Handbook Part 2 - International Mycological Association
Handbook Part 2 - International Mycological Association
Handbook Part 2 - International Mycological Association
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PS4-417-0435<br />
Characterization of Cdk-related kinases from Blastocladiella emersonii during its life cycle<br />
K. F. Ribichich, S. L. Gomes<br />
Departamento de Bioquímica - Instituto de Química - Universidade de São Paulo, São Paulo - SP, Brazil<br />
Cyclin-dependent kinases are key enzymes responsible for the control of the cell cycle progression. While in fungi, only<br />
one Cdk, named Cdk1, has been described as directly involved in the control of cell cycle transitions, in animals at<br />
least two Cdks have been indicated and members from two distinct groups could be involved in cell cycle control in<br />
plants. One characteristic of chytridiomycetes is their growth as a coenocyte without citokynesis until their entry in the<br />
sporulation phase, or briefly thereafter. Although cell cycle control mechanisms seem to be conserved in eukaryotes,<br />
mechanisms and control points of the transition from a nucleated cell to a multinucleated cell that is finally divided<br />
into cellular compartments remain obscure. As a first step towards understanding the role of the cell cycle in the life<br />
cycle of the chytridiomycete B. emersonii, we report the identification of two putative Cdk or Cdk-related kinase (Crk)<br />
cDNAs and their characterization by sequence analysis and mRNA levels. The deduced protein sequences from the<br />
isolated cDNAs present the conserved motifs of Cdks, but the characteristic PSTAIRE motif in their cyclin-binding<br />
domain is divergent. Both Crk genes showed pre-translational regulation, but with different mRNA profiles along the<br />
fungus life cycle. Post-translational regulation is a characteristic of Cdks, with one plant group described as<br />
transcriptionally regulated. Cdk associated activity was also investigated in immunoprecipitates obtained with<br />
polyclonal antiserum produced against one of the Crks and in co-precipitates with p13Suc1, a high-affinity Cdk<br />
regulatory subunit that associates with active enzyme complex. Both patterns of histone H1 phosphorylation showed<br />
kinase activity throughout the life cycle with a reduction at the end of sporulation, which accompanied the mRNA<br />
profile. Moreover, both activities were inhibited with purvalanol A, a selective and potent Cdk inhibitor. This is the first<br />
report of putative Cdks of a chytridiomycete, which appear to be different in sequence and expression from those<br />
described for other fungi.<br />
PS4-418-0449<br />
Genes involved in sclerotial differentiation in Sclerotinia sclerotiorum.<br />
R. J. Weld 1, C. C. Eady 2, H. J. Ridgway 1<br />
1 National Centre for Advanced Bio-Protection Technologies, Lincoln University, Canterbury, New Zealand, 2 New<br />
Zealand Institute for Crop and Food Research, Lincoln, Canterbury, New Zealand<br />
Sclerotinia sclerotiorum is a ubiquitous, necrotrophic, ascomycetous fungus that infects over 400 plant species<br />
including many economically important crop species. During its lifecycle this highly successful pathogen can form<br />
hardy resting structures called sclerotia that allow it to over-winter in the soil. The questions we are interested in<br />
answering are: What molecular pathways co-ordinate sclerotium formation? And are such mechanisms homologous<br />
to conidiogenesis regulation pathways in unrelated filamentous fungi? A starting point for this work has been, firstly, to<br />
identify genes differentially expressed during sclerotial initiation and secondly, to find Sclerotinia genes that are<br />
homologous to genes known to regulate conidiogenesis in other fungi. We are currently using expression knock-down<br />
to test the effect of these genes on sclerotial morphogenesis.<br />
AFLP differential display was used to compare genes expressed during sclerotium formation in a wild type strain with<br />
gene expression in a non-sclerotial UV mutant. Candidate genes homologous to known conidiation regulatory genes<br />
were identified from the Sclerotinia sclerotiorum genome sequence<br />
(http://www.broad.mit.edu/annotation/fungi/sclerotinia_sclerotiorum/). Northern blots were used for analysis of<br />
expression during sclerotial initiation. Agrobacterium-mediated transformation and RNAi are being used to specifically<br />
disrupt expression of the candidate genes.<br />
AFLP and Northern analysis identified several genes that are specifically expressed during sclerotial morphogenesis.<br />
These genes include genes putatively involved in sugar transport and secondary metabolism. One of the genes<br />
identified by AFLP (800AT) has sequence similarity to the fluffy (fl) gene which is the major regulator of conidiation in<br />
Neurospora crassa. 800AT amino acid sequence was 28% identical to fl (over 50% of the protein). There was 62%<br />
identity between the fl DNA binding domain and the corresponding domain on 800AT including residues important for<br />
DNA binding. We are currently using RNAi-induced silencing to examine the effect of loss of expression of the putative<br />
fl homologue on sclerotial morphogenesis.<br />
The development of a transformation system based on Agrobacterium tumefaciens has provided the ideal tool for<br />
creation of gene knock-outs and knock-downs in S. sclerotiorum. We have identified a candidate gene with<br />
sequence similarity to the N. crassa fluffy gene. This novel gene contains the DNA binding domain, basic region and<br />
middle homology region typical of a GAL4-type transcription factor and is differentially expressed during sclerotial<br />
formation. As some types of sclerotia are considered to be evolutionarily derived from conidiogenous tissue, we<br />
anticipate considerable homology between regulation of conidiogenesis and sclerotia formation at a molecular<br />
level.<br />
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