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1 Introduction

Fusarium oxysporum strains that are specialised on specific host plants are classified into formae speciales (ff. spp.) (singular forma specialis, abbr. f. sp.), such as Fusarium oxysporum f. sp. asparagi (asparagus); f. sp. cubense (banana); f. sp. dianthi (carnation); f. sp. lycopersici (tomato); f. sp. melonis (melon); f. sp. niveum (watermelon); f. sp. pisi (pea); f. sp. zingiberi (ginger); f. sp. vasinfectum (cotton); f. sp. medicaginis (Medicago); f. sp. ciceris (chickpea); f. sp. citri (orange); f. sp. cucumerinum (cucumber) and f. sp. conglutinans (canola and Brassica crops). While most of the above cause vascular wilts, not all formae speciales are primarily vascular pathogens, but cause foot, root rot, crown or bulb rots such as F. oxysporum f. sp. radicis-lycopersici (Agrios 2005).

Fusarium wilts are most destructive under warm conditions and thus particularly to horticultural production in greenhouses or in tropical climates. For example, Fusarium oxysporum f. sp. cubense (Foc) causes Panama disease on banana. Bananas are the world’s most popular fruit (FAO: www.fao.org) and have an estimated value of $44 billion globally (Ploetz 2015). In the 1950s the race 1 strain of Foc wiped out almost all banana production in South America and subsequently spread to other banana-growing regions of the world. Due to their susceptibility to Foc, the commercial Gros Michel banana cultivars were replaced by race 1-resistant Cavendish cultivars. However, the Cavendish variety is now under threat by Foc TR4 (tropical race 4) (reviewed by Ploetz 2015). Also of major concern is F. oxysporum f. sp. ciceris, which is a major pathogen of chickpea, the second most important legume crop worldwide with countries of tropical/sub-tropical South Asia by far the largest producers (FAO: www.fao.org). Typically this chickpea pathogen causes yield losses of 10–15 %, but complete loss can occur under conducive conditions (Trapero-Casas and Jiménez-Díaz 1985; Abera et al. 2011; Sharma et al. 2014).

2 Disease Symptoms and Pathogen Movement

F. oxysporum causes a number of symptoms depending on plant species, but common symptoms include leaf vein clearing, epinasty, wilting, stunting, yellowing of older leaves, browning of vascular tissue, necrosis and plant death (Agrios 2005). Its saprophytic ability enables it to survive in the soil between crop cycles in infected plant debris. The fungus can survive either as mycelium or as asexual spores: microconidia, macroconidia and chlamydospores (Agrios 2005). To initiate its life cycle (Fig. 1), the pathogen often directly infects the plants by entering through root tips, wounds or natural openings at lateral root initials. The pathogen then invades the root cortex first and then the xylem tissue, potentially blocking water movement leading to the appearance of wilting. The fungus will stay in xylem vessels (and some surrounding cells) as long as the plant is alive and move to other cells when the plant is dead so it can sporulate at or near the plant surface (Agrios 2005). The fungus sporulates on the dead tissue where these spores can initiate new infection cycles. The pathogen often spreads within short distances through irrigation water and through the use of contaminated equipment. It is also possible for the fungus to spread over long distances through infected plant material or contaminated soil. Therefore, hygiene (disinfection of planting materials/equipment) and quarantine measures (e.g. inhibiting the transfer of infected plant and soil material from one region to another) can be effective to stop the disease spreading although it is often quite difficult to eradicate the fungus from the soil as its chlamydospores can survive there for decades. To manage this disease, the use of resistant cultivar crop rotation with non-host plants is often recommended (Agrios 2005).

Fig. 1
figure 1

F. oxysporum life cycle. Shown is a schematic of F. oxysporum life cycle as represented by F. oxysporum strain Fo5176 infecting wild-type Arabidopsis (Col-0)

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3 Pathogen Infection Strategies

Pathogenic and non-pathogenic strains of F. oxysporum exist, both of which colonise host roots albeit to different degrees depending on the host but with initial root penetration favoured through wounds or at natural openings at the base of lateral root initials (Beckman 1987; Gordon and Martyn 1997; Recorbet et al. 2003; Michielse and Rep 2009; Kidd et al. 2011; Ma 2014; Perez-Nadales et al. 2014). Pathogenic strains have evolved to overcome host defence and cause disease. In such infected plants, wilting and eventual death occur largely as a result of water stress caused by proliferating spore and hyphae clogging the xylem vessels of roots and the stem and the action of secreted fungal proteins and toxins potentially blocking water movement and enhancing the appearance of wilting. The secreted molecules can differentially affect leaf and root tissues. For example, in roots toxins can initiate excessive division of parenchyma cells that encompass the xylem resulting in the collapse of xylem vessels or restricting their water flow, while the movement of toxins to leaves can affect chlorophyll synthesis (Di Pietro et al. 2003; Agrios 2005; Czymmek et al. 2007; Ramírez-Suero et al. 2010; Perez-Nadales et al. 2014; Li et al. 2015; Wang et al. 2015).

