Bacterial cyclic beta-(1,2)-glucan acts in systemic suppression of plant immune responses

Abstract

Although cyclic glucans have been shown to be important for a number of symbiotic and pathogenic bacterium-plant interactions, their precise roles are unclear. Here, we examined the role of cyclic beta-(1,2)-glucan in the virulence of the black rot pathogen Xanthomonas campestris pv campestris (Xcc). Disruption of the Xcc nodule development B (ndvB) gene, which encodes a glycosyltransferase required for cyclic glucan synthesis, generated a mutant that failed to synthesize extracellular cyclic beta-(1,2)-glucan and was compromised in virulence in the model plants Arabidopsis thaliana and Nicotiana benthamiana. Infection of the mutant bacterium in N. benthamiana was associated with enhanced callose deposition and earlier expression of the PATHOGENESIS-RELATED1 (PR-1) gene. Application of purified cyclic beta-(1,2)-glucan prior to inoculation of the ndvB mutant suppressed the accumulation of callose deposition and the expression of PR-1 in N. benthamiana and restored virulence in both N. benthamiana and Arabidopsis plants. These effects were seen when cyclic glucan and bacteria were applied either to the same or to different leaves. Cyclic beta-(1,2)-glucan-induced systemic suppression was associated with the transport of the molecule throughout the plant. Systemic suppression is a novel counterdefensive strategy that may facilitate pathogen spread in plants and may have important implications for the understanding of plant-pathogen coevolution and for the development of phytoprotection measures.

Figure 1.

Infection of N. benthamiana with Xcc Strains. (A) A 1.3-kb fragment of chromosomal DNA containing a 5′ portion of the ndvB gene and the 3′ end of the ynaJ gene was amplified by PCR using Xcc chromosomal DNA as a template and cloned in pGEM T-Easy (Promega). The 2-kb fragment containing the Spc^r (Ω) cassette from pHP45 Ω (Fellay et al., 1989) was ligated as a SmaI fragment into pGEM T-Easy digested with SmaI within the ndvB gene. The resulting ndvB∷Spc^r allele was cloned as a 3.3-kb NotI fragment into the sacB suicide vector pJQ200 KS digested with NotI. (B) DNA gel blot (left) and TLC (right) analysis of Xcc strains for cyclic glucan production. Genomic DNA was prepared from the wild-type strain 8004 and the ndvB⁻ mutant. Genomic DNA was digested with SacI and separated on a 0.8% agarose gel. The blot was probed with the 0.6-kb DNA fragment indicated in Figure 1A. TLC was performed with crude extracts from wild-type 8004, ndvB⁻, ndvB⁺, and pure cyclic β-(1,2)-glucan as a standard. The plate was then stained with 5% sulfuric acid in ethanol and heated at 120°C for 15 min. (C) Symptoms in N. benthamiana leaves at 5 d after inoculation with a 10⁷ cfu/mL suspension of wild-type Xcc strain 8004, ndvB⁻, and ndvB⁺. (D) Growth of Xcc strains in N. benthamiana leaves. The mean and sd from three separate measurements of bacterial numbers are given for each time point. (E) RNA gel blot analysis of the expression of the defense-related gene PR-1 in response to Xcc strains 8004, ndvB⁻, and ndvB⁺.

Figure 2.

Callose Deposition in N. benthamiana Leaves Is Associated with Resistance and Is Suppressed by the Xcc Extracellular Cyclic β-(1,2)-Glucan. (A) to (D) N. benthamiana leaves after inoculation with strains of Xcc stained for callose deposits (white dots) observed by fluorescence microscopy: Xcc strain 8004 (A); Xcc ndvB⁻ strain (B); Xcc ndvB⁻ strain inoculated after cyclic β-(1,2)glucan pretreatments (C); and Xcc ndvB⁺ strain (D). Bars = 200 μm. (E) Average numbers of callose deposits per field of view (0.45 mm²). Error bars represent sd values from three leaves of each plant and three independent experiments. Differences between the response to the ndvB⁻ mutant after water pretreatment and all other treatments were significant as assessed by Student's t test at P < 0.001. dpi, days after inoculation.

Figure 3.

Xcc Extracellular Cyclic β-(1,2)-Glucan Compromises the Disease Resistance in N. benthamiana. (A) and (B) Disease symptoms on N. benthamiana leaves preinfiltrated with water (A) or purified cyclic β-(1,2)-glucan (50 μg/mL) (B) and subsequently infected with a 10⁷ cfu/mL suspension of the Xcc ndvB⁻ strain. (C) Effects of cyclic glucan and water (control) pretreatments on Xcc ndvB⁻ strain growth. The mean and sd of three separate measurements of bacterial numbers are given for each time point. (D) Effects of cyclic glucan and water (control) pretreatments on PR-1 gene expression induced by the Xcc ndvB⁻ strain. Transcript levels were analyzed at 24 h after bacterial inoculation by RNA gel blot. (E) Effects of cyclic glucan and water (control) pretreatments on Xcc wild-type (strain 8004) growth. Bacterial numbers were measured immediately after bacterial inoculation and at 3 d after inoculation. Error bars represent sd values. Data sets marked with asterisks are significantly different from control (water-pretreated leaves) as assessed by Student's t test (P < 0.001).

Figure 4.

The Xcc Cyclic β-(1,2)-Glucan Suppression Effect is Dose-Dependent. Leaves of 4-week-old plants were preinfiltrated with either different concentrations of Xcc cyclic β-(1,2)-glucan or water at 24 h before either the same (A) or distant (B) leaves were inoculated with a 10⁷ cfu/mL suspension of the Xcc ndvB mutant strain. Numbers of mutant bacteria were assessed immediately upon inoculation and 3 d later. The mean and sd of three separate measurements of bacterial numbers are given. Data sets marked with asterisks are significantly different from control (water-pretreated leaves) as assessed by Student's t test at P < 0.001.

