Scazzocchio Fungal Biology and Biotechnology 2014, 1:7http://www.fungalbiolbiotech.com/content/1/1/7REVIEW Open AccessFungal biology in the post-genomic eraClaudio Scazzocchio1,2Abstract

In this review I give a personal perspective of how fungal biology has changed since I started my Ph. D. in 1963. At thattime we were working in the shadow of the birth of molecular biology as an autonomous and reductionistic discipline,embodied in Cricks central dogma. This first period was methodologically characterised by the fact that we knew whatgenes were, but we could not access them directly. This radically changed in the 70s-80s when gene cloning, reversegenetics and DNA sequencing become possible. The next generation sequencing techniques have produced a furtherqualitative revolutionary change. The ready access to genomes and transcriptomes of any microbial organism allowsold questions to be asked in a radically different way and new questions to be approached. I provide examples chosensomewhat arbitrarily to illustrate some of these changes, from applied aspects to fundamental problems such as theorigin of fungal specific genes, the evolutionary history of genes clusters and the realisation of the pervasiveness ofhorizontal transmission. Finally, I address how the ready availability of genomes and transcriptomes could change thestatus of model organisms.Looking back in aweWe enter the post-genomic era when it is simpler andcheaper to re-sequence a whole genome to identify a pointmutation rather than follow the classical path of geneticmapping and subsequent gene cloning and sequencing. Weare already there.I started working with fungi in the autumn of 1963,

which I realise with horror is already more than fifty yearsago. It was not my intention to work with a fungus, but towork on the control of gene expression in any appropriateorganism. I had discovered the operon model through im-probable circumstances that took me to the InternationalCongress of Biochemistry in Moscow in 1961, whereFranois Jacob gave one of the main lectures. What fasci-nated me and determined my subsequent scientific questwas the logical process by which a regulation circuit couldbe constructed through dominance and epistasis relation-ships; the formal correctness of the circuit being independ-ent of its material basis.Even more improbable circumstances landed me in

Cambridge, where John Pateman and David Cove were in-vestigating the regulation of nitrate assimilation in Asper-gillus nidulans, two of a handful of scientists workingCorrespondence: c.scazzocchio@imperial.ac.uk1Department of Microbiology, Imperial College, London SW7 2AZ, UK2Institut de Gntique et Microbiologie, CNRS UMR 8621, UniversitParis-Sud, Orsay 91405, France

2014 Scazzocchio; licensee BioMed CentralCommons Attribution License (http://creativecreproduction in any medium, provided the orDedication waiver (http://creativecommons.orunless otherwise stated.within the Jacob-Monod framework in a eukaryotic organ-ism. I have recently written about those early days [1,2].My fellow research student Andy Darlington and I

were given the task of investigating purine utilisationand its regulation. The link with the work of Pateman andCove was that two key enzymes of nitrate and purine as-similation share the molybdenum containing cofactor theyhad recently discovered [3]. In 1965 I published my firstfungal article, which was also my first article on purine as-similation in A. nidulans [4]. This year, I was a co-author ofan article also dealing with purine assimilation in this sameorganism [5]. Sticking for over fifty years to the same prob-lem and organism seems, by almost any standard, a case ofobsessional fidelity. I did work on many other subjects inthose fifty odd years, but almost without exception, I stuckwith A. nidulans as a model organism. For the 1965 article,we isolated, by random mutagenesis, mutations in theintracellular steps of the conversion of purines to ammo-nium, including those in the positively-acting transcriptionfactor. The techniques used in this and a few followingarticles were those of classical genetics and relatively un-sophisticated biochemistry, complemented occasionally byimmunology [4,6-8]. For the 2014 article we sequencedsome of the mutations described in 1965 and 1968, afteramplifying the cognate genes by PCR, we inactivated a fewgenes by homologous recombination, we constructed GFPfusions to investigate the cellular localisation of the en-zymes we first assayed in 196368 [4,6], we investigated theLtd. This is an Open Access article distributed under the terms of the Creativeommons.org/licenses/by/4.0), which permits unrestricted use, distribution, andiginal work is properly credited. The Creative Commons Public Domaing/publicdomain/zero/1.0/) applies to the data made available in this article,

mailto:c.scazzocchio@imperial.ac.ukhttp://creativecommons.org/licenses/by/4.0http://creativecommons.org/publicdomain/zero/1.0/

Scazzocchio Fungal Biology and Biotechnology 2014, 1:7 Page 2 of 18http://www.fungalbiolbiotech.com/content/1/1/7pathway in a whole order of fungi (the Eurotiales), we un-covered paralogues of unknown specificity for some of thepurine utilisation enzymes, we established that within theEurotiales the hydrolysis of allantoic acid is usually cata-lysed by the classical allantoicase but occasionally by a com-pletely different enzyme [9], whose cognate gene wasalmost certainly horizontally transferred from bacteria [5].I have indulged in this personal reminiscence to contrast

the technologies used in 196365 with those of 2014: Genecloning, PCR, fungal reverse genetics, DNA sequencing,GFP fusions allowing the study of protein localisationin vivo, the ready availability of structures for orthologuesof the enzymes we were studying, which allowed educatedguesses on paralogue specificities, and last but not least, thepublic availability of hundreds of fungal (and thousands ofbacterial) genomes which allow one to speculate on moreor less probable evolutionary scenarios.All these possibilities were undreamt off when I started

my Ph. D. The first time I heard about DNA being thegenetic material was in a series of lectures given in 1958 byHans Tuppy, a co-worker of Fred Sanger on the sequencingof the insulin molecule, who went on to sequence thepeptidic hormone oxytocin and later cytochrome C. Thisexpert on protein sequencing pondered whether we wouldunravel one day the genetic code by sequencing both pro-teins (which could be done at the time with lots of work)and DNA. He was very pessimistic about the latter.In 1963, the reduction of the classical gene to its mo-

lecular avatar was almost complete. This accomplish-ment could be called the first revolution in molecularbiology, or better still the scientific revolution that gavebirth to molecular biology. We could date this processfrom the first article pertaining to the one gene-one en-zyme concept [10] and the establishment of DNA as thedeterminant of capsular antigens in Diplococcus pneu-moni [11] to the establishment of co-linearity betweengenes and proteins [12,13], not forgetting the conver-gence of the genetic [14] and biochemical approaches tothe deciphering of the genetic code [15].A few details were missing. We were working within the

framework of what Crick called in 1959 the central dogma[16]. We knew that the genes we worked with were DNA;we knew they encoded proteins, we knew the code; how-ever we could not access or manipulate the genes directly.The techniques available were still those of classical genet-ics. The molecular reduction of the gene was in the concep-tual background, not in the operations we performed. Itwas a persistent ghost, not a helpful jinni. In 1968, GuntherStent published an article, That was the Molecular Biology,that was [17] which is related in more than one way to theconcept of the End of History. I will not discuss here thisarticle in detail, but in a nutshell Stent proclaimed the endof molecular biology. He stated that all we had to do was toiron out details, dot the Is, as they say. Little did he know.The second revolution in Molecular Biology startedabout 1973 and it is still with us. While the first revolu-tion borrowed concepts [18] and mainly techniquesfrom outside the field (ultracentrifugation, electrophor-esis, chromatography, X ray diffraction), this secondrevolution took root in developments within the field.Restriction enzymes, ligases, reverse transcriptases,DNA polymerases, allowed the jinni to escape from thejar, that is, to intervene directly on the structure of thegenetic material. The epistemological consequence ofthis second revolution was to deconstruct the iso-morphism between the formal gene and its molecularsubstratum, the DNA sequence. This is another story,which I hope to discuss in detail elsewhere. It also com-pleted the conceptual unification of the biological sci-ences initiated with the rediscovery of Mendels laws in1900. It had the unforeseen consequence of transform-ing research in Molecular Biology from a convivial,though intensive, labour-light discipline into an autis-tic, thought-light, labour intensive pursuit.This second revolution entered the fungal research

community with the establishment of transformationtechniques for Saccharomyces cerevisi [19,20]. Thisearly technical development is at the basis of the he-gemony of the S. cerevisi research community, whichcould, on its own, constitute an interesting chapter of thesociology of science. Transformation of three other modelorganisms Neurospora crassa [21], Schizosaccharomycespombe [22] and A. nidulans [23,24] followed.There is a paradox underlying what I have called the

