Fusarium oxysporum effector clustering version 2: An updated pipeline to infer host range.

The fungus Fusarium oxysporum is infamous for its devastating effects on economically important crops worldwide. F. oxysporum isolates are grouped into formae speciales based on their ability to cause disease on different hosts. Assigning F. oxysporum strains to formae speciales using non-experimental procedures has proven to be challenging due to their genetic heterogeneity and polyphyletic nature. However, genetically diverse isolates of the same forma specialis encode similar repertoires of effectors, proteins that are secreted by the fungus and contribute to the establishment of compatibility with the host. Based on this observation, we previously designed the F. oxysporum Effector Clustering (FoEC) pipeline which is able to classify F. oxysporum strains by forma specialis based on hierarchical clustering of the presence of predicted putative effector sequences, solely using genome assemblies as input. Here we present the updated FoEC2 pipeline which is more user friendly, customizable and, due to multithreading, has improved scalability. It is designed as a Snakemake pipeline and incorporates a new interactive visualization app. We showcase FoEC2 by clustering 537 publicly available F. oxysporum genomes and further analysis of putative effector families as multiple sequence alignments. We confirm classification of isolates into formae speciales and are able to further identify their subtypes. The pipeline is available on github: https://github.com/pvdam3/FoEC2.

The root-invading pathogen Fusarium oxysporum targets pattern-triggered immunity using both cytoplasmic and apoplastic effectors.

Plant pathogens use effector proteins to promote host colonisation. The mode of action of effectors from root-invading pathogens, such as Fusarium oxysporum (Fo), is poorly understood. Here, we investigated whether Fo effectors suppress pattern-triggered immunity (PTI), and whether they enter host cells during infection. Eight candidate effectors of an Arabidopsis-infecting Fo strain were expressed with and without signal peptide for secretion in Nicotiana benthamiana and their effect on flg22-triggered and chitin-triggered reactive oxidative species (ROS) burst was monitored. To detect uptake, effector biotinylation by an intracellular Arabidopsis-produced biotin ligase was examined following root infection. Four effectors suppressed PTI signalling; two acted intracellularly and two apoplastically. Heterologous expression of a PTI-suppressing effector in Arabidopsis enhanced bacterial susceptibility. Consistent with an intracellular activity, host cell uptake of five effectors, but not of the apoplastically acting ones, was detected in Fo-infected Arabidopsis roots. Multiple Fo effectors targeted PTI signalling, uncovering a surprising overlap in infection strategies between foliar and root pathogens. Extracellular targeting of flg22 signalling by a microbial effector provides a new mechanism on how plant pathogens manipulate their host. Effector translocation appears independent of protein size, charge, presence of conserved motifs or the promoter driving its expression.

The genome of opportunistic fungal pathogen Fusarium oxysporum carries a unique set of lineage-specific chromosomes.

Fusarium oxysporum is a cross-kingdom fungal pathogen that infects plants and humans. Horizontally transferred lineage-specific (LS) chromosomes were reported to determine host-specific pathogenicity among phytopathogenic F. oxysporum. However, the existence and functional importance of LS chromosomes among human pathogenic isolates are unknown. Here we report four unique LS chromosomes in a human pathogenic strain NRRL 32931, isolated from a leukemia patient. These LS chromosomes were devoid of housekeeping genes, but were significantly enriched in genes encoding metal ion transporters and cation transporters. Homologs of NRRL 32931 LS genes, including a homolog of ceruloplasmin and the genes that contribute to the expansion of the alkaline pH-responsive transcription factor PacC/Rim1p, were also present in the genome of NRRL 47514, a strain associated with Fusarium keratitis outbreak. This study provides the first evidence, to our knowledge, for genomic compartmentalization in two human pathogenic fungal genomes and suggests an important role of LS chromosomes in niche adaptation.

Use of Comparative Genomics-Based Markers for Discrimination of Host Specificity in Fusarium oxysporum.