Figure 5.

Time Dependence for Cyclic β-(1,2)-Glucan to Establish Susceptibility to the Xcc ndvB Mutant. Leaves of 4-week-old plants were preinfiltrated with either Xcc cyclic β-(1,2)-glucan (50 μg/mL) or water, and either the same (A) or distant (B) leaves were inoculated with a 10⁷ cfu/mL suspension of the Xcc ndvB mutant strain at different times after infiltration. Numbers of mutant bacteria were assessed immediately upon inoculation and 3 d later. The mean and sd of three separate measurements of bacterial numbers are given. Data sets marked with asterisks are significantly different from control (water-pretreated leaves) as assessed by Student's t test (P < 0.001).

Figure 6.

Xcc Wild-Type Strain 8004 Suppresses Disease Resistance in a Systemic Fashion in N. benthamiana. Leaves of 4-week-old plants were preinfiltrated with a 10⁷ cfu/mL suspension of either Xcc wild-type strain 8004 or ndvB mutant strain at 24 h before distant leaves were inoculated with a 10⁷ cfu/mL suspension of the ndvB mutant strain. Numbers of mutant bacteria were assessed immediately upon inoculation and 3 d later. The mean and sd of three separate measurements of bacterial numbers are given. Data sets marked with asterisks are significantly different from control (Xcc 8004-pretreated) leaves as assessed by Student's t test (P < 0.001).

Figure 7.

Xcc Cyclic β-(1,2)-Glucan Is Spread Systemically and Induces Systemic Disease Susceptibility in N. benthamiana. (A) to (D) Leaves of 4-week-old plants were preinfiltrated with either water ([A] and [B]) or 50 μg/mL purified cyclic β-(1,2)-glucan ([C] and [D]) and then inoculated 24 h later with a 10⁷ cfu/mL suspension of the Xcc ndvB mutant strain either in the same leaves ([A] and [C]) or in distant leaves ([B] and [D]). Photographs were taken at 5 d after infection. (E) RNA gel blot analysis of PR-1 gene expression in N. benthamiana leaves treated as in (A) to (D). D.L., distant leaves; T.L., same leaves. Data shown are representative of those obtained from three independent experiments. (F) Quantification of [¹⁴C]cyclic β-(1,2)-glucan in local and distant leaves. Error bars represent sd from three independent experiments. (G) Comparison of the behavior on size-exclusion chromatography on a BioGel P4 column of the ¹⁴C-labeled material from distant leaves of N. benthamiana (squares) and authentic extracellular [¹⁴C]cyclic β-(1,2)-glucan (triangles). (H) TLC analysis of ¹⁴C-labeled materials from distant N. benthamiana leaves. Column 1, purified extracellular [¹⁴C]cyclic β-(1,2)-glucan from bacterial culture; column 2, ¹⁴C-labeled material from distant leaves of N. benthamiana; column 3, partial acid digestion of purified extracellular [¹⁴C]cyclic β-(1,2)-glucan from bacterial culture; column 4, partial acid digestion of ¹⁴C-labeled material from distant leaves of N. benthamiana; column 5, total acid digestion of purified extracellular [¹⁴C]cyclic β-(1,2)-glucan from bacterial culture; column 6, total acid digestion of ¹⁴C-labeled material from distant leaves of N. benthamiana.

Figure 8.

Xcc Cyclic β-(1,2)-Glucan Compromises the Disease Resistance in Arabidopsis. (A) Symptoms in Arabidopsis plants at 6 d after inoculation by dipping with a 10⁷ cfu/mL suspension of Xcc strains 8004 (wild type), ndvB⁻, and ndvB⁺. (B) Growth of different Xcc strains in Arabidopsis plants. The bacterial population was determined as cfu per gram fresh weight, as described in Methods. The mean and sd of three separate measurements of bacterial numbers are given for each time point. (C) Disease symptoms on Arabidopsis plants infiltrated with water or purified cyclic β-(1,2)-glucan (50 μg/mL) and subsequently infected with the Xcc ndvB⁻ mutant. Four-week-old plants of Arabidopsis were submerged in a solution of Xcc cyclic glucan (50 μg/mL) for 1 min followed by vacuum infiltration, and 24 h later the entire plants were inoculated with the Xcc ndvB⁻ mutant (10⁷ cfu/mL) by dipping. Symptoms were assessed at 6 d after dip-inoculation. (D) Growth of the Xcc ndvB⁻ mutant in Arabidopsis plants treated as shown in (C). Numbers of mutant bacteria were assessed immediately upon dip-inoculation and 3 d later. The mean and sd of three separate measurements of bacterial numbers are given. Data sets marked with asterisks are significantly different from control (water-pretreated leaves) as assessed by Student's t test (P < 0.001). (E) Local and systemic symptoms on Arabidopsis leaves preinfiltrated with water or purified cyclic β-(1,2)-glucan (50 μg/mL) and subsequently infected with the Xcc ndvB⁻ mutant (10⁷ cfu/mL). One leaf of each plant was submerged in a solution of cyclic β-(1,2)-glucan (50 μg/mL) in an Eppendorf tube and infiltrated under vacuum. Twenty-four hours later, the entire plants were inoculated by dipping. Water-pretreated plants were used as controls. Disease symptoms were observed at 6 d after dip-inoculation.