hegemony of S. cerevisi. The success of S. cerevisi asa model is not based on its similarities with other eu-karyotes, but on its differences. One could even say,from an eukaryotist point of view, on its deficiencies.It has easily available replication origin sequences, whichas we readily learnt to our chagrin, do not function inother eukaryotes. It has an autonomous nuclear plasmid.It shows no heterologous recombination, easily allowinggene replacement procedures. Last but not least its strik-ingly tiny centromeres allow the engineering of single-copy stable plasmids. Workers with other organisms hadto struggle mightily to offset the eukaryotic perfection oftheir models (see for example [25-28].The third revolution started more quietly, almost un-

announced. A forerunner of what was to come was the de-termination of the sequence of the 5 of the lacZ mRNA,all 39 nt of it [29]. The first whole, massive sequencescome from Sangers lab; bacteriophage X174 (5375 nt,[30], human mitochondrial DNA (16569 bp, [31]. Ourmodest contribution to the not yet born science of genom-ics was the almost complete sequence of the A. nidulansmitochondrial DNA (app 34 kb [32]a.The early history of whole organism genome sequen-

cing, from Hmophilus influenz in 1995, Saccharomyces

Scazzocchio Fungal Biology and Biotechnology 2014, 1:7 Page 3 of 18http://www.fungalbiolbiotech.com/content/1/1/7cerevisi in 1996, Cnorhabditis elegans in 1998, Dros-ophila melanogaster in 1999, Arabidopsis thaliana in 2000to the public announcement of the human draft genomein 2000 (http://www.youtube.com/watch?v=slRyGLmt3qc)is too well known to be repeated here. For a time, thecompletion of every single genome led to public an-nouncements to the press, editorials in Science and/orNature, each genome was a scientific and mediatic event.No longer so. These genomes were sequenced by varia-tions of the Sanger di-deoxy method. It looked at the timeas though only the genome of a few model organismswould be obtained, and this in turn would reinforce theiruse as models. I remember a meeting in 1996 where weargued heatedly whether we should go for the genome se-quencing of A. nidulans or Neurospora crassa.What are called, next generation sequencing methods,

depart in different ways from the Sanger procedures.What is important here is that their implementation di-minished from about 2008 the cost and time scale ofwhole genome sequences by orders of magnitude [33]. AnNIH site shows a graph recording the cost per megabasefrom about 5292 $US in 2001 to about 5 cents in 2013, orusing a different parameter, the cost of sequencing a singlehuman genome, from slightly under 100 million $US in2001 to about 5000 $US in 2013 (http://www.genome.gov/sequencingcosts/).There are at the time of writing 384 complete fungal ge-

nomes at http://genome.jgi.doe.gov/fungi/fungi.info.html,increasing almost by the hour. The Saccharomyces data-base contains genomes of 28 different strains of S. cerevi-si. We are getting to the point that if you isolate a newstrain of a fungus, let alone a new species, the first thingyou do is to get its genome sequenced. Massive parallel se-quence techniques also led to the development of RNA-seq, by which we can, together with the genome, know thetranscriptome; and this in several growth conditions or de-velopmental stages (see for fungal examples [34,35].At the onset of the genomic revolution the selection of

the organisms to be sequenced was guided by their statusas model systems, the exception being that the humangenome was obtained before the mouse one, which wassurely a political rather than a scientific choice. Therefollowed, before the crucial date of 2008, organisms thatwere important as pathogens or because of their industrialapplications (eg. Candida albicans and Aspergillus fumiga-tus among the former Aspergillus niger and Phanerochtechrysosoporium among the latter).Among all the present and foreseeable consequences

of the second phase of the genomic revolution (from theinflexion point of 2008), there is one which I cannothelp mentioning. More and more genomes are becomingavailable not because they have behind them huge re-search communities or industrial or medical lobbies butbecause they represent crucial nodes in the tree of life.Thus we have available the genome of the sea squirtCiona intestinalis, the only extant member of the placo-zoa (Trichoplax adherens) of a coral, of a comb-jelly, ofa sponge, of the Coelacanth, of the Platypus. A specificprogramme, Origins of multicellularity is aimed atobtaining full genomes at the root of the opisthokonta(animals and fungi plus sister groups) with already availablegenomes of choanoflagellata, filasterea, icthyosporea, apu-sozoa, (http://www.broadinstitute.org/annotation/genome/multicellularity_project/MultiHome.html). Thus we canbuild phylogenies based not only on a few transcribedgene differences, but on whole concatenation of se-quences, genome organisation, synteny and intron-exonorganisation.It will be impossible to give a complete and systematic

account about how the post-genomic revolution is chan-ging and will change fungal biology: I simply try to givea few examples, which have caught my interest and im-agination, necessarily these choices will be somewhatsubjective and arbitrary.

Genome inspired biotechnology: enzymesFungi have been used for a long time as sources of extra-cellular (and in some cases intracellular) enzymes. Theavailability of whole genomes allows the search of enzymeswith enhanced properties or altered specificities. Obviousexamples are enzymes related to cellulose, chitin and lig-nin degradation. To identify enzymes with new, promisingspecificities, the availability of structures, or, as a secondbest, structural models, are of paramount importance. Therelative dearth of protein structures is a limiting factor.There are more than 100,000 protein structures publicallyavailable, as compared to 175 twenty years ago. However,the methodologies to obtain them, while improving stead-ily, with a clear upturn about 1993, have not undergone asimilar revolutionary change to that embodied by nextgeneration sequencing methods (http://www.protein-structures.com/Structure/Structure/proteinstructure-data-bases.html).To draw an example from our recent work, we have iden-

tified a uniquely fungal enzyme, xanthine -ketoglutaratedependent dioxygenase (XanA, [36,37]) Genes encoding thisenzyme are present as an alternative or in addition to theclassical M0CO (molybdenum cofactor-containing xanthinedehydrogenase, which is universally conserved). In the ge-nomes of Penicillia, but not of Aspergillus, we have identi-fied paralogues which almost certainly have a differentsubstrate specificity [5]. As dioxygenases are known tobreakdown aromatic compounds, including herbicides [38],a broad investigation of these paralogue specificities wouldbe of interest. The ascomycete Amorphotheca (Hormoconis,Cladosporium) resin has been isolated as a contaminantof jet fuel. It both degrades and it produces hydrocarbons.It obviously has some extraordinary metabolic capabilities

http://www.youtube.com/watch?v=slRyGLmt3qchttp://www.genome.gov/sequencingcosts/http://www.genome.gov/sequencingcosts/http://genome.jgi.doe.gov/fungi/fungi.info.htmlhttp://www.broadinstitute.org/annotation/genome/multicellularity_project/MultiHome.htmlhttp://www.broadinstitute.org/annotation/genome/multicellularity_project/MultiHome.htmlhttp://www.proteinstructures.com/Structure/Structure/proteinstructure-databases.htmlhttp://www.proteinstructures.com/Structure/Structure/proteinstructure-databases.htmlhttp://www.proteinstructures.com/Structure/Structure/proteinstructure-databases.html

Scazzocchio Fungal Biology and Biotechnology 2014, 1:7 Page 4 of 18http://www.fungalbiolbiotech.com/content/1/1/7[39]. A genomic search revealed four paralogues of XanA(as opposed to the standard one in most members of thePezizomycotina). One paralogue is the obvious orthologueof XanA. The other three paralogues are necessarily Fe++

