Decreased abundance of type III secretion system-inducing signals in Arabidopsis mkp1 enhances resistance against Pseudomonas syringae

Abstract

Genes encoding the virulence-promoting type III secretion system (T3SS) in phytopathogenic bacteria are induced at the start of infection, indicating that recognition of signals from the host plant initiates this response. However, the precise nature of these signals and whether their concentrations can be altered to affect the biological outcome of host-pathogen interactions remain speculative. Here we use a metabolomic comparison of resistant and susceptible genotypes to identify plant-derived metabolites that induce T3SS genes in Pseudomonas syringae pv tomato DC3000 and report that mapk phosphatase 1 (mkp1), an Arabidopsis mutant that is more resistant to bacterial infection, produces decreased levels of these bioactive compounds. Consistent with these observations, T3SS effector expression and delivery by DC3000 was impaired when infecting the mkp1 mutant. The addition of bioactive metabolites fully restored T3SS effector delivery and suppressed the enhanced resistance in the mkp1 mutant. Pretreatment of plants with pathogen-associated molecular patterns (PAMPs) to induce PAMP-triggered immunity (PTI) also restricts T3SS effector delivery and enhances resistance by unknown mechanisms, and the addition of the bioactive metabolites similarly suppressed both aspects of PTI. Together, these results demonstrate that DC3000 perceives multiple signals derived from plants to initiate its T3SS and that the level of these host-derived signals impacts bacterial pathogenesis.

Fig. 1.

Delivery and production of the type III effector AvrPto by DC3000 is restricted in mkp1 plants in an MPK6-dependent manner. (A) WT, mkp1, and mkp1 mpk6 plants were infected with 1 × 10⁸ cfu/mL DC3000 expressing AvrPto fused to an adenylate cyclase reporter (AvrPto-CyaA). cAMP levels were measured in plants 3 h postinfection (PI). Graphed data are means ± SE of percent cAMP relative to cAMP in WT plants. Data shown were pooled from four independent experiments, each with six samples per genotype; n = 24. (B) Serial-dilution plating of bacteria isolated from infected plants 3 h postinfection. Graphed data are means ± SE; n = 6. Each sample comprises three infected plants. (C) qRT-PCR analysis of avrPto transcripts in plants infected with 5 × 10⁸ cfu/mL DC3000 for 4 h. avrPto transcript levels were normalized to the levels of bacterial RpoD and 16S rRNA transcripts detected in each sample. Each sample was RNA extracted from three infected plants. Six samples were analyzed for each genotype with technical replication of each. Graphed data are means ± SE of percent avrPto transcripts relative to mean avrPto transcript levels in infected WT plants; n = 12. Asterisks in A and C denote significant differences based on Student's t-test: *P < 0.05, **P < 0.01.

Fig. 2.

Soluble signals in Arabidopsis exudates strongly enhance the expression of T3SS-associated genes in DC3000 and are genetically regulated by MKP1 and MPK6. (A) Arabidopsis exudate was mixed with DC3000 in minimal medium with or without 50 mM fructose. qRT-PCR analysis of hrpL and avrPto transcripts was performed using samples isolated at the indicated times. Graphed data are means ± SE; n = 4. ◆, −fructose/−exudate; □, +fructose/−exudate; ▲, +fructose/+exudate. **P < 0.01, ***P < 0.001 based on a t test comparison of +fructose/−exudate and +fructose/+exudate treatments. (B) Immunoblot of AvrPto from DC3000 treated as described in A. CBB, Coomassie Brilliant Blue staining. (C) DC3000 was incubated with WT or mkp1 exudate, and hrpL (Left) and avrPto (Right) transcript levels were measured by qRT-PCR 2 and 24 h posttreatment, respectively. Graphed data are means ± SE; n = 4. *P < 0.05. (D) Immunoblot of AvrPto in DC3000 24 h posttreatment with WT, mkp1, or mkp1 mpk6 exudate. A cross-reacting (x-react) band shows equal loading.

Fig. 3.

Abundance of virulence-inducing metabolites in Arabidopsis exudates is genetically regulated by MKP1 and MPK6. (A) Identification of metabolites present in WT, mkp1, and mkp1 mpk6 exudates by GC-MS. Three biological replicates were analyzed with technical replicates for each. All metabolites shown were significantly decreased in mkp1 versus WT exudates by pairwise t tests; P < 0.05. Metabolites in bold were significantly different in mkp1 and mkp1 mpk6 exudates; P < 0.05. Graphed data are means + SE of log₂-transformed peak area values for mutant/WT; n = 6. (B) DC3000 was incubated with 200 μM of individual metabolites in minimal medium plus 50 mM fructose. (Upper) Immunoblot of AvrPto in bacteria 4 h posttreatment. (Lower) Coomassie Brilliant Blue (CBB) staining to confirm equal loading. gba, 4-guanidinobutyric acid; ggl, glucosylglycerol; pga, pyroglutamic acid; 4-hba, 4-hydroxybenzoic acid; tea, triethanolamine.

Fig. 4.

T3SS-inducing metabolites suppress both mkp1- and elf26-mediated inhibition of type III effector delivery. (A) WT and mkp1 plants were infected with DC3000 expressing AvrPto-CyaA as in Fig. 1 in the presence or absence of 50 μM each of citric acid (cit), aspartic acid (asp), and/or 4-hydroxybenzoic acid (4-hba) or with a non–T3SS-inducing mixture of 50 μM each of serine (ser), threonine (thr), and valine (val). Graphed data are means ± SE of cAMP levels 4 h postinfection; n = 8. (B) WT and mkp1 plants were infected with DC3000 in the presence or absence of the indicated metabolites at concentrations of 100 μM each. Graphed data are means ± SE of bacteria 24 h postinfection; n = 12. (C) WT and efr-2 plants were treated with 1 μM elf26 or a mock control for 24 h. (Upper) WT or elf26-treated plants were infected as described in Fig. 1 in the presence or absence of 50 μM of each of the indicated metabolites. cAMP levels were measured 4 h postinfection. Graphed data are means ± SE of percent cAMP relative to cAMP in infected WT plants; n = 8. (Lower) WT or elf26-treated plants were infected with DC3000 for 30 h in the presence or absence of 100 μM of each of the indicated metabolites. Graphed data are means ± SE of bacteria; n = 12. Asterisks denote t test comparison with mock-treated mkp1 in A and B, and with mock-treated WT in C; *P < 0.05, **P < 0.01, *** P < 0.001. ns, not significant.