Chapter One - Fungal Genomics: Sequencing and Annotation

The genus Colletotrichum contains a large number of phytopathogenic fungi that produce enormous economic losses around the world. The effect of horizontal gene transfer (HGT) has not been studied yet in these organisms. Inter-Kingdom HGT into fungal genomes has been reported in the past but knowledge about the HGT between plants and fungi is particularly limited. We describe a gene in the genome of several species of the genus Colletotrichum with a strong resemblance to subtilisins typically found in plant genomes. Subtilisins are an important group of serine proteases, widely distributed in all of the kingdoms of life. Our hypothesis is that the gene was acquired by Colletotrichum spp. through (HGT) from plants to a Colletotrichum ancestor. We provide evidence to support this hypothesis in the form of phylogenetic analyses as well as a characterization of the similarity of the subtilisin at the primary, secondary and tertiary structural levels. The remarkable level of structural conservation of Colletotrichum plant-like subtilisin (CPLS) with plant subtilisins and the differences with the rest of Colletotrichum subtilisins suggests the possibility of molecular mimicry. Our phylogenetic analysis indicates that the HGT event would have occurred approximately 150–155 million years ago, after the divergence of the Colletotrichum lineage from other fungi. Gene expression analysis shows that the gene is modulated during the infection of maize by C. graminicola suggesting that it has a role in plant disease. Furthermore, the upregulation of the CPLS coincides with the downregulation of several plant genes encoding subtilisins. Based on the known roles of subtilisins in plant pathogenic fungi and the gene expression pattern that we observed, we postulate that the CPLSs have an important role in plant infection.

BackgroundThe fungal pathogen Histoplasma capsulatum is thought to be the most common cause of fungal respiratory infections in immunocompetent humans, yet little is known about its biology. Here we provide the first genome-wide studies to experimentally validate its genome annotation. A functional interrogation of the Histoplasma genome provides critical support for continued investigation into the biology and pathogenesis of H. capsulatum and related fungi.ResultsWe employed a three-pronged approach to provide a functional annotation for the H. capsulatum G217B strain. First, we probed high-density tiling arrays with labeled cDNAs from cells grown under diverse conditions. These data defined 6,172 transcriptionally active regions (TARs), providing validation of 6,008 gene predictions. Interestingly, 22% of these predictions showed evidence of anti-sense transcription. Additionally, we detected transcription of 264 novel genes not present in the original gene predictions. To further enrich our analysis, we incorporated expression data from whole-genome oligonucleotide microarrays. These expression data included profiling under growth conditions that were not represented in the tiling experiment, and validated an additional 2,249 gene predictions. Finally, we compared the G217B gene predictions to other available fungal genomes, and observed that an additional 254 gene predictions had an ortholog in a different fungal species, suggesting that they represent genuine coding sequences.ConclusionsThese analyses yielded a high confidence set of validated gene predictions for H. capsulatum. The transcript sets resulting from this study are a valuable resource for further experimental characterization of this ubiquitous fungal pathogen. The data is available for interactive exploration at http://histo.ucsf.edu.

Magnetotactic bacteria (MTB), which contain nano-scale membrane bound crystals of magnetic iron minerals, are of great interest in microbiology, biomineralization, advanced magnetic materials and bio-geosciences. However, no application has been exploited on a commercial scale due to the fastidious lifestyle of most cultured MTB. Acidithiobacillus ferrooxidans is a facultatively aerobic bacterium which can synthesize intracellular electron dense magnetite magnetosomes. Although the mass cultivation of A. ferrooxidans was performed facilely, the optimal conditions for cell growth and magnetosome formation remain unknown. In this study, transmission electronmicroscopy (TEM) and inductively coupled plasma atomic emission spectrometer (ICP-AES) were used to observe magnetosomes and measure iron content of A. ferrooxidans, respectively. The effects of ferrous sulfate concentration, ammonium sulfate concentration, oxygen concentration, pH and temperature on the cell growth and magnetosome formation were systematically investigated. Additionally, the orthogonal design of experiments were carried out, and the optimum conditions for cell growth (FeSO4 120 mM, (NH4)2SO4 3.0 g/L, temperature 30°C, initial pH 1.75, and 20% loadings) and magnetosome formation (FeSO4 160 mM, (NH4)2SO4 2.4 g/L, temperature 20°C, initial pH 1.75, and 50% loadings) were not identical. Key words: Magnetotactic bacteria, Acidithiobacillus ferrooxidans, magnetosome formation, orthogonal design, transmission electronmicroscopy, optimization.