oxygenases as the iron binding site is conserved, but thesubstrate binding residues are not (Figure 1, left panel).This may provide an interesting insight into the hydrocar-bon degrading enzymes of this organism. While the gen-ome has been available for some time, to date nogenomic-based research has been published for thisorganism.Figure 1 Divergence and horizontal transmission of XanA homologue(labelled Amore plus the accession number in the JGI database) with the cAmore_142784 is the orthologue of XanA, note strict conservation of all fuThe putative orthologue of Phytophthora parasitica (gi|568015616|gb|ETL89binding residues are, as expected, conserved in all paralogues. Alignment carrA maximum likelihood rooted tree of putative orthologues of XanA representAscomycetes, Saccharomycotina, Purple, Ascomycetes, TaphrinomycotinaOomycetes. Note the anomalous position of U. maydis. The conservationthe Pezizomycotina is a clear mark of horizontal transmission. R. minuta, R(Ustilaginomycotina); C. cinerea, Coprinopsis cinerea, C. neoformans, Crypto(Leotiomycetes); A. nidulans, Aspergillus nidulans, (Eurotiomycetes); C. gray(Sordariomycetes); C. berberidis, Curcubitaria berberidis, (Dothideomycetes); W.pombe, (Taphrinomycotina); P. blakesleeanus, Phycomyces blakesleeanus, M. circKluyveromyces lactis, Y. lipolytica, Yarrowia lipolytica (Saccharomycotina). We havin other cases we chose the closest homologue to XanA within the taxon. Aliserver/ , Curation with BMG1 [41], both with defaults parameters, tree generatratio test [43]. Circular tree redrawn with Figtree (http://tree.bio.ed.ac.uk/softwThe cytochrome P450 monoxygenase superfamily (CYP)members are involved in many different steps in both pri-mary and secondary metabolism including the biosynthesisof ergosterol. Lanosterol 14 -demethylase (CP51), a P450enzyme is the target of azole antifungals (see below). TheP450 superfamily has been surveyed in 47 completed fungalgenomes suggesting a complex pattern of gene duplicationand loss with where numbers of cognate genes varyingfrom one (Eremothecium cymbalari) to 153 (Aspergil-lus flavus), distributed among 15 phylogenetic clades[44]. While this work provides an insight into thes. Left panel: Alignment of the paralogues of Amorphotheca resinharacterised enzymes of A. nidulans and S. pombe [36,37,40].nctional residues only for this paralogue among A. resin sequences.793.1) also shows conservation of all functional residues. The Fe++

ied out with MAFT (G-INS-i) visualisation with Box-shade. Right panel:ing different fungal taxons. Green: Ascomycetes, Pezizmycotina, Blue:. Olive green: Mucoromycotina, Black: Basidiomycota. Red: P. parasitica,of crucial residues together with the position of P. parasitica withinhodotorula minuta, Pucciniomycotina; U. maydis, Ustilago maydis,coccus neoformans, (Agaricomycotina); A. resin, Amorphotheca resini, Cladonia grayi, (Lecaranomycetes); N. crassa, Neurospora crassa,mikol, Wilcoxina mikol (Pezizomycetes), S. pombe, Schizosaccharomycesinelloindes (Mucoromycotina), D. hansenii, Debaromyces hansenii, K. lactis,e included species where some experimental work was extant [36], andgnments carried out with MAFT (G-INS-i, ), http://mafft.cbrc.jp/alignment/ed with PhyML [42], digits in nodes are aLRTs (Approximate Likelihoodare/figtree/).

http://mafft.cbrc.jp/alignment/server/http://mafft.cbrc.jp/alignment/server/http://tree.bio.ed.ac.uk/software/figtree/

Scazzocchio Fungal Biology and Biotechnology 2014, 1:7 Page 5 of 18http://www.fungalbiolbiotech.com/content/1/1/7evolution of this superfamily in the fungi, it does notprovide many clues as to new substrate specificities. Anumber of structures for P450 proteins are available inthe RCSB PDB databank, and one can imagine dockingstudies, combined with high throughput methods inwhich the activity of enzymes expressed from constitu-tive promoters is assayed for libraries of substrates. Thisis quite feasible as throughput assays of P450 activitiesare available.

Genome inspired biotechnology: secondary metabolitesSecondary metabolites made by fungi range from theprovidential (-lactam antibiotics) to the fiendish (afla-toxin). Many secondary metabolites are non-ribosomalpeptides or polyketides, and moreover the genes involvedin their synthesis are clustered (see following sections).Thus it is relatively simple to recognise in genomes thoseclusters involved in their synthesis, as they usually includeone or more multimodular enzymes. There is no articleon fungal genomics that does not include an account ofhow many possible secondary metabolite clusters arepresent. A number of bioinformatic methods have beendevised to detect secondary metabolite gene clusters[45-47]. Just one illustration of these possibilities is theidentification of the gene cluster responsible for the syn-thesis of the first-line therapeutic agent pneumocandin inthe genome of Glarea lozoyensis [48].It is clear that the number of secondary metabolites that

a fungus can potentially produce is much higher that thoseproduced under laboratory conditions. Thus the availabil-ity of fungal genomes presents us with two challenges.Firstly, how do we activate the synthesis of a specific sec-ondary biosynthetic pathway? Secondly, once we have pro-duced a secondary metabolite, what is its biologicalactivity? The activity we may detect in the lab is not neces-sarily the one which the fungus uses for its own unknownaims. Lovastatin is made by Aspergillus terreus, which Iam sure does not care about the cholesterol levels of com-pulsive hamburger eaters, even if it may care about sterolsynthesis of its ecological competitors. Suffice to say thatin the Aspergilli, each of the early sequenced species car-ries in its genome 3040 putative secondary metabolitebiosynthetic gene clusters, and that there is not muchoverlap in the secondary predicted metabolomes in speciesof the same genus [49]. A similar situation is extant in theFusaria and Cochliobolus species: among the former,F. fujikuroi could potentially synthesise secondary metabo-lites belonging to 45 different families. Of these 1317clusters involve polyketide synthases, but only three arecommon to all the Fusaria analysed [50,51].Activation of silent clusters could be obtained through

overexpression of specific regulatory genes, which fre-quently can be identified because they are clustered withthe biosynthetic genes. Deletion or mutation of broaddomain regulators such as chromatin modifying proteins(erroneously called epigenetic methods) and/or modifica-tion of environmental conditions have also been used tode-repress the expression of secondary metabolite path-ways. Among the latter, specific mention must made ofco-cultivation methods, as pioneered by Brakhage and co-workers, in which a given fungus is co-cultured with otherorganisms with which it may interact in its (presumably)natural environment, that is to say obliging the fungus tocare, to use the simile spelled above [52]. The reader is re-ferred to the reviews of Brakhage and Schroeckh and Kimat al., [49,53] for a detailed breakdown of these methodsand for the identification of novel secondary metabolitesin the Aspergilli.While omics constitute a qualitative expansion in the

possibilities of secondary metabolite identification, nosimilar revolution has occurred in methods to screen thebiological action of an entirely novel natural product.While some screens such as antimicrobial activities, arestraightforward, and can be trusted to robots, others areless so. Antitumor activity screening even if more labori-ous is possible using cell lines in culture. An entirely newmetabolite may be an anti-depressant or a contraceptive,but we may never know, unless we use an adequatescreen. It is interesting how a PubMed search yields moreand more studies based on the use of plants and fungi infolk-medicine. In the absence of rational screen methods,this can be considered a reasonable, preliminary screen.

Genome inspired therapies- pathogen genomicsFungi are major plant pathogens. Some are strict special-ists, such as Ustilago maydis (maize) or Magnaporthaeoryz (rice), others such as Botrytis cinerea are muchmore eclectic in their choices. Fusarium species hostsrange from cucumbers to humans. Not surprisingly, theimportance of fungi as pathogens made them primarytargets for whole genome sequencing. The life cycle andinteraction with the host as some of these pathogensembody problems of basic biological importance andnot surprisingly, those pathogens which were amenableto direct and/or reverse genetic techniques had alreadybecome models organism in their own right in the pre-genomic era. It is not possible to describe here how theavailability of genomes and transcriptomes has changedthe study of plant infections by fungi. As in other as-pects of fungi biology, a shift from the specific to theglobal is taking place, in which it is possible to analysethe changes in gene activity of both the parasite and thehost (Cairns et al. for a review dealing with both plantand human pathogen transcriptomes [54]). I will justpinpoint some somewhat arbitrarily selected examples ofhow the omics revolution is changing our way ofstudying fungal pathogens. The population structure ofthe pathogen is addressed by whole genome sequencing

