05/07/10

The organisms I work with

How I ended up working with filamentous fungi and specifically Fusarium sp.

The first time I “met” Fusarium was in 2003 during my time as a theme student with Associated Professor Morten Grell and Professor Henriette Giese. I was immediately fascinated by this strange organism and its beautiful red mycelium. The project, which I conducted in collaboration with Jens A. Andersson (now Ph.D.), centered on the ability of different fungicides to induce autophagy. During the late stages of the project we were granted access to the Fusarium graminearum PH-1 genome sequence prior to its annotation and publication, a fascinating experience for a couple of undergrads. We used the raw sequences to identify one of the key elements of the autophagy apparatus (ATG8/AUT7) by Blast analysis, and manually aligned and annotated the obtained sequences with the ortholog from yeast (none of us had any prior experience with bioinformatics). We were both fascinated by the idea that we were the very first to see this gene. This experience fuelled my interest in research and especially in Fusarium sp.  and still gives me a warm fuzzy feeling when I think of the moment when we first laid eyes on the final version of the gene.

One year later I started as a master student in Professor Henriette Gieses laboratory, on a project aimed on describing the genetic basis for regulation of the aurofusarin gene cluster in F. graminearum. At that time the group only had little experience with performing targeted gene replacements in Fusarium, the few mutants that had been constructed was generated by protoplast transformation and the "splitmarker technique" (bipartial technique). Henriette suggested that I should try Agrobacterium tumefaciens mediated transformation in my project. I constructed the vector for replacement of aurR1 by old fashion restriction enzyme digestion and ligase dependent cloning, a process that required four cloning steps and took me 6 months. The Agrobacterium tumefaciens mediated transformation of the construct into Fusarium graminearum, surprisingly worked the first time and yielded many transformants. The project eventually ended up with a nearly complete description of the genes in the aurofusarin gene cluster and a theory for the biosynthesis of aurofusarin.

Since the release of the F. graminearum genome sequence many scientists have shifted their research to include this fungus or to focus entirely on it. However, I do not feel that the area is getting crowded, as 50 % of the identified genes in F. graminearum encodes proteins that do not show sequence similarity to any previously characterized proteins. To this day  the majority of the published papers still focus on subjects that have been thoroughly covered in other fungi - I will therefore encourage all researchers to include some of the genes with unknown functions in their studies, when possible. 

Good things about working with Fusarium:

The genus Fusarium (teleomorph: Gibberella)

 History and phylogenetics of the genus

The ascomycete genus Fusarium comprised over 1000 species before 1935, when Wollenweber & Reinkings laid the foundation for modern Fusarium taxonomy (Wollenweber & Reinking 1935). By comparing morphology and the reproductive characteristics of isolates on six different media, they reduced the number to 65 species, divided into 16 sections, based on the presence/absence and shape of microconidia, macroconidia and chlamydospores. The same year Raillo (1935) showed that some of the used characters were unsuitable for classification due to large variation within single isolates. With this new information Bilai (1950) reduced the number of species from 65 to 26, but this taxonomy did not win a wide acceptance. Snyder et al. (1957) further reduced the total number to 9 species by introducing a third name (cultivar) to the species name, indicating which host organism the subspecies showed pathogenity towards. The two classification schemes presented by Wollenweber & Reinking and Snyder et al. has been combined by Booth (1971) and later by Nelson et al. (1983) ultimately resulting in a 30 species system.

There is still debate on which characters are suitable for classification and therefore also on the number of species. The NCBI taxonomical database lists 47 Fusarium species, of which 37 are placed within the Gibberella fujikuroi species complex (http://www.ncbi.nlm.nih.gov/). To date only few species has been analysed with respect to rDNA sequence, as has been the standard for other genera (Samuels et al. 2001), (Tan & Niessen 2003). Future application of rDNA-based classification might offer a satisfying classification scheme for the taxonomy of the Fusarium genus, but until then, differences in morphological character-states will still be the deciding factor for identification and classification of Fusarium sp..

F. graminearum, F. pseudograminearum and F. culmorum

Fusarium graminearum, F. pseudograminearum and F. culmorum all belongs to the section "Discolor" in the Booth taxonomy. This section is characterized by thick-walled, distinctly septated macroconidia, with the ability to produce chlamydospores, but not microconidia (Booth 1971).

