Deciphering the Role of Cytochrome P450 Monooxygenases in Resistance to <em>Phytophthora sojae</em> in Soybean: Genomic Insight into Glyceollin Biosynthesis

Root and stem rot, caused by the oomycete Phytophthora sojae, is a devastating disease in soybean that results in billions of dollars of annual losses worldwide. As a defence mechanism, soybean produces the phytoalexin glyceollin, which has demonstrated efficacy against multiple pathogens, including P. sojae. Interestingly, five steps in glyceollin biosynthesis are catalyzed by cytochrome P450 enzymes, highlighting the vital role of this class of enzymes in the defence against pathogen infection. My study thus aimed to identify and characterize the P450 genes (GmP450s) involved in glyceollin biosynthesis. Through bioinformatic analyses, I identified 346 GmP450s in the soybean genome. Using a multi-transcriptome and gene co-expression analysis strategies, 21 P450 candidates including members from the CYP73 and CYP81 families were selected for further study. The CYP73 family includes cinnamate 4 hydroxylase (C4H), which is the first P450 enzyme in the phenylpropanoid pathway (catalyzing the conversion of trans-cinnamic acid to p-coumaric acid). In this study, I identified four C4H genes in the soybean genome, three of which are functionally active. The CYP81 family, on the other hand, encompasses isoflavone hydroxylases (IFHs), which participate in the hydroxylation of isoflavone aglycons in legumes and, which initiate glyceollin production. The soybean genome contains 12 CYP81 candidates, nine of which are functional with diverse hydroxylation capabilities with soybean isoflavone aglycons. Catalytically efficient GmC4H and GmIFH enzymes have been identified and have great potential as molecular tools in metabolic engineering applications. Finally, a transcriptome study was undertaken to explore the differential temporal dynamics of GmP450s and glyceollin biosynthetic pathway genes in two soybean lines that differ in their resistance to P. sojae. The results suggest that the partially resistant line exhibits a stronger plant innate immunity compared to the susceptible cultivar. The study of P450s and the glyceollin biosynthesis pathway has provided new insights into their production and regulation and has highlighted the presence of important side branches in the isoflavonoid pathway in soybean.