Scazzocchio Fungal Biology and Biotechnology 2014, 1:7 Page 6 of 18http://www.fungalbiolbiotech.com/content/1/1/7of different isolates, while RNAseq can be used to inves-tigate the gene expression patterns of both pathogenand host, aiming to understand the mechanism ofpathogenesis and the immune response of the host.While a number of fungi are specific animal and/or hu-

man pathogens, the main public health concern has beenthe rise in opportunist pathogen infection in immunode-pressed patients, the main culprits being Candida (mainlyC. albicans and C. glabrata) and Aspergillus species (mainlyA. fumigatus), but new species within and outside thesegenus appearing with increasing frequency. A recent reviewquotes a total of >2,000,000 life threatening fungal infec-tions/year, with Aspergillus, Candida, Cryptococcus andPneumocystis as the major worldwide opportunistic patho-gens and with mortalities reaching varying between 20 and90% [55].In the nineteen century, infection by A. fumigatus was

an exotic occupational disease, while it could be arguedthat at present the fungal opportunistic infections (withthe notable exception of AIDS-related infections), includ-ing invasive Aspergillosis are mainly iatrogenic diseases,brought about by the use of immunosuppression in trans-plant patients. These high mortality figures depend bothon diagnostic problems and on the inefficacy of antifun-gals, including the appearance of resistant strains. Of allthe first-line antimycotic drugs, only the echinocandinstarget a specific fungal metabolic step, 1,3 -glucan syn-thase. The hope is, that by identifying essential specificfungal genes, not present in the host, we should be able todesign specific inhibitors. As a first step, one could try toestablish a repertoire, within specific fungal genes, of thosethat are essential. Two different studies address this prob-lem for A. fumigatus, one by heterologous transposition[56], the other by creating conditional lethals with the useof a regulatable promoter [57]. We are presented herewith a similar problem to the one discussed above in rela-tion with secondary metabolite synthesis. Finding essentialgenes is not difficult; the strategies used are an expansionat the genomic level, with the increased sophisticationbrought about by new technologies, of the searches for re-cessive lethal mutations carried on since the 1940s inDrosophila melanogaster. Another question is, once a fun-gal specific essential gene has been identified, to design anefficient inhibitor for the cognate protein product. Thiscould be searched blindly using high-throughput tech-niques and/or rationally if we know something about thebiochemistry and physiology of the protein we are tryingto inhibit. As far as I know, no new potential antimycoticagent has yet been discovered through this strategy. A re-cent article addresses the possible differences detected inthe relevant genomes between the cation channels ofpathogenic fungi, and those of their hosts [58], but there isa long way from uncovering a primary sequence differenceto designing of a specific channel inhibitor.Candida albicans, the most frequent fungal pathogen,is a diploid. A haplo-insufficiency test has been devised,in which one allele is inactivated and heterozygous dele-tion mutants are screened for increased sensitivity tobatteries of compounds. In principle all genes in thegenome can be thus tested against any number of com-pounds [59]. Using a variation of this test, high through-put method and molecular modelling, a novel family ofnon-azole inhibitors of ergosterol synthesis was identi-fied [60]. I have seen no publications following up thesefindings in experimental animal models.Mucormycoses are relatively rare, but on the increase

among immunodepressed patients, as secondary infec-tions of severe wounds and also in patients treated foriron toxicity resulting from renal failure [61]. Thegenome of Rhizopus delemar shows an ancient wholegenome duplication, followed by expansion of specificgenes and the presence of four genes encoding sporecoat homologue proteins (CotH), specific of the Mucor-ales. CotH proteins are ligands of GRP78, a chaperonewhich also can be localised at the surface of endothelialcells, also explaining the cell specificity of infection bymembers of this order [62]. While the association ofCotH proteins with GRP78 was discovered by con-ventional co-precipitation methods, the knowledge thatthis association is limited to Mucorales, results directlyfrom the availability of numerous genomes of thatorder. A recent review speculates as to whether theGRP78/CotH interaction could be a therapeutic targetspecific for Mucormycoses, a promising post-genomicpossibility [61].Emergent fungal diseases not only concern immuno-

suppressed humans. In recent years, widespread epizoo-noses affecting wildlife have become pervasive. Thecauses of emergent zoonoses are not restricted to fungalpathogens, and whichever their immediate infectiouscause, a crucial problem is to understand how the re-cent emergencies are connected with human activitiesleading to changes in ecosystems. If the fungal humanopportunistic diseases are iatrogenic, the emerging fun-gal zoonoses are more generally anthropogenic, as en-vironmental and climatic changes have been blamed fortheir recent appearance. Among the fungal agents,Batrachochytrium dendrobatidis (Chytridiomycota) isdecimating frogs and toads while Pseudogymnoascus(Geomyces) destructans (Myxotrichace) affect bats(see Eskew and Todd for a parallel of these emergentdiseases [63]) and Nosema species (Microsporidia, seebelow) kills bees and has been blamed as a cause of col-ony collapse disorder (CCD), where worker bees sud-denly disappear from a beehive [64]. Whole genomesare available for these pathogens and for the Chytridio-mycota and microsporidia also for several other speciesof the cognate phyla.

Scazzocchio Fungal Biology and Biotechnology 2014, 1:7 Page 7 of 18http://www.fungalbiolbiotech.com/content/1/1/7Out of ~ 6000 extant species of amphibians 35% are men-aced, while about 159 may already be extinct (http://www.iucnredlist.org/initiatives/amphibians/analysis). While thecauses are surely complex, Chytridiomycosis is a majorcontributing factor. B. dendrobatidis was identified as a le-thal frog pathogen as recently as 1998. Since then it hasbeen reported world wide, affecting a wide variety of am-phibian hosts. A sudden emergence of a new disease, affect-ing a wide variety of species implies either a sudden changein pathogen virulence (such as the acquisition of new genesby horizontal transmission, see below) or environmentalfactors which upset a previous pathogen/host equilibrium[65]. As for other pathogen host/interactions next gener-ation genomics and transcriptomics have been used to in-vestigate both the nature of the pathogen and the responseof the host. A phylogeny, based on whole genome sequen-cing of 49 different samples of B. dendrobatidis shows thatdifferent lineages of the fungus long predated the emer-gence of the panzootic upbrake. One clade, the GlobalPanzootic Lineage, was seen as quite heterogeneous andemerging as long between 10,000 to 40,000 years ago [66].The data are consistent with a scenario in which there hasbeen no drastic change in the pathogen; but wide geo-graphical distribution after (or coincident with) the onset ofthe panzootic outbreak.

Fungal phylogeny and taxonomyThe idea that protein sequences could form the backboneof a new molecular phylogeny, is co-val with the closingof what I have called the first revolution in molecular biol-ogy. In 1965, long before DNA sequencing became a real-ity, Zuckerkandl and Pauling proposed the concept of amolecular phylogeny based on protein sequences [67].Molecular phylogeny was actually started even before theonset of DNA sequencing, by 5S and later 16S RNA fin-gerprinting, leading in 1976 to the three kingdoms of lifeproposal of Fox and Woese [68]. It took a few years beforethe sequencing of bacterial 16 S rRNA (and eventuallyeukaryotic 18 S) was established as a method of choice formolecular phylogeny. The transition from the one genemethod to the present emphasis on whole genome-basedphylogeny mirrors the transition from the second molecu-lar biology revolution to the present genomic revolution.Data derived from whole genome sequencing is now

been almost routinely used to address phylogeneticproblems from the kingdom to the species level, such asaddressing the unresolved problems of the position ofthe Glomeromycotina and whether the zygomycetesare indeed monophyletic or a polyphyletic group (see forexample Liu et al., 2009 [69]). I am aware of three stud-ies that have addressed the phylogeny of the fungal king-dom through whole genomic data. The older studycomprised 42 genomes and addressed mainly relation-ships among the ascomycetes, reflecting the availability ofwhole genomes at the time [70]. Later, an un-conventionalmethodology the compositional vector method was usedto establish a phylogeny comprising 82 different completedgenomes [71]. In the more recent study, 99 complete ge-nomes and ESTs from 109 species were used to constructphylogenies [72]. Figure 2, taken from the above referencesummarises the phylogenetic relationships within the fungiand of the fungi with their nearest sister phyla. Note theambiguous placement of the microsporidia (which arefungi, see below), in relation to the nuclearia (which aresurely not, but were not represented by whole genomes) isnot resolved in this comprehensive study. Since the sem-inal work of Ebersberger et al. [72], a number of new ge-nomes such as that of Rozella allomycis (which allowedplacing the microsporidia, see below) have become avail-able and more will in the near future. While this may notmake much of a change in clades that are well represented,it may influence or resolve the positioning of others whereonly one or two species were extant at the time of the ana-lysis of Ebersberger et al. [72].Encephalitozoon cuniculi, a human pathogen, is a