The polyphyletic nature of many formae speciales of Fusarium oxysporum prevents molecular identification of newly encountered strains based on conserved, vertically inherited genes. Alternative molecular detection methods that could replace labor- and time-intensive disease assays are therefore highly desired. Effectors are functional elements in the pathogen-host interaction and have been found to show very limited sequence diversity between strains of the same forma specialis, which makes them potential markers for host-specific pathogenicity. We therefore compared candidate effector genes extracted from 60 existing and 22 newly generated genome assemblies, specifically targeting strains affecting cucurbit plant species. Based on these candidate effector genes, a total of 18 PCR primer pairs were designed to discriminate between each of the seven Cucurbitaceae-affecting formae speciales When tested on a collection of strains encompassing different clonal lineages of these formae speciales, nonpathogenic strains, and strains of other formae speciales, they allowed clear recognition of the host range of each evaluated strain. Within Fusarium oxysporum f. sp. melonis more genetic variability exists than anticipated, resulting in three F. oxysporum f. sp. melonis marker patterns that partially overlapped with the cucurbit-infecting Fusarium oxysporum f. sp. cucumerinum, Fusarium oxysporum f. sp. niveum, Fusarium oxysporum f. sp. momordicae, and/or Fusarium oxysporum f. sp. lagenariae For F. oxysporum f. sp. niveum, a multiplex TaqMan assay was evaluated and was shown to allow quantitative and specific detection of template DNA quantities as low as 2.5 pg. These results provide ready-to-use marker sequences for the mentioned F. oxysporum pathogens. Additionally, the method can be applied to find markers distinguishing other host-specific forms of F. oxysporumIMPORTANCE Pathogenic strains of Fusarium oxysporum are differentiated into formae speciales based on their host range, which is normally restricted to only one or a few plant species. However, horizontal gene transfer between strains in the species complex has resulted in a polyphyletic origin of host specificity in many of these formae speciales This hinders accurate and rapid pathogen detection through molecular methods. In our research, we compared the genomes of 88 strains of F. oxysporum with each other, specifically targeting virulence-related genes that are typically highly similar within each forma specialis Using this approach, we identified marker sequences that allow the discrimination of F. oxysporum strains affecting various cucurbit plant species through different PCR-based methods.

Adaptation to the Host Environment by Plant-Pathogenic Fungi.

Many fungi can live both saprophytically and as endophyte or pathogen inside a living plant. In both environments, complex organic polymers are used as sources of nutrients. Propagation inside a living host also requires the ability to respond to immune responses of the host. We review current knowledge of how plant-pathogenic fungi do this. First, we look at how fungi change their global gene expression upon recognition of the host environment, leading to secretion of effectors, enzymes, and secondary metabolites; changes in metabolism; and defense against toxic compounds. Second, we look at what is known about the various cues that enable fungi to sense the presence of living plant cells. Finally, we review literature on transcription factors that participate in gene expression in planta or are suspected to be involved in that process because they are required for the ability to cause disease.

The tomato I gene for Fusarium wilt resistance encodes an atypical leucine-rich repeat receptor-like protein whose function is nevertheless dependent on SOBIR1 and SERK3/BAK1.

We have identified the tomato I gene for resistance to the Fusarium wilt fungus Fusarium oxysporum f. sp. lycopersici (Fol) and show that it encodes a membrane-anchored leucine-rich repeat receptor-like protein (LRR-RLP). Unlike most other LRR-RLP genes involved in plant defence, the I gene is not a member of a gene cluster and contains introns in its coding sequence. The I gene encodes a loopout domain larger than those in most other LRR-RLPs, with a distinct composition rich in serine and threonine residues. The I protein also lacks a basic cytosolic domain. Instead, this domain is rich in aromatic residues that could form a second transmembrane domain. The I protein recognises the Fol Avr1 effector protein, but, unlike many other LRR-RLPs, recognition specificity is determined in the C-terminal half of the protein by polymorphic amino acid residues in the LRRs just preceding the loopout domain and in the loopout domain itself. Despite these differences, we show that I/Avr1-dependent necrosis in Nicotiana benthamiana depends on the LRR receptor-like kinases (RLKs) SERK3/BAK1 and SOBIR1. Sequence comparisons revealed that the I protein and other LRR-RLPs involved in plant defence all carry residues in their last LRR and C-terminal LRR capping domain that are conserved with SERK3/BAK1-interacting residues in the same relative positions in the LRR-RLKs BRI1 and PSKR1. Tyrosine mutations of two of these conserved residues, Q922 and T925, abolished I/Avr1-dependent necrosis in N. benthamiana, consistent with similar mutations in BRI1 and PSKR1 preventing their interaction with SERK3/BAK1.

