Elucidation of Septoria tritici x wheat interactions using GUS-expressing isolates

Isolate ISR398 of Septoria tritici (which produces none to few pycnidia on the wheat cv. Seri 82 and high coverage on cv. Shafir) and isolate ISR8036 (which is virulent on both cultivars) were genetically cotransformed using the selectable marker gene hph, which confers resistance to hygromycin B (hygB), and the reporter gene uidA, encoding beta-glucuronidase (GUS). Most of the genetically transformed isolates (98.8%) produced similar pycnidial coverage on seedlings of 'Seri 82' and 'Shafir' as the two wild-type isolates. Southern analysis of 25 randomly selected hygB(R)GUS(+) transformants probed with the uidA sequence revealed multiple insertion sites. GUS activity was determined fluorimetrically by measuring the conversion of 4-methylumbelliferyl beta-D-glucuronide (MUG) to 4-methylumbelliferone (MU). The high GUS-expressing transformants 398D97 and 8036E27 were used to elucidate fungal development within inoculated leaf tissue by using GUS activity to estimate the fungal proteins content in planta. Increase in fungal biomass was recorded in 'Shafir' inoculated with the GUS-expressing transformants 398D97 and 8036E27 following a 12-day latent period. A 15-day latent period was recorded in 'Seri 82' inoculated with 8036E27, whereas an 18-day latent period was recorded on 'Seri 82' inoculated with 398D97 and the two mixtures 398D97 + ISR8036 and ISR398 + 8036B27. The rate of fungal development and the estimated level of fungal proteins at the pycnidia maturation stage was high in leaves of 'Shafir' and moderate to low on 'Seri 82', even in cases in which no significant differences were recorded in pycnidial coverage. An endogenous capacity to hydrolyze beta-1,4-D-glucuronidase was recorded in leaves inoculated with wild-type isolates. The latent periods in MU production of the uidA-expressing transformants mimicked those recorded for the wild-type isolates. However, at all stages, the levels of MU produced in wheat inoculated with wild-type isolates were markedly lower than those produced by GUS-expressing transformants. The mode of interaction (compatible or incompatible) determined the onset of the induction, rate, and level of enzyme production.