4,5-DOPA dioxygenase (DODA) was originally believed to be involved only in the biosynthesis of betalain, a pigment found in plants in the Caryophyllales. However, DODA-like genes have been identified in many plants that do not produce betalain, including Nicotina benthamiana, which is a plant in the Solanales. Expression of DODA-like gene, NbDODAL1, by N. benthamiana was induced in leaves following wounding, ethylene and benzothiadiazole treatments. Transcript levels of NbDODAL1 were also significantly induced in compatible interactions of wild type N. benthamiana with Colletotrichum destructivum, Colletotrichum orbiculare or Pseudomonas syringae pv. tabaci, as well as transgenic N. benthamiana expressing Pto in an incompatible interaction with P. syringae pv. tabaci expressing avrPto. Virus-induced gene silencing of NbDODAL1 resulted in increased susceptibility of N. benthamiana to C. destructivum, C. orbiculare or P. syringae pv. tabaci, but did not affect the hypersensitive response to P. syringae pv. tabaci expressing avrPto. This is the first demonstration of a role for DODA-like protein. NbDODAL1 may be involved in many aspects of plant metabolism, one of which is basal resistance, perhaps through the metabolism of aromatic compounds contributing to antimicrobial or antioxidative activity.