Effector profiles distinguish formae speciales of Fusarium oxysporum.

Formae speciales (ff.spp.) of the fungus Fusarium oxysporum are often polyphyletic within the species complex, making it impossible to identify them on the basis of conserved genes. However, sequences that determine host-specific pathogenicity may be expected to be similar between strains within the same forma specialis. Whole genome sequencing was performed on strains from five different ff.spp. (cucumerinum, niveum, melonis, radicis-cucumerinum and lycopersici). In each genome, genes for putative effectors were identified based on small size, secretion signal, and vicinity to a "miniature impala" transposable element. The candidate effector genes of all genomes were collected and the presence/absence patterns in each individual genome were clustered. Members of the same forma specialis turned out to group together, with cucurbit-infecting strains forming a supercluster separate from other ff.spp. Moreover, strains from different clonal lineages within the same forma specialis harbour identical effector gene sequences, supporting horizontal transfer of genetic material. These data offer new insight into the genetic basis of host specificity in the F. oxysporum species complex and show that (putative) effectors can be used to predict host specificity in F. oxysporum.

Comparative genomics of Fusarium oxysporum f. sp. melonis reveals the secreted protein recognized by the Fom-2 resistance gene in melon.

Development of resistant crops is the most effective way to control plant diseases to safeguard food and feed production. Disease resistance is commonly based on resistance genes, which generally mediate the recognition of small proteins secreted by invading pathogens. These proteins secreted by pathogens are called 'avirulence' proteins. Their identification is important for being able to assess the usefulness and durability of resistance genes in agricultural settings. We have used genome sequencing of a set of strains of the melon wilt fungus Fusarium oxysporum f. sp. melonis (Fom), bioinformatics-based genome comparison and genetic transformation of the fungus to identify AVRFOM2, the gene that encodes the avirulence protein recognized by the melon Fom-2 gene. Both an unbiased and a candidate gene approach identified a single candidate for the AVRFOM2 gene. Genetic complementation of AVRFOM2 in three different race 2 isolates resulted in resistance of Fom-2-harbouring melon cultivars. AvrFom2 is a small, secreted protein with two cysteine residues and weak similarity to secreted proteins of other fungi. The identification of AVRFOM2 will not only be helpful to select melon cultivars to avoid melon Fusarium wilt, but also to monitor how quickly a Fom population can adapt to deployment of Fom-2-containing cultivars in the field.

The Role of Pathogen-Secreted Proteins in Fungal Vascular Wilt Diseases.

A limited number of fungi can cause wilting disease in plants through colonization of the vascular system, the most well-known being Verticillium dahliae and Fusarium oxysporum. Like all pathogenic microorganisms, vascular wilt fungi secrete proteins during host colonization. Whole-genome sequencing and proteomics screens have identified many of these proteins, including small, usually cysteine-rich proteins, necrosis-inducing proteins and enzymes. Gene deletion experiments have provided evidence that some of these proteins are required for pathogenicity, while the role of other secreted proteins remains enigmatic. On the other hand, the plant immune system can recognize some secreted proteins or their actions, resulting in disease resistance. We give an overview of proteins currently known to be secreted by vascular wilt fungi and discuss their role in pathogenicity and plant immunity.

The AVR2-SIX5 gene pair is required to activate I-2-mediated immunity in tomato.

Plant-invading microbes betray their presence to a plant by exposure of antigenic molecules such as small, secreted proteins called 'effectors'. In Fusarium oxysporum f. sp. lycopersici (Fol) we identified a pair of effector gene candidates, AVR2-SIX5, whose expression is controlled by a shared promoter. The pathogenicity of AVR2 and SIX5 Fol knockouts was assessed on susceptible and resistant tomato (Solanum lycopersicum) plants carrying I-2. The I-2 NB-LRR protein confers resistance to Fol races carrying AVR2. Like Avr2, Six5 was found to be required for full virulence on susceptible plants. Unexpectedly, each knockout could breach I-2-mediated disease resistance. So whereas Avr2 is sufficient to induce I-2-mediated cell death, Avr2 and Six5 are both required for resistance. Avr2 and Six5 interact in yeast two-hybrid assays as well as in planta. Six5 and Avr2 accumulate in xylem sap of plants infected with the reciprocal knockouts, showing that lack of I-2 activation is not due to a lack of Avr2 accumulation in the SIX5 mutant. The effector repertoire of a pathogen determines its host specificity and its ability to manipulate plant immunity. Our findings challenge an oversimplified interpretation of the gene-for-gene model by showing requirement of two fungal genes for immunity conferred by one resistance gene.

