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Plant pathogens employ secreted proteins, among which are effectors, to manipulate and colonize their hosts. A large fraction of effectors is translocated into host cells, where they can suppress defense signaling. Bacterial pathogens directly inject effectors into host cells via the type three secretion system, but it is little understood how eukaryotic pathogens, such as fungi, accomplish this critical process and how their secreted effectors enter host cells. The root-infecting fungus Fusarium oxysporum (Fo) secrets numerous effectors into the extracellular space. Some of these, such as Foa3, function inside the plant cell to suppress host defenses. Here, we show that Foa3 suppresses pattern-triggered defense responses to the same extent when it is produced in planta irrespective of whether the protein carries the PR1 secretory signal peptide or not. When a GFP-tagged Foa3 was targeted for secretion it localized, among other locations, to mobile subcellular structures of unknown identity. Furthermore, like the well-known cell penetrating peptide Arginine 9, Foa3 was found to deliver an orthotospovirus avirulence protein-derived peptide into the cytosol, resulting in the activation of the matching resistance protein. Finally, we show that infiltrating Foa3 into the apoplast results in strong suppression of the pattern-triggered immune responses, potentially indicating its uptake by the host cells in absence of a pathogen.

Fusarium oxysporum (Fo) is best known as a host‐specific vascular pathogen causing major crop losses. Most Fo strains, however, are root endophytes potentially conferring endophyte‐mediated resistance (EMR). EMR is a mechanistically poorly understood root‐specific induced resistance response induced by endophytic or nonhost pathogenic Fo strains. Like other types of induced immunity, such as systemic acquired resistance or induced systemic resistance, EMR has been proposed to rely on the activation of the pattern‐triggered immunity (PTI) system of the plant. PTI is activated upon recognition of conserved microbe‐associated molecular patterns (MAMPs) of invading microbes. Here, we investigated the role of PTI in controlling host colonization by Fo endophytes and their ability to induce EMR to the tomato pathogen Fo f. sp. lycopersici (Fol). Transgenic tomato and Arabidopsis plants expressing the Fo effector gene Avr2 are hypersusceptible to bacterial and fungal infection. Here we show that these plants are PTI‐compromised and are nonresponsive to bacterial‐ (flg22) and fungal‐ (chitosan) MAMPs. We challenged the PTI‐compromised tomato mutants with the EMR‐conferring Fo endophyte Fo47, the nonhost pathogen Fom (a melon pathogen), and with Fol. Compared to wild‐type plants, Avr2‐tomato plants became hypercolonized by Fo47 and Fom. Surprisingly, however, EMR towards Fol, induced by either Fo47 or Fom, was unaffected in these plants. These data show that EMR‐based disease resistance is independent from the conventional defence pathways triggered by PTI, but that PTI is involved in restricting host colonization by nonpathogenic Fo isolates., Whereas MAMP‐triggered immunity restricts growth of pathogenic and endophytic Fusarium oxysporum, it does not restrict the endophyte‐mediated resistance conferred by the latter.

The fungus Fusarium oxysporum (Fo) is widely known for causing wilt disease in over 100 different plant species. Endophytic interactions of Fo with plants are much more common, and strains pathogenic on one plant species can even be beneficial endophytes on another species. However, endophytic and beneficial interactions have been much less investigated at the molecular level, and the genetic basis that underlies endophytic versus pathogenic behavior is unknown. To investigate this, 44 Fo strains from non-cultivated Australian soils, grass roots from Spain, and tomato stems from United States were characterized genotypically by whole genome sequencing, and phenotypically by examining their ability to symptomlessly colonize tomato plants and to confer resistance against Fusarium Wilt. Comparison of the genomes of the validated endophytic Fo strains with those of 102 pathogenic strains revealed that both groups have similar genomes sizes, with similar amount of accessory DNA. However, although endophytic strains can harbor homologs of known effector genes, they have typically fewer effector gene candidates and associated non-autonomous transposons (mimps) than pathogenic strains. A pathogenic ‘lifestyle’ is associated with extended effector gene catalogs and a set of “host specific” effectors. No candidate effector genes unique to endophytic strains isolated from the same plant species were found, implying little or no host-specific adaptation. As plant-beneficial interactions were observed to be common for the tested Fo isolates, the propensity for endophytism and the ability to confer biocontrol appears to be a predominant feature of this organism. These findings allow prediction of the lifestyle of a Fo strain based on its genome sequence as a potential pathogen or as a harmless or even beneficial endophyte by determining its effectorome and mimp number.