Plant pathology has a long history of categorizing fungal pathogens according to discrete nutritional lifestyles. While this is expedient for practical management efforts, this categorization often implies that disease is a defining property of the microbe, despite increasing ecological, genomic, and experimental evidence which challenges this implication. To describe the many plant-associated fungi that break this mold, we suggest adopting an encompassing term. Here, we hypothesize that facultative pathogenicity, where fungi colonize plants asymptomatically and induce disease only under specific conditions, is not an exception but the dominant state of many plant-pathogenic fungi. We further suggest that an overreliance on obligate biotrophs and other model organisms has biased conceptual frameworks and prevented our recognition of the ecological flexibility of many fungal plant pathogens. Drawing on phylogenetic, genomic, and ecological data, we show that many plant-associated fungi shift dynamically along a continuum from endophytism to necrotrophy. This evidence is synthesized across diverse fungal taxa demonstrating widespread asymptomatic colonization of hosts and highlights how environmental, developmental, genetic, and biochemical cues catalyze transitions to disease. Reframing our thinking about many of these fungi as facultative pathogens challenges prevailing concepts of pathogenic lifestyle classification, nonhost resistance, and experimental design in plant pathology. Recognizing disease as one possible outcome, rather than a defining trait, of plant-fungal interactions invites a conceptual shift towards studying the ecological and physiological conditions that govern these associations, with diverse implications for the study of these fungi and the management of their resulting diseases.

Fungi are major causes of human, animal and plant disease. Human fungal infections can be fatal, but there are limited options for therapy, and resistance to commonly used anti-fungal drugs is widespread. The genomes of many fungi have recently been sequenced, allowing identification of proteins that may become targets for novel therapies. We examined the genomes of human fungal pathogens for genes encoding homologues of cation channels, which are prominent drug targets. Many of the fungal genomes examined contain genes encoding homologues of potassium (K+), calcium (Ca2+) and transient receptor potential (Trp) channels, but not sodium (Na+) channels or ligand-gated channels. Some fungal genomes contain multiple genes encoding homologues of K+ and Trp channel subunits, and genes encoding novel homologues of voltage-gated Kv channel subunits are found in Cryptococcus spp. Only a single gene encoding a homologue of a plasma membrane Ca2+ channel was identified in the genome of each pathogenic fungus examined. These homologues are similar to the Cch1 Ca2+ channel of Saccharomyces cerevisiae. The genomes of Aspergillus spp. and Cryptococcus spp., but not those of S. cerevisiae or the other pathogenic fungi examined, also encode homologues of the mitochondrial Ca2+ uniporter (MCU). In contrast to humans, which express many K+, Ca2+ and Trp channels, the genomes of pathogenic fungi encode only very small numbers of K+, Ca2+ and Trp channel homologues. Furthermore, the sequences of fungal K+, Ca2+, Trp and MCU channels differ from those of human channels in regions that suggest differences in regulation and susceptibility to drugs.

The Japanese pear pathotype of Alternaria alternata produces the host-specific AK-toxin and causes black spot of Japanese pear. AK-toxin is produced by mycelia grown in nutritional media and by conidia germinated in water. We have previously identified two homologous genes, AKT3-1 and AKT3-2, in the genomic region controlling toxin biosynthesis. From gene-targeting experiments, we demonstrated that AKT3-2, at least, is essential for toxin biosynthesis. However, functional characterization of AKT3-1 was unsuccessful, because of the presence of multiple copies in the genome. Expression of these genes was studied in strains transformed with vectors p3-1GUS-2 and p3-2GUS-2, which contain the β-glucuronidase (GUS) reporter gene fused to the AKT3-1 and AKT3-2 promoters, respectively. GUS activity was detected in mycelia of p3-1GUS-2 and p3-2GUS-2 transformants grown in potato dextrose broth, indicating that AKT3-1 and AKT3-2 both are expressed under optimal growth conditions in an artificial medium. Because the toxin is not stored in dormant conidia, early expression of the toxin biosynthetic genes during conidial germination is crucial for successful invasion and colonization of susceptible leaf tissues. In cytological observation of germinated conidia on cellulose membranes, stable GUS activity was detected in germ tubes of both transformants during germ-tube emergence and elongation, showing the transcription of AKT3-1 and AKT3-2 during an early stage of infection.

