Fig 1.
RxL21 expression in planta causes enhanced susceptibility to biotrophic and necrotrophic pathogens.
(A) Transgenic Arabidopsis expressing RxL21 under a 35S promoter (RxL21a/b) were challenged with Hpa isolates Noks1 and Maks9 and sporangiophores counted at 4 dpi. Col-0 WT and 35S::GUS (Col-0 background) were used as controls. (Noks1 n = 45, Maks9 n = 55). (B) RxL21a/b were challenged with B. cinerea and lesion area measured 72 h post infection (n = 24). Box plots show the median, upper and lower quartiles, whiskers show the upper and lower extremes of the data. Letters indicate significant difference using a Kruskal Wallis test with p < 0.05. Experiments were repeated with similar results.
Fig 2.
The EAR motif of RxL21 interacts with the CTLH domain of TPL.
(A) RxL21 co-localizes with TPL in the nucleus. GFP:RxL21 and RFP:TPL were expressed transiently in N. benthamiana. Scale bars are 10 μm. (B) TPL Interacts with the EAR domain of RxL21 in yeast. RxL21 was cloned without the signal peptide and lacking the EAR motif (RxL21ΔEAR1; Δ402–409 and HaRxL21ΔEAR2; Δ360–409), without the N-terminus (RxL21ΔN; Δ1–36) and RxLR-DEER motif (RxL21ΔRxLR; Δ1–51) and combinations of the above. Amino acid locations of deletion constructs are indicated. Deleting the EAR motif at the C-terminus of RxL21 abolishes interaction with TPL by Y2H. Successful mating is indicated by growth on media lacking Leucine and Tryptophan (-LW). Growth on media additionally lacking Histidine (-LWH) indicates GAL1::HIS3 reporter gene activation and protein-protein interaction. 3AT is used to increase stringency in a concentration dependent manner. Growth on media additionally lacking Adenine (-LWHA) indicates activation of the GAL2::ADE2 reporter gene. AD; activation domain. DB; DNA binding domain. (C) Deleting the CTLH domain of TPL (TPLΔCTLH; Δ25–91) abolishes interaction with RxL21 in yeast. Interaction (indicated by growth on–LWH +1mM 3AT) was observed between RxL21 and TPL but not between RxL21 and TPLΔCTLH. All Y2H was repeated at least twice with similar results. (D) TPL interacts with RxL21 and the interaction requires the EAR motif in planta. TPL:HA, TPLΔCTLH:HA together with RxL21 and RxL21 EAR motif mutants RxL21ΔEAR, RxL21-FMFTF and RxL21-AMATA (under control of an estradiol-inducible promoter and N-terminal Myc tag) were transiently expressed in N. benthamiana leaves. RxL21 expression was induced by 30μM β-estradiol 24 hr prior to harvesting. c-myc beads were used for immunoprecipitation (IP). HA antibody was used for TPL and TPLΔCTLH immunoblots (IB) and c-myc antibody was used to detect RxL21, RxL21ΔEAR and the respective EAR motif mutants.
Fig 3.
The EAR motif is necessary for RxL21 virulence function in planta.
Transgenic lines expressing RxL21 under control of a 35S promoter and N-terminal HA tag (HA:21#1/2 and HA:21ΔEAR#1/2) were compared to Col-4 WT and HA:GFP (Col-4 background) controls during infection with (A) Hpa isolate Noks1 (sporangiophore counts per seedling at 4 dpi). Letters indicate significance using a Kruskal Wallis test, p < 0.05, n = 45 (B) B. cinerea. Lesion area was measured at 72 hpi (letters indicate significance determined by a one way ANOVA, p < 0.05, n = 24). Error bars display 95% CE. (C) Estradiol inducible lines expressing RxL21 and RxL21ΔEAR fused to an N-terminal Myc tag (Est:21#2/9 and Est:21ΔEAR#1/2) were infected with Hpa 18 hours after induction with 30 μM β-estradiol or mock treatment (n = 100 per treatment). (D) The same lines with estradiol or mock treatment were subsequently infected with B. cinerea. Lesion area was measured at 72 hpi (n = 30). Letters in (C) and (D) indicate significant difference between treatments using a 2-way ANOVA and Bonferroni’s multiple comparison test. P < 0.05. Whiskers show data range. Experiments were repeated with similar results.
