Comparative secretome analysis of Striga and Cuscuta species identifies candidate virulence factors for two evolutionarily independent parasitic plant lineages.

BACKGROUND: Many parasitic plants of the genera Striga and Cuscuta inflict huge agricultural damage worldwide. To form and maintain a connection with a host plant, parasitic plants deploy virulence factors (VFs) that interact with host biology. They possess a secretome that represents the complement of proteins secreted from cells and like other plant parasites such as fungi, bacteria or nematodes, some secreted proteins represent VFs crucial to successful host colonisation. Understanding the genome-wide complement of putative secreted proteins from parasitic plants, and their expression during host invasion, will advance understanding of virulence mechanisms used by parasitic plants to suppress/evade host immune responses and to establish and maintain a parasite-host interaction. RESULTS: We conducted a comparative analysis of the secretomes of root (Striga spp.) and shoot (Cuscuta spp.) parasitic plants, to enable prediction of candidate VFs. Using orthogroup clustering and protein domain analyses we identified gene families/functional annotations common to both Striga and Cuscuta species that were not present in their closest non-parasitic relatives (e.g. strictosidine synthase like enzymes), or specific to either the Striga or Cuscuta secretomes. For example, Striga secretomes were strongly associated with 'PAR1' protein domains. These were rare in the Cuscuta secretomes but an abundance of 'GMC oxidoreductase' domains were found, that were not present in the Striga secretomes. We then conducted transcriptional profiling of genes encoding putatively secreted proteins for the most agriculturally damaging root parasitic weed of cereals, S. hermonthica. A significant portion of the Striga-specific secretome set was differentially expressed during parasitism, which we probed further to identify genes following a 'wave-like' expression pattern peaking in the early penetration stage of infection. We identified 39 genes encoding putative VFs with functions such as cell wall modification, immune suppression, protease, kinase, or peroxidase activities, that are excellent candidates for future functional studies. CONCLUSIONS: Our study represents a comprehensive secretome analysis among parasitic plants and revealed both similarities and differences in candidate VFs between Striga and Cuscuta species. This knowledge is crucial for the development of new management strategies and delaying the evolution of virulence in parasitic weeds.

Human mesenchymal stem cell secretomes: Factors affecting profiling and challenges in clinical application.

The promising field of regenerative medicine is thrilling as it can repair and restore organs for various debilitating diseases. Mesenchymal stem cells are one of the main components in regenerative medicine that work through the release of secretomes. By adopting the use of the secretome in cell-free-based therapy, we may be able to address the challenges faced in cell-based therapy. As one of the components of cell-free-based therapy, secretome has the advantage of a better safety and efficacy profile than mesenchymal stem cells. However, secretome has its challenges that need to be addressed, such as its bioprocessing methods that may impact the secretome content and its mechanisms of action in clinical settings. Effective and standardization of bioprocessing protocols are important to ensure the supply and sustainability of secretomes for clinical applications. This may eventually impact its commercialization and marketability. In this review, the bioprocessing methods and their impacts on the secretome profile and treatment are discussed. This improves understanding of its fundamental aspects leading to potential clinical applications.

Three-Dimensionally Cultured Jaw Periosteal Cells Attenuate Macrophage Activation of CD4+ T Cells and Inhibit Osteoclastogenesis.

The implementation of a successful therapeutic approach that includes tissue-engineered grafts requires detailed analyses of graft-immune cell interactions in order to predict possible immune reactions after implantation. The phenotypic plasticity of macrophages plays a central role in immune cell chemotaxis, inflammatory regulation and bone regeneration. The present study addresses effects emanating from JPC-seeded ß-TCP constructs (3DJPCs) co-cultivated with THP-1 derived M1/M2 macrophages within a horizontal co-culture system. After five days of co-culture, macrophage phenotype and chemokine secretion were analyzed by flow cytometry, quantitative PCR and proteome arrays. The results showed that pro-inflammatory factors in M1 macrophages were inhibited by 3DJPCs, while anti-inflammatory factors were activated, possibly affected by the multiple chemokines secreted by 3D-cultured JPCs. In addition, osteoclast markers of polarized macrophages were inhibited by osteogenically induced 3DJPCs. Functional assays revealed a significantly lower percentage of proliferating CD4+ T cells in the groups treated with secretomes from M1/M2 macrophages previously co-cultured with 3DJPCs compared to controls without secretomes. Quantifications of pit area resorption assays showed evidence that supernatants from 3DJPCs co-cultured with M1/M2 macrophages were able to completely suppress osteoclast maturation, compared to the control group without secretomes. These findings demonstrate the ability of 3D cultured JPCs to modulate macrophage plasticity.