3.1 Pathogen Versus Non-pathogen

The ability of both pathogenic and non-pathogenic isolates to colonialise and penetrate the roots of hosts and non-hosts (Olivain et al. 2006; Ma 2014) suggests following colonisation plants adequately defend themselves against most F. oxysporum isolates, likely due to their recognition of conserved fungal molecules called microbe-associated molecular patterns (MAMPs) (also known as pathogen-associated molecular patterns (PAMPs) as they are present in pathogens). These include molecules such as chitin and β-glucan. PAMPs are typically recognised at the plant cell surface by membrane-bound receptor kinases and receptor-like proteins called pattern recognition receptors (PRRs) and induce PAMP-triggered immunity (PTI). PTI can also be triggered by host-derived products of infection called damage-associated molecular patterns (DAMPs) (e.g. plant cell wall fragments). Non-pathogenic F. oxysporum isolates would be recognised by these receptors; however, some isolates have become pathogenic by producing host-specific effectors that suppress or overcome PTI resulting in effector-triggered susceptibility (ETS). These effectors may mask MAMPs, manipulate host cell physiology or modify, inhibit or remove host immune response targets. Although an increasing list of candidate F. oxysporum effectors have been identified, relatively few F. oxysporum effectors have been functionally characterised. These are discussed in detail in further sections. Under selective pressure, plants have evolved receptors (resistance (R) proteins) to recognise specific effectors (avirulence (Avr) gene products) and mount resistance in a process termed effector-triggered immunity (ETI). ETI only occurs when specific F. oxysporum f. sp. isolates, known as races, express Avr products recognised by the corresponding host receptor, and unlike a classical ETI response of hypersensitive cell death to biotrophic pathogens, ETI in known F. oxysporum Avr–R-gene responses results in callose deposition, the vascular accumulation of phenolics, tyloses and gels (Takken and Rep 2010; De Coninck et al. 2015). See recent reviews for overviews of PTI and ETI triggered against plant–fungal pathogens (Win et al. 2012; van Schie and Takken 2014; Lo Presti et al. 2015).

3.2 Origins of Pathogenicity

3.2.1 Evolution of Pathogenicity

As stated above, pathogenic strains of F. oxysporum are classified into formae speciales (ff. spp.) based on the host species they cause disease on. For example, F. oxysporum f. sp. lycopersici (Fol) causes disease on tomato (Solanum lycopersicum) but no other plant species. While it was assumed isolates of a f. sp. arose through descent from a monophyletic origin, it has been demonstrated for some that this is not the case and that their genetic heterogeneity is polyphyletic in origin (Gordon and Martyn 1997; O’Donnell et al. 1998; Michielse and Rep 2009). That is, pathogenicity on a specific host may have arisen independently several times.

The polyphyletic origins of host specificity observed in some f. sp. can be explained by the recent demonstration of whole chromosome horizontal transfer. Experimentally it was shown a so-called pathogenicity chromosome containing most known effectors from a virulent Fol isolate was transferred to a non-pathogenic isolate, conferring its virulence on tomato (Ma et al. 2010). While horizontal gene transfer (HGT) has been demonstrated amongst many fungi, this was one the first demonstrations of whole chromosome transfer conferring host-specific pathogenicity. This pathogenicity chromosome could also transfer to another f. sp. (melonis); however, virulence of this isolate on tomato was not conferred suggesting other genetic content defines disease-causing host specificity.

3.2.2 Genomic Organisation of Pathogenicity Components

The sequencing of F. oxysporum genomes and their comparative analysis amongst ff. spp. and other fusaria has allowed identification of chromosomes and gene content geared towards pathogenicity. For example, the 15 chromosomes of the reference F. oxysporum genome (Fol race 2 isolate 4287) can be divided into “core” and “lineage specific” (Ma et al. 2010). Core chromosomes are conserved across fusaria and contain genes required for normal growth and metabolism, while lineage-specific chromosomes are absent or poorly conserved across fusaria or other fungi and lack house-keeping genes. For this reason, the latter chromosomes are also often referred to as “conditionally dispensable” or “accessory”.