Plants exist in close association with uncountable numbers of microorganisms around, on, and within them. Some of these endophytically colonize plant roots. The colonization of roots by certain symbiotic strains of plant-associated bacteria and fungi results in these plants performing better than plants whose roots are colonized by only the wild populations of microbes. We consider here crop plants whose roots are inhabited by introduced organisms, referring to them as Enhanced Plant Holobionts (EPHs). EPHs frequently exhibit resistance to specific plant diseases and pests (biotic stresses); resistance to abiotic stresses such as drought, cold, salinity, and flooding; enhanced nutrient acquisition and nutrient use efficiency; increased photosynthetic capability; and enhanced ability to maintain efficient internal cellular functioning. The microbes described here generate effects in part through their production of Symbiont-Associated Molecular Patterns (SAMPs) that interact with receptors in plant cell membranes. Such interaction results in the transduction of systemic signals that cause plant-wide changes in the plants’ gene expression and physiology. EPH effects arise not only from plant-microbe interactions, but also from microbe-microbe interactions like competition, mycoparasitism, and antibiotic production. When root and shoot growth are enhanced as a consequence of these root endophytes, this increases the yield from EPH plants. An additional benefit from growing larger root systems and having greater photosynthetic capability is greater sequestration of atmospheric CO2. This is transferred to roots where sequestered C, through exudation or root decomposition, becomes part of the total soil carbon, which reduces global warming potential in the atmosphere. Forming EPHs requires selection and introduction of appropriate strains of microorganisms, with EPH performance affected also by the delivery and management practices.

The fungal kingdom comprises some of the most devastating plant pathogens. Sequencing the genomes of fungal pathogens has shown a remarkable variability in genome size and architecture. Population genomic data enable us to understand the mechanisms and the history of changes in genome size and adaptive evolution in plant pathogens. Although transposable elements predominantly have negative effects on their host, fungal pathogens provide prominent examples of advantageous associations between rapidly evolving transposable elements and virulence genes that cause variation in virulence phenotypes. By providing homogeneous environments at large regional scales, managed ecosystems, such as modern agriculture, can be conducive for the rapid evolution and dispersal of pathogens. In this Review, we summarize key examples from fungal plant pathogen genomics and discuss evolutionary processes in pathogenic fungi in the context of molecular evolution, population genomics and agriculture.

Plant growth promoting rhizobacteria (PGPR) are known to confer disease resistance to plants. Bacillus sp. JS demonstrated antifungal activities against five fungal pathogens in in vitro assays. To verify whether the volatiles of Bacillus sp. JS confer disease resistance, tobacco leaves pre-treated with the volatiles were damaged by the fungal pathogen, Rhizoctonia solani and oomycete Phytophthora nicotianae. Pre-treated tobacco leaves had smaller lesion than the control plant leaves. In pathogenesis-related (PR) gene expression analysis, volatiles of Bacillus sp. JS caused the up-regulation of PR-2 encoding β-1,3-glucanase and acidic PR-3 encoding chitinase. Expression of acidic PR-4 encoding chitinase and acidic PR-9 encoding peroxidase increased gradually after exposure of the volatiles to Bacillus sp. JS. Basic PR-14 encoding lipid transfer protein was also increased. However, PR-1 genes, as markers of salicylic acid (SA) induced resistance, were not expressed. These results suggested that the volatiles of Bacillus sp. JS confer disease resistance against fungal and oomycete pathogens through PR genes expression.