Intracellular nucleotide‐binding leucine‐rich repeat (NLR)‐type immune receptors are a fundamental part of plant immune systems. As infection occurs at foci, activation of immune responses is typically non‐uniform and non‐synchronized, hampering the systematic dissection of their cellular effects and determining their phasing. We investigated the potato NLR Rx1 using the CESSNA (Controlled Expression of effectors for Synchronized and Systemic NLR Activation) platform. CESSNA‐mediated Potato virus X coat protein (CP) expression allowed the monitoring of Rx1‐mediated immune responses in a quantitative and reproducible manner. Rx1 was found to trigger a reactive oxygen species (ROS) burst and ion leakage within 1 h and a change in autofluorescence within 2 h after the induction of CP production. After 2 h, HIN1 expression was increased and single‐stranded DNA (ssDNA) damage and loss of cellular integrity became apparent, followed by double‐stranded DNA (dsDNA) damage after 3 h and increased PR‐1a, LOX, ERF1 and AOX1B expression and cell death at 4 h. Nuclear exclusion of Rx1 resulted in increased basal levels of ROS and permitted Rx1 activation by an Rx1‐breaking CP variant. In contrast, nuclear‐targeted Rx1 showed diminished basal ROS levels, and only avirulent CP could trigger a compromised ROS production. Both nuclear‐excluded and nuclear‐targeted Rx1 triggered a delayed ion leakage compared with non‐modified Rx1, suggesting that ion leakage and ROS production originate from distinct signalling pathways. This work offers novel insights into the influence of Rx1 localization on its activity, and the interplay between Rx1‐triggered processes.

Summary Many plant intracellular immune receptors mount a hypersensitive response (HR) upon pathogen perception. The concomitant localized cell death is proposed to trap pathogens, such as viruses, inside infected cells, thereby preventing their spread. Notably, extreme resistance (ER) conferred by the potato immune receptor Rx1 to potato virus X (PVX) does not involve the death of infected cells. It is unknown what defines ER and how it differs from HR‐based resistance. Interestingly, Rx1 can trigger an HR, but only upon artificial (over)expression of PVX or its avirulence coat protein (CP). Rx1 has a nucleocytoplasmic distribution and both pools are required for HR upon transient expression of a PVX‐GFP amplicon. It is unknown whether mislocalized Rx1 variants can induce ER upon natural PVX infection. Here, we generated transgenic Nicotiana benthamiana producing nuclear‐ or cytosol‐restricted Rx1 variants. We found that these variants can still mount an HR. However, nuclear‐ or cytosol‐restricted Rx1 variants can no longer trigger ER or restricts viral infection. Interestingly, unlike the mislocalized Rx1 variants, wild‐type Rx1 was found to compromise CP protein accumulation. We show that the lack of CP accumulation does not result from its degradation but is likely to be linked with translational arrest of its mRNA. Together, our findings suggest that translational arrest of viral genes is a major component of ER and, unlike the HR, is required for resistance to PVX., Significance Statement It is unknown how antiviral immunity is conferred in the case of extreme resistance. We show that extreme resistance correlates with a translational arrest of viral transcripts and can be uncoupled from the hypersensitive response, which was observed to not be required for viral immunity.

Although the vascular pathogen Fusarium oxysporum is notorious for being the causal agent of Fusarium wilt disease, the vast majority of F. oxysporum strains are harmless soil and root colonizers. The latter F. oxysporum’s are often endophytes colonizing roots intracellularly without negatively affecting plant fitness. Actually, most of them, like Fo47, are beneficial providing biological control to various root pathogens. Interestingly, also pathogenic F. oxysporum inoculated on a resistant host (i.e., avirulent F. oxysporum f. sp. lycopersici) can reduce susceptibility to virulent F. oxysporum strains via a mechanism called “cross protection.” It has been hypothesized that cross protection is based on activation of a resistance protein of the host upon recognition of a cognate avirulence (Avr) protein of the pathogen. Currently, it is unknown whether the biocontrol activity of F. oxysporum endophytes utilizes similar mechanisms as cross protection conferred by avirulent pathogens, and whether both provide a quantitative similar level of protection. Here, we show that in tomato biocontrol activity of the Fo47 endophyte to the pathogen F. oxysporum f. sp. lycopersici is more effective than cross protection induced by avirulent F. oxysporum f. sp. lycopersici strains activating either I, I-2, or both resistance proteins upon recognition of Avr1 or the Avr2/Six5 pair, respectively. These findings imply that cross protection and biological control utilize different mechanisms to reduce susceptibility of the host to subsequent infections.

Endophytic microbes conferring biocontrol are an eco-friendly alternative to control diseases in crops. Unfortunately, the use of endophytes to control diseases is not yet widespread as their application in agricultural settings is challenging and the outcome variable. Translating strains that perform well under laboratory conditions to the field poses several challenges. One is large scale inoculum production in a cost-effective manner. Here, we developed a framework to scale up inoculum production of Fusarium oxysporum 47 (Fo47), assess inoculum viability and its performance in the field and effects on potato yield and performance. The Fo47 endophyte is a well-described biocontrol agent, isolated from disease suppressive soils in the 1980’s. Using mung bean medium, we could routinely produce ≈7 × 108 spores/mL. Using 60 mL of 107 spores/mL per tuber we could re-isolate the fungus 79 days after application from 60 to 70% of the inoculated plants in a large-scale potato field trial (Clenze, Germany). Furthermore, this protocol can be used to assess Fo47 biocontrol potential under field conditions. The presence of the fungus did not negatively affect plant yield or starch production and did not increase susceptibility to endemic pathogens.