The effector protein Avr2 of the xylem-colonizing fungus Fusarium oxysporum activates the tomato resistance protein I-2 intracellularly.

To promote host colonization, many plant pathogens secrete effector proteins that either suppress or counteract host defences. However, when these effectors are recognized by the host's innate immune system, they trigger resistance rather than promoting virulence. Effectors are therefore key molecules in determining disease susceptibility or resistance. We show here that Avr2, secreted by the vascular wilt fungus Fusarium oxysporum f. sp. lycopersici (Fol), shows both activities: it is required for full virulence in a susceptible host and also triggers resistance in tomato plants carrying the resistance gene I-2. Point mutations in AVR2, causing single amino acid changes, are associated with I-2-breakingFol strains. These point mutations prevent recognition by I-2, both in tomato and when both genes are co-expressed in leaves of Nicotiana benthamiana. Fol strains carrying the Avr2 variants are equally virulent, showing that virulence and avirulence functions can be uncoupled. Although Avr2 is secreted into the xylem sap when Fol colonizes tomato, the Avr2 protein can be recognized intracellularly by I-2, implying uptake by host cells.

The presence of a virulence locus discriminates Fusarium oxysporum isolates causing tomato wilt from other isolates.

Fusarium oxysporum is an asexual fungus that inhabits soils throughout the world. As a species, F. oxysporum can infect a very broad range of plants and cause wilt or root rot disease. Single isolates of F. oxysporum, however, usually infect one or a few plant species only. They have therefore been grouped into formae speciales (f.sp.) based on host specificity. Isolates able to cause tomato wilt (f.sp. lycopersici) do not have a single common ancestor within the F. oxysporum species complex. Here we show that, despite their polyphyletic origin, isolates belonging to f.sp. lycopersici all contain an identical genomic region of at least 8 kb that is absent in other formae speciales and non-pathogenic isolates, and comprises the genes SIX1, SIX2 and SHH1. In addition, SIX3, which lies elsewhere on the same chromosome, is also unique for f.sp. lycopersici. SIX1 encodes a virulence factor towards tomato, and the Six1, Six2 and Six3 proteins are secreted in xylem during colonization of tomato plants. We speculate that these genes may be part of a larger, dispensable region of the genome that confers the ability to cause tomato wilt and has spread among clonal lines of F. oxysporum through horizontal gene transfer. Our findings also have practical implications for the detection and identification of f.sp. lycopersici.

Colonization of the Arabidopsis rhizosphere by fluorescent Pseudomonas spp. activates a root-specific, ethylene-responsive PR-5 gene in the vascular bundle.

Plants of which the roots are colonized by selected strains of non-pathogenic, fluorescent Pseudomonas spp. develop an enhanced defensive capacity against a broad spectrum of foliar pathogens. In Arabidopsis thaliana, this rhizobacteria-induced systemic resistance (ISR) functions independently of salicylic acid but requires responsiveness to jasmonic acid and ethylene. In contrast to pathogen-induced systemic acquired resistance (SAR), ISR is not associated with systemic changes in the expression of genes encoding pathogenesis-related (PR) proteins. To identify genes that are specifically expressed in response to colonization of the roots by ISR-inducing Pseudomonas fluorescens WCS417r bacteria, we screened a collection of Arabidopsis enhancer trap and gene trap lines containing a transposable element of the Ac/Ds system and the GUS reporter gene. We identified an enhancer trap line (WET121) that specifically showed GUS activity in the root vascular bundle upon colonization of the roots by WCS417r. Fluorescent Pseudomonas spp. strains P. fluorescens WCS374r and P. putida WCS358r triggered a similar expression pattern, whereas ISR-non-inducing Escherichia coli bacteria did not. Exogenous application of the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC) mimicked the rhizobacteria-induced GUS expression pattern in the root vascular bundle, whereas methyl jasmonic acid and salicylic acid did not, indicating that the Ds element in WET121 is inserted in the vicinity of an ethylene-responsive gene. Analysis of the expression of the genes in the close vicinity of the Ds element revealed AtTLP1 as the gene responsible for the in cis activation of the GUS reporter gene in the root vascular bundle. AtTLP1 encodes a thaumatin-like protein that belongs to the PR-5 family of PR proteins, some of which possess antimicrobial properties. AtTLP1 knockout mutant plants showed normal levels of WCS417r-mediated ISR against the bacterial leaf pathogen Pseudomonas syringae pv. tomato DC3000, suggesting that expression of AtTLP1 in the roots is not required for systemic expression of ISR in the leaves. Together, these results indicate that induction of AtTLP1 is a local response of Arabidopsis roots to colonization by non-pathogenic fluorescent Pseudomonas spp. and is unlikely to play a role in systemic resistance.

