Comparative Transcriptomics of Fusarium graminearum and Magnaporthe oryzae Spore Germination Leading up To Infection.

For fungal plant pathogens, the germinating spore provides the first interaction with the host. Spore germlings move across the plant surface and use diverse penetration strategies for ingress into plant surfaces. Penetration strategies include pressurized melanized appressoria, which facilitate physically punching through the plant cuticle, and nonmelanized appressoria, which penetrate with the help of enzymes or cuticular damage to breach the plant surface. Two well-studied plant pathogens, Fusarium graminearum and Magnaporthe oryzae, are typical of these two modes of penetration. We applied comparative transcriptomics to Fusarium graminearum and Magnaporthe oryzae to characterize the genetic programming of the early host-pathogen interface. Four sequential stages of development following spore localization on the plant surface, from spore swelling to appressorium formation, were sampled for each species on culture medium and on barley sheaths, and transcriptomic analyses were performed. Gene expression in the prepenetration stages in both species and under both conditions was similar. In contrast, gene expression in the final stage was strongly influenced by the environment. Appressorium formation involved the greatest number of differentially expressed genes. Laser-dissection microscopy was used to perform detailed transcriptomics of initial infection points by F. graminearum. These analyses revealed new and important aspects of early fungal ingress in this species. Expression of the trichothecene genes involved in biosynthesis of deoxynivalenol by F. graminearum implies that toxisomes are not fully functional until after penetration and indicates that deoxynivalenol is not essential for penetration under our conditions. The use of comparative gene expression of divergent fungi promises to advance highly effective targets for antifungal strategies. IMPORTANCE Fusarium graminearum and Magnaporthe oryzae are two of the most important pathogens of cereal grains worldwide. Despite years of research, strong host resistance has not been identified for F. graminearum, so other methods of control are essential. The pathogen takes advantage of multiple entry points to infect the host, including breaches in the florets due to senescence of flower parts and penetration of the weakened trichome bases to breach the epidermis. In contrast, M. oryzae directly punctures leaves that it infects, and resistant cultivars have been characterized. The threat of either pathogen causing a major disease outbreak is ever present. Comparative transcriptomics demonstrated its potential to reveal novel and effective disease prevention strategies that affect the initial stages of disease. Shedding light on the basis of this diversity of infection strategies will result in development of increasingly specific control strategies.

Comparative Genomics and Transcriptomics During Sexual Development Gives Insight Into the Life History of the Cosmopolitan Fungus Fusarium neocosmosporiellum.

Fusarium neocosmosporiellum (formerly Neocosmospora vasinfecta) is a cosmopolitan fungus that has been reported from soil, herbivore dung, and as a fruit- and root-rot pathogen of numerous field crops, although it is not known to cause significant losses on any crop. Taking advantage of the fact that this species produces prolific numbers of perithecia in culture, the genome of F. neocosmosporiellum was sequenced and transcriptomic analysis across five stages of perithecium development was performed to better understand the metabolic potential for sexual development and gain insight into its life history. Perithecium morphology together with the genome and transcriptome were compared with those of the plant pathogen F. graminearum, a model for studying perithecium development. Larger ascospores of F. neocosmosporiellum and their tendency to discharge as a cluster demonstrated a duality of dispersal: the majority are passively dispersed through the formation of cirrhi, while a minority of spores are shot longer distances than those of F. graminearum. The predicted gene number in the F. neocosmosporiellum genome was similar to that in F. graminearum, but F. neocosmosporiellum had more carbohydrate metabolism-related and transmembrane transport genes. Many transporter genes were differentially expressed during perithecium development in F. neocosmosporiellum, which may account for its larger perithecia. Comparative analysis of the secondary metabolite gene clusters identified several polyketide synthase genes that were induced during later stages of perithecium development. Deletion of a polyketide synthase gene in F. neocosmosporiellum resulted in a defective perithecium phenotype, suggesting an important role of the corresponding metabolite, which has yet to be identified, in perithecium development. Results of this study have provided novel insights into the genomic underpinning of development in F. neocosmosporiellum, which may help elucidate its ability to occupy diverse ecological niches.