The lineage-specific chromosomes of Fol refer to chromosomes 3, 6, 14 and 15 and telomere-proximal parts of chromosomes 1 and 2. These chromosomes are enriched in rapidly evolving genes and in transposable elements (TEs), remarkably accounting for nearly 75 % of all TEs in the Fol genome with Chr 14 comprised of 87 % TEs (Ma et al. 2010; Schmidt et al. 2013; Sperschneider et al. 2015). Further, only 20 % of genes on these chromosomes can be functionally classified and are enriched for genes related to pathogenicity such as known and putative effectors, fungal transcription factors and genes with roles in signal transduction and secondary metabolism.

The smaller lineage-specific chromosome 14 is referred to as the “pathogenicity” chromosome as it contains the majority of known Fol in planta expressed effectors and its horizontal transfer of pathogenicity to a non-pathogenic isolate (Michielse et al. 2009a; Ma et al. 2010; de Sain and Rep 2015). Interestingly, the most virulent of the newly created pathogenic isolates following HGT also contained additional parts of the lineage-specific chromosomes 3 and 6 (Ma et al. 2010). Loss of pathogenicity or virulence is also associated with the spontaneous loss of all or parts of Fol Chr 14 (Rep et al. 2004, 2015). This gain and loss of genetic material are likely associated with the enrichment of transposable and/or repetitive elements on the lineage-specific chromosomes surrounding effectors and other pathogenicity-related genes (Ma et al. 2010; Schmidt et al. 2013). The impact of transposable element activity combined with horizontal gene/chromosome transfer may facilitate the rapid modification of genetic material and ability for F. oxysporum to cause disease on so many diverse hosts.

With the advent of short-read sequencing technology, the list of available F. oxysporum genomes is increasing at a solid rate and covers ff. spp. causing disease over a range of economically important crops such as banana, brassicas, melons, cotton and legumes (Table 1). This not only facilitates the prediction of effectors and other pathogenicity components but also enables genome-wide analyses and comparative studies. For example, it was suggested the Fol (4287) effector Avr3 and its homologous pseudogene may undergo accelerated evolution (Rep 2005). Unbiased whole-genome comparative analysis of diversifying selection between Fol 4287 and another f. sp., conglutinans Fo5176, indeed identified Avr3, as well as other candidate effectors, as undergoing diversifying selection (Sperschneider et al. 2015). Even small modifications in avirulence proteins can affect their recognition by host receptors (e.g. a single amino acid change in Fol SIX3 (Avr2) confers a loss of recognition by the host receptor I-2 (Immunity-2), but interestingly does not affect its virulence phenotype (Houterman et al. 2009)). Comparative genomic analysis of ff. spp. pathogenic to three different legume species enabled the discovery of several effector candidates and a previously unrecognised gene region specifically conserved amongst legume-infecting isolates (Williams et al. 2016). These types of analyses expedite the identification of effectors responsible for inciting disease on specific hosts, an area of research that will hopefully identify the genetic determinants for classifying an isolate into a f. sp.

Table 1 F. oxysporum genomes deposited at NCBI. Accessed 11-06-2015
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3.3 Pathogenicity Machinery

To invade and initiate disease on a host, pathogenic F. oxysporum secrete an arsenal of enzymes, toxins, secondary metabolites and effectors. Effectors suppress or overcome PTI to induce host susceptibility, and while typically classified as host specific, a broader definition of effectors includes many molecules such as toxins (e.g. fusaric acid), degradative enzymes and even PAMPs/MAMPs (Hogenhout et al. 2009; Stergiopoulos and de Wit 2009; Dong et al. 2014; Pusztahelyi et al. 2015). This is supported by the finding that genes encoding some of the latter molecules are induced upon plant contact. Large-scale fungal mutagenesis and xylem sap proteomics facilitated the initial discovery of F. oxysporum effectors and pathogenicity-related proteins, but more recently comparative genomics and high-coverage in planta transcriptome sequencing (RNA-seq) have increased the rate of candidate effector identification across ff. spp. The rate-limiting step here is still functional characterisation which is best studied in knockout and mutant lines.

3.3.1 General Pathogenicity Machinery

Like other pathogenic plant–fungal pathogens, the genomes of F. oxysporum ff. spp. are enriched in genes encoding plant cell wall-degrading enzymes (CWDEs) (Ma et al. 2010; Zhao et al. 2013; Williams et al. 2016) and are known to secrete these enzymes during host colonisation (Beckman 1987; Roncero et al. 2003). These include polygalacturonases, pectate lyases, xylanases and proteases and act by degrading cell walls and membranes, releasing nutrient sources such as sugars (Yadeta and Thomma 2013). While these enzymes play key roles in pathogenicity, are expressed during infection and likely contribute to virulence, individual gene knockouts have failed to produce altered disease phenotypes, which is expected in multi-gene families like these where functional redundancy may exist (Di Pietro et al. 2003; Recorbet et al. 2003; McFadden et al. 2006; Guo et al. 2014; Kubicek et al. 2014). Functional analysis therefore requires the generation of at least double deletions, for example, as shown in a Fol polygalacturonase and endopolygalacturonase double mutant (Δpg1Δpgx6) which exhibited reduced virulence on tomato (Ruiz et al. 2015).