Changes in Rubisco activase gene expression and polypeptide content in Brachypodium distachyon

A monooxygenase gene was isolated from a biocontrol strain of Trichoderma hamatum and its role in biocontrol was investigated. The gene had homologues in other fungal genomes, but was not closely related to any fully characterised gene. The T. hamatum monooxygenase gene was expressed specifically in response to the plant pathogens Sclerotinia sclerotiorum, Sclerotinia minor and Sclerotium cepivorum, but not in response to Botrytis cinerea or T. hamatum. Expression of the gene did not occur until contact had been made between the two fungal species. Homologues in T. atroviride and T. virens showed similar expression patterns. Expression of the gene in response to S. sclerotiorum was influenced by pH, with a peak of expression at pH 4, and was subject to nitrogen catabolite repression. Disruption of the monooxygenase gene did not affect the growth or morphology of T. hamatum, but caused a decrease in its ability to inhibit the growth and sclerotial production of S. sclerotiorum. The monooxygenase gene had a role in the antagonistic activity of Trichoderma species against specific fungal plant pathogens and is therefore a potentially important factor in biocontrol by Trichoderma species.

Methionine synthases (MetH) catalyse the methylation of homocysteine (Hcy) with 5-methyl-tetrahydrofolate (5, methyl-THF) acting as methyl donor, to form methionine (Met) and tetrahydrofolate (THF). This function is performed by two unrelated classes of enzymes that differ significantly in both their structures and mechanisms of action. The genomes of plants and many fungi exclusively encode cobalamin-independent enzymes (EC.2.1.1.14), while some fungi also possess proteins from the cobalamin-dependent (EC.2.1.1.13) family utilised by humans. Methionine synthase's function connects the methionine and folate cycles, making it a crucial node in primary metabolism, with impacts on important cellular processes such as anabolism, growth and synthesis of proteins, polyamines, nucleotides and lipids. As a result, MetHs are vital for the viability or virulence of numerous prominent human and plant pathogenic fungi and have been proposed as promising broad-spectrum antifungal drug targets. This review provides a summary of the relevance of methionine synthases to fungal metabolism, their potential as antifungal drug targets and insights into the structures of both classes of MetH.

Expression of a synthetic gene for improved protein quality in transformed potato plants

Cobalt complex with histidine ligand was synthesized and physico-chemically characterized by solubility testing, melting point, UV-spectra and FTIR. The synthesized metal complex was evaluated for in-vitro antibacterial and antifungal activity against the multidrug resistant pathogens, such as Pseudomonas aeruginosa, Vibrio cholerae, Salmonella typhi, E. coli, Klebsiella pneumonia, Staphylococcus aureus, Aspergillus niger, Aspergillus flavus and Candida albicans. The metal complex showed the significant antibacterial and antifungal activity comparison with commercial antibiotics. Further work can be extended through In-silico docking of this metal complex and bacterial, fungal genome which can be given a better idea about genome metal interaction in molecular level.

Identification of pathogenic fungi isolated from clinical specimens in clinical microbiology laboratories is primarily based on observing fungal phenotypic structures under the microscope and performing biochemical tests for fungal cultures. This conventional method is very time-consuming and labor-dependent. It usually requires several weeks for the fungi to grow sufficiently on culture media, and the identification processes on fungal phenotypic structure rely very much on experienced staff. Therefore, a more accurate and rapid method for pathogenic fungal identification is necessary for clinical laboratories to get abreast of modern development.
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\nGene sequencing and phylogenetic analysis targeting the internal transcribed spacer (ITS) region in the fungal genomes are the most commonly used molecular methods for fungal identification. Because of the optimal inter and intra-species variation property of the ITS region, it can act as the DNA barcode to identify fungi to the species level. In this study, 33 clinical fungal isolates were identified by both phenotypic method and ITS sequencing. The results showed that 23 isolates were successfully identified to thespecies level by both phenotypic and molecular methods. Moreover, five isolates were only identified to the genus level by phenotypic method, but they could be successfully identified to the species level by ITS sequencing. However, five isolates have not been differentiated because there were mismatched results from phenotypic and sequencing methods. It may be due to the limitation of sequencing method on some fungal species. Building up a more comprehensive database or setting up a standard platform to guide the molecular process may help improve the performance of molecular method.
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\nTo conclude, molecular method is a rapid and reliable way for fungal identification because ITS region acts as the DNA barcode for pathogenic fungi.