Category Archives: ascomycota

Methylation to the max!

A new paper from the Zilberman lab at UC Berkeley shows the application of high throughput sequencing to the study of DNA methylation in eukaryotes.  They generate an huge data set of whole genome methylation patterns in several plants, animals, and five fungi including early diverging Zygomycete.

The work was performed using Bisulfite sequencing (Illumina) to capture methylated DNA, RNA-Seq of mRNA. The also performed some ChIP-Seq of H2A.Z on pufferfish to look at the nucleosome positioning in that species. For aligning the reads, they used BowTie to align the bisulfite sequences (though I’d be curious how a new aligner, BRAT, designed for Bisulfite seq reads would perform) to the genome.  They also sequenced mRNA via RNA-Seq to assay gene expression for some of the species.

They find several interesting patterns in animal and fungal genomes.  I’ll highlight one in the fungi. They find an unexpected pattern in U. reesii of reduced CGs in repeats, which shows signatures of a RIP-like process, are also methylated.  This finding is also consistent with observations in Coccidioides (Sharpton et al, Genome Res 2009) that showed depleted CGs pairs in repeats.  Since the phenomenon is also found in Coccidioides genomes this methylation of some repeats is likely not unique to U. reesii but may be important in recent evolution of the Onygenales fungi or the larger Eurotiales fungi.  There are several other interesting findings with the first such study that shows methylation data for Zygomycete fungi and a basidiomycete close to my heart, Coprinopsis.  It will be interesting is to dig deeper into this data and see how the patterns of methylation compare to other genomic features and the mechanisms regulating methylation process.

Zemach, A., McDaniel, I., Silva, P., & Zilberman, D. (2010). Genome-Wide Evolutionary Analysis of Eukaryotic DNA Methylation Science DOI: 10.1126/science.1186366

I’ll have the truffles and huitlacoche

Black TruffleA couple of papers should have captured your attention lately in the realm of fungal genomics.

One is the publication of the genome of the black truffle Tuber melanosporum. This appears as an advanced publication at Nature (OA by virtue of Nature’s agreement on genome papers) along with a NYT writeup and is a tasty exploration of the genome of an ascomycete ectomycorrhizal (ECM) fungus. There are several gems in there including the differences in transposable element content, content of gene families related to carbohydrate metabolism. This genome helps open the doorway for exploring the several independent origins of ECM in both ascomycete and basidiomycete fungi.

I’ll also point out there is some work on the analysis of mating type locus found in this genome has applied aspects suggesting that inoculation of roots with both mating types may increase truffle yields in truffle farms. Evidence for sexual reproduction is also discovered from this genome analysis based on the sexual cycle genes present and the structure of the MAT locus.  Much like what was revealed in the genome analysis of the previously ‘asexual’ species Aspergillus fumigatus (and later reconstitution of a sexual cycle), the Tuber genome has the potential for mating and is a heterothallic (outcrossing) fungus based on its mating type locus -just like many other filamentous Ascomycete species.

A second paper I encourage you take a look at (those with a Science subscription) is from Virginia Walbot’s lab on the formation of tumors by U. maydis in Maize. These tumors end up destroying the corn but can produce a delicious (to some) dish that is huitlacooche. The idea that the fungus is co-opting the host system by secreting proteins that acted in the same way as native proteins and that it has a tissue or organ specific repertoire was one that her lab has been pursuing. U. maydis can grow inside corn without detection and  the formation of tumors seems to be a manipulation of the plant as much as it is the pathogen directly taking resources from the plant.  It reminds me a bit of the production of secondary metabolites that can control plant growth like gibberellins produced by fungi.  This kind of manipulation and also ability to evade detection suggests a pretty specific set of controls that prevent the fungus from doing the wrong thing at the wrong time (to avoid detection). So they set out to see if there are a set of organ specific genes that the fungus uses during infection that would suggest a very host-specific strategy by this corn smut.

