Proteome analysis of Phytophthora cinnamomi, the causal agent of Dieback

Phytophthora cinnamomi is a nasty pathogen that causes significant environmental and agricultural destruction. In this thesis, proteomic analysis was used to obtain a biochemical snapshot of the organism to understand its contributors to pathogenicity. The mode of action of phosphite, the only chemical used to combat dieback disease was also elucidated as there is a growing pressure of chemical resistance

Suppression and triggering of Arabidopsis immunity by Albugo species

Albugo species are obligate biotrophic phytopathogens. Like other biotrophs, they are anticipated to secrete effectors that can suppress or trigger plant defenses; the nature of Albugo effectors is currently unknown. Sequencing of A. laibachii isolate Nc14 (AlNc14) genome reveals 13032 genes encoded in a ~37 Mb genome. We analyze the effector complement of AlNc14 and find known effector classes but also classes unique to A. laibachii. Experiments reveal that CHXCs are a novel class of effectors that suppress host defense. We functionally characterize two predicted AlNc14 effectors in detail; CHXC1 a potential core effector conserved in other oomycete species, and SSP6, a fast-evolving effector specific to A. laibachii. CHXC1 encodes a nuclear localized HECT E3 ligase homolog, which suppresses host defenses dependent on cys651. We find 7 variants of SSP6 that are under diversifying selection. Two highly expressed variants SSP6-2c and SSP6-A are plasma membrane localized when expressed in planta. Interestingly, SSP6-2c but not SSP6-A, is able to enhance growth of P. infestans race blue 13 and suppress flg22-dependent ROS production. In Arabidopsis cells we find SSP6-2c localizes around AlNc14 haustoria. We propose that AlNc14 secretes the effectors SSP6-2c and CHXC1 into the plant cell to suppress defense and promote infection. Current methods to screen for virulence of effector candidates predominantly rely on measuring growth of bacterial pathogens. Quantitative assessment of resistance and susceptibility to eukaryotic pathogens is more difficult. We develop a semi-automated high-throughput system for assaying Hpa growth. We investigate the genetic basis of resistance to Albugo in Arabidopsis. We find that resistance to AlNc14 is linked to RAC1 and RAC3 in Ksk-1. In contrast, resistance to A. candida Nc2 (AcNc2) is linked to WRR4 in Col-0, Col-5 and Ksk-1. A second dominant locus, WRR5a/b in Col-5 also confers resistance to AlNc2. Thus, different R-genes and presumably different effectors govern resistance to AlNc14 and AcNc2.

Epidemiology and Evolution of Fungal Pathogens in Plants and Animals

International audienc

Molecular studies on compatibility in the mutualistic plant root-Piriformospora indica interaction