member of the microsporidia, which made it into thehistorical record as the second fungal species to be se-quenced [73]. The taxonomical relationships of themicrosporidia had been controversial for quite sometime. Members of this phylum are obligate intracellularparasites of all metazoan phyla and even of some pro-tists. In common with other widely different organisms(Giardia lamblia, Trichomonas vaginalis, Entamba his-tolytica) microsporidia lack mitochondria. While it wasspeculated that these very diverse organisms representeda basal, premitochondrial kingdom of eukaryotes,called Archeozoa, it is now clear that in every single caserecorded, the loss of mitochondria is secondary, and thatstructures akin to mitochondria are present (hydrogeno-somes, mitosomes) and some typically mitochondrialgenes reside in the nuclear genomes [74]. However, phy-logenies based on single genes (such as those encodingHSP70 and tubulins) casted doubt on this placement,suggesting a relation to fungi, which was finally sup-ported by the whole genome sequencing of E. cuniculi[73]. The placing of microsporidia as a basal group ofthe fungi involved two independent studies in which se-quences derived in one study from 6 and in another casefrom 9 genomes, were compared by a variety of globalmethods with multiple genomes of fungi from otherphyla [75,76]. In the light of these studies whether weplace microsporidia within fungi or outside fungi as asister group may be a matter of taste, where we definesomewhat arbitrarily what makes a fungus a fungus.However these quite robust phylogenies lead to anotherproblem: if the loss of mitochondria (and other typicaleukaryotic landmarks such a the Golgi, peroxisomes,70S rather than 80S ribosomes, 9 + 2 microtube

http://www.iucnredlist.org/initiatives/amphibians/analysishttp://www.iucnredlist.org/initiatives/amphibians/analysis

Figure 2 A view of fungal phylogeny. This figures is Sup Figure nine of Ref [72], obtained by whole genomic/ ESTs comparison (see text). Fordefinition of the different data sets, see original article. The original legend is reproduced below. The phylogenetic backbone of the fungi basedon 15 datasets. The numbers of species represented by each leaf are given in parenthesis for the data sets fungi_1 and fungi fungi_2,respectively. A *denotes those instances where either one or both species are absent from data set fungi_2 and are represented only in thesupertree based on fungi_2A. A - indicates that a taxon is entirely missing in a data set. Colors highlight major systematic groups of the fungi(Ascomycota: red; Basidiomycota: blue; Mucoromycotina: magenta; Glomeromycota: purple; Entomophthoromycotina: yellow; Blastocladiomycota:marine; Chytriodiomycota/Neocallimastigomycota: green). Given the tentativeness in our reconstruction of the basal fungal relationships we keepthe network structure for this part of the fungal backbone tree. Contractions of the dashed branches result in the topology that is suggested byour refined analysis of the early branching fungi with data set fungi_3.

Scazzocchio Fungal Biology and Biotechnology 2014, 1:7 Page 8 of 18http://www.fungalbiolbiotech.com/content/1/1/7structures) is secondary, there must necessarily be or-ganisms that are basal to both microsporidia and (other)fungi, unless by some unlucky event, these basal organ-isms are all extinct. A possible hint at the origin ofmicrosporidia is its phylogentic clustering at the base ofthe fungal tree of life, with the only sequenced species ofthe Cryptomycota, Rozella allomycis, an obligate parasiteof the water-mold Allomyces (Blastocladiomycota). Thisorganism is the only member of a clade known throughrRNA environmental sampling which can be grown inculture [77]. R. allomycis has mitochondria with atrimmed 12 kb mitochondrial genome. The publishedphylogeny is based on 200 genes, but it may be worthrepeating this phylogeny using as an out-group a mem-ber of the nuclearia (supposed to be the sister group offungi [69]) when a genome becomes available, and/orthe already available Fonticula alba, a slime mouldphylogenetically related to the nuclearia [78].

The origin of specific fungal genesA gene is fungal specific until new genomes reveal it ina non-fungal organism. In a preceding section, I have re-ferred to XanA, the xanthine -ketoglutarate dependentdioxygenase as a fungal novelty. In our original articlewe limited our searches to the then available ascomy-cetes and basidiomycetes [36]. The availability of ge-nomes throughout the fungal tree of life could allow usto pin-point the node where the postulated gene dupli-cation, which originated a xanA-like gene, occurred. Asearch on the species represented at the JGI revealed

Scazzocchio Fungal Biology and Biotechnology 2014, 1:7 Page 9 of 18http://www.fungalbiolbiotech.com/content/1/1/7orthologous proteins, besides in the ascomycetes and ba-sidiomycetes, in all the available genomes of the Mucoro-mycotina, but in no other fungal taxon. Two possibilitiescan account for this result, one is an ancient duplicationof another -ketoglutarate dependent dioxygenase encod-ing gene, the other being a horizontal transfer event froma non-fungal organism, both scenarios affecting a com-mon ancestor of the Dikarya and the Mucoromycotina. Asearch in the NCBI database does not reveal any bacterialpossible homologue, which could be at the origin of aXanA-like enzyme. This contrasts with another purinedegradation enzyme, an alternative to the classical allan-toicase, where horizontal transmission from bacteria tosome fungi is almost certain [5]. However, strict ortholo-gues of XanA, showing as much as 70% identity and withall crucial residues conserved are present in all sequencedstrains of Phytophthora parasitica, in P. infestans and P.soj (see Figure 1, left panel) A maximum likelihood treeplaces the sequences of Phyothphtora sp. (only one in-cluded, Figure 1, right panel) within the Pezizomycotinarather that as an out-group, which strongly suggest anhorizontal transmission from fungi to oomycetes ratherthan the other way around (see following section).Zn-DNA binding motifs belong to several different clas-

ses. One, the nuclear receptor class, is unique to meta-zoans, and it has not been found even in the closest sistergroups. Analogously the Cys6Zn2 (Zn binuclear clusters)are often quoted as exclusively fungal DNA binding pro-teins [79]. I investigated, using accessible databases,whether the Cys6Zn2 motif is present in all fungi. The re-sults are shown in Figure 3 top panel, where the numberof Cys6Zn2 containing proteins is shown for representa-tives of different fungal taxa. While no homologue ispresent in any of the microsporidia, just one is present inRozella allomycis, the only sequenced representative ofthe Cryptomycota. I searched then the genome of Fonti-cula alba, the nearest relative of the nuclearia, the sistergroup to fungi, where I found two large proteins con-taining typical Zn binuclear cluster motifs (Figure 3 bot-tom panel). A search in http://pfam.xfam.org/ for motifZn_clus (PF00172) lead to some surprises (some ofthese were reported previously [80]). Cys6Zn2 clustersare present in number of non-fungal organisms, somemay make some sense while others do not. In Capsas-pora owczarzaki, (Filasteria) an opisthokont, which be-longs to a sister group of both fungi and metazoa [81],there are 7 bona fide Zn-cluster proteins. They arepresent in the Dictyosteliidea, with one or two Cys6Zn2representatives/species. These findings are consistentwith a scenario in which proteins carrying this motifwere present in the base of the Unikonts (comprisingopisthokonts, and ambozoa, including Dictyosteliidea)and were lost in some taxa and expanded in others. Theexpansion of this protein family in fungi seemed to havehappened at the base of the Dikarya. However otheroccurrences are more difficult to account for, such as thepresence of one protein in two diatoms (Thalassiosirapseudonana and Phaeodactylum tricornutum, howeverthe sequence is non-canonical in the latter species) and ina brown alga (Ectocarpus siliculosus). A number ofproteins detected in Hordeum vulgare var. vulgare, and inno other plant should perhaps not worry us, they aretypical fungal proteins, some showing as much as89% identity with a protein of Exophiala aquamarina(Pezizomycotina) and are most likely due to fungal DNAcontamination. But such trivial explanation cannotaccount for the 24 canonical Cys6Zn2 proteins present inEctocarpus siliculosus or the 71 proteins carrying a Znbinuclear cluster recorded for Ngleria gruberi (Percolozoa,Heterolobosea), a fascinating organism which alternatesbetween a flagellate and amoebic form (http://genome.jgipsf.org/Naegr1/download/Naegr_differentiation.mov)and which is phylogenetically as far from fungi as anyother eukaryote could be. The Ngleria Cys6Zn2 pro-teins look quite different from their fungal counterparts,and present a variety of architectures. It looks like theorganism took up the Cys6Zn2 finger motif and used itfor its own ends. We would dearly like to know whatthese ends are. The expansion of Cys6Zn2 proteins inDikarya is most likely due to their recruitment to regulatea diversified primary and secondary metabolism, includ-ing the ability to utilise the most disparate substrates assole nitrogen and/or carbon sources. Ngleria gruberi,on the other hand is a predator who phagocytes bacteria,a style of life very different from that of saprophyticfungi.