In this paper the authors evaluate the role of fungal genes specifically expressed in infection of different organs and also the role of secreted proteins in colonization of the organs.  In what is impressive and elegant work, the authors show through the use of microarrays and genetics that there is plant tissue specific gene expression of U. maydis – so infections in leaves express a different set of genes than those in seedlings.  Genetic and phenotypic evaluation of fungal strains with knockouts of sets of the predicted secreted proteins was able to confirm a role for specific secreted proteins that previously may have not had any discernible phenotype. They infect strains with knockouts of sets of genes that encode secreted proteins and compare the virulence when these strains infect individual organs of the maize host.  They showed there is significantly different virulence in the various tissues for a some of the mutants suggesting an organ-specific role for virulence of secreted proteins. They also go on to show that some of this organ specific infection requires organ-specific gene expression by evaluating maize mutants and the ability of the fungus to infect different organs.

Future work will hopefully followup to see what these secreted proteins are manipulating in the host and how they either enable virulence by protecting the pathogen, avoiding detection by turning of host responses, or co-opting host gene networks in some other way.

Martin F, Kohler A, Murat C, Balestrini R, Coutinho PM, Jaillon O, Montanini B, Morin E, Noel B, Percudani R, Porcel B, Rubini A, Amicucci A, Amselem J, Anthouard V, Arcioni S, Artiguenave F, Aury JM, Ballario P, Bolchi A, Brenna A, Brun A, Buée M, Cantarel B, Chevalier G, Couloux A, Da Silva C, Denoeud F, Duplessis S, Ghignone S, Hilselberger B, Iotti M, Marçais B, Mello A, Miranda M, Pacioni G, Quesneville H, Riccioni C, Ruotolo R, Splivallo R, Stocchi V, Tisserant E, Viscomi AR, Zambonelli A, Zampieri E, Henrissat B, Lebrun MH, Paolocci F, Bonfante P, Ottonello S, & Wincker P (2010). Périgord black truffle genome uncovers evolutionary origins and mechanisms of symbiosis. Nature PMID: 20348908

Skibbe DS, Doehlemann G, Fernandes J, & Walbot V (2010). Maize tumors caused by Ustilago maydis require organ-specific genes in host and pathogen. Science (New York, N.Y.), 328 (5974), 89-92 PMID: 20360107

Hey there fluffy

ResearchBlogging.orgI spy a picture of Neurospora growing on the cover of Genetics this month.  The cover highlights the results from the work of the lab of Luis Corrochano who works on  light regulation in a variety of systems like Neurospora and Phycomyces.  This work describes their work on the fluffy gene which regulates conidiation (production of conidia or asexual spores). They show that an important interplay between an inducer of light response, the White Collar Complex (WCC), and the FLD protein on fluffy.  The data from indicate hat FLD represses fluffy as a response to dark but that this repression is removed in response to light through the action of WCC.

Olmedo, M., Ruger-Herreros, C., & Corrochano, L. (2009). Regulation by Blue Light of the fluffy Gene Encoding a Major Regulator of Conidiation in Neurospora crassa Genetics, 184 (3), 651-658 DOI: 10.1534/genetics.109.109975

A cacophony of comparative genomics papers

A nice series of comparative genomics articles have been published in the last few weeks. The pace of genome sequencing has accelerated to the point that we have lots of sequencing projects coming from individual labs and small consortia not necessarily from genome centers. We are seeing a preview of what next (2nd) generation sequencing will enable and can start to imagine what happens when even cheaper 3rd generation sequencing technologies are applied. I’m behind in reviewing these papers for you, dear reader, but I hope you’ll click through and take a look at some of these papers if you are interested in the topics.

In the following set of papers we have some nice examples of comparative genomics of closely related species and among a clade of species. The papers mentioned below include our work on the human pathogens Coccidioides and Histoplasma (Sharpton et al) studied at several evolutionary distances, a study on Saccharomycetaceae (Souciet et al) clade of yeast species, and a comparison of two species of Candida (Jackson et al): the commensal and opportunistic fungal pathogen Candida albicans with a very closely related species Candida dubliensis.  There is also a nice comparison of strains of Saccharomyces cerevisiae looking at effects of domestication and examples of horizontal transfer.

There is also a report of de novo sequencing of a filamentous fungus using several approaches, traditional Sanger sequencing, 454, and Illumina/Solexa (DiGuistini et al).

Finally, a paper from a few months ago (Ma et al), gives a fantastic look at one of the early branches in the fungal tree – the Mucorales (formerly Zygomycota) – via the genome of Rhizopus oryzae.  This paper is a really excellent example of what we can learn about a group of species by contrasting genomic features in the early branches in the tree with the more well studied Ascomycete and Basidiomycete fungi.  More genome sequences will help us build on these findings and clarify if some of the observations are unique to the lineage or universal aspects of the earliest fungi.

