Effector Discovery Research Articles

Combinatorial effector targeting (COMET) for transcriptional modulation and locus-specific biochemistry.

Understanding how human gene expression is coordinately regulated by functional units of proteins across the genome remains a major biological goal. Here, we present COMET, a high-throughput screening platform for combinatorial effector targeting for the identification of transcriptional modulators. We generate libraries of combinatorial dCas9-based fusion proteins, containing two to six effector domains, allowing us to systematically investigate more than 110,000 combinations of effector proteins at endogenous human loci for their influence on transcription. Importantly, we keep full proteins or domains intact, maintaining catalytic cores and surfaces for protein-protein interactions. We observe more than 5800 significant hits that modulate transcription, we demonstrate cell type specific transcriptional modulation, and we further investigate epistatic relationships between our effector combinations. We validate unexpected combinations as synergistic or buffering, emphasizing COMET as both a method for transcriptional effector discovery, and as a functional genomics tool for identifying novel domain interactions and directing locus-specific biochemistry.

Paths of Least Resistance: Unconventional Effector Secretion by Fungal and Oomycete Plant Pathogens.

Effector secretion by different routes mediates the molecular interplay between host plant and pathogen, but mechanistic details in eukaryotes are sparse. This may limit the discovery of new effectors that could be utilized for improving host plant disease resistance. In fungi and oomycetes, apoplastic effectors are secreted via the conventional endoplasmic reticulum (ER)-Golgi pathway, while cytoplasmic effectors are packaged into vesicles that bypass Golgi in an unconventional protein secretion (UPS) pathway. In Magnaporthe oryzae, the Golgi bypass UPS pathway incorporates components of the exocyst complex and a t-SNARE, presumably to fuse Golgi bypass vesicles to the fungal plasma membrane. Upstream, cytoplasmic effector mRNA translation in M. oryzae requires the efficient decoding of AA-ending codons. This involves the modification of wobble uridines in the anticodon loop of cognate tRNAs and fine-tunes cytoplasmic effector translation and secretion rates to maintain biotrophic interfacial complex integrity and permit host infection. Thus, plant-fungal interface integrity is intimately tied to effector codon usage, which is a surprising constraint on pathogenicity. Here, we discuss these findings within the context of fungal and oomycete effector discovery, delivery, and function in host cells. We show how cracking the codon code for unconventional cytoplasmic effector secretion in M. oryzae has revealed AA-ending codon usage bias in cytoplasmic effector mRNAs across kingdoms, including within the RxLR-dEER motif-encoding sequence of a bona fide Phytophthora infestans cytoplasmic effector, suggesting its subjection to translational speed control. By focusing on recent developments in understanding unconventional effector secretion, we draw attention to this important but understudied area of host-pathogen interactions. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Genome and Transcriptome Sequence Resources and Effector Repertoire of Pythium myriotylum Drechsler.

Genome and Transcriptome Sequence Resources and Effector Repertoire of Pythium myriotylum Drechsler.

Proteinaceous Effector Discovery and Characterization in Plant Pathogenic Colletotrichum Fungi.

Anthracnose caused by plant pathogenic Colletotrichum fungi results in large economic losses in field crop production worldwide. To aid the establishment of plant host infection, Colletotrichum pathogens secrete numerous effector proteins either in apoplastic space or inside of host cells for effective colonization. Understanding these effector repertoires is critical for developing new strategies for resistance breeding and disease management. With the advance of genomics and bioinformatics tools, a large repertoire of putative effectors has been identified in Colletotrichum genomes, and the biological functions and molecular mechanisms of some studied effectors have been summarized. Here, we review recent advances in genomic identification, understanding of evolutional characteristics, transcriptional profiling, and functional characterization of Colletotrichum effectors. We also offer a perspective on future research.

Bacterial Spot of Tomato and Pepper in Africa: Diversity, Emergence of T5 Race, and Management.

Bacterial spot disease was first reported from South Africa by Ethel M. Doidge in 1920. In the ensuing century after the initial discovery, the pathogen has gained global attention in plant pathology research, providing insights into host–pathogen interactions, pathogen evolution, and effector discovery, such as the first discovery of transcription activation-like effectors, among many others. Four distinct genetic groups, including Xanthomonas euvesicatoria (proposed name: X. euvesicatoria pv. euvesicatoria), Xanthomonas perforans (proposed name: X. euvesicatoria pv. perforans), Xanthomonas gardneri (proposed name: Xanthomonas hortorum pv. gardneri), and Xanthomonas vesicatoria, are known to cause bacterial spot disease. Recently, a new race of a bacterial spot pathogen, race T5, which is a product of recombination between at least two Xanthomonas species, was reported in Nigeria. In this review, our focus is on the progress made on the African continent, vis-à-vis progress made in the global bacterial spot research community to provide a body of information useful for researchers in understanding the diversity, evolutionary changes, and management of the disease in Africa.

