Unique gene Pmhyp controlling melanization of pycnidia in Phoma medicaginis

Phoma medicaginis (syn. Ascochyta medicaginicola Qchen & L. Cai) causes spring black stem and leaf spot of alfalfa and the model legume Medicago truncatula. Phoma medicaginis produces uninucleate conidia in melanized pycnidia and is genetically tractable through Agrobacterium tumefaciens-mediated transformation (ATMT), which can result in insertional mutants. One T-DNA-tagged mutant, P1A17 produced conidia in non-melanized (hyaline) pycnidia. Pycnidial melanization recovered if the mutant was supplemented with melanin precursors or allowed to age. DNA sequences flanking the insertion did not predict any disrupted open reading frames (ORF) unless a Coccidioides prediction algorithm was used. Pmhyp gene was expressed in the wild type, but not the mutant, and has not been annotated in any genomes, to date. Expression of two conserved genes flanking the T-DNA disrupted Pmhyp was unchanged from the wild type. Knockout of Pmhyp strain displayed same cultural phenotype (non-melanized pycnidia). Complementation of Pmhyp strains with wild type PmHYP partially recovered pycnidial melanization. Both knockout and complementation transformants were confirmed using RT-PCR and southern blot analysis. Taken together, PmHYP appears to be a novel regulator of pycnidium specific melanization.