I hope you enjoy!

Novo, M., Bigey, F., Beyne, E., Galeote, V., Gavory, F., Mallet, S., Cambon, B., Legras, J., Wincker, P., Casaregola, S., & Dequin, S. (2009). Eukaryote-to-eukaryote gene transfer events revealed by the genome sequence of the wine yeast Saccharomyces cerevisiae EC1118 Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.0904673106 (via J Heitman)

Jackson, A., Gamble, J., Yeomans, T., Moran, G., Saunders, D., Harris, D., Aslett, M., Barrell, J., Butler, G., Citiulo, F., Coleman, D., de Groot, P., Goodwin, T., Quail, M., McQuillan, J., Munro, C., Pain, A., Poulter, R., Rajandream, M., Renauld, H., Spiering, M., Tivey, A., Gow, N., Barrell, B., Sullivan, D., & Berriman, M. (2009). Comparative genomics of the fungal pathogens Candida dubliniensis and C. albicans Genome Research DOI: 10.1101/gr.097501.109

DiGuistini, S., Liao, N., Platt, D., Robertson, G., Seidel, M., Chan, S., Docking, T., Birol, I., Holt, R., Hirst, M., Mardis, E., Marra, M., Hamelin, R., Bohlmann, J., Breuil, C., & Jones, S. (2009). De novo genome sequence assembly of a filamentous fungus using Sanger, 454 and Illumina sequence data. Genome Biology, 10 (9) DOI: 10.1186/gb-2009-10-9-r94 (open access)

Sharpton, T., Stajich, J., Rounsley, S., Gardner, M., Wortman, J., Jordar, V., Maiti, R., Kodira, C., Neafsey, D., Zeng, Q., Hung, C., McMahan, C., Muszewska, A., Grynberg, M., Mandel, M., Kellner, E., Barker, B., Galgiani, J., Orbach, M., Kirkland, T., Cole, G., Henn, M., Birren, B., & Taylor, J. (2009). Comparative genomic analyses of the human fungal pathogens Coccidioides and their relatives Genome Research DOI: 10.1101/gr.087551.108 (open access)

Souciet, J., Dujon, B., Gaillardin, C., Johnston, M., Baret, P., Cliften, P., Sherman, D., Weissenbach, J., Westhof, E., Wincker, P., Jubin, C., Poulain, J., Barbe, V., Segurens, B., Artiguenave, F., Anthouard, V., Vacherie, B., Val, M., Fulton, R., Minx, P., Wilson, R., Durrens, P., Jean, G., Marck, C., Martin, T., Nikolski, M., Rolland, T., Seret, M., Casaregola, S., Despons, L., Fairhead, C., Fischer, G., Lafontaine, I., Leh, V., Lemaire, M., de Montigny, J., Neuveglise, C., Thierry, A., Blanc-Lenfle, I., Bleykasten, C., Diffels, J., Fritsch, E., Frangeul, L., Goeffon, A., Jauniaux, N., Kachouri-Lafond, R., Payen, C., Potier, S., Pribylova, L., Ozanne, C., Richard, G., Sacerdot, C., Straub, M., & Talla, E. (2009). Comparative genomics of protoploid Saccharomycetaceae Genome Research DOI: 10.1101/gr.091546.109 (open access)

Ma, L., Ibrahim, A., Skory, C., Grabherr, M., Burger, G., Butler, M., Elias, M., Idnurm, A., Lang, B., Sone, T., Abe, A., Calvo, S., Corrochano, L., Engels, R., Fu, J., Hansberg, W., Kim, J., Kodira, C., Koehrsen, M., Liu, B., Miranda-Saavedra, D., O’Leary, S., Ortiz-Castellanos, L., Poulter, R., Rodriguez-Romero, J., Ruiz-Herrera, J., Shen, Y., Zeng, Q., Galagan, J., Birren, B., Cuomo, C., & Wickes, B. (2009). Genomic Analysis of the Basal Lineage Fungus Rhizopus oryzae Reveals a Whole-Genome Duplication PLoS Genetics, 5 (7) DOI: 10.1371/journal.pgen.1000549 (open access)