Two other classes of secreted effector proteins found in F. oxysporum are the necrosis and ethylene-inducing-like proteins (NLPs) and lysine motifs (LysMs). Nep1 was first identified in F. oxysporum culture filtrates, but NLPs are present in other fungi as well as oomycetes and even bacteria (Bailey 1995; Pemberton and Salmond 2004; Bae et al. 2006; Böhm et al. 2014; Oome et al. 2014). LysM effectors contain the LysM carbohydrate-binding domain that mediates recognition of fungal chitin, an essential component of the fungal cell wall, and is found in some membrane-localised plant receptors (Gust et al. 2012; Kombrink and Thomma 2013). It is proposed that LysM effectors (most well characterised in Cladosporium fulvum and Verticillium pathogens) contribute to virulence through mechanisms such as suppression of chitin-triggered PTI. For example, by protecting fungal hyphae from hydrolytic plant enzymes or to scavenge hydrolytically derived chitin oligomers produced during invasion and subsequently avoid or delay host detection (Kombrink and Thomma 2013). Further, knockouts in several Fol chitin synthase genes are associated with a loss of pathogenicity phenotype or reduced virulence (reviewed in Michielse and Rep 2009). Fol also produces enzymes that neutralise host-produced chitinases that bind chitin. A recent study identified a secreted metalloprotease and a serine protease that were responsible for the cleavage of chitinases. When the genes encoding these enzymes were deleted, the mutant showed reduced virulence against tomato, suggesting that these enzymes are important for fungal virulence (Karimi Jashni et al. 2015). Although not functionally characterised in F. oxysporum, LysM domain-containing proteins are present in most if not all ff. spp. (Thatcher unpublished) with some expressed in planta (Williams et al. 2016). As effectors are often defined by the absence of detectable orthologous proteins outside the genus, the wide distribution of NLPs and LysMs suggests these are best designated as PAMPs (Thomma et al. 2011).

Other F. oxysporum proteins found to be secreted during infection include a catalase-peroxidase, a serine protease and the oxidoreductase Orx1 which is a homologue of the Ave1 avirulence protein from Verticillium dahliae. These proteins were detected in the xylem sap of Fol-infected tomato plants, suggesting they are important for infection (Houterman et al. 2007; Schmidt et al. 2013). Some enzymes such as catalase-peroxidase, galactosidase and chitinase might also contribute to the strong virulence of Foc TR4 (Sun et al. 2014).

3.3.2 F. oxysporum Signal Transduction Machinery Involved in Pathogen Virulence

Signalling processes and the coordinated control of F. oxysporum pathogenicity machinery have been shown in several cases to be critical for host colonisation, penetration or virulence. Components of signal transduction such as kinases and transcription factors are expressed during host infection, and in several cases, their targeted gene knockouts show reduced pathogenicity (Guo et al. 2014; Michielse et al. 2009a, b). For example, mutants of G-protein-coupled receptor subunits α (FGA1, FGA2) and β (FGB1) are impaired in or have lost pathogenicity in Fol and F. oxysporum f. sp. cucumerinum (Jain et al. 2002, 2003, 2005). Mutants of the Fol mitogen-activated protein kinase (MAPK) genes FMK1 and SNF1 (Di Pietro et al. 2001; Michielse et al. 2009b) are impaired in root penetration and pathogenicity (see reviews by Di Pietro et al. 2003; Michielse and Rep 2009). The constitutively expressed Fol F-box gene FRP1 may function in SCF-mediated ubiquitination processes and is required for pathogenicity as knockouts are non-pathogenic and unable to colonise roots (Duyvesteijn et al. 2005; Jonkers and Rep 2009).

Several transcription factors with roles in pathogenicity have been functionally characterised. For example, a knockout of the zinc finger XlnR is severely impaired in extracellular xylanase activity (Calero-Nieto et al. 2007). The transcription factor gene FOW2 encoding a Zn(II)2Cys6 family transcriptional regulator appears conserved amongst F. oxysporum ff. spp. and in Fol, and F. oxysporum f. sp. melonis is required for colonisation and pathogenicity (Imazaki et al. 2007; Michielse et al. 2009b). And another transcription factor (SGE1, SIX gene expression 1) is not required for root colonisation or penetration, but is essential for pathogenicity in Fol where its expression is upregulated during infection of tomato roots and is required for expression of most secreted Fol effectors as discussed in the following section (Michielse et al. 2009a).