Fusarium spp. cause severe economic damage in many crops, exemplified by Panama Disease of banana or Fusarium head blight of wheat. Plants sense immunogenic patterns (termed elicitors) at the cell surface to initiate pattern-triggered immunity (PTI). Knowledge of fungal elicitors and corresponding plant immune-signaling is incomplete but could yield valuable sources of resistance.- We characterized Arabidopsis thaliana PTI responses to a peptide elicitor fraction present in several Fusarium spp. and employed a forward-genetic screen using plants containing a cytosolic calcium reporter to isolate fusarium elicitor reduced elicitation (fere) mutants.- We mapped the causal mutation in fere1 to the leucine-rich repeat receptor-like kinase MDIS1-INTERACTING RECEPTOR-LIKE KINASE 2 (MIK2) and confirmed a crucial role of MIK2 in fungal elicitor perception. MIK2-dependent elicitor responses depend on known signaling components and transfer of AtMIK2 is sufficient to confer elicitor sensitivity to Nicotiana benthamiana.- Arabidopsis senses Fusarium elicitors by a novel receptor complex at the cell surface that feeds into common PTI pathways. These data increase mechanistic understanding of PTI to Fusarium and place MIK2 at a central position in Arabidopsis elicitor responses.

Although the vascular pathogen

Specificity in the plant immune system is mediated by Resistance (R) proteins. Most R genes encode intracellular NLR-type immune receptors and these pathogen sensors require helper NLRs to activate immune signaling upon pathogen perception. Resistance conferred by many R genes is temperature sensitive and compromised above 28°C. Many Solanaceae R genes, including the potato NLR Rx1 conferring resistance to Potato Virus X (PVX), have been reported to be temperature labile. Rx1 activity, like many Solanaceae NLRs, depends on helper-NLRs called NRC's. In this study, we investigated Rx1 resistance at elevated temperatures in potato and in Nicotiana benthamiana plants stably expressing Rx1 upon rub-inoculation with GFP-expressing PVX particles. In parallel, we used susceptible plants as a control to assess infectiousness of PVX at a range of different temperatures. Surprisingly, we found that Rx1 confers virus resistance in N. benthamiana up to 32°C, a temperature at which the PVX::GFP lost infectiousness. Furthermore, at 34°C, an Rx1-mediated hypersensitive response could still be triggered in N. benthamiana upon PVX Coat-Protein overexpression. As the Rx1-immune signaling pathway is not temperature compromised, this implies that at least one N. benthamiana helper NRC and its downstream signaling components are temperature tolerant. This finding suggests that the temperature sensitivity for Solanaceous resistances is likely attributable to the sensor NLR and not to its downstream signaling components.

Summary 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.

Interactions between plants and the root-colonizing fungus Fusarium oxysporum (Fo) can be neutral, beneficial, or detrimental for the host. Fo is infamous for its ability to cause wilt, root-, and foot-rot in many plant species, including many agronomically important crops. However, Fo also has another face; as a root endophyte, it can reduce disease caused by vascular pathogens such as Verticillium dahliae and pathogenic Fo strains. Fo also confers protection to root pathogens like Pythium ultimum, but typically not to pathogens attacking above-ground tissues such as Botrytis cinerea or Phytophthora capsici. Endophytes confer biocontrol either directly by interacting with pathogens via mycoparasitism, antibiosis, or by competition for nutrients or root niches, or indirectly by inducing resistance mechanisms in the host. Fo endophytes such as Fo47 and CS-20 differ from Fo pathogens in their effector gene content, host colonization mechanism, location in the plant, and induced host-responses. Whereas endophytic strains trigger localized cell death in the root cortex, and transiently induce immune signaling and papilla formation, these responses are largely suppressed by pathogenic Fo strains. The ability of pathogenic strains to compromise immune signaling and cell death is likely attributable to their host-specific effector repertoire. The lower number of effector genes in endophytes as compared to pathogens provides a means to distinguish them from each other. Co-inoculation of a biocontrol-conferring Fo and a pathogenic Fo strain on tomato reduces disease, and although the pathogen still colonizes the xylem vessels this has surprisingly little effect on the xylem sap proteome composition. In this tripartite interaction the accumulation of just two PR proteins, NP24 (a PR-5) and a β-glucanase, was affected. The Fo-induced resistance response in tomato appears to be distinct from induced systemic resistance (ISR) or systemic acquired resistance (SAR), as the phytohormones jasmonate, ethylene, and salicylic acid are not required. In this review, we summarize our molecular understanding of Fo-induced resistance in a model and identify caveats in our knowledge.