Sequencing wine spoilage yeast

There is an article in Wine Spectator (Seen on the JGI feed) on sequencing the wine spoilage yeast bruxellensis (correct name is now Dekkera bruxellensis) which adds the not-so-excellent taste of “sweaty horse” to wines.  There is already some survey sequencing done by Ken Wolfe and Jurge Piskur’s groups so a full genome sequencing project will help work out how this yeast is able to out compete Saccharomyces and cause dramatic wine spoilage.  This is also relevant on the bio-fuel side since this yeast can also taint an ethanol bio-reactor.  It is an interesting ecology inside the wine bottle and this competition for resources can lead to bad tasting wine. The competition presumably originated in some form in the rotting fruit where these yeasts compete for space and use different approaches in their niche including the fermentation process which produces the revered ethanol by-product and helps establish a chemical-warfare driven landgrab.  The ethanol also helps prevent and of course this has implications for the Drosophila (Sophophora) flies that land there and eat yeast. They needed a good way to overcome the ethanol like the well studied Adh gene.

For your reading pleasure

Too much on my plate as of late, so I’m woefully behind on posting much on interesting papers or news.  Here’s a short list of links and papers that are worth a look though.

  • “Evolution of pathogenicity and sexual reproduction in eight Candida genomes” published (Nature)
  • NYT Science article sort of summarizing the good, bad, and ugly of fungi and human interactions
  • Attempts to save amphibians from chytridiomycosis “Riders of a Modern-Day Ark” (PLoS Biology)
  • Looks like Scott Baker with the JGI are in the process of resequencing several classical mutant strains of Phycomyces, Neurospora and Cochliobolus, Cryphonectria for sequence-based mapping of mutants (i.e. here and here and here).

How do I name thee?
In a letter to the editor to the journal Nature, regarding the recently discovered/induced sexual stage in Aspergillus fumigatus, David Hawksworth argues that using the separate names for sexual (teleomorph) and asexual (anamorph) stages is confusing and unnecessary in this context.  The name Neosartorya fumigata is given to the sexual stage which was produced from two individuals which were both A. fumigatus. The letter writer makes the point that referring to a new name for the sexual stage when we already know what its anamorph is seems superfluous and overly confusing. He gives the analogy of Aspergillus nidulans where its teleomorph Emericella nidulans is “largely ignored”.

The double names for something which is the same species (i.e. has the same genomic sequence) is certainly a confusing aspect of mycology. It stems from the morphological description of species and that before DNA or molecular approaches to identification it was difficult to connect the anamorph and teleomorph stages unless you could induce the entire lifecycle in the laboratory. I think that the same name for homologous structures from different phyla is also equally confusing, but necessary aspect of how things are currently named and classified.

What researchers should described the sample/individual they are using for experiments in their manuscripts is important to avoid confusion and for readers so I think Prof Hawksworth makes an important point especially when discussing something where the anamorphs and teleomorphs are unified. Certainly an agreed upon protocol here would be quite helpful of what to preferably use when the stages have been connected.

Hawksworth, D. (2009). Separate name for fungus’s sexual stage may cause confusion Nature, 458 (7234), 29-29 DOI: 10.1038/458029c

Aspergillus has a posse


Shepard Fairley has gotten alot of notice lately for his Obama art that has been replicated pretty much everywhere. I mocked up a homage to his earlier street art — here we’ll discuss the growing Aspergillus genome posse.

But the work from mainly the JCVI, Broad Institute, JGI, NITE, and Sanger centre has generated an excellent collection of genome sequences for the Eurotiales clade (feel free to get a login for the wiki and add other that are missing).  The Aspergillus community now has a AGD – Aspergillus Genome Database project that includes a curator of genome annotation (they are hiring) and presumably literature in the SGD and CGD model of curation.

I think a lot of other projects have a Posse too (or maybe just a loosely organized band) in terms of a community of people working on related species and willing to work together to coordinate.  As these sort of “clade” databases start to develop we will have better clusters of information that can be mapped among multiple species.

Eventually I hope this will spur efforts for more coordinated genome databases for comparative genomic and transfer of known gene and functional information between experimental systems.  The efforts really require coordination or centralization of the data so that gene models can be updated as well as orthologs and phylogenomic inference of function.