Chromosome-level genome assembly and manually-curated proteome of model necrotroph Parastagonospora nodorum Sn15 reveals a genome-wide trove of candidate effector homologs, and redundancy of virulence-related functions within an accessory chromosome

BackgroundThe fungus Parastagonospora nodorum causes septoria nodorum blotch (SNB) of wheat (Triticum aestivum) and is a model species for necrotrophic plant pathogens. The genome assembly of reference isolate Sn15 was first reported in 2007. P. nodorum infection is promoted by its production of proteinaceous necrotrophic effectors, three of which are characterised – ToxA, Tox1 and Tox3.ResultsA chromosome-scale genome assembly of P. nodorum Australian reference isolate Sn15, which combined long read sequencing, optical mapping and manual curation, produced 23 chromosomes with 21 chromosomes possessing both telomeres. New transcriptome data were combined with fungal-specific gene prediction techniques and manual curation to produce a high-quality predicted gene annotation dataset, which comprises 13,869 high confidence genes, and an additional 2534 lower confidence genes retained to assist pathogenicity effector discovery. Comparison to a panel of 31 internationally-sourced isolates identified multiple hotspots within the Sn15 genome for mutation or presence-absence variation, which was used to enhance subsequent effector prediction. Effector prediction resulted in 257 candidates, of which 98 higher-ranked candidates were selected for in-depth analysis and revealed a wealth of functions related to pathogenicity. Additionally, 11 out of the 98 candidates also exhibited orthology conservation patterns that suggested lateral gene transfer with other cereal-pathogenic fungal species. Analysis of the pan-genome indicated the smallest chromosome of 0.4 Mbp length to be an accessory chromosome (AC23). AC23 was notably absent from an avirulent isolate and is predominated by mutation hotspots with an increase in non-synonymous mutations relative to other chromosomes. Surprisingly, AC23 was deficient in effector candidates, but contained several predicted genes with redundant pathogenicity-related functions.ConclusionsWe present an updated series of genomic resources for P. nodorum Sn15 – an important reference isolate and model necrotroph – with a comprehensive survey of its predicted pathogenicity content.

The hop downy mildew pathogen Pseudoperonospora humuli.

Pseudoperonospora humuli is an obligate biotrophic oomycete that causes downy mildew, one of the most devastating diseases of cultivated hop, Humulus lupulus. Downy mildew occurs in all production areas of the crop in the Northern Hemisphere and Argentina. The pathogen overwinters in hop crowns and roots, and causes considerable crop loss. Downy mildew is managed by sanitation practices, planting of resistant cultivars, and fungicide applications. However, the scarcity of sources of host resistance and fungicide resistance in pathogen populations complicates disease management. This review summarizes the current knowledge on the symptoms of the disease, life cycle, virulence factors, and management of hop downy mildew, including various forecasting systems available in the world. Additionally, recent developments in genomics and effector discovery, and the future prospects of using such resources in successful disease management are also discussed.TaxonomyClass: Oomycota; Order: Peronosporales; Family: Peronosporaceae; Genus: Pseudoperonospora; Species: Pseudoperonospora humuli.Disease symptomsThe disease is characterized by systemically infected chlorotic shoots called “spikes". Leaf symptoms and signs include angular chlorotic lesions and profuse sporulation on the abaxial side of the leaf. Under severe disease pressure, dark brown discolouration or lesions are observed on cones. Infected crowns have brown to black streaks when cut open. Cultivars highly susceptible to crown rot may die at this phase of the disease cycle without producing shoots. However, foliar symptoms may not be present on plants with systemically infected root systems.Infection processPathogen mycelium overwinters in buds and crowns, and emerges on infected shoots in spring. Profuse sporulation occurs on infected tissues and sporangia are released and dispersed by air currents. Under favourable conditions, sporangia germinate and produce biflagellate zoospores that infect healthy tissue, thus perpetuating the infection cycle. Though oospores are produced in infected tissues, their role in the infection cycle is not defined.ControlDowny mildew on hop is managed by a combination of sanitation practices and timely fungicide applications. Forecasting systems are used to time fungicide applications for successful management of the disease.Useful Websites https://content.ces.ncsu.edu/hop‐downy‐mildew (North Carolina State University disease factsheet), https://www.canr.msu.edu/resources/michigan‐hop‐management‐guide (Michigan Hop Management Guide), http://uspest.org/risk/models (Oregon State University Integrated Plant Protection Center degree‐day model for hop downy mildew), https://www.usahops.org/cabinet/data/Field‐Guide.pdf (Field Guide for Integrated Pest Management in Hops).