The plant immune system employs resistance (R) genes to detect the presence of pathogenic microbes by the avirulence (Avr) factors they produce. Whereas some R-genes confer extreme resistance, completely blocking pathogen proliferation, others act later during infection and allow initial microbial multiplication in the host. We hypothesized that transmembrane R-proteins – as opposed to intracellular R-proteins - may recognize and arrest pathogen entry earlier in the infection process. In tomato, two transmembrane R-proteins (I and I-3) and one intracellular R-protein (I-2) have been identified conferring resistance against the same pathogen: the soil borne fungus Fusarium oxysporum f.sp. lycopersici (Fol). We found that in all root inoculations with Fol on resistant tomato plants, the fungus was able to reach the vasculature of the stem. However, the extent of host vasculature colonization was less than that in susceptible plants. This indicates that a complete blockade of fungal ingress does not occur in any of the incompatible interactions. However, resistance mediated by the intracellular R-protein I-2 allowed more extensive fungal colonization than resistance mediated by the transmembrane R-proteins I or I-3. The first phases of invasion – penetration of the root epidermis, cortex colonization and early stage xylem colonization – are unaffected by R-gene mediated resistance of the host. We suggest that all 3 R-proteins only limit Fol colonization after the fungus has reached the xylem tissues, and that I and I-3 act prior to and/or are more effective than I-2 in reducing pathogen proliferation and spread.

Phytohormones, such as salicylic acid (SA), ethylene (ET) and jasmonic acid (JA), play key roles in plant defence following pathogen attack. The involvement of these hormones in susceptibility following Fusarium oxysporum (Fo) infection has mostly been studied in Arabidopsis thaliana. However, Fo causes vascular wilt disease in a broad range of crops, including tomato (Solanum lycopersicum). Surprisingly little is known about the involvement of these phytohormones in the susceptibility of tomato towards Fo f. sp. lycopersici (Fol). Here, we investigate their involvement by the analysis of the expression of ET, JA and SA marker genes following Fol infection, and by bioassays of tomato mutants affected in either hormone production or perception. Fol inoculation triggered the expression of SA and ET marker genes, showing the activation of these pathways. NahG tomato, in which SA is degraded, became hypersusceptible to Fol infection and showed stronger disease symptoms than wild-type. In contrast, ACD and Never ripe (Nr) mutants, in which ET biosynthesis and perception, respectively, are impaired, showed decreased disease symptoms and reduced fungal colonization on infection. The susceptibility of the def1 tomato mutant, and a prosystemin over-expressing line, in which JA signalling is compromised or constitutively activated, respectively, was unaltered. Our results show that SA is a negative and ET a positive regulator of Fol susceptibility. The SA and ET signalling pathways appear to act synergistically, as an intact ET pathway is required for the induction of an SA marker gene, and vice versa.

Interactions between plants and the root-colonizing fungus Fusarium oxysporum (Fo) can be neutral, beneficial, or detrimental for the host. Fo is infamous for its ability to cause wilt, root-, and foot-rot in many plant species, including many agronomically important crops. However, Fo also has another face; as a root endophyte, it can reduce disease caused by vascular pathogens such as Verticillium dahliae and pathogenic Fo strains. Fo also confers protection to root pathogens like Pythium ultimum, but typically not to pathogens attacking above-ground tissues such as Botrytis cinerea or Phytophthora capsici. Endophytes confer biocontrol either directly by interacting with pathogens via mycoparasitism, antibiosis, or by competition for nutrients or root niches, or indirectly by inducing resistance mechanisms in the host. Fo endophytes such as Fo47 and CS-20 differ from Fo pathogens in their effector gene content, host colonization mechanism, location in the plant, and induced host-responses. Whereas endophytic strains trigger localized cell death in the root cortex, and transiently induce immune signaling and papilla formation, these responses are largely suppressed by pathogenic Fo strains. The ability of pathogenic strains to compromise immune signaling and cell death is likely attributable to their host-specific effector repertoire. The lower number of effector genes in endophytes as compared to pathogens provides a means to distinguish them from each other. Co-inoculation of a biocontrol-conferring Fo and a pathogenic Fo strain on tomato reduces disease, and although the pathogen still colonizes the xylem vessels this has surprisingly little effect on the xylem sap proteome composition. In this tripartite interaction the accumulation of just two PR proteins, NP24 (a PR-5) and a β-glucanase, was affected. The Fo-induced resistance response in tomato appears to be distinct from induced systemic resistance (ISR) or systemic acquired resistance (SAR), as the phytohormones jasmonate, ethylene, and salicylic acid are not required. In this review, we summarize our molecular understanding of Fo-induced resistance in a model and identify caveats in our knowledge.

