Differential expression of biphenyl synthase gene family members in fire-blight-infected apple 'Holsteiner Cox'

Abstract

Fire blight, caused by the bacterium Erwinia amylovora, is a devastating disease of apple (Malus × domestica). The phytoalexins of apple are biphenyls and dibenzofurans, whose carbon skeleton is formed by biphenyl synthase (BIS), a type III polyketide synthase. In the recently published genome sequence of apple 'Golden Delicious', nine BIS genes and four BIS gene fragments were detected. The nine genes fall into four subfamilies, referred to as MdBIS1 to MdBIS4. In a phylogenetic tree, the BIS amino acid sequences from apple and Sorbus aucuparia formed an individual cluster within the clade of the functionally diverse type III polyketide synthases. cDNAs encoding MdBIS1 to MdBIS4 were cloned from fire-blight-infected shoots of apple 'Holsteiner Cox,' heterologously expressed in Escherichia coli, and functionally analyzed. Benzoyl-coenzyme A and salicoyl-coenzyme A were the preferred starter substrates. In response to inoculation with E. amylovora, the BIS3 gene was expressed in stems of cv Holsteiner Cox, with highest transcript levels in the transition zone between necrotic and healthy tissues. The transition zone was the accumulation site of biphenyl and dibenzofuran phytoalexins. Leaves contained transcripts for BIS2 but failed to form immunodetectable amounts of BIS protein. In cell cultures of apple 'Cox Orange,' expression of the BIS1 to BIS3 genes was observed after the addition of an autoclaved E. amylovora suspension. Using immunofluorescence localization under a confocal laser-scanning microscope, the BIS3 protein in the transition zone of stems was detected in the parenchyma of the bark. Dot-shaped immunofluorescence was confined to the junctions between neighboring cortical parenchyma cells.

Figure 1.

Biosynthesis of the biphenyl and dibenzofuran scaffolds.

Figure 2.

Neighbor-joining tree of type III PKSs. CHSs and functionally divergent PKSs from angiosperms are highlighted in blue and red, respectively. Bold letters mark BIS1 to BIS4 and CHS from cv Holsteiner Cox. Numbers at the forks are bootstrap values from 1,000 replicates. ACS, Acridone synthase; ALS, aloesone synthase; BAS, benzalacetone synthase; BBS, bibenzyl synthase; CTAS, 4-coumaroyl triacetic acid lactone synthase; DpgA, 3,5-dihydroxyphenylacetate synthase; HKS, hexaketide synthase; HTAS, hexanoyl triacetic acid lactone synthase; PhID, acetylphloroglucinol synthase; 2-PS, 2-pyrone synthase; RppA, 1,3,6,8-tetrahydroxynaphthalene synthase; STS, stilbene synthase; STCS, stilbene carboxylate synthase; VAS, valerophenone synthase.

Figure 3.

Dissection of an E. amylovora-inoculated shoot of cv Holsteiner Cox (6 d postinoculation) into five zones for separate analysis.

Figure 4.

RT-PCR amplification of BIS1 to BIS4 transcripts in stems (transition zone) and leaves (first living leaf from the top) of cv Holsteiner Cox 6 d after E. amylovora inoculation, and in cell cultures of cv Cox Orange 9 h after treatment with an autoclaved suspension of E. amylovora. Profilin transcript levels served as control for use of equal RNA template amounts.

Figure 5.

Determination by real-time PCR of relative BIS3 (A) and BIS2 (B) transcript levels in stem segments and leaves, respectively, of cv Holsteiner Cox 6 d after E. amylovora inoculation. mRNA isolation from zone 1 and leaves 1 and 2, which were necrotic, was not feasible. Control mRNA was obtained from stems and leaves of mock-inoculated shoots. sds are indicated (n = 3).

Figure 6.

GC-MS analysis of biphenyls and dibenzofurans present in the transition zone of fire-blight-infected stems of cv Holsteiner Cox. The phytoalexins were separated as trimethylsilyl derivatives. Stars indicate constitutive compounds also detected in mock-inoculated shoots. IS, Internal standard (4-phenylphenol).

Figure 7.

Concentrations of biphenyls and dibenzofurans in the transition zone of fire-blight-infected stems of cv Holsteiner Cox. Data are mean values ± sd of four independent experiments.

Figure 8.

Immunoblotting after SDS-PAGE of BIS1 to BIS4 (100 ng) and protein extracts (50 μg) from stems and leaves of cv Holsteiner Cox 6 d after E. amylovora inoculation and wounding only.

Figure 9.

Immunofluorescence localization of BIS protein in cross sections of stems of cv Holsteiner Cox 6 d after E. amylovora inoculation. A and D, Cross sections of the transition zone (zone 2) after incubation with anti-BIS IgG (A) and preimmune IgG (D). B, Detail from A displaying cortex parenchyma and phloem. The arrows indicate fluorescent dots between phloem ray cells and phloem parenchyma cells. C, Detail from A displaying a single cortical parenchyma cell with dot-shaped immunofluorescence at the junctions with the neighboring cells. ep, Epidermis; pa, parenchyma; co, collenchyma; ph, phloem; pr, phloem ray.