Methyl Salicylate Glucosylation Regulates Plant Defense Signaling and Systemic Acquired Resistance

Abstract

Plant systemic acquired resistance (SAR) provides an efficient broad-spectrum immune response to pathogens. SAR involves mobile signal molecules that are generated by infected tissues and transported to systemic tissues. Methyl salicylate (MeSA), a molecule that can be converted to salicylic acid (SA), is an essential signal for establishing SAR, particularly under a short period of exposure to light after pathogen infection. Thus, the control of MeSA homeostasis is important for an optimal SAR response. Here, we characterized a uridine diphosphate-glycosyltransferase, UGT71C3, in Arabidopsis (Arabidopsis thaliana), which was induced mainly in leaf tissue by pathogens including Pst DC3000/avrRpt2 (Pseudomonas syringae pv tomato strain DC3000 expressing avrRpt2). Biochemical analysis indicated that UGT71C3 exhibited strong enzymatic activity toward MeSA to form MeSA glucosides in vitro and in vivo. After primary pathogen infection by Pst DC3000/avrRpt2, ugt71c3 knockout mutants exhibited more powerful systemic resistance to secondary pathogen infection than that of wild-type plants, whereas systemic resistance in UGT71C3 overexpression lines was compromised. In agreement, after primary infection of local leaves, ugt71c3 knockout mutants accumulated significantly more systemic MeSA and SA than that in wild-type plants. whereas UGT71C3 overexpression lines accumulated less. Our results suggest that MeSA glucosylation by UGT71C3 facilitates negative regulation of the SAR response by modulating homeostasis of MeSA and SA. This study unveils further SAR regulation mechanisms and highlights the role of glucosylation of MeSA and potentially other systemic signals in negatively modulating plant systemic defense.

Figure 1.

Expression of UGT71C3 in wild type Arabidopsis is induced by SA and Pst DC3000/avrRpt2. A and B, RT-qPCR analysis of UGT71C3 expression induced by SA and Pst DC3000/avrRpt2 treatments. Mock treatments are DMSO solvent (A) and 10-mm MgCl₂ solution (B). The value for wild type under mock treatment for 0 h was set at “1.” UGT71C3 levels were detected and normalized to ACTIIN2. Data are means ≤ sd of three biological replicates (**P < 0.01, *P < 0.05, Student’s t test). C and D, GUS staining of UGT71C3Pro:GUS transgenic seedlings after Pst DC3000/avrRpt2 (C) and MgCl₂ (Mock, D) inoculation. Bars = 1 mm (upper) and 200 μm (lower).

Figure 2.

The glucosylating activity of UGT71C3 toward MeSA but not SA. A, SDS-PAGE detection of the purified recombinant UGT71C3-GST fusion protein. Proteins were visualized by Coomassie blue staining. UGT71C3: UGT71C3 fusion protein. B, No reaction product from SA was found by HPLC analysis. C, Potential reaction product MeSAG from MeSA was found by HPLC analysis. UDP-Glc was used as the sugar donor. Heat-inactivated UGT71C3 and GST were used as the negative controls in these enzymatic reactions. D, Identity of reaction products from MeSA was further confirmed by LC-MS analysis under positive ion mode. E, Retention time of authentic standard MeSAG in HPLC analysis. F, Ion peaks of authentic standard MeSAG in LC-MS analysis under positive ion mode.

Figure 3.

Induced expression of UGT71C3 by MeSA and the assays of UGT71C3 enzyme activity in vivo. A, RT-qPCR analysis of UGT71C3 expression induced by MeSA treatment. Mock treatment is DMSO solvent. The value of the wild type under mock treatment for 0 h was set at 1. Data are means ± sd of three biological replicates (**P < 0.01, Student’s t test). B, GUS staining of UGT71C3Pro:GUS expression after MeSA inoculation. Bars = 1 mm (upper) and 200 μm (lower). C, The glucosyltransferase activities of the crude protein extracts for the MeSAG (MeSAG) formation from 2-week–old UGT71C3 transgenic plants, wild type, and mutant lines. A quantity of 0.1 mg of crude proteins from each sample was used in these assays. OE-6 and ko-1 were used as representatives for overexpression lines and mutant lines, respectively. D and E, HPLC profiling of MeSAGs from wild type, overexpression lines (OE-6, OE-10), and mutant lines (ko-1 used as the representative). “St” in (C), (D), and (E) represents the authentic standard of MeSAG. For (D) and (E), the plant tissues were incubated with MeSA before the extraction process. To monitor the recovery rate, 2-methoxybenzoic acid was used as a reference in these assays. WT, wild type.

Figure 4.