3.3.3 Effectors

While general machinery necessary for host colonisation tends to be expressed constitutively, genes necessary for pathogenicity and virulence are typically only expressed upon plant contact (lowly or not expressed under axenic conditions) (Rep 2005). The most well-characterised effectors from F. oxysporum belong to a class termed the secreted in xylem or SIX effectors, first identified in the xylem sap proteome of tomato plants infected with Fol, with roles in virulence and/or avirulence determined for some depending on the host genotype (Rep et al. 2004, 2005; Houterman et al. 2007; de Sain and Rep 2015). So far, 14 families of SIX proteins have been identified (Rep et al. 2004; Houterman et al. 2007; van der Does and Rep 2007; Lievens et al. 2009; Ma et al. 2010; Rep and Kistler 2010; Schmidt et al. 2013), and these are typically only found in F. oxysporum isolates, although some, such as SIX6, are present in other fungi such as Colletotrichum species (Gawehns et al. 2014). The SIX effectors were originally thought to be unique to Fol but have since been identified in several F. oxysporum ff. spp. with some sharing high levels of sequence identity (Lievens et al. 2009; Meldrum et al. 2012; Thatcher et al. 2012a; Laurence et al. 2015; Schmidt et al. 2016). For example, the Arabidopsis infecting isolate Fo5176 contains a highly conserved SIX4 homologue, only differing from the Fol SIX4 by two amino acids (Thatcher et al. 2012a). Interestingly, in the tomato pathosystem, Fol SIX4 (Avr1) is not required for general virulence but acts by suppressing ETI mediated by two resistance genes (immunity-2 (I-2) and immunity-3 (I-3)), whereas in Arabidopsis lacking immunity resistance genes, Fo5176 SIX4 is required for full virulence (Rep et al. 2005; Houterman et al. 2008; Thatcher et al. 2012a). Fol SIX4 (Avr1), as well as Fol SIX6, can also suppress cell death triggered by I-2 (Gawehns et al. 2014).

Similar to most known fungal effectors, SIX proteins are small and generally cysteine rich and most contain a signal peptide for secretion (Houterman et al. 2007; Schmidt et al. 2013), but apart from these characteristics, they share little similarity with each other and other known fungal proteins (Rep 2005). Secreted into apoplast or xylem, the cysteine-rich nature of these extracellular proteins creates disulphide bridges that stabilises the protein against protease degradation (Takken and Rep 2010). The majority of Fol SIX genes reside on pathogenicity Chr 14 or in some cases on other dispensable chromosomes and are located within transposon-rich regions often associated with miniature transposable elements (MITE) present in their promoters (Ma et al. 2010; Schmidt et al. 2013). Some are even co-located at the same loci and share common promoters (e.g. SIX3 (Avr2) and SIX5) and may also physically interact with each other at the protein level (Schmidt et al. 2013; Ma et al. 2015).

For most SIX effectors, their expression requires the core-chromosome-encoded transcription factor Sge1 (SIX gene expression 1) (Michielse et al. 2009a). The expression profiles of SIX genes from other F. oxysporum ff. spp. confirm that most are either highly in planta inducible or only expressed in planta (McFadden et al. 2006; van der Does et al. 2008; Thatcher et al. 2012a; Gawehns et al. 2014; Guo et al. 2014; Williams et al. 2016). In planta gene expression has also been used in other F. oxysporum ff. spp. to identify putative effectors (e.g. f. sp. cubense, f. sp. vasinfectum, f. sp. medicaginis (McFadden et al. 2006; Guo et al. 2014; Williams et al. 2016)), and the associated presence of MITEs helped identify the F. oxysporum f. sp. melonis avirulence protein AvrFOM2 that is recognised by the melon resistance gene Fom-2 (Schmidt et al. 2016).

4 Host Resistance

The genetic and molecular F. oxysporum–plant interaction is best understood in the tomato pathosystem where R-gene resistance is available (Takken and Rep 2010), with other model pathosystems in Arabidopsis thaliana and Medicago truncatula also studied (Diener and Ausubel 2005; Lichtenzveig et al. 2006; Berrocal-Lobo and Molina 2008; Ramírez-Suero et al. 2010; Lyons et al. 2015a; Rispail et al. 2015). The following sections will discuss the findings from studying host resistance to F. oxysporum.