Warhorses in soil bioremediation: Seed biopriming with PGPF secretome to phytostimulate crop health under heavy metal stress.

The fungal symbiosis with the plant root system is importantly recognized as a plant growth promoting fungi (PGPFs), as well as elicitor of plant defence against different biotic and abiotic stress conditions. Thus PGPFs are playing as a key trouper in enhancing agricultural quality and increased crop production and paving a way towards a sustainable agriculture. Due to increased demand of food production, the over and unscientific usage of chemical fertilizers has led to the contamination of soil by organic and inorganic wastes impacting on soil quality, crops quality effecting on export business of agricultural products. The application of microbial based consortium like plant growth promoting fungi is gaining worldwide importance due to their multidimensional activity. These activities are through plant growth promotion, induction of systemic resistance, disease combating and detoxification of organic and inorganic toxic chemicals, a heavy metal tolerance ability. The master key behind these properties exhibited by PGPFs are attributed towards various secretory biomolecules (secondary metabolites or enzymes or metabolites) secreted by the fungi during interaction mechanism. The present review is focused on the multidimensional role PGPFs as elicitors of Induced systemic resistance against phytopathogens as well as heavy metal detoxifier through seed biopriming and biofortification methods. The in-sights on PGPFs and their probable mechanistic nature contributing towards plants to withstand heavy metal stress and stress alleviation by activating of various stress regulatory pathways leading to secretion of low molecular weight compounds like organic compounds, glomalin, hydrophobins, etc,. Thus projecting the importance of PGPFs and further requirement of research in developing PGPFs based molecules and combining with trending Nano technological approaches for enhanced heavy metal stress alleviations in plant and soil as well as establishing a sustainable agriculture.

Deep into the Apoplast: Grapevine and Plasmopara viticola Proteomes Reveal the Secret Beneath Host and Pathogen Communication at 6 h After Contact.

The apoplast is the first hub of plant-pathogen communication where pathogen effectors are recognized by plant defensive proteins and cell receptors, thus activating signal transduction pathways. As a result of this first contact, the host triggers a defense response that involves the modulation of extra- and intracellular proteins. In grapevine-pathogen interactions, little is known about the trafficking between extra- and intracellular spaces. Grapevine is an economically important crop that relies on heavy fungicide use to control several diseases, and a deeper knowledge on the activation of its immune response is crucial to define new control strategies. In this study, we focused on the first 6 h postinoculation with Plasmopara viticola to evaluate grapevine proteome modulation in the apoplast. The in planta P. viticola proteome was also assessed to enable a deeper understanding of plant-pathogen communication. Our results showed that several plant mechanisms are triggered in the tolerant grapevine cultivar Regent after inoculation, such as oomycete recognition, plant cell wall modifications, reactive oxygen species signaling, and secretion of proteins to disrupt oomycete structures. On the other hand, P. viticola proteins related to development and virulence were the most predominant. This pioneer study highlights the early dynamics of cellular communication in grapevine defense that leads to the successful establishment of an incompatible interaction.

Can mesenchymal stem/stromal cells and their secretomes combat bacterial persisters?

The increasing number of life-threatening infections caused by persister bacteria is associated with various issues, including antimicrobial resistance and biofilm formation. Infections due to persister cells are often difficult to suppress without the use of last-resort antibiotics. Throughout the world, bacterial persistence and resistance create an unmet clinical demand for the exploration of newly introduced therapeutic approaches. Mesenchymal stem / stromal cells (MSCs) have an antimicrobial activity to protect against bacterial infections, including those caused by bacterial persisters. MSCs have substantial potential to secrete antimicrobial peptides (AMPs), including cathelicidin, beta-defensins, lipocalin-2, hepcidin, indoleamine 2,3-dioxygenase (IDO), cysteine proteases, and inducible nitric oxide synthases (iNOS). MSCs possess the potential to contribute to innate immunity by regulating the immune response. Recently, MSCs and their secreted components have been reported to improve antimicrobial activity. Bactericidal activity by MSCs and their secretomes has been shown to be mediated in part by the secretion of AMPs. Even though they were discovered more than 80 years ago, therapeutic options for persisters are restricted, and there is an urgent need for alternative treatment regimens. Hence, this review intends to critically assess the current literature on the effects of MSCs and their secretomes on persister bacteria. MSCs and their secretome-based therapies could be preferred as an up-and-coming approach to reinforce the antimicrobial efficiency in persister infections.