Yeast population genomics
I have cheered the Sanger-Wellcome SGRP group work to generate multiple Saccharomyces cerevisiae and S. paradoxus strain genome sequences.   The group had previously submitted a version of the manuscript to Nature precedings and it is now published in Nature AOP showing that submitting to a preprint server doesn’t necessarily hurt your manuscript getting published…  The research groups explored the impact of domestication (as was also recently done for the sake and soy sauce worker fungus, Aspergillus oryzae) on the Saccharomyces genome by comparing individuals from wild strains of S. paradoxus.

This paper addressed several challenges including methodology for light genome sequencing for population genomics. This data represents in a way, a pilot project on for genome resequencing projects and using draft genome sequencing with next generation sequencing tools. Of course with the pace of sequencing technology development, any project more than a couple months old will be using outdated technology it seems, but this work represents some important progress.  Tools like MAQ were also developed and tuned as part of the project.  In addition to the methods development it also provided a new look at evolutionary dynamics of a well-studied fungus.

Genome assembly
The authors apply several different quality controls and utilize a new tool called PALAS (Parallel ALignment and ASsembly)  to assemble all the strains at the same time using a graph-based approach that utilized the reference genome sequences for each species. This is different than a full-blown WGA approach like PCAP, Phusion or Arachne because this is deliberately low-coverage sequencing pass.  The authors are trying impute missing sequence via Ancestral Recombination Graphs as implemented in the Margarita system.   They also use MAQ to align sequence from Illumina/Solexa sequencing to these assemblies made by PALAS.

Since this project was on two species of SaccharomycesS. cerevisiae and S. paradoxus they needed good reference assemblies for each of these species. The previously availably S.paradoxus assembly wasn’t complete enough for this study so they did an addition 4.3 X coverage with sanger/ABI sequencing and 80X coverage with Illumina.

Population genomics and domestication

The sequencing data also provided a framework for population genetic investigations. Some simple findings showed that geographic isolates within each species were more genetically similar to each other.  The main geographic regions of samples for S.paradoxus data included the UK, American, and Far East samples, some of which had been analyzed in a very nice study on Chromosome III.  For the S. cerevisiae samples there were individuals from around Europe, at least 10 European wine strains, Malaysian, Sake brewing strains, West Africa, and North America. From these data it was possible to discover that there are several of strains with mosiac genomes meaning that pieces of the genome match best with the sake fermentation strains and other parts from the wine/European samples.

Efforts to detect the effects of natural selection that may be linked to domestication of these strains explored two different approaches. The McDonald-Kreitman test did not identify any loci under positive selection while Tajima’s D was negative in the S.cerevisiae global and wine strain populations indicating an excess of singleton polymorphisms – though they draw little conclusions from that.  The authors also observed a sharper decay of linkage disequilibrium in S.cerevisiae (half maximum of 3kb) than S.paradoxus (half maximum 9kb) suggesting that S.cerevisiae is recombining more, either due to increased opportunities or a great frequency of recombination events when it does.

In context of the paper title and the idea of exploring the effects of domestication on the genome, the authors observe that the standard paradigm that ‘domesticated’ species have lower diversity levels is simply not the case in these samples.  This isn’t to say there isn’t evidence of the selection for fermentation production from these strains based on the stress response conditions they were tested on, but that there is still ample evidence of maintaining diversity within the populations presumably through various amounts of outcrossing.

We are also interested in these results as we apply similar questions to population genomics of the human pathogenic fungus Coccidioides where 14 strains have been sequenced with sanger sequencing technology.  Hopefully some of these lessons will resonate in our analyses and also that this era of population genomics will see ever more extensive collections to address aspects of migration, phylogeography, and local adaptations within populations of fungi and other microbes.

Gianni Liti, David M. Carter, Alan M. Moses, Jonas Warringer, Leopold Parts, Stephen A. James, Robert P. Davey, Ian N. Roberts, Austin Burt, Vassiliki Koufopanou, Isheng J. Tsai, Casey M. Bergman, Douda Bensasson, Michael J. T. O’Kelly, Alexander van Oudenaarden, David B. H. Barton, Elizabeth Bailes, Alex N. Nguyen, Matthew Jones, Michael A. Quail, Ian Goodhead, Sarah Sims, Frances Smith, Anders Blomberg, Richard Durbin, Edward J. Louis (2009). Population genomics of domestic and wild yeasts Nature DOI: 10.1038/nature07743