Plant immunity suppression via PHR1-RALF-FERONIA shapes the root microbiome to alleviate phosphate starvation (2022)
Figure 3

Figure 3. PHR1-mediated RALF activation disrupts FLS2-BAK1 complex formation to inhibit immunity.

(A) The PSR inhibits flg22-induced MAPK activation in a PHR1-dependent manner. Col-0 and phr1 mutant seedlings grown in either HP or LP media were treated with flg22 for 15 min and roots were harvested. The MAPK level was determined with anti-pMAPK, and β-actin was used as a loading control.

(B) PHR1 negatively regulates FLS2-BAK1 complex formation under Pi-starvation conditions. Col-0, phr1 mutant seedlings were grown in either HP or LP media for 5 days, then lysing roots into protoplasts, transformed with water or PHR1-Myc plasmid and treated with flg22 for 6 hours. The amount of immunoprecipitated FLS2 and coimmunoprecipitated BAK1 were determined using anti-FLS2 and anti-BAK1 antibodies, respectively. Preimmune serum (IgG) was used as negative control.

(C) PHR1 enhances the growth of Pto DC3000 under Pi-starvation conditions. Col-0, phr1 mutant and PHR1-Myc overexpression seedlings grown in either HP or LP media were root inoculated (by soaking) with a bacterial suspension (OD600 = 0.2) for 2 min, and the number of bacteria from individual roots was quantified at 5 dpi. The data shown indicate the means ± SDs (n = 18, n refers to the number of roots per group). The different lowercase letters indicate statistical significance (one-way ANOVA).

(D) Application of RALF23/34 disrupted FLS2-BAK1 complex formation in phr1 mutants. phr1 mutants grown in LP media were treated with water, 1 μM flg22, 1 μM RALF23, 1 μM RALF34 or both flg22 and RALF for 6 hours. The amount of immunoprecipitated FLS2 and coimmunoprecipitated BAK1 were determined using anti-FLS2 and anti-BAK1 antibodies, respectively. IgG was used as a negative control.

(E) RALF23/34 promoted the growth of Pto DC3000 in phr1 mutants. Col-0 and phr1 mutant seedlings grown in LP media were root inoculated (by soaking) with a bacterial suspension (OD600 = 0.2) for 2 min, and the number of bacteria from individual root was quantified at 5 dpi. The data shown indicate the means ± SDs (n = 18, n refers to the number of roots per group). The different lowercase letters indicate statistical significance (one-way ANOVA).

(F) FER negatively regulates FLS2-BAK1 complex formation under Pi-starvation conditions. Col-0, fer-4 mutant and FER/fer-4 complementation lines seedlings were grown in either HP or LP media for 5 days and treated with flg22 for 6 hours. The amount of immunoprecipitated FLS2 and coimmunoprecipitated BAK1 was determined using anti-FLS2 and anti-BAK1 antibodies, respectively. Preimmune serum (IgG) was used as a negative control.

(G) LP promoted the growth of Pto DC3000 in fls2 and bak1 mutants. Col-0, fls2, bak1 and fer-4 mutant seedlings grown in either HP or LP media were inoculated (by soaking) with a GFP-labelled bacterial suspension (OD600 = 0.2) for 2 min, and the number of bacteria from individual root was quantified at 5 dpi. The data shown indicate the means ± SDs (n = 18, n refers to the number of roots per group). The different lowercase letters indicate statistical significance (one-way ANOVA).

Data information: All experiments were repeated three times with similar results.