Birth of three stowaway-like MITE families via microhomology-mediated miniaturization of a Tc1/Mariner element in the yellow fever mosquito.

Eukaryotic genomes contain numerous DNA transposons that move by a cut-and-paste mechanism. The majority of these elements are self-insufficient and dependent on their autonomous relatives to transpose. Miniature inverted repeat transposable elements (MITEs) are often the most numerous nonautonomous DNA elements in a higher eukaryotic genome. Little is known about the origin of these MITE families as few of them are accompanied by their direct ancestral elements in a genome. Analyses of MITEs in the yellow fever mosquito identified its youngest MITE family, designated as Gnome, that contains at least 116 identical copies. Genome-wide search for direct ancestral autonomous elements of Gnome revealed an elusive single copy Tc1/Mariner-like element, named as Ozma, that encodes a transposase with a DD37E triad motif. Strikingly, Ozma also gave rise to two additional MITE families, designated as Elf and Goblin. These three MITE families were derived at different times during evolution and bear internal sequences originated from different regions of Ozma. Upon close inspection of the sequence junctions, the internal deletions during the formation of these three MITE families always occurred between two microhomologous sites (6-8 bp). These results suggest that multiple MITE families may originate from a single ancestral autonomous element, and formation of MITEs can be mediated by sequence microhomology. Ozma and its related MITEs are exceptional candidates for the long sought-after endogenous active transposon tool in genetic control of mosquitoes.

Role of Fig1, a component of the low-affinity calcium uptake system, in growth and sexual development of filamentous fungi.

The function of Fig1, a transmembrane protein of the low-affinity calcium uptake system (LACS) in fungi, was examined for its role in the growth and development of the plant pathogen Fusarium graminearum. The Δfig1 mutants failed to produce mature perithecia, and sexual development was halted prior to the formation of perithecium initials. The loss of Fig1 function also resulted in a reduced vegetative growth rate. Macroconidium production was reduced 70-fold in the Δfig1 mutants compared to the wild type. The function of the high-affinity calcium uptake system (HACS), comprised of the Ca(2+) channels Mid1 and Cch1, was previously characterized for F. graminearum. To better understand the roles of the LACS and the HACS, Δfig1 Δmid1, Δfig1 Δcch1, and Δfig1 Δmid1 Δcch1 double and triple mutants were generated, and the phenotypes of these mutants were more severe than those of the Δfig1 mutants. Pathogenicity on wheat was unaffected for the Δfig1 mutants, but the Δfig1 Δmid1, Δfig1 Δcch1, and Δfig1 Δmid1 Δcch1 mutants, lacking both LACS and HACS functions, had reduced pathogenicity. Additionally, Δfig1 mutants of Neurospora crassa were examined and did not affect filamentous growth or female fertility in a Δfig1 mating type A strain, but the Δfig1 mating type a strain failed to produce fertile fruiting bodies. These results are the first report of Fig1 function in filamentous ascomycetes and expand its role to include complex fruiting body and ascus development.

Sexual development and ascospore discharge in Fusarium graminearum.