Proteinaceous effector discovery and characterization in filamentous plant pathogens.

The complicated interplay of plant–pathogen interactions occurs on multiple levels as pathogens evolve to constantly evade the immune responses of their hosts. Many economically important crops fall victim to filamentous pathogens that produce small proteins called effectors to manipulate the host and aid infection/colonization. Understanding the effector repertoires of pathogens is facilitating an increased understanding of the molecular mechanisms underlying virulence as well as guiding the development of disease control strategies. The purpose of this review is to give a chronological perspective on the evolution of the methodologies used in effector discovery from physical isolation and in silico predictions, to functional characterization of the effectors of filamentous plant pathogens and identification of their host targets.

Nature's genetic screens: using genome-wide association studies for effector discovery.

Nature's genetic screens: using genome-wide association studies for effector discovery.

Comparative genomics and prediction of conditionally dispensable sequences in legume-infecting Fusarium oxysporum formae speciales facilitates identification of candidate effectors.

BackgroundSoil-borne fungi of the Fusarium oxysporum species complex cause devastating wilt disease on many crops including legumes that supply human dietary protein needs across many parts of the globe. We present and compare draft genome assemblies for three legume-infecting formae speciales (ff. spp.): F. oxysporum f. sp. ciceris (Foc-38-1) and f. sp. pisi (Fop-37622), significant pathogens of chickpea and pea respectively, the world’s second and third most important grain legumes, and lastly f. sp. medicaginis (Fom-5190a) for which we developed a model legume pathosystem utilising Medicago truncatula.ResultsFocusing on the identification of pathogenicity gene content, we leveraged the reference genomes of Fusarium pathogens F. oxysporum f. sp. lycopersici (tomato-infecting) and F. solani (pea-infecting) and their well-characterised core and dispensable chromosomes to predict genomic organisation in the newly sequenced legume-infecting isolates. Dispensable chromosomes are not essential for growth and in Fusarium species are known to be enriched in host-specificity and pathogenicity-associated genes. Comparative genomics of the publicly available Fusarium species revealed differential patterns of sequence conservation across F. oxysporum formae speciales, with legume-pathogenic formae speciales not exhibiting greater sequence conservation between them relative to non-legume-infecting formae speciales, possibly indicating the lack of a common ancestral source for legume pathogenicity. Combining predicted dispensable gene content with in planta expression in the model legume-infecting isolate, we identified small conserved regions and candidate effectors, four of which shared greatest similarity to proteins from another legume-infecting ff. spp.ConclusionsWe demonstrate that distinction of core and potential dispensable genomic regions of novel F. oxysporum genomes is an effective tool to facilitate effector discovery and the identification of gene content possibly linked to host specificity. While the legume-infecting isolates didn’t share large genomic regions of pathogenicity-related content, smaller regions and candidate effector proteins were highly conserved, suggesting that they may play specific roles in inducing disease on legume hosts.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2486-8) contains supplementary material, which is available to authorized users.

Effector discovery in the fungal wheat pathogen Zymoseptoria tritici.

Fungal plant pathogens, such as Zymoseptoria tritici (formerly known as Mycosphaerella graminicola), secrete repertoires of effectors to facilitate infection or trigger host defence mechanisms. The discovery and functional characterization of effectors provides valuable knowledge that can contribute to the design of new and effective disease management strategies. Here, we combined bioinformatics approaches with expression profiling during pathogenesis to identify candidate effectors of Z. tritici. In addition, a genetic approach was conducted to map quantitative trait loci (QTLs) carrying putative effectors, enabling the validation of both complementary strategies for effector discovery. In planta expression profiling revealed that candidate effectors were up-regulated in successive waves corresponding to consecutive stages of pathogenesis, contrary to candidates identified by QTL mapping that were, overall, expressed at low levels. Functional analyses of two top candidate effectors (SSP15 and SSP18) showed their dispensability for Z. tritici pathogenesis. These analyses reveal that generally adopted criteria, such as protein size, cysteine residues and expression during pathogenesis, may preclude an unbiased effector discovery. Indeed, genetic mapping of genomic regions involved in specificity render alternative effector candidates that do not match the aforementioned criteria, but should nevertheless be considered as promising new leads for effectors that are crucial for the Z. tritici-wheat pathosystem.