4.1 Transcriptome Studies

Plant responses to F. oxysporum infection have been studied using genome-wide expression profiling using microarray and RNA-seq analyses (see Table 2 for examples). Most of the earlier efforts investigated defence responses occurring in the leaves. A recent study that comparatively analysed defence responses triggered by Fusarium infection revealed that the infection triggers expression from separate classes of defence-associated genes in the roots and shoots (leaves or rosettes), suggesting that different physiological and defence-associated processes might be operational in these tissues (Lyons et al. 2015a). Plant development and flowering time seem to have a major effect on F. oxysporum disease symptom expression. It was shown recently that diverse Arabidopsis ecotypes and various mutants affected in flowering time also show altered disease development (Lyons et al. 2015b). In particular, late flowering time is associated with increased disease resistance. It was speculated that delayed senescence as a result of late flowering could be a reason explaining this delay in disease progression.

Table 2 Recent transcriptome, metabolome and proteome studies analysing F. oxysporum infection
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Other studies (Table 2) have compared differentially expressed genes between resistant and susceptible genotypes to determine what makes the plant resistant or susceptible to infection. For instance, Xue et al. (2015a) recently compared resistant and susceptible bean plants, while Bai et al. (2013) looked at resistant and susceptible banana cultivars. As a result, large numbers of genes corresponding to certain defence categories have been identified. These studies have certainly provided useful candidates that can be further studied functionally, and if their association with disease resistance is confirmed, they may be useful targets for marker-assisted selection studies. However, it should be remembered that some of the host genes induced by the pathogen may also be associated with susceptibility.

Interestingly, a recent study comparing transcriptomes of banana roots inoculated with either race 1 or tropical race 4 shows that both Foc race 1 and Foc TR4 triggered similar gene expression profiles in banana roots, despite their differing pathogenicity/virulence (Li et al. 2013a). Following F. oxysporum Fo5176 infection, we have also analysed the root transcriptomes of wild-type Arabidopsis plants and Arabidopsis overexpressing the Fo5176 SIX4 effector (arrays conducted on root tissue from Col-0 or 35sSIX4 plants (Thatcher et al. 2012a) 4 days postinoculation, pathogen infection and microarray analysis conducted as described previously (Kidd et al. 2009), microarray data deposited at NCBI under accession number GSE75928). This process identified genes downregulated >1.5-fold in the effector overexpression plants to be enriched in genes associated with oxidative stress and wound/defence responses suggesting virulence function of the SIX4 effector is associated with modifying host-signalling processes.

4.2 Genetics of Host Resistance in Arabidopsis

Analysis of mutants affected in disease resistance against F. oxysporum has identified a number of genes that regulate resistance or susceptibility in Arabidopsis. So far a number of transcription factors altering disease resistance to F. oxysporum have been identified. This has also helped in the development of a model that explains host susceptibility or resistance. In particular, the SA signalling pathway seems to be required for increased resistance, while F. oxysporum seems to exploit the JA signalling pathway to cause disease. The evidence for this comes from the observation that Arabidopsis JA signalling mutants such as coi1, myc2 and pft1 but not JA biosynthesis mutants show increased resistance to F. oxysporum (Anderson et al. 2004; Thatcher et al. 2009; Kidd et al. 2009). The esr1-1 (enhanced stress response 1) mutant defective in a KH domain containing RNA-binding protein (At5g53060) also confers increased resistance to F. oxysporum. Similar to other JA signalling genes that make Arabidopsis susceptible to F. oxysporum infection, ESR1 seems to modulate JA responses as well (Thatcher et al. 2015). It is possible that pathogen-produced JA-like compounds secreted by the pathogen activate the host’s JA signalling pathway, which then promotes senescence (Thatcher et al. 2009; Cole et al. 2014). In the banana–Foc interaction, fusaric acid secreted by Foc also seems to play a role in promoting senescence (Dong et al. 2014). Transgenic expression of JA-responsive transcription factors such as ethylene response factors (ERFs) can also positively contribute to disease inhibition by modulating defence gene expression without promoting senescence. For instance, overexpression of ERF1 in Arabidopsis increases F. oxysporum resistance by altering the expression of defence-related genes (Berrocal-Lobo and Molina 2004). Similarly, another Arabidopsis ERF transcription factor, ERF14, is required for wild-type resistance to F. oxysporum in Arabidopsis as erf14 loss-of-function mutants show reduced defence gene expression and increased susceptibility to this pathogen (Onate-Sanchez et al. 2007).

