Category Archives: functional

On the content of (petri plate) Media

ResearchBlogging.orgAn avid reader pointed out that I was not entirely thorough in describing that we don’t enough about the V8 agar media that is used to induce mating in Cryptococcus. In fact a great deal of work on mating in this fungus had focused on identifying what pathways are induced by V8 agar that induce mating.  It was shown that inositol stimulates mating through use of defined media containing inositol (Xue et al, 2007).  This paper interestingly explores plant-fungal interactions and Cryptococcus suggesting that mating may occur preferentially on plants in cases where inositol is abundant.

It is also worth noting that V8 media contains a high level of copper ions and it was also pointed out to me that Jef Edman’s lab showed that melanin mutants have mating defects, and both phenotypes are suppressed by copper. And more recently (Lin et al, PLOS Genetics 2006) found that alleles of the Mac1 copper regulated transcription factor are a QTL influencing hyphal growth and melanin production, and showed that copper can enhance hyphal growth.

So the role of copper and interplay with V8 agar media and how this induces mating is actually quite known.

C XUE, Y TADA, X DONG, J HEITMAN (2007). The Human Fungal Pathogen Cryptococcus Can Complete Its Sexual Cycle during a Pathogenic Association with Plants Cell Host & Microbe, 1 (4), 263-273 DOI: 10.1016/j.chom.2007.05.005

Sex in fungi: MAT locus cloned from a Zygomycete

On the cover of this week’s Nature is a picture of Phycomyces blakesleeanus Nature Coverhighlighting the discovery of the MAT locus in this Zygomycete fungus from Alex Idnurm and Joe Heitman and colleagues. While it was previously known that Zygomycetes (the Orange lineage represented by R. oryzae in the tree below) mate, the specific locus has until now, never been discovered. The authors in this study identified the MAT locus through a sequence search looking for HMG-box genes knowing that these are found the Mating Type locus in Basidiomycetes and Ascomycetes. They confirmed the identity through a through set of experiments that included PCR, sequencing and crosses of (+) and (-) strains of P. blakesleeanus, and Southern blots.

Continue reading Sex in fungi: MAT locus cloned from a Zygomycete

Yes, Ecology can improve Genomics

Blogging on Peer-Reviewed ResearchFew organisms are as well understood at the genetic level as Saccharomyces cerevisiae. Given that there are more yeast geneticists than yeast genes and exemplary resources for the community (largely a result of their size), this comes as no surprise. What is curious is the large number of yeast genes for which we’ve been unable to characterize. Of the ~6000 genes currently identified in the yeast genome, 1253 have no verified function (for the uninclined, this is roughly 21% of the yeast proteome). Egads! If we can’t figure this out in yeast, what hope do we have in non-model organisms?Lourdes Peña-Castillo and Timothy R. Hughes discuss this curious observation and its cause in their report in Genetics.

Continue reading Yes, Ecology can improve Genomics

Gene knockouts in Candida parapsilosis

Cparapsilosis from G.ButlerA recent paper “Targeted gene deletion in Candida parapsilosis demonstrates the role of secreted lipase in virulence”, from the Nosanchuk lab at Yeshiva University, shows the role of secreted lipases in virulence of this pathogen. C. parapsilosis is second only to the evolutionarily closely related commensal Candida albicans as worldwide cause of invasive candidiasis. This paper demonstrates a knockout system using selectable marker which confers resistance to the drug Nourseothricin. The authors sought to delete the adjacent and convergently-transcribed lipase genes CpLIP1 and CpLIP2 and characterize the phenotype of the lipase deficient mutants as blood-borne C. parapsilosis infections are in a lipid rich environment.

Through a series of experiments testing growth in rich media, media with olive-oil, and in infection models they showed that the importance of lipase activity. The knockout strain was unable to grow efficiently on YNB media+olive oil indicating that these two genes are the only ones capable of lipase activity. The murine infection experiments indicated that the knockout could be cleared in 4 days while the WT and reconstituted were cleared in 7. The authors acknowledge some limitations in the infection model in that it does not fully recapitulate an invasive candidiasis because mice were infected intravenously so the role of endothelial cell invasion was tested in vivo.

