Transcriptome Analyses of the Nematode-trapping Fungus Monacrosporium haptotylum

Detta är en avhandling från Margareta Tholander Mikrobiologisk ekologi Ekologihuset Sölvegatan 37 223 62 Lund

Sammanfattning: Nematode-trapping fungi are soil living organisms with the ability to infect and kill nematodes. These fungi have developed specialized infection structures, traps for the capture of nematodes. To be able to identify genes in nematode-trapping fungi that are involved in the infection of nematodes cDNA microarrays were constructed. The cDNA microarrays were printed with cDNA reporers obtained from expressed sequence tag (EST sequencing of four different cDNA libraries representing the mycelium and traps of the nematode-trapping fungus Monacrosporium haptotylum, as well as the fungus infecting the nematode Caenorhabditis elegans for 4 h and 24 h, respectively. In total, 8,466 EST seqences were generated from these libraries. Following an assembly of these sequences, 3,518 clones were amplified and printed on the array, 2,822 of fungal, 540 of worm, and 156 of unknown origin. In order to identify genes that are expressed and regulated during the development of traps, total RNA was isolated from knobs and mycelium and hybridized to the cDNA array. In spite of the fact that knobs and mycelium were grown in the same medium a total of 23.3% (657 of 2,822 of the gene representatives were differentially expressed in knobs as compared to mycelium. Several of the genes that were regulated in knobs showed sequence similarities to genes involved in development of plant pathogenic infection structures (appressoria). Typical for trap cells in nematodetrapping fungi is the presence of numerous dense bodies which are organelles related to peroxisomes. The transcriptional profiling of M. haptotylum traps identified one gene representative with homology to the peroxisomal membrane protein Pex11p from S. cerevisiae. This homolog was significantly upregulated in traps (knobs) as compared to mycelium. The Pex11p protein is known to have a role in peroxisome proliferation in yeast. In order to further characterize the M. haptotylum PEX11 homolog, th e full length cDNA was cloned and expressed in the yeast Hansenula polymorpha. The result showed that the M. haptotylum Pex11p could not functionally complement the pex11 mutant of H. polymorpha. In the third study, the changes in the transcripome of M. haptotylum were followed during the various stages of the infection. Isolated knobs from M. haptotylum were used in the infection of the nematode C. elegans. RNA was extracted from the infection samples at four different time-points corresponding to the varoius stages of of the infection (adhesion, penetration and digestion) and was subsequently used for hybridization to the cDNA array. RNA isolated from axenic knobs and noninfected nematodes were used as a reference sample in the hybridization of the cDNA array. In total, 58% (1,562) of the fungal genes represented on the array, were regulated in at least one of the stages of the infection. The most dramatic shift in the fungal transcriptome occurred after 4 h, a time-point associated with the penetration of the nematode.