Mesenchymal Stem Cell-Derived Extracellular Vesicles: Progress and Remaining Hurdles in Developing Regulatory Compliant Quality Control Assays.

Regenerative medicine is shaping into a new paradigm and could be the future medicine driven by the therapeutic capabilities shown by mesenchymal stem cell-derived extracellular vesicles (MSC-EVs). Despite the advantages and promises, the therapeutic effectiveness of MSC-EVs in some clinical applications is restricted due to inconsistent manufacturing process and the lack of stringent quality control (QC) measurement. In particular, QC assays which are crucial to confirm the safety, efficacy, and quality of MSC-EVs available for end use are poorly designed. Hence, in this review, characterization of MSC-EVs and quality control guidelines for biologics are presented, with special attention given to the description of technical know-how in developing QC assays for MSC-EVs adhering to regulatory guidelines. The remaining challenges surrounding the development of potency and stability of QC assays are also addressed.

Global genomic and proteomic analysis indicates co-evolution of Neisseria species and with their human host.

Neisseria, a genus from the beta-proteobacteria class, is of potential clinical importance. This genus contains both pathogenic and commensal strains. Gonorrhea and meningitis are two major diseases caused by pathogens belonging to this genus. With the increased use of antimicrobial agents against these pathogens they have evolved the antimicrobial resistance capacity making these diseases nearly untreatable. The set of anti-bacterial resistance genes (resistome) and genes associated with signal processing (secretomes) are crucial for the host-microbial interaction. With the virtue of whole-genome sequences and computational biology, it is now possible to study the genomic and proteomic riddles of Neisseria along with their comprehensive evolutionary and metabolic profiling. We have studied relative synonymous codon usage, amino acid usage, reverse ecology, comparative genomics, evolutionary analysis and pathogen-host (Neisseria-human) interaction through bioinformatics analysis. Our analysis revealed the co-evolution of Neisseria genomes with the human host. Moreover, the co-occurrence of Neisseria and humans has been supported through reverse ecology analysis. A differential pattern of the evolutionary rate of resistomes and secretomes was evident among the pathogenic and commensal strains. Comparative genomics supported the presence of virulent genes in both pathogenic and commensal strains of the select genus. Our analysis also indicated a transition from commensal to pathogenic Neisseria strains through the long run of evolution.

Perivascular Stem Cell-Derived Cyclophilin A Improves Uterine Environment with Asherman's Syndrome via HIF1α-Dependent Angiogenesis.

Asherman's syndrome (AS) is characterized by intrauterine adhesions or fibrosis resulting from scarring inside the endometrium. AS is associated with infertility, recurrent miscarriage, and placental abnormalities. Although mesenchymal stem cells show therapeutic promise for the treatment of AS, the molecular mechanisms underlying its pathophysiology remain unclear. We ascertained that mice with AS, like human patients with AS, suffer from extensive fibrosis, oligo/amenorrhea, and infertility. Human perivascular stem cells (hPVSCs) from umbilical cords repaired uterine damage in mice with AS, regardless of their delivery routes. In mice with AS, embryo implantation is aberrantly deferred, which leads to intrauterine growth restriction followed by no delivery at term. hPVSC administration significantly improved implantation defects and subsequent poor pregnancy outcomes via hypoxia inducible factor 1α (HIF1α)-dependent angiogenesis in a dose-dependent manner. Pharmacologic inhibition of HIF1α activity hindered hPVSC actions on pregnancy outcomes, whereas stabilization of HIF1α activity facilitated such actions. Furthermore, therapeutic effects of hPVSCs were not observed in uterine-specific HIF1α-knockout mice with AS. Secretome analyses of hPVSCs identified cyclophilin-A as the major paracrine factor for hPVSC therapy via HIF1α-dependent angiogenesis. Collectively, we demonstrate that hPVSCs-derived cyclophilin-A facilitates HIF1α-dependent angiogenesis to ameliorate compromised uterine environments in mice with AS, representing the major pathophysiologic features of humans with AS.

Current Status of Angiogenic Cell Therapy and Related Strategies Applied in Critical Limb Ischemia.