In addition, it was reported that auxin signalling and biosynthesis mutants show increased susceptibility to F. oxysporum as a number of auxin mutants show altered F. oxysporum resistance (Kidd et al. 2011). A F. oxysporum strain genetically modified to produce increased levels of auxin shows hypervirulence (Cohen et al. 2002), further suggesting that auxin is associated with increased disease. However, how auxin promotes disease susceptibility is currently unknown. One possibility is that auxin signalling and transport are required for lateral root formation and increased lateral root formation may provide a higher number of infection sites. F. oxysporum is known to infect the plant lateral root initials and root tips that are also auxin-rich regions. Interestingly a recent study showed that volatiles produced by F. oxysporum improve plant growth and were dependent on a functional auxin signalling pathway in Arabidopsis (Bitas et al. 2015) (Table 3).

Table 3 Arabidopsis genes that regulate resistance or susceptibility to the F. oxysporum strain Fo5176
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4.3 Deployment of Resistance Genes and Marker-Based Selection Approaches

In several crops resistance against specific pathogenic f. sp. or races of F. oxysporum have been identified enabling researchers to develop molecular markers that can be used for germplasm-screening purposes (Jimenez-Gasco et al. 2004; reviewed Michielse and Rep 2009; Sharma et al. 2014; Schmidt et al. 2016). However, only a handful of the underlying R-genes have been cloned (Table 4), the majority of which isolated from tomato are based on monogenetic resistance conferring classical gene-for-gene-mediated interactions. Plant resistance genes can be divided into two main categories, the leucine-rich repeat (LRR) and intracellular nucleotide-binding site (NBS)-LRR-containing R proteins, with the latter mediating recognition of intracellular pathogen-derived signals (Martin et al. 2003). Some transmembrane LRR proteins also have an intracellular protein kinase (PK) domain and belong to the larger class of receptor-like protein kinases (RLKs). The extracellular LRR domain of LRR-TM and LRR-TM-PK proteins is thought to function as receptors for extracellular pathogen-derived signals such as conserved pathogen molecules (MAMPs) and damage-associated molecules.

Table 4 Summary of cloned or well-characterised F. oxysporum resistance genes
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For some R proteins, their cellular localisation has been determined. For example, the cytosolic R-protein I-2 from tomato mainly localises to xylem tissues of roots, stems and leaves, where it intracellularly perceives the Fol effector SIX3 (Avr2) (Mes et al. 2000; Houterman et al. 2009; Gawehns et al. 2014; Ma et al. 2015). The tomato I-3 protein is a plasma membrane-bound receptor with a cytoplasmic kinase domain and an extracellular S-domain (Catanzariti et al. 2015). Interestingly, I-3 gene expression is higher in leaf tissues compared to root or stem tissues where initial stages of Fol infection take place. Like I-3, I-7 also contains an extracellular recognition domain suggesting SIX1 (Avr3) and Avr7 are recognised at the cell surface and may not be taken up by host plant cells (Catanzariti et al. 2015; Gonzalez-Cendales et al. 2015).

In the Arabidopsis pathosystem, several resistance loci have been identified using various f. sp. such as f. sp. conglutinans, f. sp. raphani and f. sp. matthioli (Diener and Ausubel 2005). Crosses between the F. oxysporum f. sp. matthioli-resistant Col-0 ecotype and the susceptible Ty-0 ecotype identified six resistance loci (RFO1–6) with RFO1 the largest contributor to resistance encoding a WALL-ASSOCIATED KINASE-LIKE KINASE (WAKL22) that provides resistance to three isolates of F. oxysporum (Diener and Ausubel 2005), while RFO2 and RFO3 encode a receptor-like protein and a receptor-like kinase, respectively, which have undergone duplication in the parent ecotypes (Cole and Diener 2013; Shen and Diener 2013). Identification of RFO2 also leads to a role for tyrosine-sulphated peptide signalling in the F. oxysporum interaction (Shen and Diener 2013). Therefore the identification and characterisation of R-genes effective against F. oxysporum provide an opportunity to understand effective resistance strategies against this pathogen. Transcriptome analysis of F. oxysporum infected Arabidopsis also identified significant upregulation of several receptor-associated genes including a wall-associated kinase-like gene, lectin receptor kinases, receptor-like protein kinase 1 and TIR-NBS-LRR genes suggesting roles in resistance (Zhu et al. 2013). Using a comparative transcriptome approach between resistant and susceptible Chinese cabbage (Brassica rapa var. pekinensis), Shimizu et al. (2014) were also able to narrow a single dominant R-gene down to two possible candidates encoding TIR-NBS-LRRs.

