A Medicago truncatula Cytochrome p450 gene Plays a Role in Penetration Resistance Against Nonhost Pathogen Phakopsora pachyrhizi

Asian Soybean Rust (ASR), caused by Phakopsora pachyrhizi, is an economically important disease of soybean. The model legume species, Medicago truncatula which is a nonhost to P. pachyrhizi, shows incompatible interactions with this fungus. In order to identify genes involved in durable nonhost resistance against ASR, we screened ca. 2,500 M. truncatula Tnt1 insertion lines upon inoculation with P. pachyrhizi and identified several mutants with an altered response to ASR. One such mutant, epr (enhanced penetration of rust), showed small germ tubes and enhanced penetration upon germination of P. pachyrhizi urediniospores on leaf surfaces compared to wild-type R108. Scanning electron microscopy revealed defects in cuticle formation on leaf surfaces of epr mutant leaves, thus indicating the role of leaf surface topology in altering rust pre-infection structure formation. Genetic co-segregation analysis identified a Tnt1 insertion in the exon region of the Cytochrome p450 gene which co-segregates with the epr phenotype. Transcript analysis of the Cytochrome p450 gene detected a negligible transcript in leaves but significant transcript abundance in leaf epidermis, indicating its role in leaf surface structure formation. Furthermore, five independent mutant alleles with Tnt1 insertions at different locations of Cytochrome p450 gene showed similar phenotype. Polyester analysis by GC-MS revealed more abundance of decanoic acid and reduced abundance of 10,16-dihydroxy decanoic acid in epr mutant leaves compared to wild-type leaves indicating a role of this gene as in-chain hydroxylase. The 10,16-dihydroxy decanoic acid is a major monomer of cutin. Hence, Epr is responsible for penetration resistance in M. truncatula against nonhost fungal pathogens such as P. pachyrhizi.