New insights into fungal evolution: horizontaltransmissionThe appearance of a typical fungal protein in diatoms andits expansion in Ngleria gruberi could be accounted forif Cys6Zn2 proteins existed at the root of eukaryotes,while suffering episodes of loss and expansion. An alterna-tive scenario is that at one stage horizontal gene trans-mission has occurred between a fungus or a fungalancestor and more remote organisms such as diatomsand a Ngleria ancestor. There are a number of such pos-sible scenarios; the most parsimonious one would be theappearance of Cys6Zn2 proteins in an ur-unikont, followedby episodes of loss (including in the ancestor of metazoans)and expansion, with horizontal transfer to (some) diatomsand Ngleria species. Occams razor is not always an ap-propriate tool, thus any other scenario shuffling verticaland horizontal descent events can be envisaged.It is arguable that the most fascinating concept arising

from the availability of multiple genomes is the awarenessof horizontal transmission between phylogenetically dis-tant organisms as a motor of evolution. A new paradigm

http://pfam.xfam.org/http://genome.jgipsf.org/Naegr1/download/Naegr_differentiation.movhttp://genome.jgipsf.org/Naegr1/download/Naegr_differentiation.mov

Figure 3 Comparison of Cys6Zn2 in different organisms. Top panel: Number of Cys6Zn2 transcription factors in representative species ofdifferent fungal taxa, or in same cases in the only available species of the taxon. Search carried out in the JGI fungal database (http://genome.jgi-psf.org/programs/fungi/index.jsf) with PFAM motif PF00172. Bottom panel: Alignment of a number of Cys6Zn2 motifs. Motifs corresponding to three wellstudied proteins (GAL4, NirA, AlcR) which bind to different DNA sequences are included. In red representatives of non-fungal Zn cluster proteins. ForF. alba, the nearest sister species to the fungi available, the Zn clusters of both extant proteins are included. To the right of the sequence the totalnumber of proteins of the species comprising canonical Cys6Zn2 clusters are recorded. Proteins which do not comprise all the conserved cysteines arenot included in this count. Note that the homologue of T. pseudonana included has an extension in the third loop of similar nature to that of AlcR ofA. nidulans. See text for complete names of non-fungal species. Searches carried out in JGI, NCBI, http://pfam.xfam.org/, alignment carried out withMAFT (G-INS-i) visualisation with Box-shade.

Scazzocchio Fungal Biology and Biotechnology 2014, 1:7 Page 10 of 18http://www.fungalbiolbiotech.com/content/1/1/7is being born, one in which organisms are not only relatedby descent, but also by common ecologies. Perhaps weshould be looking at the Cys6Zn2 proteins of aquaticfungi, to find the nearest progenitors of the diatoms andNgleria proteins.In a simplified Darwinian view of the world, in which evo-

lution is variation followed by natural selection, horizontaltransmission is a drastic mechanism of variation, in which aready-made gene or group of genes is inserted into an aliengenome, creating a whole new series of possible interactionsat all levels, from the transcriptome to the metabolome tocommunity ecological interactions. Such an occurrence maybe at the root of the appearance of new pathogens [82].In the prehistory of the concept of horizontal trans-mission, Pealva and co-workers [83] (see Brakhageet al. [84] for a more recent account) proposed that abil-ity to produce -lactam antibiotics was transmitted twiceindependently from Streptomyces species to fungi. Sincethen, horizontal transmission of secondary metabolitegenes has become a household concept in fungal biology(see below).While some instances of transfer from bacteria to

fungi were discovered incidentally, a whole genome ap-proach revealed 713 transferred genes from bacteria tofungi, derived from a minimum of 235 individual events.These events affected mostly the Pezizomycotina, but all

http://genome.jgi-psf.org/programs/fungi/index.jsfhttp://genome.jgi-psf.org/programs/fungi/index.jsfhttp://pfam.xfam.org/

Scazzocchio Fungal Biology and Biotechnology 2014, 1:7 Page 11 of 18http://www.fungalbiolbiotech.com/content/1/1/7analysed groups were affected, including Saccharomyco-tina, Taphrinomycotina and Basidiomycota, and morebasal groups such as Microsporidia, Chytridiomycota andMucoromycotina. Particularly interesting is the fact thatmore than one transmission event affected similar genesor pathways, genes encoding an arsenate detoxificationenzyme were independently transferred to Yarrowialipolytica (Saccharomycotina) and to Rhizopus oryzae(Mucoromycotina), catalase and amino acid racemasegenes were multiply transferred to different phylogenetic-ally distant species [85].Reports of intra-kingdom transfer of the biosynthetic

clusters encoding secondary metabolism biosyntheticgenes are not yet a flood but rather a steady flow. Thesynthesis of fumosisin apparently travelled from a mem-ber of the Sordariomycetes to Aspergillus niger, [86] italso jumped about among the Fusaria [87]. Intra-fungaltravel of genes have been summarised by Richards[88,89]. The latter review confirmed 323 bacterial trans-fers to fungi and 9 events of intra-kingdom transfer, in-cluding genes involved in both primary and secondarymetabolism.The biosynthesis, considered as restricted to some As-

pergilli, of the related metabolites aflatoxin and sterig-matocystin is arguably the most thoroughly studiedsecondary metabolic process [90]. A gene cluster en-codes all enzymes involved in the synthesis together withthe specific transcription regulatory gene aflR (54 kb,23 genes in A. nidulans which produces sterigmatocys-tin, 67 kb, 26 genes in Aspergillus flavus which pro-duces aflatoxin). A whole genome survey, involving 94species has revealed the presence of an orthologous clus-ter in Podospora anserina, but not in other sequencedSordariomycetes The A. nidulans and P. anserina clus-ters show quite striking intra-cluster synteny [91].Dothistromin, another secondary metabolite chemically

related to aflatoxin and sterigmatocystin, shares with thelatter a common intermediate. Dothistromin is producedby the pine pathogen Dothistroma septosporum and a fewother Dothideomycetes. Results of Bradshaw et al. [92], to-gether with those of Slot and Rokas 2011 suggest a storyof horizontal transfer of the whole cluster from an ances-tor of A. nidulans to P. anserina and independently to anancestral member of the Dothideomycetes followed by ep-isodes of cluster fragmentation with recruitment of add-itional biosynthetic genes. Sterigmatocystin productionhas been detected in other widely diverse ascomycetes[93], thus other episodes of horizontal transmission of thecluster may have occurred. However, there is another pos-sible extreme scenario involving only vertical transmissionfrom an ascomycete ancestor followed by multiple lossand fragmentation. A number of arguments make this sce-nario highly improbable for the A. nidulans/P. anserinaorthologous cluster [91].Evolutionary stories as just stories (some would sayjust so stories), and a number of combinations of hori-zontal and vertical transfer episodes are possible, whichmay be become more or less improbable when a com-prehensive phylogeny of many diverse aflatoxin/sterig-matocystin/dothistromin biosynthesis occurrences isundertaken.There is no a priori reason why horizontal transfer

should be limited to bacterial-fungal and fungal-fungaltransfer. While the numbers of plant genomes availableincrease, so do the opportunities to investigate plant-fungal gene transfers. This is a matter of some interest,given the large number of fungal plant pathogens andsymbionts. An automated comparison of 6 plant, speciesand 46 fungal species resulted in only 9 strong candi-dates for gene transfer between plants and fungi ofwhich 5 were from fungi to plants and 4 from plants tofungi. Two of the fungal genes transferred to plants pos-sibly originated from prokaryotes. This study was neces-sarily limited by the genomes available at the time. Inparticular, only three genomes outside the Dikarya wereavailable. Only one ectomycorrhizal fungus (Laccaria bi-color) could be included. The genome of Glomus intrar-adices, an arbucular mychorrrizal fungus, was not yetavailable. Within these necessary limits the authors con-clude that plant/fungal transfer events are rare and an-cient [94]. One can look forward to a newer survey inwhich a number of basal fungal species now becomingavailable is included.Ernest Rutherford is famously supposed to have said, All

science is either physics or stamp collecting. Molecularbiology can be construed to represent a transition fromstamp collecting to physics. Genome gazing, however so-phisticated the bioinformatic methods we may use, suspi-ciously resembles a reversion to stamp collecting. We havecollected a number of instances of intra and inter-kingdomhorizontal gene transfer events. Perhaps we could say, thatthere is nothing wrong with stamp collecting in biology andthat all science starts with or goes through stamp-collectingphases.While we can speculate on the evolutionary importance

of horizontal gene transfer, we are in the dark regarding themechanism of such transfer in eukaryotes, more strikinglyso in organisms such as fungi which acquire nutrientsthrough extracellular rather than intracellular digestion.How does intact DNA get in? How and how often does itescape nucleases? How does it go through the nuclearmembrane? Evoking transposons only displaces the prob-lem, as to how a transposon carrying a given gene travelsfrom one organism to the other. Phylogenomics tells usthat horizontal transfer even if not rampant is much morethan a naturalist curiosity. It opens a whole new field of en-quiry pertaining to the mechanism(s) of inter-organismalDNA mobilisation.