In the original article, there was an error. The amount of xylem sap protein used for nLC-MS/MS analysis was incorrectly depicted; instead of 540 µg of protein 60 µg of protein was TCA precipitated and used for SDS-polyacrylamide gel electrophoresis. A correction has been made to the MATERIALS AND METHODS section, in the sub-section Sample Preparation for nLC-MS/MS: Potential fungal spores were removed from the sap by centrifugation at 800 ×g for 10 min. Xylem sap proteins were concentrated by passing 12 ml of cleared sap through Amicon Ultra-15 Filter Units (Millipore). After centrifugation at 2500 ×g for 15–30 min retentates containing the proteins were recovered. A BCA (bicinchoninic acid) assay (ThermoFischer) was performed to determine the protein concentration. Based on BCA quantification, a volume containing 60 µg of protein was trichloroacetic acid/aceton-precipitated and the pellet was resuspended in SDS loading buffer (2% SDS, 10% glycerol, 60 mM TRIS-HCl pH 6.8, 5% β-mercaptoethanol, 0.01% bromophenol blue), heated at 98◦ C for 5 min and loaded on a 12% SDS-polyacrylamide gel. Following a short electrophoresis, the proteins were stained overnight at 4◦ C with Commassie PageBlue (ThermoFischer). The bands containing the proteins were excised and cysteine reduction and alkylation of the proteins was performed by adding 10 mM DTT pH 8 (incubation at 60◦ C for 1 h) and 20 mM iodoacetamide pH 8 (incubation at room temperature in the dark for 30 min). Protein-containing gel slices were chopped into pieces of approximately 1 mm2 and transferred to 1.5 ml low-binding tubes (Protein LoBind microcentrifuge tubes, Eppendorf). Tryptic in-gel digestion was performed overnight by adding 50 µl of 5 ng/µl Trypsin Sequencing Grade (Sigma-Aldrich). In-house prepared µcolumns were set up by adding C18 Empore disk and LichroprepC18 column material into a 200 µl pipette tip and the tryptic peptides were eluted from the µcolumn with 50 µl of 50% acetonitrile. Acetonitrile content was reduced to

Root endophytes can confer resistance against plant pathogens by direct antagonism or via the host by triggering induced resistance. The latter response typically relies on jasmonic acid (JA)/ethylene (ET)-depended signaling pathways, but can also be triggered via salicylic acid (SA)-dependent signaling pathways. Here, we set out to determine if endophyte-mediated resistance (EMR), conferred by the Fusarium endophyte Fo47, against Fusarium wilt disease in tomato is mediated via SA, ET or JA. To test the contribution of SA, ET, and JA in EMR we performed bioassays with Fo47 and Fusarium oxysporum f. sp. lycopersici in tomato plants impaired in SA accumulation (NahG), JA biosynthesis (def1) or ET-production (ACD) and -sensing (Nr). We observed that the colonization pattern of Fo47 in stems of wildtype plants was indistinguishable from that of the hormone mutants. Surprisingly, EMR was not compromised in the lines affected in JA, ET, or SA signaling. The independence of EMR on SA, JA, and ET implies that this induced resistance-response against Fusarium wilt disease is distinct from the classical Induced Systemic Resistance (ISR) response, providing exciting possibilities for control of wilt diseases independent of conventional defense pathways.

A recent study finds that the Arabidopsis DM1 and DM2d proteins physically interact and trigger autoimmunity in plants. The DM1-DM2d interaction pattern differs from that of known immune receptor pairs, portraying the versatility in NLR functioning.

Plant NLR proteins enable the immune system to recognize and respond to pathogen attack. An early consequence of immune activation is transcriptional reprogramming. Some NLRs have been shown to act in the nucleus and interact with transcription factors. The Rx1 NLR protein of potato binds and distorts double-stranded DNA. However, the components of the chromatin-localized Rx1 complex are largely unknown. Here, we report a physical and functional interaction between Rx1 and NbDBCP, a bromodomain-containing chromatin-interacting protein. NbDBCP accumulates in the nucleoplasm and nucleolus, interacts with chromatin, and redistributes Rx1 to the nucleolus in a subpopulation of imaged cells. Rx1 overexpression reduces the interaction between NbDBCP and chromatin. NbDBCP is a negative regulator of Rx1-mediated immune responses to potato virus X (PVX), and this activity requires an intact bromodomain. Previously, Rx1 has been shown to regulate the DNA-binding activity of a Golden2-like transcription factor, NbGlk1. Rx1 and NbDBCP act synergistically to reduce NbGlk1 DNA binding, suggesting a mode of action for NbDBCP’s inhibitory effect on immunity. This study provides new mechanistic insight into the mechanism by which a chromatin-localized NLR complex co-ordinates immune signaling after pathogen perception., The Rx1 NLR protein of potato binds and distorts double-stranded DNA. This study demonstrates physical and functional interaction between Rx1 and NbDBCP, a bromodomain-containing chromatin-interacting protein. NbDBCP is a negative regulator of Rx1-mediated immune responses to potato virus X and acts in concert with Rx1 to reduce the DNA-binding activity of the immune-activating Golden2-like transcription factor, NbGlk1.