Fig 4.
TPL family members function in immunity against a biotrophic and a necrotrophic pathogen.
(A) tpl-tpr1-tpr4 plants showed enhanced susceptibility to Hpa isolate Noks1 measured by sporangiophore counts per seedling at 4 dpi compared to Col-0 WT and 35s::GUS controls. Letters indicate significance using a Kruskal Wallis test, n = 45, p < 0.05. (B) Enhanced susceptibility of tpl-tpr1-tpr4 to B. cinerea infection was also observed as determined by lesion area at 48 hpi (n = 24, letters indicate significance using a one-way ANOVA and a Tukey test, p < 0.05) and (C) visual inspection of sporulation. Here we show representative leaf images at 96h post infection. Scale bar is 1 cm.
Fig 5.
RNAseq identifies differentially expressed genes in RxL21 compared to RxL21ΔEAR transgenic lines.
RNAseq was performed on HA-tagged RxL21 and RxL21ΔEAR-expressing transgenic lines. (A) The number of up- and down-regulated genes among differentially expressed genes under mock and flg22 treatment. DEGs were defined as having a log2 fold change ≥ 1 or ≤-1, and a BH adjusted p-value of < 0.05. (B) Venn diagram shows differentially expressed genes between RxL21 and RxL21ΔEAR after mock and flg22 treatment with an overlap of 84 upregulated and 29 down regulated genes. (C) Eight genes were selected for validation in estradiol (Est) inducible RxL21 and RxL21ΔEAR expressing lines and Col-0 WT. We treated Arabidopsis seedlings with 30 μM of β-estradiol for 18 hours. For Est:21 and Est:21ΔEAR, data were obtained from 2 independent lines each with 3 biological replicates and expression was normalised to Arabidopsis tubulin gene. Black circles are individual data points and bars denote the mean ± SE of target gene expression. Letters indicate significant differences (P < 0.05) (One-way ANOVA with Tukey’s honest significance difference).
Fig 6.
Genes that are bound by TPR1 are overrepresented in genes repressed by RxL21.
(A) Arabidopsis genes with repressed expression in HA:RxL21 lines compared to HA:21ΔEAR lines show weak TPR1 binding. Metaplots display the TPR1 enrichment around the transcription start site (TSS) on genes regulated by RxL21 (BH adjusted p-value < 0.05 and log2-fold-change ≥1, with or without flg22) or control genes without evidence for RxL21 dependent expression regulation (RxL21 control). TPR1 bound genes defined in Griebel et al. (2020) were used as a positive control (red line). On the y-axis is mean read count for the TRP1-GFP ChIP samples normalized to the input samples. TPR1 ChIP and input samples were scaled based on the number of mapped reads. TES = transcription end site. (B) ChIP-qPCR of RxL21-repressed genes. Two-week old seedlings, overexpressing RxL21 or RxL21ΔEAR with an N-terminal myc tag and under the control of an estradiol inducible promoter, were treated with 30 μM β-estradiol to induce RxL21 expression 18 hrs before harvesting and cross-linking with 1% formaldehyde. ChIP assays were performed with an anti-Myc antibody. In the ChIP–qPCR, the enrichment of immunoprecipitated DNA was normalized by the percent input method (signals obtained from ChIP samples were divided by signals obtained from an input sample). Error bar represents ± SE of four technical repeats. Arabidopsis Actin 2 was included as a control but no amplification was detected after 40 cycles. The experiment was repeated with similar results.