Scazzocchio Fungal Biology and Biotechnology 2014, 1:7 Page 12 of 18http://www.fungalbiolbiotech.com/content/1/1/7New insights into fungal biology: gene clusteringI have worked for a good part of my scientific career ontwo primary metabolism gene clusters of A. nidulans,the nitrate assimilation gene cluster [2,95,96] and theproline assimilation gene cluster [97,98], while initiatingthe work on the alc gene cluster, then continued by BettyFelenbok and co-workers [99]. A situation diametricallyopposed to clustering is found for the purine utilisationpathway of A. nidulans where none of the 17 genes encod-ing enzymes or transporters of this pathway is clusteredwith any other [100]. I have always wondered why the ni-trate assimilation genes are clustered in A. nidulans anddispersed in N. crassa and the proline assimilation genesare completely clustered in A. nidulans and dispersed in S.cerevisi and why we see within the same organism clus-tering in some catabolic pathways and not in others. Ourability to interrogate a large number of genomes may begiving some insights into these old questions.In the previous section I have mentioned that secondary

metabolites genes are usually clustered. An attractive idea isthat these clustered genes share a common chromatinorganisation [101]. Chromatin proteins and chromatinmodifying proteins have an important role in secondarymetabolism gene expression [90,101-104], however evi-dence for a specific chromatin (or heterochromatin) spe-cific structure of secondary metabolism clusters is wanting.Many secondary metabolism gene-clusters are located insub-telomeric positions [105], but we really do not knowwhether they are subject to sub-telomeric heterochromaticsilencing of the type described for D. melanogaster or S.pombe [106]. The attractive simple model of facultative het-erochromatisation of secondary metabolism gene clustersduring vegetative growth, for which I am partly responsible,may well be an oversimplification.It has been proposed that clustering of secondary me-

tabolite genes is a result not of selective pressure arisingfrom the necessity of co-regulation, but rather that thewhole cluster behaves like a selfish DNA segment thatpersist through horizontal transmission [107], even if wehave no hint why some DNA segments may be moreprone to horizontal transmission than others. Necessarily,once a cluster is transferred, another level of selection act-ing on the phenotype of the whole organism will be oper-ating. But this second level of selection only cares aboutthe selective value of the metabolites resulting from thepathway and eventually about their toxicity (see below).To borrow a terminology from linguistics, when dis-

cussing gene clustering two types of explanations arepossible: diachronic (historical) explanations, concernedwith the origin of the cluster and synchronic (functional)explanations, concerned with its expression and regula-tion here and now. A paradigmatic example of a dia-chronic explanation is that of Wong and Wolfe in Birthof gene cluster by adaptive gene relocation [108].Clustering of six genes involved in allantoin utilisation isdemonstrated to be a relatively recent novelty appearingat one specific stage of the evolution of the genus Sac-charomyces. This novelty coincides with the ability togrow in anaerobic conditions and with the inability toutilise urate as a nitrogen source (a process that gener-ates reactive oxygen species), due to the concomitantloss of the genes encoding urate oxidase and the urate/xanthine transporter [108,109]. Not surprisingly threeother genes (orthologues of xanA, uaX and uaW of A.nidulans, [100]), also necessary for xanthine and urateutilisation, are lost together with the appearance of theallantoin utilisation cluster (my own unpublished obser-vations). An allantoin specific transporter gene, DAL4 ,integrated in the cluster, originated from a duplication ofthe uracil transporter gene FUR4, concomitantly withthe birth of the cluster [108].The clustering of three genes involved in nitrate assimi-

lation (encoding the transporter, nitrate reductase, and ni-trite reductase) in a number of fungi (including Eurotialesamong the Ascomycetes and at least some Basidiomy-cetes) but not in others, has been interpreted as a resulton horizontal transmission of the whole cluster from anOomycete to the ancestor of Dikarya or perhaps even earl-ier, as the genes (but not as a cluster) are present inMucoromycotina [110]. Episodes of de-clustering wouldhave repeatedly occurred among the Dikarya.The assimilation of nitrate has been studied in detail in

three ascomycetes, A. nidulans [2,95,96], N. crassa [111]and a member of the Saccharomycotina, Pichia angusta(Hansenula polymorpha). In the latter organism the genesof the nitrate utilisation pathway are completely clustered.This cluster comprises not only the three genes mentionedabove, but also two Cys6Zn2 transcription factors (Yna1and Yna2 [112]), which are different from the orthologouspathway-specific A. nidulans NirA and N. crassa NIT4transcription factors. The regulatory patterns of A. nidu-lans and N. crassa are very similar, not withstanding thefact that no clustering is extant in N. crassa. Figure 4 com-pares the clusters of A. nidulans and P. angusta. I can seeno obvious explanation for the assimilation of two noveltranscription factor genes into the cluster of Pichia angu-sta. While horizontal transmission is a suitable explan-ation for the ancestral presence of the cluster, there is noclear rationale either for de-clustering or for assimilationof new genes into the cluster. The cluster has been func-tionally characterised in another member of the Saccharo-mycotina, Arxula (Blastobotrys) adeninivorans, where itincludes two transporter genes but not the transcriptionfactor genes [113]. Arxula is a basal clade of the Saccharo-mycotina [114], which supports a secondary clustering ofthe regulatory genes occurring after the divergence ofArxula and Pichia. Complete clustering of the nitrate as-similation pathway is found in yet another nitrate utilising

Figure 4 Comparison of the nitrate assimilation gene cluster in A. nidulans and P. angusta. White, nitrate transporter, yellow nitratereductase, blue nitrite reductase, green transcription factors. In A. nidulans a second transporter gene (ntrB) and the nirA transcription factor geneare in the same chromosome (VIII) as the gene cluster but not genetically linked to it or to each other.

Scazzocchio Fungal Biology and Biotechnology 2014, 1:7 Page 13 of 18http://www.fungalbiolbiotech.com/content/1/1/7member of the Saccharomycotina, Kuraishia capsulata[115]. The cluster includes two regulatory genes, whichare however surprisingly different from Yna1 and Yan2,and this in spite of the phylogenetic proximity of the twospecies. A search in the genome of A. adeninivorans failedto find any possible orthologues of Yna1, Yna2, the tworegulatory genes from K. capsulata or NirA/NIT4. It willbe interesting to know which transcription factor(s) hasbeen recruited to regulate this pathway in A. adeninivorans.Rokas and co-workers suggested a different evolution-

ary rationale for clustering [116-118]. They propose thatclustering of the primary metabolism genes of fungi oc-curs when one of the products of the metabolism istoxic. This is the case for galactose utilisation, whereGalactose-1-phsophate is toxic, and clustering of threeenzymes of galactose metabolism has occurred inde-pendently twice within the Saccharomycotina, and oncein the basidiomycetes, with a probable horizontal trans-fer from a Candida species to Schizosaccharomyces [116].Tyrosine utilisation as nitrogen source involves the verytoxic intermediate fumarylacetoacetate, The production ofbetaine from choline also involves a toxic intermediate(betaine aldehyde). Clustering is seen in all these pathways.I could add that 1 pyrroline-5-carboxylate, the product ofproline oxidation, is converted non-enzymatically to glu-tamic semialdehyde which is highly toxic [119], and nitrite,the product of nitrate reduction in the nitrate assimilationpathway is also toxic [120] (see above, the cognate genesin these pathways are clustered in some organisms but notin others).The selective advantage of clustering is embodied by the

fact that if the genes are clustered, the probability of loos-ing just the one gene encoding the enzyme responsible forthe detoxification of a toxic intermediate becomes auto-matically lower. If the cluster is lost as a whole, a catabolicpathway is lost but no toxic intermediate is accumulated.The selective advantage resides in this all or none situ-ation. However there are counter examples: uric acid, al-lantoin and allantoic acid are highly toxic in A. nidulans[121] and as mentioned above, no clustering is seen forthe genes of the purine degradation pathway in this organ-ism [100]. It can be argued that selective pressures differfrom one organism to another, from one pathway to theother. If we want to avoid a circular argument, we need in-dependent, ideally experimental evidence, pertaining tothese proposed different selective pressures.I think this discussion illustrates both the virtues and

limitation of the genome gazing approach: it can surelysuggest correlations, which may be supported or disprovedby additional examples. It should lead to evolutionary ex-periments, to check if under challenging conditions birthof a cluster or de-clustering occurs. Work based ongenomics seems to be excellent to generate diachronic hy-potheses; we also need experiments to access the syn-chronic level of explanation.