Critical limb ischemia (CLI) constitutes the most severe form of peripheral arterial disease (PAD), it is characterized by progressive blockade of arterial vessels, commonly correlated to atherosclerosis. Currently, revascularization strategies (bypass grafting, angioplasty) remain the first option for CLI patients, although less than 45% of them are eligible for surgical intervention mainly due to associated comorbidities. Moreover, patients usually require amputation in the short-term. Angiogenic cell therapy has arisen as a promising alternative for these "no-option" patients, with many studies demonstrating the potential of stem cells to enhance revascularization by promoting vessel formation and blood flow recovery in ischemic tissues. Herein, we provide an overview of studies focused on the use of angiogenic cell therapies in CLI in the last years, from approaches testing different cell types in animal/pre-clinical models of CLI, to the clinical trials currently under evaluation. Furthermore, recent alternatives related to stem cell therapies such as the use of secretomes, exosomes, or even microRNA, will be also described.

hTERT-Driven Immortalization of RDEB Fibroblast and Keratinocyte Cell Lines Followed by Cre-Mediated Transgene Elimination.

The recessive form of dystrophic epidermolysis bullosa (RDEB) is a crippling disease caused by impairments in the junctions of the dermis and the basement membrane of the epidermis. Using ectopic expression of hTERT/hTERT + BMI-1 in primary cells, we developed expansible cultures of RDEB fibroblasts and keratinocytes. We showed that they display the properties of their founders, including morphology, contraction ability and expression of the respective specific markers including reduced secretion of type VII collagen (C7). The immortalized keratinocytes retained normal stratification in 3D skin equivalents. The comparison of secreted protein patterns from immortalized RDEB and healthy keratinocytes revealed the differences in the contents of the extracellular matrix that were earlier observed specifically for RDEB. We demonstrated the possibility to reverse the genotype of immortalized cells to the state closer to the progenitors by the Cre-dependent hTERT switch off. Increased ß-galactosidase activity and reduced proliferation of fibroblasts were shown after splitting out of transgenes. We anticipate our cell lines to be tractable models for studying RDEB from the level of single-cell changes to the evaluation of 3D skin equivalents. Our approach permits the creation of standardized and expandable models of RDEB that can be compared with the models based on primary cell cultures.

Activation of TLR4 of Mesenchymal Stem Cells Enhances the Regenerative Properties of Their Secretomes.

Mesenchymal stem cells (MSC), like macrophages, can be polarized in vitro. In particular, activation of type 4 Toll-like receptor in MSC leads to the appearance of the so-called "proinflammatory" MSC phenotype (MSC1). We showed that secretome (conditioned media) of MSC1 can affect the wound healing processes: promote healing and modulate exudative inflammation and subsequent fibroplastic processes in the damaged area. These effects of secretomes of polarized MSC were superior to those of intact MSC.

Progresses on bacterial secretomes enlighten research on Mycoplasma secretome.

Bacterial secretome is a comprehensive catalog of bacterial proteins that are released or secreted outside the cells. They offer a number of factors that possess several significant roles in virulence as well as cell to cell communication and hence play a core role in bacterial pathogenesis. Sometimes these proteins are bounded with membranes giving them the shape of vesicles called extracellular vesicles (EVs) or outer membrane vesicles (OMVs). Bacteria secrete these proteins via Sec and Tat pathways into the periplasm. Secreted proteins have found to be important as diagnostic markers as well as antigenic factors for the development of an effective candidate vaccine. Recently, the research in the field of secretomics is growing up and getting more interesting due to their direct involvement in the pathogenesis of the microorganisms leading to the infection. Many pathogenic bacteria have been studied for their secretome and the results illustrated novel antigens. This review highlights the secretome studies of different pathogenic bacteria in humans and animals, general secretion mechanisms, different approaches and challenges in the secretome of Mycoplasma sp.

Insights into the functionality of fungal community during the large scale aerobic co-composting process of swine manure and rice straw.

Composting is a cost-efficient method of transferring various unstable and complex organic matters into a stable and humus-like substance, during which various fungus play a critical role in the decomposition of organic matters. In this study, the rice straw and swine manure co-composting were carried out in a pilot-scale, and the evolution of various biochemical parameters and fungi community were detected at different time points. The results showed that most of the parameters fluctuated strongly at the thermophilic phase (THP), and the Canonical Correlation Analysis (CCA) results showed that Mycothermus spp. and Aspergillus spp. were with abundances of 47.82% and 3.51%, respectively, which were considered as the core fungi during the composting process. In addition, five culturable thermophilic filamentous fungi were isolated from the samples obtained at the high temperature stage, among which Aspergillus fumigatus were considered as the core specie at this special phase. The capacity of lignocellulose degradation of this strains was also evaluated by analyzing the secretomes in a coculture group with rice straw and crystalline cellulose as carbon sources, and the identified proteins illustrated that the enzymes were chiefly secreted by A. fumigatus in both treatments, with the abundances of 91.41% and 85.19%, respectively.

