GUS transformation of the maize fungal endophyte Fusarium moniliforme

Visual markers detectable by histochemical staining have been developed for analysing the time course and tissue specificity of maize infections by Fu sarium moniliforme. Three F. moniliforme strains, RRC 374, MRC 826 and RRC PAT, were transformed with a plasmid, pHPG, containing the gusA reporter ge ne which codes for P-glucuronidase (GUS) and the hph gene for hygromycin re sistance as the selectable marker. Introduction of plasmid DNA into germina ting conidia yielded 1.2 x 10(-7) transformants per conidium; expression of both gusA and hph was however, transient. Stable transformants were obtain ed using protoplasts as the recipient but transformation frequency was redu ced. Southern blot and PCR analyses confirmed incorporation of pHPG into th e genome of all three F. moniliforme strains with gusA properly inserted in MRC 826 and RRC PAT, but apparently disrupted in RRC 374. The growth patte rn for transformed F. moniliforme isolates and the parental wild types foll owed a sigmoid curve on minimal and enriched media. Hygromycin totally inhi bited growth for wild type isolates, but not of transformants. Transformed isolates maintained the ability to infect the maize plant. Thus, this study is the first report of F. moniliforme transformed with a visibly detectabl e reporter gene to use for analysing this endophyte-host interaction of wor ld-wide importance to animal and human health.