Cooperative Research Centre for Tropical Plant Pathology, The University of Queensland, Brisbane, 4072, Australia

Background and objectives
The genus Colletotrichum includes pathogens that infect a wide range of agricultural plant species grown in the tropics. Despite their importance, there is little known at the molecular level about the processes employed by Colletotrichum species to infect their hosts. We have previously reported that the glutamine synthetase (GS) gene of C. gloeosporioides was highly expressed at the early stages of infection of leaves of the tropical legume Stylosanthes guianensis and was also strongly expressed in axenic culture under conditions of nitrogen (N) starvation [1]. In most organisms the abundance of GS acts a biochemical marker for conditions of N starvation. Taken together, these observations suggest that C. gloeosporioides may be subjected to N limitation during the early stages of infection of S. guianensis, and it is also possible that N deprivation may act as a signal for the expression of other genes necessary for pathogenicity. In the present work we have tested this hypothesis, and describe an essential pathogenicity gene of C. gloeosporioides termed CgDN3 that is induced in axenic culture by N starvation and is also expressed at early stages of infection. Importantly, disruption of the CgDN3 gene leads to a loss of pathogenicity and the production of a strong hypersensitive resistance response in the inoculated host.

Results and conclusions
Eight cDNA clones corresponding to C. gloeosporioides genes that are induced by N starvation in axenic culture were isolated by differential hybridization. One of these genes, termed CgDN3, was chosen for detailed investigation because Northern analysis and RT-PCR analyses indicated that it was expressed at early stages of infection of the host S. guianensis. The sequence of the CgDN3 cDNA suggested that it encodes a small basic peptide of 74 amino acids that contains a 24-amino-acid signal sequence for secretion of a 50-amino-acid mature protein. Comparison with sequences in the databases failed to find a homologue for the product of the CgDN3. The putative promoter sequence of the gene contained motifs found in the promoters of other fungal genes that are regulated by nitrogen supply. To test the role of the CgDN3 gene in infection, the CgDN3 gene was disrupted using a gene replacement strategy that substituted a region of CgDN3 including promoter and coding sequences with a hygromycin resistance gene cassette. Two transformants were confirmed as mutants in the CgDN3 gene by Southern hybridization analysis. Northern analysis of one of the mutants confirmed that no transcript corresponding to CgDN3 was being produced in mycelia subjected to N starvation. The mutants also had a characteristic phenotype in culture, with occasional production of some thick hyphae with pigmentation and the spontaneous production of appressoria. The mutants produced spores with a normal appearance and appressoria were formed after the inoculation of plants. However, the CgDN3 mutants were not able to proceed with infection, and the normal mass of mycelial growth, formation of acervuli and blighting of host tissue normally associated with anthracnose on S. guianensis were not evident. Microscopic analysis revealed small clusters of necrotic host cells at penetration sites, and small brown flecks were visible on host leaves, suggesting that these mutants elicited a hypersensitive response.

The CgDN3 gene is essential for pathogenicity and may have a role in the suppression of a host hypersensitive response (HR) reaction during attempted infection. The CgDN3 gene of C. gloeosporioides appears to represent a new class of pathogenicity gene in phytopathogenic fungi.

1. Stephenson S-A, Green JR, Manners JM, Maclean DJ, 1997. Current Genetics 31, 447-454.