Plants have developed diverse strategies for protection against the threat of invading pathogens. In order to improve their performance as well as to evade abiotic and biotic stresses, one strategy of plants is to establish associations with beneficial microbial organisms. Piriformospora indica is a root interacting fungus, which transfers several benefits to colonized plants like a better tolerance to various biotic and abiotic stresses, as well as an improved plant growth and yield. P. indica colonizes a broad range of monocot and dicot plants. This broad host range indicates that P. indica has developed efficient strategies to overcome innate immune responses and to manipulate the metabolism in different plants. This is even more intriguing as the fungus was shown to follow an initial biotrophic colonisation strategy at which penetrated cells are living. Plant colonizing microbes are known to secrete proteins (also called effectors) in order to modify host physiology and modulate plant defense mechanisms and, hence, confer compatibility. The aim of this study was to identify P. indica effector proteins as well as plant compatibility factors that are involved in the manipulation of those processes required for successful fungal establishment in planta. Therefore, two different strategies were followed. In the first approach, the so-called yeast signal sequence trap (YSST) assay was established. As the result of YSST, several plant genes were identified that are known to be involved in stress responses and cell wall development. These genes were shown to have a specific expression in barley roots during P. indica colonisation. In addition, a fungal gene was identified that does not show any similarities to other sequences deposited in public databases. The identified P. indica protein (PIALH43) carries a signal peptide and was shown to be induced during barley root colonisation. Interestingly, PIALH43 harbours a highly conserved C-terminal RING finger motif. In silico protein modelling of PIALH43 confirmed a 3D structural overlap and verified the accurate conformation of the E2 binding residues when compared with known human and plant ubiquitin ligases. Moreover, E3 ligase activity of PIALH43 was confirmed in vitro. Currently, PIALH43 is overexpressed in planta and in P. indica in order to study its function in mutualistic root colonisation. In a second approach, a simplified subtraction-based assay, designated Transcript Subtractive Hybridization (TSH), was established to identify and study plant compatibility factors in the barley-P. indica interaction. The subtraction assay delivered various differentially regulated genes. These genes are known to be involved in stress responses, phytohormone- and secondary metabolism, autophagy, and protein processing. Among the up-regulated candidates was a gene encoding S-adenosylmethionine synthetase 2, which is thought to be involved in the synthesis of ethylene. De novo synthesis of ethylene during root colonization was verified by quantifying the ethylene precursor 1-aminocyclopropane 1-carboxylic acid (ACC) in barley and by cytologically monitoring GUS accumulation in ACC synthase reporter plants of Arabidopsis. In addition, the effects of ethylene precursor ACC or ethylene antagonist 1-methylcyclopropene (MCP) was determined. In these pharmacological experiments, barley plants were about 40% less colonised by P. indica after application of MCP while treatment with ACC resulted in significant increase (~ 60%) in colonization. To further elucidate the impact of ethylene on plant root colonization by P. indica, genetic analyses were performed with Arabidopsis mutants altered in ethylene synthesis and signaling at early biotrophic (~ 3 dai) and later cell death-associated colonization phases (~ 14 dai). In accordance with the studies in barley, Arabidopsis mutants ctr1-1 (constitutive ethylene signaling) and eto1-1 (ethylene overproducer) exhibited a significant increase in fungal colonization (especially at later interaction stages), while a reduced colonization was observed in ein2-1 (ethylene insensitive). In summary, ethylene might function as general plant compatibility factor in the plant-P. indica system.Pflanzen haben verschieden Strategien entwickelt, um sich vor den Bedrohungen durch eindringende Pathogene zu schützen. Um ihre Leistungsfähigkeit zu verbessern und abiotischem und biotischem Stress zu entgehen, besteht eine Strategie der Pflanzen darin, Verbindungen mit nützlichen Mikroorganismen einzugehen. Piriformospora indica ist ein Pilz, der mit Pflanzenwurzeln interagiert und der besiedelten