Characterization of a gene (sp1) encoding a secreted protein from Leptosphaeria maculans, the blackleg pathogen of Brassica napus

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Abstract
SUMMARY A gene (sp1) encoding a 12.3 kDa protein with a predicted secretion signal has been characterized from Leptosphaeria maculans, the dothideomycete that causes blackleg disease of canola (Brassica napus). This protein (SP1) contains four cysteine residues and shows a high sequence similarity to proteins from other ascomycetes. L. maculans sp1 has been placed on genetic and physical maps. This gene is expressed during the infection of B. napus cotyledons 10 days post-inoculation, coinciding with detection of the constitutively expressed fungal gene, beta-tubulin. L. maculans sp1, along with opsin and glyceraldehyde phosphate dehydrogenase, is light regulated. A recombinant SP1 protein expressed in Escherichia coli and a crude protein fraction secreted by L. maculans induced an autofluorescence response on B. napus leaves. The sp1 gene was mutated by targeted gene disruption whereby a hygromycin resistance gene was inserted. Such mutants caused similar-sized lesions on B. napus cotyledons as those caused by the wild-type isolate, indicating that sp1 is not crucial for pathogenicity of L. maculans on B. napus. This is the first report of disruption of this gene in any fungus.