A small, cysteine-rich protein secreted by Fusarium oxysporum during colonization of xylem vessels is required for I-3-mediated resistance in tomato.

A 12 kDa cysteine-rich protein is secreted by Fusarium oxysporum f. sp. lycopersici during colonization of tomato xylem vessels. Peptide sequences obtained with mass spectrometry allowed identification of the coding sequence. The gene encodes a 32 kDa protein, designated Six1 for secreted in xylem 1. The central part of Six1 corresponds to the 12 kDa protein found in xylem sap of infected plants. A mutant that had gained virulence on a tomato line with the I-3 resistance gene was found to have lost the SIX1 gene along with neighbouring sequences. Transformation of this mutant with SIX1 restored avirulence on the I-3 line. Conversely, deletion of the SIX1 gene in a wild-type strain results in breaking of I-3-mediated resistance. These results suggest that I-3-mediated resistance is based on recognition of Six1 secreted in xylem vessels.

A tomato xylem sap protein represents a new family of small cysteine-rich proteins with structural similarity to lipid transfer proteins.

The coding sequence of a major xylem sap protein of tomato was identified with the aid of mass spectrometry. The protein, XSP10, represents a novel family of extracellular plant proteins with structural similarity to plant lipid transfer proteins. The XSP10 gene is constitutively expressed in roots and lower stems. The decline of XSP10 protein levels in tomato infected with a fungal vascular pathogen may reflect breakdown or modification by the pathogen.

Different signalling pathways contribute to the control of GPD1 gene expression by osmotic stress in Saccharomyces cerevisiae.

Yeast cells respond to a shift to higher osmolarity by increasing the cellular content of the osmolyte glycerol. This response is accompanied by a stimulation of the expression of genes encoding enzymes in the glycerol production pathway. In this study the osmotic induction of one of those genes, GPD1, which encodes glycerol-3-phosphate dehydrogenase, was monitored in time course experiments. The response is independent of the osmolyte and consists of four apparent phases: a lag phase, an initial induction phase, a feedback phase and a sustained long-term induction. Osmotic shock with progressively higher osmolyte concentrations caused a prolonged lag phase. Deletion of HOG1, which encodes the terminal protein kinase of the high osmolarity glycerol (HOG) response pathway, led to an even longer lag phase and drastically lower basal and induced GPD1 mRNA levels. However, the induction was only moderately diminished. Overstimulation of Hog1p by deletion of the genes for the protein phosphatases PTP2 and PTP3 led to higher basal and induced mRNA levels and a shorter lag phase. The protein phosphatase calcineurin, which mediates salt-induced expression of some genes, does not appear to contribute to the control of GPD1 expression. Although GPD1 expression has so far not been reported to be controlled by a general stress response mechanism, heat-shock induction of the GPD1 mRNA level was observed. However, unregulated protein kinase A activity, which strongly affects the general stress response, only marginally altered the mRNA level of GPD1. The osmotic stimulation of GPD1 expression does not seem to be mediated by derepression, since deletion of the SSN6 gene, which encodes a general repressor, did not significantly alter the induction profile. A hypoosmotic shock led to a transient 10-fold drop of the GPD1 mRNA level. Neither the HOG nor the protein kinase C pathway, which is stimulated by a decrease in external osmolarity, is involved in this effect. It was concluded that osmotic regulation of GPD1 expression is the result of an interplay between different signalling pathways, some of which remain to be identified.