Defense phenotypes of the 35S::UGT71C3 transgenic plants and ugt71c3 mutants. A, SAR response to avirulent pathogen infection in wild type, 35S::UGT71C3 transgenic plants, and ugt71c3 mutants. The lower leaves of 5–6-week–old plants were primarily inoculated with avirulent bacteria (Pst DC3000/avrRpt2 suspended in 10-mm MgCl₂). At 2 d after primary infection, upper leaves were inoculated with virulent bacteria (Pst DC3000). Photographs of disease resistance phenotypes of upper leaves were taken 5 d after infection of upper leaves with pathogens. Bars = 0.5 cm. B, Trypan blue staining showing the necrosis in upper leaves infected with pathogens as described in (A). Bars = 0.5 cm. C, Growth of Pst DC3000 in upper leaves of wild type, 35S::UGT71C3 transgenic plants, and ugt71c3 mutants infected with pathogens. At first, the lower leaves of 5–6-week–old plants were primarily inoculated with avirulent bacteria (Pst DC3000/avrRpt2 suspended in 10-mm MgCl₂) or with 10-mm MgCl₂ as mock treatment. At 2 d after primary infection, upper leaves were inoculated with virulent bacteria (Pst DC3000). The in planta bacterial titers were determined 3 d postinoculation of upper leaves. Data represent the mean of 10 independent samples with sd of three biological replicates (**P < 0.01, Student’s t test). WT, wild type.

Figure 5.

The induced expression of SAR-related genes in systemic leaves of UGT71C3 overexpression lines, ugt71c3 mutants, and wild type. Three lower leaves on each plant were inoculated with Pst/avrRpt2 (1 × 10⁶ CFU/mL in 10-mm MgCl₂) or mock-treated with 10-mm MgCl₂. After 2 d, total RNA was extracted from the upper untreated systemic leaves and analyzed for the expression of indicated genes using RT-qPCR. Expression was normalized against constitutively expressed ACTIN2. The value of wild-type plants under mock-treated condition was set at 1.0. Data = means ± sd of three biological replicates (Student’s t test, *P < 0.05, **P < 0.01). WT, wild type.

Figure 6.

Profiling of MeSA contents in mutant lines and overexpression lines. A, Profiling of MeSA levels using a GC system for upper leaves of wild type, mutant lines, and overexpression lines 24 h after inoculation of lower leaves with Pst/avrRpt2. “St” represents the authentic standard of MeSA. B, MeSA contents in upper leaves of wild type, mutant line, and overexpression lines after 10-mm MgCl₂ (mock) or Pst/avrRpt2 treatment for 24 h. Asterisks denote statistically significant difference of a particular line compared with that in the wild type. Data = means ± sd. All experiments were repeated three times with similar results (**P < 0.01, Student’s t test). WT, wild type.

Figure 7.

Profiling of SA contents in mutant lines and overexpression lines during SAR. A, HPLC profiling of free SA levels in upper leaves of wild type, mutant lines (ko-1 and ko-2), and overexpression lines (OE-6 and OE-10) 48 h after inoculation of lower leaves with Pst /avrRpt2. B, Quantification of free SA levels in upper leaves of wild type, mutant lines, and overexpression lines 48 h after inoculation of lower leaves with 10-mm MgCl₂ (Mock) or Pst/avrRpt2. C, HPLC profiling of total SA levels in upper leaves of wild type, mutant lines (ko-1 and ko-2), and overexpression lines (OE-6 and OE-10) 48 h after inoculation of lower leaves with Pst /avrRpt2. D, Quantification of total SA levels in upper leaves of wild type, mutant lines, and overexpression lines 48 h after inoculation of lower leaves with 10 mm MgCl₂ (Mock) or Pst /avrRpt2. “St” represents the authentic standard of SA. All experiments were repeated three times with similar results. Data are means ≥ sd (Student’s t test, **P < 0.01). WT, wild type.

Figure 8.

Profiling of SA contents in mutant lines and overexpression lines after treatment with MeSA. A, HPLC profiling of free SA levels in upper leaves of wild type, mutant lines (ko-1 and ko-2), and overexpression lines (OE-6 and OE-10) 48 h after inoculation of lower leaves with 1 mg/L MeSA. B, Quantification of free SA levels in upper leaves of wild type, mutant lines, and overexpression lines 48 h after inoculation of lower leaves with 1 mg/L MeSA or DMSO solvent (Mock). C, HPLC profiling of total SA levels in upper leaves of wild type, mutant lines (ko-1 and ko-2), and overexpression lines (OE-6 and OE-10) 48 h after inoculation of lower leaves with 1 mg/L MeSA. D, Quantification of total SA levels in upper leaves of wild type, mutant lines, and overexpression lines 48 h after treatment of lower leaves with 1 mg/L MeSA or DMSO solvent (Mock). “St” represents the authentic standard of SA. All experiments were repeated three times with similar results. Data = means ≥ sd (Student’s t test, **P < 0.01). WT, wild type.

Figure 9.

Working model for the negative regulation of plant systemic defense through MeSA glucosylation. When plants are infected by some pathogens, MeSA levels rise rapidly in the primary infected plant tissues. UGT71C3 is up-regulated due to the induction of pathogens and MeSA. MeSA glucosylation is thus accelerated, resulting in less MeSA being transported to the uninoculated systemic tissues. In the systemic tissues, MeSA is further glucosylated by UGT71C3, resulting in further reduced MeSA levels and thus a reduced level of SA, which is converted from MeSA. Thereby, the expression of pathogen-related proteins decreases and defense responses are attenuated. The downward arrows mean the decrease of MeSA and SA levels or down-regulation of PR genes. AtMES, Arabidopsis methylesterases; BSMT1, SA methyl transferase 1.