1.10.6
ISOLATION AND CHARACTERIZATION OF A TRIHYDROPHOBIN GENE OF CLAVICEPS PURPUREA AND CELL-SPECIFIC LOCALIZATION OF MRNA DURING INFECTION OF RYE

T CORREIA1, V GARRE2, P STELLAMANNS1, KB TENBERGE1 and P TUDZYNSKI1

1Institut für Botanik, Westf. Wilhelms-Universität, Schlossgarten 3, D-48149 Münster, Germany; 2Present address: Departamento de Genética y Microbiología, Facultad de Biología, Universidad de Murcia, E-30071, Murcia, Spain.

Background and objectives
Hydrophobins are unique fungal extracellular proteins that produce amphipathic films at interfaces and mediate contact to hydrophobic surfaces. During the screening for genes expressed in alkaloid producing cultures of Claviceps fusiformis, a gene has been identified the derived polypeptide of which contained three hydrophobin domains [1]. The corresponding protein has been isolated from axenic culture, identified by partial sequencing and biochemically characterized; it is a new type of hydrophobin, named trihydrophobin. The pathogenic ascomycete Claviceps purpurea causes ergot disease in grasses and cereals and, due to the alkaloid content of sclerotia, ergotism in livestock. Since hydrophobins are known to be important in phytopathogenicity, the current research aimed at the identification, isolation and characterization of a hydrophobin gene of this important ergot fungus. Expression analyses included cell-specific localization of hydrophobin mRNA during infection of rye to elucidate presence and function of the hydrophobin in the pathogenic interaction.

Results and conclusions
A gene encoding a comparable type of hydrophobin was isolated from a cDNA library of C. purpurea and the corresponding genomic copy was cloned and characterized. According to the derived amino acid sequence, the C. purpurea hydrophobin CPTH1 has an overall homology of about 40% to the C. fusiformis trihydrophobin. Its biochemical properties and hydropathy characteristic are typical of class two hydrophobins. The open reading frame of 1.8 kb comprises six introns, two in each hydrophobin domain. The molecular mass was estimated at 45 kDa. Using RT-PCR and Northern blotting analysis, the expression of cpth1 was studied in axenic and parasitic culture. The transcription of cpth1 was significantly repressed by carbon. Specific mRNA was isolated from different infection phases.

Semithin cryosections proved to be suitable for microscopical gene expression analysis using immune-mediated alkaline-phosphatase staining for detection of digoxigenin-labeled cRNA probes in the nonradioactive in situ hybridization technique [2]. While permeabilization by proteinase K had only a minor effect, the inclusion of detergent into the hybridization solutions enhanced specific RNA-RNA hybridization under maximum stringency. Hydrophobin mRNA was detected in fungal cells growing in axenic culture. In the disease cycle, hydrophobin transcripts were localized in abundance during vegetative fructification in conidiophores that actively produced conidia. No signals were observed in sclerotial hyphae during formation of the alkaloid-containing ergots. Notably, in situ hybridization experiments resulted in specific signals during early infection and colonization phases in the external mycelia and in hyphae penetrating the host epidermal layer.

The presumed role of the hydrophobin gene product in ergot pathogenicity is discussed with respect to the described spatio-temporal distribution of the hydrophobin transcripts. Ongoing work includes gene-directed mutagenesis to prove the function of the hydrophobin in the plant-pathogen interaction.

References
1. Arntz C, Tudzynski P, 1997. Current Genetics 31, 357-360.
2. Tenberge KB, Stellamanns P, Plenz G, Robenek H, 1998. European Journal of Cell Biology 75 (in press).