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 3 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 salicoylcoenzyme 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.

Biphenyl 4-Hydroxylases Involved in Aucuparin Biosynthesis in Rowan and Apple Are Cytochrome P450 736A Proteins

“…Real-time PCR was used to quantify Md(GD)B4H mRNA amounts in cv Golden Delicious stems after inoculation with E. amylovora. As observed previously with cv Holsteiner Cox, which is used as a model system (Chizzali et al, 2012a), infection with the fire blight pathogen caused the typical shepherd's crook symptom at the shoot tip (Vanneste, 2000) and resulted in the formation of a transition zone that was framed by the necrotic and the healthy stem segments (Fig. 6A).…”

“…Recently, MdBIS3 was immunochemically localized to the junctions between neighboring cortical parenchyma cells in the transition zone of fire blight-infected apple stems, suggesting an association of the enzyme with plasmodesmata in primary pit fields (Chizzali et al, 2012a. Thus, it is tempting to speculate that the aucuparin biosynthetic enzymes may be assembled into a metabolon, which allows for an efficient transfer of biosynthetic intermediates between the active sites in close proximity (Winkel, 2004;Jørgensen et al, 2005).…”

“…Md(GD)B4H expression levels in fire blight-infected stems of cv Golden Delicious. A, Inoculation of cv Golden Delicious shoots with E. amylovora led to the formation of a transition zone between the necrotic and healthy stem segments, as observed previously with cv Holsteiner Cox stems (Chizzali et al, 2012a. B, Quantitative real-time PCR was used to determine Md(GD)B4H expression levels 6 d post inoculation.…”

“…In apple, BIS is encoded by a gene family consisting of four differentially regulated subfamilies (Chizzali et al, 2012a). MdBIS3 is expressed in stems upon fire blight infection.…”

“…6A). Md(GD)B4H expression in E. amylovora-infected stems of cv Golden Delicious was measured in relation to that observed in mock-inoculated control stems 6 d post inoculation, based on the previous observations with cv Holsteiner Cox (Chizzali et al, 2012a). A single PCR product was amplified using gene-specific primers for MdB4H, and no primer dimers were observed.…”