Plant immunity in plant-aphid interactions.

Aphids are economically important pests that cause extensive feeding damage and transmit viruses. While some species have a broad host range and cause damage to a variety of crops, others are restricted to only closely related plant species. While probing and feeding aphids secrete saliva, containing effectors, into their hosts to manipulate host cell processes and promote infestation. Aphid effector discovery studies pointed out parallels between infection and infestation strategies of plant pathogens and aphids. Interestingly, resistance to some aphid species is known to involve plant resistance proteins with a typical NB-LRR domain structure. Whether these resistance proteins indeed recognize aphid effectors to trigger ETI remains to be elucidated. In addition, it was recently shown that unknown aphid derived elicitors can initiate reactive oxygen species (ROS) production and callose deposition and that these responses were dependent on BAK1 (BRASSINOSTERIOD INSENSITIVE 1-ASSOCIATED RECEPTOR KINASE 1) which is a key component of the plant immune system. In addition, BAK-1 contributes to non-host resistance to aphids pointing to another parallel between plant-pathogen and – aphid interactions. Understanding the role of plant immunity and non-host resistance to aphids is essential to generate durable and sustainable aphid control strategies. Although insect behavior plays a role in host selection and non-host resistance, an important observation is that aphids interact with non-host plants by probing the leaf surface, but are unable to feed or establish colonization. Therefore, we hypothesize that aphids interact with non-host plants at the molecular level, but are potentially not successful in suppressing plant defenses and/or releasing nutrients.

Avirulence effector discovery in a plant galling and plant parasitic arthropod, the Hessian fly (Mayetiola destructor).

Highly specialized obligate plant-parasites exist within several groups of arthropods (insects and mites). Many of these are important pests, but the molecular basis of their parasitism and its evolution are poorly understood. One hypothesis is that plant parasitic arthropods use effector proteins to defeat basal plant immunity and modulate plant growth. Because avirulence (Avr) gene discovery is a reliable method of effector identification, we tested this hypothesis using high-resolution molecular genetic mapping of an Avr gene (vH13) in the Hessian fly (HF, Mayetiola destructor), an important gall midge pest of wheat (Triticum spp.). Chromosome walking resolved the position of vH13, and revealed alleles that determine whether HF larvae are virulent (survive) or avirulent (die) on wheat seedlings carrying the wheat H13 resistance gene. Association mapping found three independent insertions in vH13 that appear to be responsible for H13-virulence in field populations. We observed vH13 transcription in H13-avirulent larvae and the salivary glands of H13-avirulent larvae, but not in H13-virulent larvae. RNA-interference-knockdown of vH13 transcripts allowed some H13-avirulent larvae to escape H13-directed resistance. vH13 is the first Avr gene identified in an arthropod. It encodes a small modular protein with no sequence similarities to other proteins in GenBank. These data clearly support the hypothesis that an effector-based strategy has evolved in multiple lineages of plant parasites, including arthropods.

Resequencing and Comparative Genomics of Stagonospora nodorum: Sectional Gene Absence and Effector Discovery

Stagonospora nodorum is an important wheat (Triticum aestivum) pathogen in many parts of the world, causing major yield losses. It was the first species in the large fungal Dothideomycete class to be genome sequenced. The reference genome sequence (SN15) has been instrumental in the discovery of genes encoding necrotrophic effectors that induce disease symptoms in specific host genotypes. Here we present the genome sequence of two further S. nodorum strains (Sn4 and Sn79) that differ in their effector repertoire from the reference. Sn79 is avirulent on wheat and produces no apparent effectors when infiltrated onto many cultivars and mapping population parents. Sn4 is pathogenic on wheat and has virulences not found in SN15. The new strains, sequenced with short-read Illumina chemistry, are compared with SN15 by a combination of mapping and de novo assembly approaches. Each of the genomes contains a large number of strain-specific genes, many of which have no meaningful similarity to any known gene. Large contiguous sections of the reference genome are absent in the two newly sequenced strains. We refer to these differences as “sectional gene absences.” The presence of genes in pathogenic strains and absence in Sn79 is added to computationally predicted properties of known proteins to produce a list of likely effector candidates. Transposon insertion was observed in the mitochondrial genomes of virulent strains where the avirulent strain retained the likely ancestral sequence. The study suggests that short-read enabled comparative genomics is an effective way to both identify new S. nodorum effector candidates and to illuminate evolutionary processes in this species.