F. culmorum with no known teleomorphic state, was first described by Smith in 1884 and initially named Fusisporium culmorum, but renamed in 1895 to its present name also by Smith. The homothallic F. graminearum (teleomorph: Gibberella zeae) was first described in 1822 by Fries E. as Sphearia zeae, renamed in 1838 by Schwabe to its present name and linked to its teleomorphic state Gibberella zeae in 1936 by Petch (based on Booth 1971). In 1969, McKnight et al. showed that the F. graminearum species comprised two distinct pathogenic forms, one infecting maize and the other infecting cereals, for the following 33 years they were designated group 1 and 2, respectively. Upon closer examination by Aoki & O’Donnall the specie was split into to distinct species, group 2 remained F. graminearum (Gibberella zeae) while group 1 was renamed F. pseudograminearum (G. coronicola) (Aoki & O’Donnell 1999a). Aoki and O’Donnelll (1999b) also showed that F. pseudograminearum is heterothallic. F. graminearum is found in Europe, North and South America, Africa and Asia, while F. pseudograminearum currently only has been found in Asia and Australia. The relative narrow distribution of F. pseudograminearum is believed to be the result of its short history as a distinct specie, and that many scientists still classify them as F. graminearum.

Fusarium fujikuroi

Fusarium fujikuroi (teleomorph: Gibberella fujikuroi) is a plant pathogen that attacks rice, causing bakanae disease. The bakanae disease is characterized by elongation of the stem (hypertrophy) and seedling blight. The hypertrophy is due to the fungus production of gibberellic acid, a plant growth hormon. The thin elongated stems are typically not able to support the weight of the panicle, why they topple over, resulting in no production of edable grains (Kawaide 2006). The F. fujikuroi mycelium is red due to a production of bikaverin, a monomeric polyketide.

Fusarium proliferatum

Fusarium proliferatum (Gibberella fujikuroi mating population D) is also a cosmopolite plant pathogen that attacks many important crops, such as rice (Desjardins et al. 1997), maize (Logrieco et al. 1995), date palm (Abdalla et al. 2000), asparagus (Elmer 1990) and onions (Stankovic et al. 2007). F. proliferatum has been reported to produce a broad range of mycotoxins, Fusaric acid (bacon et al. 1995), beaucericin (Logrieco et al. 1995), moniliformin (Marasas et al. 1984) and fumonisin B1 (Leslie et al. 1996).

Biology of Fusarium

For many of the Fusarium sp. sexual reproduction has not been observed and the genus are therefore classified as Deutoromycetes (fungi imperfecti). The fusaria species which has been linked to their respective teleomorphic stages are placed within the Ascomycetes.  The lack of a teleomorphic stage can either reflect true reduction and loss of this stage (as seen in Penicillium sp.) or can be a result of inadequate information. Assignment of species to the Deutoromycete group is solely based on the absence of the sexual/teleomorphic stage (fungi perfecti) and the group does therefore not represent a true evolutionary group (Petersen 1998).

Members of the Deutoromycetes typically show parasexuality as a mean of genomic recombination. The parasexual cycle is dependent on anastomosis (fusion) of compatible haploid mycelia, resulting in the formation of heterokaryotic mycelia (Two types of nucleus in one cytoplasma)  (Glass et al. 2003). The nuclei may in time fuse, giving rise to a diploid nucleus. This opens for the possibility of mitotic recombination between homologous chromosomes, and interchange of genetic material. The mycelium eventually becomes haploid again by random chromosome loss during successive mitotic divisions (Griffiths et al. 1999). This random chromosome loss also contribute to the formation of new genotypes (Sidhu 2002). 

Dispersal and survival of the anamorphic state are ensured through vegetative (asexual) reproduction by formation of mitospores (known in Ascomycetes as conidia). Fusarium species features three types of conidia: microconidia, macroconidia and chlamydospores.  The micro- and macroconidias are formed externally on hypha-like conidiophores, which categorizes Fusarium as a Hyphomycete. Chlamydospores are formed from normal hypha which undergoes increased growth and thickening of their cell wall (Deacon 1997).

Fusarium infection of cereals and the effects on humans 

The Fusarium genus contains some of the most important toxigenic plant pathogenic fungi. Their infection of important crops such as wheat, barley, oats, rice and maize pose a serious problem as infection leads to yield loss through lowered growth rate, reduction of grain size (Fusarium Head Blight/ tombstone/ scab (http://www.plantmanagementnetwork.org/pub/php/research/2003/fhb) and weakening of the straw (Foot rot) (Samson et al 2000), (Munkvold et al. 1998). In the US alone Fusarium outbreaks in the 1990's resulted in losses in the region of $3 billion (McMullen et al., 1997; Windels, 2000).