Root colonization by Fusarium oxysporum (Fo) endophytes reduces wilt disease symptoms caused by pathogenic Fo strains. The endophytic strain Fo47, isolated from wilt suppressive soils, reduces Fusarium wilt in various crop species such as tomato, flax, and asparagus. How endophyte-mediated resistance (EMR) against Fusarium wilt is achieved is unclear. Here, nonpathogenic colonization by Fo47 and pathogenic colonization by Fo f.sp. lycopersici (Fol) strains were assessed in tomato roots and stems when inoculated separately or coinoculated. It is shown that Fo47 reduces Fol colonization in stems of both noncultivated and cultivated tomato species. Conversely, Fo47 colonization of coinoculated tomato stems was increased compared to single inoculated plants. Quantitative PCR of fungal colonization of roots (co)inoculated with Fo47 and/or Fol showed that pathogen colonization was drastically reduced when coinoculated with Fo47, compared with single inoculated roots. Endophytic colonization of tomato roots remained unchanged upon coinoculation with Fol. In conclusion, EMR against Fusarium wilt is correlated with a reduction of root and stem colonization by the pathogen. In addition, the endophyte may take advantage of the pathogen-induced suppression of plant defences as it colonizes tomato stems more extensively.

Root endophytes can confer resistance against plant pathogens by direct antagonism or via the host by triggering induced resistance. The latter response typically relies on jasmonic acid (JA)/ethylene (ET)-depended signaling pathways, but can also be triggered via salicylic acid (SA)-dependent signaling pathways. Here, we set out to determine if endophyte-mediated resistance (EMR), conferred by the Fusarium endophyte Fo47, against Fusarium wilt disease in tomato is mediated via SA, ET or JA. To test the contribution of SA, ET, and JA in EMR we performed bioassays with Fo47 and Fusarium oxysporum f. sp. lycopersici in tomato plants impaired in SA accumulation (NahG), JA biosynthesis (def1) or ET-production (ACD) and -sensing (Nr). We observed that the colonization pattern of Fo47 in stems of wildtype plants was indistinguishable from that of the hormone mutants. Surprisingly, EMR was not compromised in the lines affected in JA, ET, or SA signaling. The independence of EMR on SA, JA, and ET implies that this induced resistance-response against Fusarium wilt disease is distinct from the classical Induced Systemic Resistance (ISR) response, providing exciting possibilities for control of wilt diseases independent of conventional defense pathways.

The ability to induce a defense response after pathogen attack is a critical feature of the immune system of any organism. Nucleotide-binding leucine-rich repeat receptors (NLRs) are key players in this process and perceive the occurrence of nonself-activities or foreign molecules. In plants, coevolution with a variety of pests and pathogens has resulted in repertoires of several hundred diverse NLRs in single individuals and many more in populations as a whole. However, the mechanism by which defense signaling is triggered by these NLRs in plants is poorly understood. Here, we show that upon pathogen perception, NLRs use their N-terminal domains to transactivate other receptors. Their N-terminal domains homo- and heterodimerize, suggesting that plant NLRs oligomerize upon activation, similar to the vertebrate NLRs; however, consistent with their large number in plants, the complexes are highly heterometric. Also, in contrast to metazoan NLRs, the N-terminus, rather than their centrally located nucleotide-binding (NB) domain, can mediate initial partner selection. The highly redundant network of NLR interactions in plants is proposed to provide resilience to perturbation by pathogens., Author summary The ability to induce defenses in response to pathogen attack is a critical feature of immunity in any organism. Nucleotide-binding leucine-rich repeat receptors (NLRs) are key players in this process and have evolved to perceive the occurrence of nonself-activities or foreign molecules. In plants, coevolution with a variety of pests and pathogens has resulted in repertoires of several hundred diverse NLRs in single individuals and many more in populations as a whole. The mechanism by which defense signaling is triggered by these NLRs is poorly understood. Here, we show that upon pathogen perception, NLRs use their N-terminal domains to transactivate other receptors. Their N-terminal domains homo- and heterodimerize, suggesting that plant NLRs oligomerize upon activation, similar to the vertebrate NLRs; however, consistent with their large number in plants, their complexes are highly heterometric. Also, in contrast to metazoan NLRs, their N-terminus, rather than their centrally located nucleotide-binding (NB) domain, can mediate initial partner selection. The highly redundant network of NLR interactions is proposed to provide resilience to perturbation by pathogens.

Pathogens use effector proteins to manipulate their hosts. During infection of tomato, the fungus Fusarium oxysporum secretes the effectors Avr2 and Six5. Whereas Avr2 suffices to trigger I-2-mediated cell death in heterologous systems, both effectors are required for I-2-mediated disease resistance in tomato. How Six5 participates in triggering resistance is unknown. Using bimolecular fluorescence complementation assays we found that Avr2 and Six5 interact at plasmodesmata. Single-cell transformation revealed that a 2xRFP marker protein and Avr2-GFP only move to neighboring cells in the presence of Six5. Six5 alone does not alter plasmodesmatal transduction as 2xRFP was only translocated in the presence of both effectors. In SIX5-expressing transgenic plants, the distribution of virally expressed Avr2-GFP, and subsequent onset of I-2-mediated cell death, differed from that in wild-type tomato. Taken together, our data show that in the presence of Six5, Avr2 moves from cell to cell, which in susceptible plants contributes to virulence, but in I-2 containing plants induces resistance.