Fusarium graminearum has become a model system for studies in development and pathogenicity of filamentous fungi. F. graminearum most easily produces fruiting bodies, called perithecia, on carrot agar. Perithecia contain numerous tissue types, produced at specific stages of perithecium development. These include (in order of appearance) formation of the perithecium initials (which give rise to the ascogenous hyphae), the outer wall, paraphyses (sterile mycelia which occupy the center of the perithecium until the asci develop), the asci, and the ascospores within the asci. The development of each of these tissues is separated by approximately 24 hours and has been the basis of transcriptomic studies during sexual development. Refer to Hallen et al. (2007) for a more thorough description of development, including photographs of each stage. Here, we present the methods for generating and harvesting synchronously developing lawns of perithecia for temporal studies of gene regulation, development, and physiological processes. Although these methods are written specifically to be used with F. graminearum, the techniques can be used for a variety of other fungi, provided that fruiting can be induced in culture and there is some synchrony to development. We have recently adapted this protocol to study the sexual development of F. verticillioides. Although individual perithecia must be hand picked in this species, because a lawn of developing perithecia could not be induced, the process worked well for studying development (Sikhakolli and Trail, unpublished). The most important function of fungal fruiting bodies is the dispersal of spores. In many of the species of Ascomycota (ascus producing fungi), spores are shot from the ascus, due to the generation of turgor pressure within the ascus, driving ejection of spores (and epiplasmic fluid) through the pore in the ascus tip. Our studies of forcible ascospore discharge have resulted in development of a "spore discharge assay", which we use to screen for mutations in the process. Here we present the details of this assay. F. graminearum is homothallic, and thus can form fruiting bodies in the absence of a compatible partner. The advantage of homothallism is that crossing is not necessary to generate offspring homozygous for a particular trait, a facet that has facilitated the study of sexual development in this species. However, heterothallic strains have been generated that can be used for crossing. It is also possible to cross homothallic strains to obtain mutants for several genes in one strain. This is done by coinoculating one Petri dish with 2 strains. Along the meeting point, the majority of perithecia will be recombinant (provided a mutation in one of the parent strains does not inhibit outcrossing). As perithecia age, they exude ascospores en masse instead of forcibly discharging them. The resulting spore exudate (called a cirrhus) sits at the tip of the perithecium and can easily be removed for recovery of individual spores. Here we present a protocol to facilitate the identification of recombinant perithecia and the recovery of recombinant progeny.

Fusarium graminearum from expression analysis to functional assays.

Fusarium graminearum, the causal agent of head blight of wheat, was the third filamentous fungus to have a completed genome sequence. Since the release of the genome sequence in 2003, F. graminearum has become a model for studies of genomics and transcriptomics, mycotoxins, fungal population genetics, gene function, and sexual development. Herein we present the techniques we have used in our laboratory to perform expression analyses on life cycle stages of F. graminearum and techniques to functionally characterize those genes identified as potentially interesting.

Mid1, a mechanosensitive calcium ion channel, affects growth, development, and ascospore discharge in the filamentous fungus Gibberella zeae.

The role of Mid1, a stretch-activated ion channel capable of being permeated by calcium, in ascospore development and forcible discharge from asci was examined in the pathogenic fungus Gibberella zeae (anamorph Fusarium graminearum). The Δmid1 mutants exhibited a >12-fold reduction in ascospore discharge activity and produced predominately abnormal two-celled ascospores with constricted and fragile septae. The vegetative growth rate of the mutants was ∼50% of the wild-type rate, and production of macroconidia was >10-fold lower than in the wild type. To better understand the role of calcium flux, Δmid1 Δcch1 double mutants were also examined, as Cch1, an L-type calcium ion channel, is associated with Mid1 in Saccharomyces cerevisiae. The phenotype of the Δmid1 Δcch1 double mutants was similar to but more severe than the phenotype of the Δmid1 mutants for all categories. Potential and current-voltage measurements were taken in the vegetative hyphae of the Δmid1 and Δcch1 mutants and the wild type, and the measurements for all three strains were remarkably similar, indicating that neither protein contributes significantly to the overall electrical properties of the plasma membrane. Pathogenicity of the Δmid1 and Δmid1Δcch1 mutants on the host (wheat) was not affected by the mutations. Exogenous calcium supplementation partially restored the ascospore discharge and vegetative growth defects for all mutants, but abnormal ascospores were still produced. These results extend the known roles of Mid1 to ascospore development and forcible discharge. However, Neurospora crassa Δmid1 mutants were also examined and did not exhibit defects in ascospore development or in ascospore discharge. In comparison to ion channels in other ascomycetes, Mid1 shows remarkable adaptability of roles, particularly with regard to niche-specific adaptation.