Handbook Part 2 - International Mycological Association

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