Plant nucleotide-binding leucine–rich repeat (NLR) proteins enable the immune system to recognize and respond to pathogen attack. An early consequence of immune activation is transcriptional reprogramming, and some NLRs have been shown to act in the nucleus and interact with transcription factors. The Rx1 NLR protein of potato is further able to bind and distort double-stranded DNA. However, Rx1 host targets that support a role for Rx1 in transcriptional reprogramming at DNA are unknown. Here, we report a functional interaction between Rx1 and NbGlk1, a Golden2-like transcription factor. Rx1 binds to NbGlk1 in vitro and in planta. NbGlk1 binds to known Golden2-like consensus DNA sequences. Rx1 reduces the binding affinity of NbGlk1 for DNA in vitro. NbGlk1 activates cellular responses to potato virus X, whereas Rx1 associates with NbGlk1 and prevents its assembly on DNA in planta unless activated by PVX. This study provides new mechanistic insight into how an NLR can coordinate an immune signaling response at DNA following pathogen perceptions.

Cell-to-cell movement of proteins through plasmodesmata is a widely-established mechanism for intercellular signaling in plants. Current techniques to study intercellular protein translocation rely on single-cell transformation using particle bombardment or transgenic lines expressing photo-inducible fluorophores. The method presented here allows visualization and objective quantification of (effector) protein movement between N. benthamiana leaf cells. Agroinfiltration is performed using a single binary vector encoding a GFP-tagged protein of interest that is either mobile or non-mobile (MP; non-MP), together with an ER-anchored mCherry. Upon creation of mosaic-like transformation patterns, cell-to-cell movement of the MP can be followed by monitoring translocation of the GFP signal from mCherry labeled transformed cells into neighboring non-transformed cells. This process can be visualized using confocal microscopy and quantified following protoplast isolation and flow cytometric cell analysis. This method overcomes the limitations of existing methods as it allows rapid and objective quantification of protein translocation without the need of creating transgenic plants.

To confer resistance against pathogens and pests in plants, typically dominant resistance genes are deployed. However, because resistance is based on recognition of a single pathogen-derived molecular pattern, these narrow-spectrum genes are usually readily overcome. Disease arises from a compatible interaction between plant and pathogen. Hence, altering a plant gene that critically facilitates compatibility could provide a more broad-spectrum and durable type of resistance. Here, such susceptibility (S) genes are reviewed with a focus on the mechanisms underlying loss of compatibility. We distinguish three groups of S genes acting during different stages of infection: early pathogen establishment, modulation of host defenses, and pathogen sustenance. The many examples reviewed here show that S genes have the potential to be used in resistance breeding. However, because S genes have a function other than being a compatibility factor for the pathogen, the side effects caused by their mutation demands a one-by-one assessment of their usefulness for application.

The host preferences of the anthropophilic mosquito species in the Anopheles gambiae complex (Diptera: Culicidae) are mediated by skin bacterial volatiles. However, it is not known whether these mosquitoes respond differentially to skin bacterial volatiles from non-human host species. In this study, the responses of two malaria mosquito species in the An. gambiae complex, Anopheles gambiae s.s. (hereafter, An. gambiae) and Anopheles arabiensis, with different host preferences, to volatiles released from skin bacteria were tested. Skin bacteria collected from human, cow and chicken skin significantly increased trap catches; traps containing bacteria collected from human skin caught the highest proportions of An. gambiae and An. arabiensis. Traps with bacteria of human origin caught a significantly higher proportion of An. gambiae than of An. arabiensis, whereas bacterial volatiles from the chicken attracted significantly higher numbers of An. arabiensis than of An. gambiae. Additionally, An. gambiae showed a specialized response to volatiles from four specific bacteria, whereas An. arabiensis responded equally to all species of bacteria tested. Skin bacterial volatiles may therefore play important roles in guiding mosquitoes with different host preferences. The identification of these bacterial volatiles can contribute to the development of an odour blend that attracts mosquitoes with different host preferences.

