A defect in nir1, a nirA-like transcription factor, confers morphological abnormalities and loss of pathogenicity in Colletotrichum acutatum

A non-pathogenic mutant of Colletotrichum acutatum, designated Ca5, exhibited epiphytic hyphal growth and did not cause lesions on strawberry plants but grew necrotrophically when inoculated directly onto wounded stolons. In the absence of an external nitrogen source, the mutant exhibited extended germ-tube growth prior to appressorium formation. The deduced product of the impaired gene (nir1) is similar to NirA, an Aspergillus nidulans transcriptional regulator of nitrogen metabolism. Inoculation of leaves with wild-type or Ca5 conidia in the presence of a preferred nitrogen source resulted in massive epiphytic hyphal production, appressorium formation and rapid symptom development. Expression of C. acutatum wild-type nitrate reductase (nit1) and glutamine synthetase (gln1) was induced by nitrate but only nit1 expression was repressed in a rich medium. nit1 transcription increased during the appressorium-production stage, indicating that nitrogen starvation constitutes a cue for the regulation of appressorium development. The presence of nit1 transcript during various phases of infection is indicative of partial nitrogen starvation in planta. cAMP-dependent protein kinase A (PKA) was determined to be a negative regulator of immediate post-germination appressoria formation in the wild-type. As inhibition of PKA activity in the nir1 mutant did not affect appressoria formation, we suggest that NIR1 acts either in parallel or downstream of the PKA pathway. Our results show that nir1 is a pathogenicity determinant and a regulator of pre-infection development under nitrogen-starvation conditions and that nitrogen availability is a significant factor in the pre-penetration phase.