Pflanze verschiedene Vorteile verschafft, zum Beispiel eine bessere Toleranz gegenüber verschiedenen biotischen und abiotischen Stressfaktoren, verbessertes Wachstum und höhere Ernteerträge. P. indica ist in der Lage, ein breites Spektrum an monokotylen und dikotylen Pflanzen zu besiedeln. Dieses breite Wirtsspektrum deutet darauf hin, dass P. indica effiziente Mechanismen entwickelt hat, um pflanzliche Immunantworten zu überwinden und den Metabolismus verschiedener Pflanzen zu manipulieren. Dies ist umso mehr erstaunlich, da gezeigt werden konnte, dass der Pilz zu Beginn der Besiedlung in einer biotrophen Phase lebende Zellen penetriert. Pflanzen besiedelnde Mikroorganismen sekretieren bekanntermaßen Proteine (so genannte Effektoren), um die Wirtsphysiologie zu verändern, die Abwehrmechanismen der Pflanze zu modulieren und schließlich Kompatibilität zu erreichen. Das Ziel dieser Untersuchungen war es, Effektorproteine von P. indica und Kompatibilitäts-faktoren der Pflanze zu identifizieren, die an manipulativen Prozessen beteiligt sind und die erfolgreiche Etablierung des Pilzes in der Pflanze ermöglichen. Hierfür wurden zwei verschiedene Strategien verfolgt. Zunächst wurde der so genannte yeast signal sequence trap (YSST) etabliert. Als Ergebnis des YSST konnten verschiedene pflanzliche Gene identifiziert werden, die an Stressantworten und Zellwandbildung beteiligt sind. Es wurde gezeigt, dass diese Gene spezifisch in Gerstenwurzeln exprimiert wurden, wenn diese mit P. indica besiedelt waren. Zusätzlich konnte ein pilzliches Gen identifiziert werden, das keinerlei Ähnlichkeiten mit bisher bekannten Sequenzen aus öffentlich zugänglichen Datenbanken aufweist. Das entdeckte P. indica-Protein (PIALH43) trägt ein Signalpeptid, es wurde gezeigt, dass es während der Besiedlung von Gerstenwurzeln exprimiert wird. Interessanterweise beinhaltet PIALH43 ein stark konserviertes, C-terminales RING-Finger Motiv. In silico Proteinmodellierung von PIALH43 bestätigte eine dreidimensinole Überlappung und wies die genaue Konformation der der E2-Bindestellen im Vergleich mit bekannten menschlichen und pflanzlichen Ubiquitin-Ligasen nach. Die E3-Ligase-Aktivität konnte in vitro bekräftigt werden. Im Moment wird PIALH43 in der Pflanze (Arabidopsis thaliana) und in P. indica überexprimiert, um seine Funktion in der mutualistischen Wurzelkolonisierung zu studieren. In einer weiteren Annäherung wurde eine vereinfachte, auf Subtraktion basierende Methode, bezeichnet als Transcript Substractive Hybridization (TSH), etabliert. Mit Hilfe dieser Methode wurden pflanzliche Kompatibilitätsfaktoren der Gerste-P. indica Interaktion identifiziert und studiert. Die Subtraktionsmethode lieferte viele verschieden regulierte Gene. Die gefundenen Gene sind bekanntermaßen in Stressantworten, Phytohormon- und Sekundärmetabolismus, Autophagie und Proteinprozessierung beteiligt. Unter den hochregulierten Kandidaten war eine S-Adenosylmethionin-Synthetase 2, bei der vermutet wird, dass sie in der Synthese von Ethylen eine Rolle spielt. Die de novo Synthese von Ethylen während der Wurzelbesiedlung wurde durch die Quantifizierung des Ethylen-Vorläufers 1-Aminocyclopropan-1-carboxylsäure (ACC) in Gerste und durch zytologische Kontrolle der Akkumulation von GUS in ACC-Synthase-Reporterpflanzen (Arabidopsis) bestätigt. Zusätzlich wurden die Effekte des Ethylen Vorläufers ACC und des Ethylen-Antagonists 1-Methylcyclopropene (MCP) auf die Besiedlung bestimmt. In diesen pharmakologischen Untersuchungen waren Gerstenpflanzen nach der Zugabe von MCP um etwa 40 % weniger mit P. indica besiedelt, während die Behandlung mit ACC zu einem signifikanten Anstieg (~ 60 %) in der Besiedlung führte. Um den Einfluss von Ethylen auf die Besiedlung von Pflanzenwurzeln durch P. indica weiter zu beleuchten, wurden genetische Untersuchungen mit Arabidopsis-Mutanten, deren Ethylensynthese und Ethylen-Signalwege verändert waren, in der frühen biotrophen (~ 3 days after inoculation, dai) und späteren Zelltod-assoziierten Phase (~ 14 dai) durchgeführt. In Übereinstimmung mit Studien in Gerste waren die Arabidopsis-Mutanten ctr1-1 (Ethylen-Signalweg konstitutiv aktiv) und eto1-1 (Ethylen-Überproduzierer) signifikant stärker besiedelt, vor allem in späteren Interaktionsstadien. Bei der Ethylen-insensitiven Mutante ein2-1 wurde hingegen eine reduzierte Besiedlung beobachtet. Zusammenfassend scheint Ethylen ein genereller Kompatibilitätsfaktor zu sein, der durch den Pilz rekrutiert wird, um verschiedene Wirtspflanzen zu besiedeln, wie hier beispielhaft an den Modellpflanzen Gerste und Arabidopsis gezeigt werden konnte