4.4 Resistance Through the Application of Biological and Chemical Agents

Given the long-term survival of F. oxysporum in the soil, attention has been given to treatments that can suppress disease. Silicon addition has been observed to provide increased tolerance to Foc in banana (Fortunato et al. 2012a, b). While the role that silicon plays in protecting plants against plant pathogens is debated, a recent study found that silicon may act by stimulating lignin and products of the phenylpropanoid pathway in infected banana plants (Fortunato et al. 2014).

Non-pathogenic isolates of F. oxysporum may also be employed to manage pathogenic isolates of F. oxysporum (Forsyth et al. 2006). For instance incompatible Foc race 1 was used to induce systemic resistance against Foc TR 4 (Wu et al. 2013). This increased resistance state was accompanied by systemic upregulation of defence-related genes such as MaNPR1A, MaNPR1B, PR1 and PR3 as well as upregulation of SA and JA pathways (Wu et al. 2013). Similar findings were found with Fo47, a protective strain of Fusarium wilt in tomato (Olivain et al. 2006). Fo47 reduces the growth of pathogenic F. oxysporum f. sp. lycopersici isolate Fol8 and induces the expression of defence genes CHI3, GLUA and PR1a in tomato (Aimé et al. 2013). Understanding the microbiome may also provide protection against F. oxysporum. Studying the microbial components of disease-suppressive soils has been a popular area of research (see Ajilogba and Babalola 2013 for research in tomato), and recent reports have focussed on the banana rhizosphere given the current global outbreak of TR4 (Huang et al. 2015; Xue et al. 2015b). A recent analysis of soils suppressive to Fusarium wilt of strawberry identified members of the Actinobacteria and the identification of a novel antifungal thiopeptide from one of these bacteria which targeted fungal cell wall biosynthesis (Cha et al. 2015). While many microbial isolates appear beneficial in suppressing the disease in particular soil types, so far those identified haven’t been sufficient to prevent disease occurrence globally, but this is a promising area of research.

4.5 Engineering of Resistance

Given that F. oxysporum infection leads to widespread cell death and necrosis on the above-ground tissues, genes that play roles in inhibiting apoptosis or cell death (namely, Bcl-xL, Ced-9) can play a role in disease resistance. Indeed, transgenic expression of apoptosis-related genes enhanced banana resistance to Foc and is undergoing field testing (Paul et al. 2011). Transgenic plants expressing a defensive gene from Nicotiana alata were recently shown to provide a quantitative resistance to Fusarium oxysporum and Verticillium dahliae in cotton (Gaspar et al. 2014). The expression of defensin chitinase and/or thaumatin-like genes from other plant species also shows promise as candidates for increasing Fusarium wilt resistance in tomato and banana (Abdallah et al. 2010; Ghag et al. 2012; Mahdavi et al. 2012; Jabeen et al. 2015).

4.6 Host-Induced Gene Silencing

Inhibiting the expression of genes involved in fungal growth and development and pathogenicity through host-delivered (host-induced) gene silencing seems to be a promising way to engineer disease resistance against F. oxysporum. In a recent study, transgenic banana plants expressing hairpin RNA against Velvet and FTF1 genes (Fusarium transcription factor 1) showed complete resistance to Foc in greenhouse bioassays (Ghag et al. 2014). In Arabidopsis, survival rates of transgenic lines expressing dsRNA against three F. oxysporum genes (FOW2, FRP1 and OPR) were found to be higher than wild-type plants (Hu et al. 2015). FOW2 encodes a Zn(II)2Cys6 TF that is required for the pathogenicity of F. oxysporum f. sp. melonis (Imazaki et al. 2007), FRP1 encodes an F-box protein involved in protein ubiquitination, which was also required for F. oxysporum f. sp. lycopersici pathogenicity, and OPR encodes a 12-oxo-phytodienoate-10-11-reductase-like protein potentially involved in JA biosynthesis in F. oxysporum (Hu et al. 2015). These studies show promising results; however, commercialisation of transgenic plants is dependent on a number of factors including regulatory (no adverse health and environmental effects), legal (e.g. patenting and licensing issues) as well as economic and social consideration (consumer acceptance). Therefore, genetic modification approaches can be difficult to commercialise under the current climate but provide potential solutions for combatting F. oxysporum.

5 Conclusion

The ubiquitous and persistent nature of F. oxysporum as well as its ability to evolve new pathogenic strains makes F. oxysporum a particularly difficult pathogen to control. Despite this, significant progress has been made in recent years in understanding the factors responsible for both virulence in the pathogen and resistance or susceptibility in the host. Building upon these studies will hopefully lead to the identification of additional resistance genes that can be implemented in crops where resistance is lacking. Hopefully, continual research may lead to protection against the current forms of F. oxysporum but ideally lead to strategies that may protect against future evolving strains.