 antiviral signaling.

a Rapid NIK1 phosphorylation induced by heat stress. Twenty-eight days-old plants were subjected to 38 °C for 0 to 3 h. NIK1 was immunoprecipitated from proteins extracts with an α-NIK1 antibody and probed with α-phosphoserine (p-Ser) and α-NIK1 antibodies. b NIK1-HA is correctly phosphorylated in response to heat. NIK1-HA-expressing lines were subjected to heat stress as in A. NIK1-HA was immunoprecipitated with an α-HA antibody and probed with α-pSer and α-NIK1 antibodies. c Heat stress mediates RPL10 phosphorylation. RPL10-GFP-overexpressing lines were heat-treated, and immunoprecipitated RPL10-GFP was probed with α-pSer and α-GFP antibodies. d Heat stress mediates LIMYB phosphorylation. YFP-LIMYB-expressing lines were heat treated for 1 h and 3 h. Immunoprecipitated LIMYB was probed with α-pSer. e Heat stress induces phosphorylation of endogenous LIMYB. Col-0 and T474D-expressing lines were heat-treated for the indicated time, LYMIB was immunoprecipitated with anti-LIMYB and probed with α-pSer. A T474D-expressing line is a positive control. f Heat stress represses PsbP and FD1 genes in a NIK1- or NIK2-dependent manner. Plants were heat-treated for 1-h and 3-h and gene expression was analyzed by qRT-PCR. Data are shown as the mean ± SE (n = 3). Asterisks indicate statistically significant differences to the untreated sample (one way ANOVA, multiple comparisons, Dunnett’s test p < 0.05). g High temperature suppresses ribosomal protein gene expression. RT-qPCR data are shown as the mean ± SE (n = 3). Asterisks indicate statistically significant differences to the untreated sample (one way ANOVA, multiple comparisons, Dunnett’s test p < 0.05). For f, g Exact p-values in the Source Data file. h Heat stress induces rapid NIK1 phosphorylation in the limyb knockout line. NIK1 was immunoprecipitated with α-NIK and immunoblotted with α-phosphothreonine, α-phosphoserine, and α-NIK antibodies. i Heat stress does not induce repression of NIK1 signaling pathway-associated marker genes in limyb-32 mutant. The expression of the marker genes RPL25 and RPL13 was examined by RT-qPCR, 1-h, and 3-h post-treatment. Data are shown as the mean ± SE (n = 3). Asterisks indicate differences from the untreated sample (one way ANOVA, multiple comparisons, Dunnett’s test p < 0.05). All the above experiments were repeated at least three times with similar results.

Fig. 8. The osmotic signal triggers the NIK1 antiviral signaling activation.

a NIK1 is phosphorylated under osmotic stress. NIK1-HA-expressing seedlings were treated with 10% PEG for the indicated intervals. The NIK1 phosphorylation was monitored by probing immunoprecipitated NIK1-HA with an α-pSer antibody. Immunoprecipitated NIK1-HA was probed with α-NIK1. b Osmotic stress induces RPL10 phosphorylation. Osmotic stress was induced in RPL10-GFP-expressing Arabidopsis seedlings, and then RPL10 phosphorylation was monitored by immunoblotting immunoprecipitated RPL10-GFP with an α-pSer antibody. c Osmotic stress-mediated repression of RP genes requires the function of NIK1 and/or NIK2. RPS25 and RPL13 expression was quantified by RT-qPCR 3-h and 24-h after PEG treatment in the indicated genotypes. A T474D-overexpressing line was used as a positive control. d Osmotic stress suppresses the photosynthetic genes PsbP and FD1 in Col-0 but not in nik1nik2 double knockouts. Transcript accumulation of PsbP and FD1 was determined by RT-qPCR 3 h and 24 h after PEG treatment. e Osmotic stress induces NIK1 phosphorylation in the limyb-32 knockout line. NIK1 was immunoprecipitated with an α-NIK antibody and probed with an α-phosphothreonine antibody and an α-phosphoserine antibody to monitor phosphorylation and with α-NIK antibody to visualize the immunoprecipitated NIK1. f, g Osmotic stress does not induce repression of NIK1 signaling pathway-associated marker genes in limyb-32 mutant. The expression of the marker genes was examined by RT-qPCR, at the time indicated in the Figure. For c, d, f, and g data are shown as the mean ± SE (n = 3). Asterisks indicate statistically significant differences from the untreated sample (one way ANOVA, multiple comparisons, Dunnett’s test p < 0.05). For c the exact p-values can be found in the Source Data file. For d p-values: FD1, Col-0 (3 h): 0.0255; Col-0 (24 h): 0.0001; NIK1- T474D-6 (24 h): 1E-11; nik-1(3 h): 1.03E-07; nik-1(24 h): 3.32E-07; nik-2 (24 h): 0.0010; p-value, PsbB, Col-0 (24 h): 5.2E-06; NIK1- T474D-6 (3 h): 2.41E-08; NIK1- T474D-6 (24 h): 2.46E-08; nik-1(3 h): 0.0001; nik-1(24 h): 0.0462; nik-2 (3 h): 0.0008). For f, g p-values are shown in the Figure. The above experiments were repeated at least twice with similar results.