Glucosinolate metabolites required for an Arabidopsis innate immune response

Abstract

The perception of pathogen or microbe-associated molecular pattern molecules by plants triggers a basal defense response analogous to animal innate immunity and is defined partly by the deposition of the glucan polymer callose at the cell wall at the site of pathogen contact. Transcriptional and metabolic profiling in Arabidopsis mutants, coupled with the monitoring of pathogen-triggered callose deposition, have identified major roles in pathogen response for the plant hormone ethylene and the secondary metabolite 4-methoxy-indol-3-ylmethylglucosinolate. Two genes, PEN2 and PEN3, are also necessary for resistance to pathogens and are required for both callose deposition and glucosinolate activation, suggesting that the pathogen-triggered callose response is required for resistance to microbial pathogens. Our study shows that well-studied plant metabolites, previously identified as important in avoiding damage by herbivores, are also required as a component of the plant defense response against microbial pathogens.

Fig. 1

IGS biosynthesis and hydrolysis are required for Flg22-induced callose formation. (A–U) Cotyledons of seedlings treated with water (A) or Flg22 (B–U) and stained with aniline blue. (A–B) Wild type. (C) fls2-c. (D) pmr4-1; traditional staining method. (E) etr1-1. (F) ein2-1. (G) myb51-1. (H) atr1-3/myb34. (I) cyp81F2-1. (J) cyp79b2 cyp79b3. (K) atr4-1/cyp83b1. (L) ugt4b1-2. (M) ref/cyp83a1. (N) trp3-1. (O) cyp71a13-3. (P) pad3-1/cyp71b15. (Q) tgg1-3 tgg2-1. (R) pen2-1. (S) pad2-1/gsh1. (T) pcs1-2. (U) pen3-1. (V-AA) Cotyledons pretreated with water (V), INA (W), ACC (X), MeJA (Y), or ABA (Z) for 24 hours and then treated with Flg22 (V–Z), or co-treated with IAN and Flg22 (AA). Shown are representative examples of 40 to 60 cotyledons from two independent experiments per genotype.

Fig. 2

Blocking IGS biosynthesis or hydrolysis depletes or elevates 4-methoxy-I3G levels, respectively. Upon Flg22 elicitation, I3G levels (A) are reduced in wild type and mutant seedlings, and 4-methoxy-I3G levels (B) are reduced in ein2-1, myb51-1, and cyp81F2-1 mutants, and elevated in pen2-1, cad1-3 (a null pcs1 allele), and pen3-1 mutants. Mean ±S.D., n=4 replicate samples. Different letters above the bars denote statistically significant differences, P<0.01, 2-tailed t-test.

Fig. 3

4-methoxy-I3G induces callose formation in IGS biosynthetic mutants. (A–I) Cotyledons of seedlings simultaneously treated with Flg22 and I3G or 4-methoxy-I3G (4M–I3G), and then stained with aniline blue. (A) Wild type. (B) fls2-c. (C) pmr4-1. (D) ein2-1. (E) myb51-1. (F) cyp81F2-1. (G) pen2-1. (H) cad1-3/pcs1. (I) pen3-1.

Fig. 4

SA pretreatment rescues Flg22-induced callose formation in pen2 and pcs1 mutants. (A–O) Cotyledons of seedlings pretreated with water (A, D), INA (B, E, J–M), or SA (C, F, H–I, and N–O) for 24 hours, treated with Flg22, and stained with aniline blue. (A–B) pen2-1. (C) pen2-2. (D–E) pcs1-2. (F) cad1-3/pcs1. (G–H) vtc2-1. (I) vtc1-1. (J) cyp812-1. (K) pen3-1. (L) pad2-1/gsh1. (M) pmr4-1. (N) pen2-2 pen2-like. (O) pcs1-1 pcs2-1. Shown are representative of 40 to 60 cotyledons from two independent experiments.

Fig. 5

A model of MAMP-triggered callose deposition in Arabidopsis.