Bushnell et al. (2003) have described the infection mechanism of F. graminearum on wheat. Briefly summarized; The spores land on spikes and colonize the exterior of the plant. During anthesis the mycelium grow into the flowers and infect through weak points, such as stomata and anthers. Following the initial colonization, the mycelium easily spreads to the epidermis and parenchyma of the flower, stigmas and anthers. In some cases, the mycelium also resides between the kernel epidermis cell wall and cuticle. This growth pattern is believed to serve as a mechanism for dispersal of the fungi (Goswamin & Kistler 2004). The mycelium’s penetration is thought to be aided by excretion of hydrolytic enzymes (cutinases, celluloses, amylases and pectinases) (Jenczionka et al. 2003).

Overvintering is achieved either in the chlamydospore state or as a saprophyte on plant debris left in the field. Schmidt & Feistritzer showed that F. culmorum can remain viable for up to 2 years buried at a depth of 50 cm (according to Booth 1971). This ability poses a substantial threat to the following year’s crop. Counter-measures for preventing reinfection, includes crop rotation and deep burial of the previous crop before seeding, with crop rotation as the most efficient of the two [http://www.planteinfo.dk/cp/graphics/Name.asp?Language=da&TaskID =4&NameID=236].

Favorable conditions for perithecia and macroconidia formation results in the dispersal of the formed ascospores (sexually formed spores) and macroconidia (asexualspores) to plants.  Ascospores and macroconidia are typically spread by wind, rain or insects (Parry et al. 1995).

Apart from field infections, F. culmorum is also known to course storage rot of sugar beet, potatoes and apples. Storage rot of cereals is typically a secondary effect of the primary field infection (Goswami & Kistler 2004).

Fusariosis

Many Fusarium species may under the right conditions turn pathogenic toward animals (invasive fusariosis). A successful invasion is dependant on depression of the victim’s immune system, as found in cancer, AIDS, bone marrow transplant patients or even in cases of a common cold (Beardall & Miller 1994). Invasion of the blood stream is quite common (in this aspect Fusarium sp. differs from other opportunistic fungal animal pathogens), but has little effect on healthy individuals. As a testimony to this a large portion of the general population has antibodies against Fusarium cell surface polysaccharides (Notermans et al. 1988). F. solani, F. oxysporum and F. moniliforme are the species most commonly causing fusariosis.
Fusarium sp. are generally soil born, but surveys has shown that Fusarium under certain conditions is more abundant in air samples than Aspergillus and that 17 % of throat samples from otherwise healthy people contain Fusarium sp.. Ocular infections by F. solani has also been observed especially in contact lens users, as 14 % of all soft contact lenses are infected with Fusarium sp. without being invasive. Fusarium infections are typically treated with Amphotericin B, natamycin and rifampicin (Nelson et al. 1994).
 

Genomics and Gene calls

Fusarium graminearum, isolate PH-1, was sequenced in 2003 as a collaboration between the Broad Institute (MIT) and the International Gibberella zeae Genomics Consortium (IGGR). The sequence is public available at the Broad Institute home page (called FG1) and it has later been annotated by the Munich Information Center for protein sequences (MIPS) and is available at Fusarium graminearum Genome DataBase (FGDB) (called FG2). The annotation resulted in approximately 14.000 gene models based on EST data and gene prediction software (Calhoun annotation system, FGENESH and manual annotation (GENEMARK, GENEFINDER, GENSCAN)), the genes are distributed on four chromosomes with a total size of 36 Mb (Broad). Approximately 50 % of the genome encode genes, giving an average gene density of 1 gene pr. 3100 bp (Güldener et al. 2006). Over 50 % of the called genes do not show similarity to any previously characterized genes/proteins, showing that there still is a long way to go for functional analysis.

The development of the F. graminearum GeneChip (Affymetix platform) allowed for a more direct annotation of the included genes based on hybridization results from individual probes, this has resulted in a third gene set (FG3), which is accessible through both FGDB and the Broad Institute (Güldener et al. 2006). Expression data obtained from the Affymetix GeneChip is public accessible through www.plexdb.org.

The genomes of Fusarium oxysporum and Fusarium verticillioides (Gibberella moniliformis)  were sequenced in 2005 and 2007, also by the Broad Institute, and are accessible through the "Fusarium Comparative Database". In 2007 Fusarium solani (Nectria haematococca) was sequenced by JGI (DOE Joint Genome Institute) and the gene calls are available through "JGI N. haematococca v2.0 Home".

The availability of four Fusarium  genomes now opens for the possibility of comparative genomics and more efficient gene calling. Currently the four genomes have been annotated independently, however when doing comparative analysis it quickly becomes apparent that the gene calls for the four genomes do not always match, as extensive synteny between the genome sequences are not always reflected in the gene calls. A project for a simultaneous gene calling for three of the species have been initiated and received funding - and we all currently awaits the release of the new gene sets.

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