Whither the model organism?Definition of model organism: The organism on which Iwork, with the exclusion of other organisms, principallythose used by others to address the same biological prob-lem as I do.The concept of the model organism implies a societal

positive feedback process, by which an organism is chosento investigate a given problem because of specific charac-teristics or accessibility to experimental manipulations;this generates a research community, whose coherenceand number strengthens the model status of the chosenorganism. Drosophila melanogaster was chosen by Morganas the organism to construct classical genetics, but bothBeadle and Ephrussi abandoned it (for Neurospora crassa

Scazzocchio Fungal Biology and Biotechnology 2014, 1:7 Page 14 of 18http://www.fungalbiolbiotech.com/content/1/1/7and S. cerevisi respectively) as unsatisfactory to identifythe primary product of the gene. The work of Beadle, asso-ciated with Tatum, lead to the one gene-one enzyme con-cept [10] and collaterally established N. crassa as a fungalmodel organism.The crowned king of fungal model organisms is with no

question S. cerevisi. I have hinted in the first section ofthis article at some of the reasons for this status. It wasthe first eukaryote where reverse genetics was possible,which in turn resulted in a research community numerousenough to afford to carry out the first eukaryotic wholegenome sequencing, followed by systematic gene inactiva-tion and GFP (green fluorescent protein) tagging, not for-getting the creation of a species specific data base, thusfurther reinforcing the model organism status.One of my favourite recent articles describes a self-

sustaining, albeit unstable mechanism of gene silencing inMucor circinelloides [122]. Whether M. circinelloides is amodel organism or not is possibly a matter of taste. Per-haps now it is becoming one. Inspired genome gazing,with or even without the assistance of sophisticated bio-informatics can lead to new discoveries, which then haveto be tested in the appropriate organisms. For example,comparing by hand, or better to say by eye, gene modelsof a biotin biosynthetic gene in different fungi my col-league Michel Flipphi realised that canonical splicing ofsome introns would lead to inactive, frame-shifted pro-teins. He then realised (again by eye) that there were innerintrons interrupting the donor sequence of the main in-tron. The experimental test for introns within introns(stwintrons, splicesosomal twin introns, a new concept)was carried out necessarily not in our life-long model A.nidulans, but in Fusarium verticilloides, Trichoderma ree-sei and Botrytis cinerea [123].It is clear that model organisms are here to stay. It

would be superfluous to attempt to replicate in otherAspergilli the wonderful work of Pealva and collabo-rators on the Golgi apparatus of A. nidulans [124].Nevertheless caution is required when extrapolatingfindings of one organism to the other, even within thesame genus as recent findings demonstrate [125]. Weneed to work with related organism, to understandmorphological differences such as why the conidia ofA. nidulans and A. fumigatus are uni-nucleate andthose of A. niger multinucleate, why the conidiophoresof A. nidulans are biseriate and those of A. fumigatusuniseriate. Not withstanding the advantages of A. nidu-lans as a model, if we want to understand why Asper-gillus versicolor can grow in the Dead Sea and thrive atpH 9.0 we have no alternative but to work directly withA. versicolor.The present post-genomic revolution is creating almost

limitless opportunities to initiate new work in a large var-iety of organisms. Search of databases, driven by aknowledge of biological and biochemical processes leadsalmost fatally to organisms far beyond the restricted wordof models. Indeed we have more genomes than peopleable to work with the cognate organisms.I have mentioned in the first section of this review

how the second revolution (gene cloning, reverse gen-etics, limited sequencing) transformed the practice ofour science. Post-genomic biology will necessarily leadto sociological changes. On the one hand we start shar-ing with experimental physicists the experience of pub-lishing articles with over one hundred authors. On theother hand, the existence of extensive public databasesallows individual isolated scientists to ask specific ques-tions, if they have a clear biological problem in mind. Inthe course of writing this review some new questionsarose and all I had to do was to address the appropriatedatabases and online calculation facilities. We could saythat the lonely scientist pottering away on his/her com-puter has a parasite/host relationship with publicallyfunded databases. The NIH and the USA Department ofEnergy fund the databases I mostly use, thus I am beingsubsidised unwittingly by the North American taxpayer.While I may consider that this is a just return for theEuropean and Latin American brain drain (these are theones that concern me directly), it also means that politicalchanges or budgetary considerations can jeopardise theexistence of major scientific facilities. This is one problemwe have to face and solve in this post-genomic era.The post-genomic era could lead to scientific hyper-

autism or to new convivial networks. I am lucky to have anumber of former students, post-docs and new colleagueswith whom to share and discuss my genomic gazing and Iwish to thank all of them warmly for the fun we had inthese few years since my official retirement.

Concluding remarksThis already lengthy review is necessarily incomplete.There are a number of aspects of fungal biology, whichsurely can be or have already been illuminated by omicswhich I did not address. I try to indicate them below. Inall fungal genomes, there are orphan genes. Those comein two flavours, those for which we have no inkling oftheir function, and those who belong to characterised genefamilies, but where we ignore the specific function. Of the300 odd Cys6Zn2 proteins of A. nidulans we only knowthe function of a few. Alternative splicing is present in thefungi, and it has already been investigated genome andtranscriptome-wise [126-128]. Metagenomics is usually apursuit of bacteriologists, but it is starting to be extendedto fungi [129]. Related to this question is whether the con-cept of the pangenome is relevant to fungi [130]. Theavailability of complete genomes has revealed mating typegenes in many fungi supposedly asexual, leading in somespecific cases to experimental verification of sexuality

Scazzocchio Fungal Biology and Biotechnology 2014, 1:7 Page 15 of 18http://www.fungalbiolbiotech.com/content/1/1/7[131-133]. Transposons of different classes are present inall fungi but their distribution is patchy, and this patchi-ness in striking for helitrons ([134] and my unpublishedobservations). An investigation of their distribution maybe quite relevant to the mechanism of horizontal trans-mission. Prions and a number of epigenetic phenomena,which are formally prion-like, have been investigated in P.anserina and S. cerevisi [122,135,136]. May genomes andtranscriptomes help reveal the existence of new prions[137] or prion-like phenomena? Perhaps the theme I re-gret the most not having included is the use of omics toinvestigate community relations between organisms.These go from inter-organism signalling, even among or-ganisms of different kingdoms [52], to the investigation ofthe dynamics of mycorrhiz [138] and to the challengingsymbiosis of fungi and algae on lichens [139]. The latter isnow opened with the completion of a number of genomes,in one particular case of the genomes of both partners.

EndnoteaPart of this effort was carried out in Hans Kuntzels la-

boratory in Gottingen. I take the opportunity to stress thecontribution of Richard Waring and Terry Brown, and theforwardlooking leadership of R. Wayne Davies to this ef-fort. Very few remember today that it was Wayne whofirst planned the complete sequencing of Chromosome IIIof S. cerevisi.

Competing interestThe author declare that he has no competing interests.

AcknowledgementsI thank George Diallinas for critical reading of the manuscript, OscarZaragoza for valuable suggestions for the section dealing with fungalpathogens, Ccile Neuvglise for her insights on nitrate utilising yeasts andhelp in accessing the Arxula adeninovorans genome and Antonis Rokas forilluminating discussions concerning gene clustering. I thank IngoEbersberger for allowing the use of Figure 2, from Ref [72].

Received: 3 September 2014 Accepted: 15 September 2014

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