Cinnamate-CoA ligase is involved in biosynthesis of benzoate-derived biphenyl phytoalexin in Malus x domestica 'Golden Delicious' cell cultures

Apple (Malus sp.) and other genera belonging to the sub-tribe Malinae of the Rosaceae family produce unique benzoic acid-derived biphenyl phytoalexins. Cell cultures of Malus domestica cv. 'Golden Delicious' accumulate two biphenyl phytoalexins, aucuparin and noraucuparin, in response to the addition of a Venturia inaequalis elicitor (VIE). In this study, we isolated and expressed a cinnamate-CoA ligase (CNL)-encoding sequence from VIE-treated cell cultures of cv. 'Golden Delicious' (M. domestica CNL; MdCNL). MdCNL catalyses the conversion of cinnamic acid into cinnamoyl-CoA, which is subsequently converted to biphenyls. MdCNL failed to accept benzoic acid as a substrate. When scab-resistant (cv. 'Shireen') and moderately scab-susceptible (cv. 'Golden Delicious') apple cultivars were challenged with the V. inaequalis scab fungus, an increase in MdCNL transcript levels was observed in internodal regions. The increase in MdCNL transcript levels could conceivably correlate with the pattern of accumulation of biphenyls. The C-terminal signal in the MdCNL protein directed its N-terminal reporter fusion to peroxisomes in Nicotiana benthamiana leaves. Thus, this report records the cloning and characterisation of a cinnamoyl-CoA-forming enzyme from apple via a series of in vivo and in vitro studies. Defining the key step of phytoalexin formation in apple provides a biotechnological tool for engineering elite cultivars with improved resistance.