Development of Computational, Visualization, and Molecular Tools for Fungal and Oomycete Community Ecology

Oomycetes are an important group of organisms with a variety of ecological roles similar to fungi. Although many are well-studied plant pathogens known for their devastating effects on agricultural systems, most are little-studied saprobes and parasites of plants and animals in nearly every ecosystem on earth. The advent of affordable and high-throughput sequencing technologies have resulted in new opportunities for the study of microbial communities, including oomycetes, as well as new challenges associated with analyzing and visualizing the vast amount of data produced. This thesis describes new tools developed for the analysis and visualization of microbial communities, with an emphasis on oomycetes, and studies into the communities of oomycetes and fungi associated with Rhododendron. The widespread adoption of high-throughput molecular community ecology methods is making large data sets classified by taxonomic information common, but additional tools to analyze and visualize these data are needed. Taxonomic classifications are hierarchical, making them much more difficult to analyze compared to typical tabular data. There are many R packages that use taxonomic data to varying degrees but there is currently no cross-package standard for how this information is encoded and manipulated. We developed the R package taxa to provide a robust and flexible solution to storing and manipulating taxonomic data in R and any application-specific information associated with it. It is meant to be a foundation for other packages to build on, so that diverse packages dealing with taxonomic information can be integrated seamlessly. One package that is built on top of taxa is metacoder. Metacoder is an R package for plotting and manipulating data classified by a taxonomy, like the abundance data associated with metabarcoding. Its primary feature is the novel tree-based visualization called “heat trees” that is used to depict data for every taxon in a taxonomy using color and size. Heat-trees provide a more informative alternative to pie charts or stacked bar charts for visualizing communities. Metacoder also provides various functions to do common tasks in microbiome research with data stored in the taxmap format supplied by the taxa package. Both of these packages are open source, version controlled, have unit tests that help detect bugs, and include extensive documentation. Although metabarcoding methods for fungal and bacterial communities are well-developed at this point, no standard and reliable method for oomycete metabarcoding exists. Every currently proposed method for oomycete metabarcoding has at least one flaw; some produce too long an amplicon for Illumina sequencers, some target only a subset of oomycete diversity, some have unacceptable levels of non-target amplification, and some have technical difficulties that make the PCR reactions unreliable. We developed a new method for oomycete metabarcoding targeting the rps10 gene and an associated reference database. Compared to one of the more popular methods currently being used, our method has better taxonomic resolution, less non-target amplification, and a more reliable PCR reaction. A reference database of rps10 sequences for many genera of oomycetes was developed for use in assigning taxonomic classifications to metabarcoding data. Finally, a website was created to host the database that supports searching the database and conducting BLAST searches. Rhododendron is a major ornamental crop in the Pacific Northwest and is known to host both mycorrhizal symbionts and plant pathogens such as Phytophthora ramorum. The fungal and oomycete microbiome in the rhizosphere of rhododendrons from Oregon nurseries was sequenced and differences among cultivars, growth conditions, and nurseries were analyzed. Few oomycetes were found, but this might have been partially due to limitations of the metabarcoding method used. Fungal species found were mostly saprobes and mutualists. Nurseries that grew plants in containers and in-field had a significantly higher diversity of fungi than those that only grew plants in containers. Microbiome composition differed significantly among growth conditions and nurseries, but not among cultivars. This body of work provides novel insights into oomycete communities and novel tools for molecular community ecology that might be of broader interest

Abstracts from the 25th Fungal Genetics Conference

Abstracts from the 25th Fungal Genetics Conferenc

A bioinformatic analysis of genes involved in stress responses in Arabidopsis thaliana