Concise Review: Optimized Strategies for Stem Cell-Based Therapy in Myocardial Repair: Clinical Translatability and Potential Limitation.

Ischemic heart diseases (IHDs) remain major public health problems with high rates of morbidity and mortality worldwide. Despite significant advances, current therapeutic approaches are unable to rescue the extensive and irreversible loss of cardiomyocytes caused by severe ischemia. Over the past 16 years, stem cell-based therapy has been recognized as an innovative strategy for cardiac repair/regeneration and functional recovery after IHDs. Although substantial preclinical animal studies using a variety of stem/progenitor cells have shown promising results, there is a tremendous degree of skepticism in the clinical community as many stem cell trials do not confer any beneficial effects. How to accelerate stem cell-based therapy toward successful clinical application attracts considerate attention. However, many important issues need to be fully addressed. In this Review, we have described and compared the effects of different types of stem cells with their dose, delivery routes, and timing that have been routinely tested in recent preclinical and clinical findings. We have also discussed the potential mechanisms of action of stem cells, and explored the role and underlying regulatory components of stem cell-derived secretomes/exosomes in myocardial repair. Furthermore, we have critically reviewed the different strategies for optimizing both donor stem cells and the target cardiac microenvironments to enhance the engraftment and efficacy of stem cells, highlighting their clinical translatability and potential limitation. Stem Cells 2018;36:482-500.

Applicability of regenerative medicine and tissue engineering for the treatment of stress urinary incontinence in female patients.

Stress urinary incontinence (SUI) is an age health-related issue that generates interest due to its considerable public health burden and the controversies surrounding treatment. It is highly prevalent affecting 30-40% of all women during their lifetime. Midurethral slings are the standard of gold standard treatment for female patients with SUI. They have excellent short-term cure rates; however, their efficacy tends to decrease over time and patients often report urinary incontinence recurrence. This paper addresses the applicability of regenerative medicine and tissue engineering for the treatment of SUI in female patients. Cell-based treatment with periurethral injection of autologous adipose or muscle-derived stem cells have been used for SUI; however, the cure rates and SUI recurrence at 1 year were 40% and 70%, respectively. Novel minimally invasive approaches, such as low-intensity extracorporeal shock wave therapies have shown promising results in SUI animal models. In addition, local injection of growth factors, chemokines, and specific antibodies have shown histological evidence of neoangiogenesis, nerve, and sphincter regeneration in rodents and nonhuman primates with SUI. The use of bioactive factors and proteins secreted by cells, which is called secretomes, have been recognized as key regulators of various mechanisms, such as immunomodulation, angiogenesis, inflammation, apoptosis, and tissue repair. Emerging therapies aiming to replace or restore tissues and organ functionality may improve the long-term efficacy and in the near future may represent the standard of care for the treatment of SUI.

Proteomic Analysis of Cancer-Associated Fibroblasts Reveals a Paracrine Role for MFAP5 in Human Oral Tongue Squamous Cell Carcinoma.

Bidirectional communication between cells and their microenvironment is crucial for both normal tissue homeostasis and tumor growth. During the development of oral tongue squamous cell carcinoma (OTSCC), cancer-associated fibroblasts (CAFs) create a supporting niche by maintaining a bidirectional crosstalk with cancer cells, mediated by classically secreted factors and various nanometer-sized vesicles, termed as extracellular vesicles (EVs). To better understand the role of CAFs within the tumor stroma and elucidate the mechanism by which secreted proteins contribute to OTSCC progression, we isolated and characterized patient-derived CAFs from resected tumors with matched adjacent tissue fibroblasts (AFs). Our strategy employed shotgun proteomics to comprehensively characterize the proteomes of these matched fibroblast populations. Our goals were to identify CAF-secreted factors (EVs and soluble) that can functionally modulate OTSCC cells in vitro and to identify novel CAF-associated biomarkers. Comprehensive proteomic analysis identified 4247 proteins, the most detailed description of a pro-tumorigenic stroma to date. We demonstrated functional effects of CAF secretomes (EVs and conditioned media) on OTSCC cell growth and migration. Comparative proteomics identified novel proteins associated with a CAF-like state. Specifically, MFAP5, a protein component of extracellular microfibrils, was enriched in CAF secretomes. Using in vitro assays, we demonstrated that MFAP5 activated OTSCC cell growth and migration via activation of MAPK and AKT pathways. Using a tissue microarray of richly annotated primary human OTSCCs, we demonstrated an association of MFAP5 expression with patient survival. In summary, our proteomics data of patient-derived stromal fibroblasts provide a useful resource for future mechanistic and biomarker studies.

