Phytoalexins of the Pyrinae: Biphenyls and dibenzofurans

Biphenyls and dibenzofurans are the phytoalexins of the Pyrinae, a subtribe of the plant family Rosaceae. The Pyrinae correspond to the long-recognized Maloideae. Economically valuable species of the Pyrinae are apples and pears. Biphenyls and dibenzofurans are formed de novo in response to infection by bacterial and fungal pathogens. The inducible defense compounds were also produced in cell suspension cultures after treatment with biotic and abiotic elicitors. The antimicrobial activity of the phytoalexins was demonstrated. To date, 10 biphenyls and 17 dibenzofurans were isolated from 14 of the 30 Pyrinae genera. The most widely distributed compounds are the biphenyl aucuparin and the dibenzofuran γ-cotonefuran. The biosynthesis of the two classes of defense compounds is not well understood, despite the importance of the fruit crops. More recent studies have revealed simultaneous accumulation of biphenyls and dibenzofurans, suggesting sequential, rather than the previously proposed parallel, biosynthetic pathways. Elicitor-treated cell cultures of Sorbus aucuparia served as a model system for studying phytoalexin metabolism. The key enzyme that forms the carbon skeleton is biphenyl synthase. The starter substrate for this type-III polyketide synthase is benzoyl-CoA. In apples, biphenyl synthase is encoded by a gene family, members of which are differentially regulated. Metabolism of the phytoalexins may provide new tools for designing disease control strategies for fruit trees of the Pyrinae subtribe.

Outside the subtribe Pyrinae, biphenyls and dibenzofurans were also found in a number of species. However, they do not function as phytoalexins, i.e., de novo formed defense compounds after microbial infection. They occur as preformed constituents (phytoanticipins), which are present before any challenge by microorganisms or herbivores and provide a constitutive barrier.
The antibacterial activity of biphenyls and dibenzofurans is less well studied [24,25]. Recently, a number of the Pyrinae-specific phytoalexins were tested for in vitro antibacterial activity against E. amylovora, the fire-blight-causing agent [12]. 3,5-Dihydroxybiphenyl was the most active compound with a minimum inhibitory concentration (MIC) of 115 μg/mL. While this concentration was bactericidal, a concentration approximately ten times lower led to 50% growth inhibition (MIC 50 = 17 μg/mL). Biphenyls exhibited somewhat stronger antibacterial activity than structurally related dibenzofurans did [12], whereas the opposite tendency was observed for antifungal activity [26]. However, more biphenyls and dibenzofurans need to be tested for their antibacterial and antifungal potentials in order to allow for reliable conclusions concerning structure-activity relationships. The array of phytoalexins accu-mulated in response to infection in a number of Pyrinae species appears to provide protection from both bacterial and fungal pathogens, such as E. amylovora and V. inaequalis, respectively. The mechanism of antimicrobial action of biphenyls and dibenzofurans has not yet been established.

Elicitor-treated cell cultures as a model system
Cell suspension cultures treated with elicitors are widely used to investigate microbe-induced processes in systems of reduced complexity, as compared to natural interactions between differentiated plants and intact pathogens [22,[27][28][29][30][31][32]. The phytoalexin response in elicitor-treated cell cultures is magnified relative to that at local infection sites of plant organs. Furthermore, the disruption of cultured cells to extract phytoalexins, as well as enzymes and transcripts, is easier than homogenization of intact, woody plants. However, cell cultures fail to provide insight into the organ and tissue specificities of the biosynthetic pathway.
We have established cell cultures of S. aucuparia as a model system for studying biphenyl and dibenzofuran formation after  elicitor treatment [22,23]. S. aucuparia cell cultures respond to the addition of elicitors with the accumulation of both biphenyls (2, 3, 6) and dibenzofurans (17,18). Simultaneous formation of the two classes of defense compounds has thus been observed with M. domestica, P. communis, and S. aucuparia, although intact S. aucuparia plants contain only biphenyls [3,[20][21][22]. The pattern of phytoalexins formed in S. aucuparia cell cultures varied with the type of elicitor added [23]. Yeast extract mainly induced the formation of aucuparin (3), whereas chitosan, although being a relatively poor elicitor, primarily stimulated the production of noraucuparin (2). Maximum phytoalexin levels were observed after the addition of autoclaved suspensions of the fire-blight bacterium, E. amylovora, and the scabcausing fungus, V. inaequalis. Eriobofuran (17) was the major inducible defense compound. The total biphenyl and dibenzofuran concentrations were 8.5 and 9.5 μg/g dry weight, respectively, and did not appreciably differ after treatment with the scab fungus and the fire-blight bacterium ( Figure 2). These two pathogens along with the powdery mildew-causing fungus are responsible for the most destructive diseases affecting the Pyrinae, which lead to significant yield losses and crop failures in apple and pear production ( Figure 3) [33].

Biosynthesis of biphenyls and dibenzofurans
The key enzyme of the biosynthetic pathway is biphenyl synthase (BIS) [22]. This type-III polyketide synthase (PKS) catalyzes the iterative condensation of benzoyl-CoA with three acetyl units from the decarboxylation of malonyl-CoA to form a linear tetraketide intermediate, which undergoes intramolecular C2→C7 aldol condensation and decarboxylative elimination of the terminal carboxyl group to give 3,5-dihydroxybiphenyl ( Figure 4). BIS activity was first detected in cell cultures of S. aucuparia treated with yeast extract as an elicitor [22]. A BIS cDNA was cloned, and the recombinant enzyme was functionally expressed in Escherichia coli and characterized [34].

Conclusion
Upon attack by pathogens, species of the Pyrinae form biphenyl and dibenzofuran phytoalexins. The biosynthesis of these two classes of defense compounds is poorly understood, although the Pyrinae include apple, pear, and related fruit trees. Plant diseases, such as fire blight, scab, and powdery mildew, lead to dramatic losses of fruits and trees. Engineering of the phytoalexin metabolism may provide new tools for enhancing disease resistance in economically important cultivars. However, this approach requires a detailed knowledge of biphenyl and dibenzofuran biosynthesis at the metabolic, enzymatic, and genetic levels. Data obtained with elicitor-treated cell cultures as a simplified experimental system lay the foundation for the study of the more complex interaction of differentiated plants and intact pathogens.