Elicitor-induced plant immunity relies on amino acids accumulation to delay the onset of bacterial virulence

Abstract

Plant immunity relies on the perception of microbe-associated molecular patterns (MAMPs) from invading microbes to induce defense responses that suppress attempted infections. It has been proposed that MAMP-triggered immunity (MTI) suppresses bacterial infections by suppressing the onset of bacterial virulence. However, the mechanisms by which plants exert this action are poorly understood. Here, we showed that MAMP perception in Arabidopsis (Arabidopsis thaliana) induces the accumulation of free amino acids in a salicylic acid (SA)-dependent manner. When co-infiltrated with Glutamine and Serine, two of the MAMP-induced highly accumulating amino acids, Pseudomonas syringae pv. tomato DC3000 expressed low levels of virulence genes and failed to produce robust infections in otherwise susceptible plants. When applied exogenously, Glutamine and Serine directly suppressed bacterial virulence and growth, bypassing MAMP perception and SA signaling. In addition, an increased level of endogenous Glutamine in the leaf apoplast of a gain-of-function mutant of Glutamine Dumper-1 rescued the partially compromised bacterial virulence- and growth-suppressing phenotype of the SA-induced deficient-2 (sid2) mutant. Our data suggest that MTI suppresses bacterial infections by delaying the onset of virulence with an excess of amino acids at the early stages of infection.

Figure 1.

The perception of flg22 induces the accumulation of amino acids in treated leaves. A) Concentrations of L-AA in eleven-day-old seedlings 24 h post-treatment (HPT) with water (mock) or flg22. B) Total L-AA in leaves of wild-type plants after infiltration of leaves with water (mock) or flg22. C) Total L-AA in leaf apoplastic washing fluids of wild-type plants treated with water (mock) or flg22, 24 HPT. D) Total L-AA in the phloem sap of wild-type leaves 24 HPT. Statistical analysis: Welch t-test (A) and t-tests for (B-D) at P values ≤0.05 (*), ≤0.01 (**), and ≤0.0001 (****). All figures show one representative experiment from at least 3 independent experiments.

Figure 2.

Amino acids that accumulate in MTI-elicited leaves modulate PstDC3000 growth and virulence in naïve wild-type plants. A) Bacterial growth 48 hours post-inoculation (HPI) of PstDC3000 alone (mock) or co-infiltrated with individual amino acids in leaves of naïve plants and normalized by the growth of PstDC3000 in mock. The number of plants used per condition is indicated atop. B) hrpL gene expression 2.5 h after inoculation of liquid HMM medium with PstDC3000 supplemented with the indicated amino acids and normalized by the expression in non-supplemented HMM medium. C) Expression of virulence genes in leaves of naïve plants 3 HPI with PstDC3000 co-infiltrated with either Gln (n = 12), Ser (n = 12), or Val (n = 18), normalized by the expression in non-supplemented PstDC3000. Statistical analysis: Brown-Forsythe and Welch ANOVA tests compared with mock (A); P values ≤0.05 (*), ≤0.01 (**), ≤0.001 (***) were adjusted by the two-stage step-up false discovery rate method (Q = 0.01). The center line indicates the median; the box extends from the 25th to 75th percentiles, and the whiskers show the entire data distribution. One sample t-tests for (B-D). Combined data of 3 (A,C) and 4 (B) independent experiments.

Figure 3.

MTI suppresses bacterial virulence and growth via Gln and Ser accumulation. A) Expression of virulence genes in naïve plants 3 hours post-inoculation (HPI) with PstDC3000 and Gln + Ser. Mean ± SEM, n = 12. B) Expression of virulence genes in flg22-treated plants 3 HPI with PstDC3000 alone (n = 36) or with PstDC3000 and Gln + Ser (n = 28). Mean ± SEM. C) relA expression in mock-treated plants 3 HPI with PstDC3000 and Gln + Ser (n = 27) or in flg22-treated plants with PstDC3000 alone (n = 11). Mean ± SEM. D) Bacterial growth 48 HPI in mock- or flg22-treated plants infiltrated with PstDC3000 alone, or mock-treated plants infiltrated with PstDC3000 and Gln + Ser. E) PstDC3682 growth 48 HPI in naïve plants co-infiltrated with Gln + Ser. F) Pst∆hrcC growth 48 HPI in naïve plants infiltrated alone or co-infiltrated with Gln+Ser. G) Bacterial growth 48 HPI in mock-treated or flg22-treated, and inoculated with PstDC3000 alone, or flg22-treated plants co-infiltrated with PstDC3000 and Gln + Ser. Statistical analysis: one-sample t-tests (A-C); Welch t-test (D); t-test (E-G). Statistically significant differences at P values ≤0.05 (*), ≤0.01 (**), ≤0.001 (***), or non-significant (ns) are shown. (A-C) combination of 3 independent experiments. (D-G) one representative experiment of at least 3 independent experiments.

Figure 4.

SA-mediated signaling contributes to the flg22-induced accumulation of L-AA and bacterial virulence suppression. A) Total L-AA in mock or flg22-treated leaves of wild-type (wt) or sid2-2 (sid2) plants 24 hours post-treatment (HPT). B) Total L-AA in leaf apoplastic washing fluids (AWF) of mock or flg22-treated wt or sid2 naïve plants 24 HPT. C) Expression of virulence genes in wt or sid2 flg22-treated plants inoculated with PstDC3000 alone and normalized by gene expression in mock-treated plants 1h or 3h post-inoculation (HPI); Mean ± SEM (n = 3); D) Expression of virulence genes in wt or sid2 naïve plants co-infiltrated with PstDC3000 and Gln + Ser; Mean ± SEM (n = 12). E) Bacterial growth in wt or sid2 naïve plants 48 HPI with PstDC3000 alone or PstDC3000 and Gln + Ser. Statistical analysis: one-way ANOVA (A,B); t-test (C-E). (A-E) one representative experiment from at least 3 independent experiments. (D) combination of 3 independent experiments. Different letters denote atop data denote statistically significant differences at P value ≤0.05 (A,B). Statistically significant differences at P values ≤0.05 (*), ≤0.01 (**), ≤0.001 (***), or non-significant (ns) are shown (C-E).

Figure 5.

High endogenous levels of Gln impact PstDC3000 virulence and growth in naïve plants. A) Concentration of total amino acids (L-AA) in leaf apoplastic washing fluids (AWF) of wild-type (wt), sid2, and sid2 x gdu1-1D naïve plants. B) Expression of virulence genes in sid2 x gdu1-1D (n = 15) naïve plants inoculated with PstDC3000 alone and normalized by the expression in sid2 plants (n = 13); Mean ± SEM C) PstDC3000 growth in wt, sid2, and sid2 x gdu1-1D naïve plants 48 HPI. Statistical analysis: Brown-Forsythe and Welch ANOVA tests (A) and (C); P values were adjusted by the two-stage step-up false discovery rate method (Q = 0.01). (A,C) one representative experiment from at least three independent experiments. (B) combination of 3 independent experiments. Different letters atop each data group denote statistically significant differences at P value ≤0.05 in (A,C). Statistically significant differences at P values ≤0.01 (**) and ≤0.001 (***) are shown in (B).