Proteomics Insights into Autophagy.

Autophagy, a conserved cellular process by which cells recycle their contents either to maintain basal homeostasis or in response to external stimuli, has for the past two decades become one of the most studied physiological processes in cell biology. The 2016 Nobel Prize in Medicine and Biology awarded to Dr. Ohsumi Yoshinori, one of the first scientists to characterize this cellular mechanism, attests to its importance. The induction and consequent completion of the process of autophagy results in wide ranging changes to the cellular proteome as well as the secretome. MS-based proteomics affords the ability to measure, in an unbiased manner, the ubiquitous changes that occur when autophagy is initiated and progresses in the cell. The continuous improvements and advances in mass spectrometers, especially relating to ionization sources and detectors, coupled with advances in proteomics experimental design, has made it possible to study autophagy, among other process, in great detail. Innovative labeling strategies and protein separation techniques as well as complementary methods including immuno-capture/blotting/staining have been used in proteomics studies to provide more specific protein identification. In this review, we will discuss recent advances in proteomics studies focused on autophagy.

Comprehensive profiling of functional attributes, virulence potential and evolutionary dynamics in mycobacterial secretomes.

Mycobacterium is an interesting genus which not only includes intimidating pathogens, associated with severe devastations globally, but also comprises of non-pathogenic eco-friendly members that detoxify environmental pollutants. Secretory proteins of the mycobacterial communities are essential components which are firmly believed to facilitate proper cross-talk and apt communication with host cellular surroundings and environmental niche. Secretory elements also play vital roles in mycobacterial pathogenesis. In the present endeavor, an extensive profiling of mycobacterial secretomes, considering both pathogenic and non-pathogenic members, has been executed. Thorough analysis on amino acid composition and functional behavior of the mycobacterial secretory proteins has also been performed. In-depth scrutiny of biosynthetic cost of the secretory proteins with respect to the non-secretory ones indicated that the genus Mycobacterium strictly follows the policy of cost-minimization among the sets of imperative secretory proteins. Comprehensive assessment of potential virulence among the key secretory components signified that the pathogenic mycobacterial members possess a larger share of potentially virulent secretory elements in comparison to their non-pathogenic counterparts. Present analysis also revealed contrasted evolutionary features of the secretomes wherein secretory proteins were found to evolve faster than non-secretory proteins in mycobacterial pathogens but not in the concerned non-pathogens. Outcomes of present investigation promise to provide novel insights into the enigma of mycobacterial pathogenesis, bioremediation and adaptation in diverse niche and aid further scientific investigations associated with concerned research area.

EffectorP: predicting fungal effector proteins from secretomes using machine learning.

Eukaryotic filamentous plant pathogens secrete effector proteins that modulate the host cell to facilitate infection. Computational effector candidate identification and subsequent functional characterization delivers valuable insights into plant-pathogen interactions. However, effector prediction in fungi has been challenging due to a lack of unifying sequence features such as conserved N-terminal sequence motifs. Fungal effectors are commonly predicted from secretomes based on criteria such as small size and cysteine-rich, which suffers from poor accuracy. We present EffectorP which pioneers the application of machine learning to fungal effector prediction. EffectorP improves fungal effector prediction from secretomes based on a robust signal of sequence-derived properties, achieving sensitivity and specificity of over 80%. Features that discriminate fungal effectors from secreted noneffectors are predominantly sequence length, molecular weight and protein net charge, as well as cysteine, serine and tryptophan content. We demonstrate that EffectorP is powerful when combined with in planta expression data for predicting high-priority effector candidates. EffectorP is the first prediction program for fungal effectors based on machine learning. Our findings will facilitate functional fungal effector studies and improve our understanding of effectors in plant-pathogen interactions. EffectorP is available at http://effectorp.csiro.au.