Hyaloperonospora arabidopsidis is an obligate biotrophic oomycete shown to cause downy mildew in Arabidopsis thaliana. The main focus of this project is examining plant stress response and the strategies employed by H. arabidopsidis to infect Arabidopsis and evade plant stress responses. Two regions of the H. arabidopsidis genome containing genes expressed in planta during infection were bioinformatically annotated. The results indicated the genes were involved in regulatory processes associated with the pathogenicity of H. arabidopsidis but not a direct role in pathogenicity. H. arabidopsidis infects its host by secreting effector proteins into the cytoplasm and apoplastic space of the host. The secretome of H. arabidopsidis was analysed to identify classes of cysteine rich apoplastic effectors. This identified 15 candidate elicitin (ELI) and elicitin-like (ELL) sequences, three Kazal-like serine protease inhibitors and four candidates similar to the protein sequences of Ppats 14 and 24, expressed during infection. A second set of aims was to identify potential signalling networks up activated during plant defence responses to infection by H. arabidopsidis using a new model developed by Beal et al (Beal, Falciani et al. 2005) to eventually engineer transcriptional networks. Unfortunately this failed due to problems with the experiment. However, it was still possible to identify signalling networks from a second microarray time course experimental data set centred on signalling networks up regulated in response to the onset of senescence, as they share overlapping signalling pathways. The modelling methodology was used to model the anthocyanin biosynthesis pathway. The model predicted the presence of AtMYB15 as a positive regulator of anthocyanin biosynthesis along with AtMYB90. Research carried out by Nichola Warner (Warner 2008) suggested that AtMYB90 was not essential for anthocyanin biosynthesis during senescence based on by comparing the phenotype of the MYB90 knock out, IM28, with the wild type (WT) Col-0 using a time course microarray. Models of networks of transcriptional regulation of the anthocyanin biosynthesis pathway for IM28 and WT implicate AtMYB29 as a positive regulator of anthocyanin biosynthesis

Doctor of Philosophy

dissertationWhole genome sequencing projects have expanded our understanding of evolution, organism development, and human disease. Now advances in secondgeneration technologies are making whole genome sequencing routine even for small laboratories. However, advances in annotation technology have not kept pace with genome sequencing, and annotation has become the major bottleneck for many genome projects (especially those with limited bioinformatics expertise). At the same time, challenges associated with genomics research extend beyond merely annotating genomes, as annotations must be subjected to diverse downstream analyses, the complexities of which can confound smaller research groups. Additionally, with improvements in genome assembly and the wide availability of next generation transcriptome data (mRNA-seq), researchers have the opportunity to re-annotate previously published genomes, which creates new difficulties for data integration and management that are not well addressed by existing tools. In response to the challenges facing second-generation genome projects, I have developed the annotation pipeline MAKER2 together with accessory software for downstream analysis and data management. The MAKER2 annotation pipeline finds repeats within a genome, aligns ESTs and cDNAs, identifies sites of protein homology, and produces database-ready gene annotations in association with supporting evidence. However MAKER2 can go beyond structural annotation to identify and integrate functional annotations. MAKER2 also provides researchers iv with the capability to re-annotate legacy genome datasets and to incorporate mRNAseq. Additionally, MAKER2 supports distributed parallelization on computer clusters, thus providing a scalable solution for datasets of any size. Annotations produced by MAKER2 can be directly loaded into many popular downstream annotation analysis and management tools from the Generic Model Organism Database Project. By using MAKER2 with these tools, research groups can quickly build genome annotations, perform analyses, and distribute their data to the wider scientific community. Here I describe the internal architecture of MAKER2, and document its computational capabilities. I also describe my work to annotate and analyze eight emerging model organism genomes in collaboration with their associated genome projects. Thus, in the course of my thesis work, I have addressed a specific need within the scientific community for easy-to-use annotation and analysis tools while also expanding our understanding of evolution and biology

28th Fungal Genetics Conference

Full abstracts from the 28th Fungal Genetics Conference Asilomar, March 17-22, 2015