This is not the first paper on targeted gene knockouts in this fungus. A paper from earlier this summer, “Development of a gene knockout system in Candida parapsilosis reveals a conserved role for BCR1 in biofilm formation”, from Geraldine Butler’s group at University College group, who work on both evolutionary and pathogenesis questions in Candida species, developed a knockout system using the same drug marker. The Butler laboratory also showed that the C. parapsilosis MAT locus, part of the sexual reproduction machinery of fungi, has degraded, consistent with the observed asexuality of these species.

The improving genetic tools for targeted disruption of loci in additional species is permitting experiments that get at the heart of what makes some fungi pathogenic. With the genome sequence of many of the relatives of the pathogens we can systematically dissect what genetic differences have a role in virulence. It will be interesting to reconstruct whether the ancestor of many of these Candida spp always had the potential for virulence or if it co-evolved with its human or other mammalian commensal lifestyle.

That was a lot of work

I’ve never worked with Magnaporthe grisea, the fungus responsible for rice blast, one of the most devastating crop diseases, but I do know that its life cycle is complicated and that knocking out roughly 61% of the genes in the genome and evaluating the mutant phenotype to infer gene function is not trivial. In their recent letter to Nature, Jeon et al did what many of us have dreamed of doing in our fungus of interest: manipulate every gene to find those that contribute to a phenotype of interest.

In their study, the authors looked for pathogenecity genes. Interestingly, the defects in appressorium formation and condiation had the strongest correlation with defects pathogenicity, suggesting that these two developmental stages are crucial for virulence. Ultimately, the authors identify 203 loci involved in pathogenecity, the majority of which have no homologous hits in the sequence databases and have no clear enriched GO functions. Impressively, this constitutes the largest, unbiased list of pathogenecity genes identified for a single species (though so of us, I’m sure, may have a problem with the term “unbiased”).

If you’d like to play with their data, the authors have made it available in their ATMT Database.

Approaching 100% coverage for GO assignments in S.pombe

A paper by Martin Aslett and Val Wood indicate that the fission yeast community is approaching 100% coverage of a GO annotation for every gene in the S. pombe genome. Only Ashbya gossypii has a smaller genome in the fungi (see a recent paper on Ashbya annotation database) and doesn’t yet have complete GO coverage. This is quite remarkable and a great dataset for studies in S. pombe and all fungi.

S. pombe taken from Paul Young’s site

My quick predictions of genes a closely related species, S. japonicus, has more than twice as many genes as S. pombe (but be over-prediction by ab initio predictors). Taken in comparison to many other fungi, S. pombe represents a streamlined and reduced genome which probably occured indepdently from reduction in the Hemiascomycetes.

Making the Revolution Work for You

In a recent Microbiology Mini-Review, Meriel Jones catalogs both the potential benefits and problems that arise from fungal genome sequencing. Using the nine genomes (being) sequenced from the Aspergillus clade, Jones addresses several issues tied to a singular theme: if we are to unlock the potential that fungal genome sequencing holds, both academically and entrepreneurially, then a more robust infrastructure that enables comparative and functional annotation of genomes must be established.

Fortunately, like any good awareness advocate, Jones points us in the direction of e-Fungi, a UK based virtual project aimed at setting up such an infrastructure. Anyone can navigate this database to either compare the stored genomic information or evaluate any fungus of interest in the light of the e-Fungi genomic data. The data appears to be precomputed, similar to IMG from JGI, so there are inherent limitations on the data that one can obtain. However, tools such as these put important data in the hands of expert mycologists that can turn the information into something biologically meaningful.

As Jones points out, this is just the beginning. If fungal genomes are to live up to their promise, they must engage more than just experts at reading genomes.