Nematode effector proteins: an emerging paradigm of parasitism

Phytonematodes use a stylet and secreted effectors to modify host cells and ingest nutrients to support their growth and development. The molecular function of nematode effectors is currently the subject of intense investigation. In this review, we summarize our current understanding of nematode effectors, with a particular focus on proteinaceous stylet-secreted effectors of sedentary endoparasitic phytonematodes, for which a wealth of information has surfaced in the past 10 yr. We provide an update on the effector repertoires of several of the most economically important genera of phytonematodes and discuss current approaches to dissecting their function. Lastly, we highlight the latest breakthroughs in effector discovery that promise to shed new light on effector diversity and function across the phylum Nematoda.

Identifying Allosteric Binding Sites in Proteins with a Two-State Go̅ Model for Novel Allosteric Effector Discovery.

Allostery is a common mechanism of controlling many biological processes such as enzyme catalysis, signal transduction, and metabolic regulation. The use of allostery to regulate protein activity is an important and promising strategy in drug discovery and biological network regulation. In order to modulate protein activity by allostery, predictive methods need to be developed to discover allosteric binding sites. In the present study, we developed a new approach to identify allosteric sites in proteins based on the coarse-grained two-state Go̅ model. Starting from the concept that allostery is a conformation population shift process, we first constructed an ensemble of two functional states of a protein and tuned the energy landscape to bias one state. We then added perturbations to a binding site and monitored the population distribution of the new ensemble. If population redistribution occurred, then the binding perturbed site was predicted as a potential allosteric site. Our approach successfully identified all the known allosteric sites in a set of test proteins. Several new allosteric sites in the test proteins were also predicted. By use of one of the new allosteric sites predicted from Escherichia coli phosphoglycerate dehydrogenase (PGDH), novel allosteric regulating molecules were screened by molecular docking and enzymatic assay. Three novel allosteric inhibitors were discovered and their binding modes were confirmed by mutation experiments and competitive assay. The IC50 of the strongest inhibitor discovered was 21 μM, which is comparable to that of the native allosteric inhibitor l-serine. The novel allosteric site discovered in PGDH is l-serine-independent, and inhibitors targeting this site can be used as novel regulators of the E. coli serine synthesis pathway. Our approach for allosteric site prediction is generally applicable and the predicted sites can be used in discovering novel allosteric regulating molecules.

Roadmap for future research on plant pathogen effectors

Bacterial and eukaryotic plant pathogens deliver effector proteins into plant cells to promote pathogenesis. Bacterial pathogens containing type III protein secretion systems are known to inject many of these effectors into plant cells. More recently, oomycete pathogens have been shown to possess a large family of effectors containing the RXLR motif, and many effectors are also being discovered in fungal pathogens. Although effector activities are largely unknown, at least a subset suppress plant immunity. A plethora of new plant pathogen genomes that will soon be available thanks to next-generation sequencing technologies will allow the identification of many more effectors. This article summarizes the key approaches used to identify plant pathogen effectors, many of which will continue to be useful for future effector discovery. Thus, it can be viewed as a 'roadmap' for effector and effector target identification. Because effectors can be used as tools to elucidate components of innate immunity, advances in our understanding of effectors and their targets should lead to improvements in agriculture.

Proposed Guidelines for a Unified Nomenclature and Phylogenetic Analysis of Type III Hop Effector Proteins in the Plant Pathogen Pseudomonas syringae

Pathovars of Pseudomonas syringae interact with their plant hosts via the action of Hrp outer protein (Hop) effector proteins, injected into plant cells by the type III secretion system (TTSS). Recent availability of complete genome sequences for a number of P. syringae pathovars has led to a significant increase in the rate of effector discovery. However, lack of a systematic nomenclature has resulted in multiple names being assigned to the same Hop, unrelated Hops designated by the same alphabetic character, and failure of name choices to reflect consistent standards of experimental confirmation or phylogenetic relatedness. Therefore, specific experimental and bioinformatic criteria are proposed for proteins to be designated as Hops. A generic Hop name structure, HopXY#pv strain, also is proposed, wherein family membership is indicated by the alphabetic characters, subgroup membership numerically, and source pathovar and strain in subscript. Guidelines are provided for phylogenetic characterization and name selection for Hops that are novel, related to previously characterized Hops, chimeras, pseudogenes, truncations, or nonexpressed alleles. Phylogenetic analyses of previously characterized Hops are described, the results of which have been used to guide their integration into the proposed nomenclature.