Pathogens secrete effector proteins to manipulate the host for their own proliferation. Currently it is unclear whether the uptake of effector proteins from extracellular spaces is a host autonomous process. We study this process using the Avr2 effector protein from Fusarium oxysporum f. sp. lycopersici (Fol). Avr2 is an important virulence factor that is secreted into the xylem sap of tomato following infection. Besides that, it is also an avirulence factor triggering immune responses in plants carrying the I-2 resistance gene. Recognition of Avr2 by I-2 occurs inside the plant nucleus. Here, we show that pathogenicity of an Avr2 knockout Fusarium (FolΔAvr2) strain is fully complemented on transgenic tomato lines that express either a secreted (Avr2) or cytosolic Avr2 (ΔspAvr2) protein, indicating that Avr2 exerts its virulence functions inside the host cells. Furthermore, our data imply that secreted Avr2 is taken up from the extracellular spaces in the presence of the fungus. Grafting studies were performed in which scions of I-2 tomato plants were grafted onto either a ΔspAvr2 or on an Avr2 rootstock. Although the Avr2 protein could readily be detected in the xylem sap of the grafted plant tissues, no I-2-mediated immune responses were induced suggesting that I-2-expressing tomato cells cannot autonomously take up the effector protein from the xylem sap. Additionally, ΔspAvr2 and Avr2 plants were crossed with I-2 plants. Whereas ΔspAvr2/I-2 F1 plants showed a constitutive immune response, immunity was not triggered in the Avr2/I-2 plants confirming that Avr2 is not autonomously taken up from the extracellular spaces to trigger I-2. Intriguingly, infiltration of Agrobacterium tumefaciens in leaves of Avr2/I-2 plants triggered I-2 mediated cell death, which indicates that Agrobacterium triggers effector uptake. To test whether, besides Fol, effector uptake could also be induced by other fungal pathogens the ΔspAvr2 and Avr2 transgenic lines were inoculated with Verticillium dahliae. Whereas ΔspAvr2 plants became hyper-susceptible to infection, no difference in disease development was found in the Avr2 plants as compared to wild-type plants. These data suggest that effector uptake is not a host autonomous process and that Fol and A. tumefaciens, but not V. dahliae, facilitate Avr2 uptake by tomato cells from extracellular spaces.

Many haematophagous ectoparasites use carbon dioxide (CO

Somatic embryogenesis receptor kinases (SERKs) are transmembrane receptors involved in plant immunity. Tomato (Solanum lycopersicum) carries three SERK members. One of these, SlSERK1, is required for Mi-1.2-mediated resistance to potato aphids (Macrosiphum euphorbiae). Mi-1.2 encodes a coiled-coil nucleotide-binding leucine-rich repeat protein that in addition to potato aphids confers resistance to two additional phloem-feeding insects and to root-knot nematodes (Meloidogyne spp.). How SlSERK1 participates in Mi-1.2-mediated resistance is unknown, and no Mi-1.2 cognate pest effectors have been identified. Here, we study the mechanistic involvement of SlSERK1 in Mi-1.2-mediated resistance. We show that potato aphid saliva and protein extracts induce the Mi-1.2 defense marker gene SlWRKY72b, indicating that both saliva and extracts contain a Mi-1.2 recognized effector. Resistant tomato cultivar Motelle (Mi-1.2/Mi-1.2) plants overexpressing SlSERK1 were found to display enhanced resistance to potato aphids. Confocal microscopy revealed that Mi-1.2 localizes at three distinct subcellular compartments: the plasma membrane, cytoplasm, and nucleus. Coimmunoprecipitation experiments in these tomato plants and in Nicotiana benthamiana transiently expressing Mi-1.2 and SlSERK1 showed that Mi-1.2 and SlSERK1 colocalize only in a microsomal complex. Interestingly, bimolecular fluorescence complementation analysis showed that the interaction of Mi-1.2 and SlSERK1 at the plasma membrane distinctively changes in the presence of potato aphid saliva, suggesting a model in which a constitutive complex at the plasma membrane participates in defense signaling upon effector binding.

Plants interact with a huge variety of soil microbes, ranging from pathogenic to mutualistic. The Fusarium oxysporum (Fo) species complex consists of ubiquitous soil inhabiting fungi that can infect and cause disease in over 120 different plant species including tomato, banana, cotton, and Arabidopsis. However, in many cases Fo colonization remains symptomless or even has beneficial effects on plant growth and/or stress tolerance. Also in pathogenic interactions a lengthy asymptomatic phase usually precedes disease development. All this indicates a sophisticated and fine-tuned interaction between Fo and its host. The molecular mechanisms underlying this balance are poorly understood. Plant hormone signaling networks emerge as key regulators of plant-microbe interactions in general. In this review we summarize the effects of the major phytohormones on the interaction between Fo and its diverse hosts. Generally, Salicylic Acid (SA) signaling reduces plant susceptibility, whereas Jasmonic Acid (JA), Ethylene (ET), Abscisic Acid (ABA), and auxin have complex effects, and are potentially hijacked by Fo for host manipulation. Finally, we discuss how plant hormones and Fo effectors balance the interaction from beneficial to pathogenic and vice versa.

Increasing numbers of infectious crop diseases that are caused by fungi and oomycetes urge the need to develop alternative strategies for resistance breeding. As an alternative for the use of resistance (R) genes, the application of mutant susceptibility (S) genes has been proposed as a potentially more durable type of resistance. Identification of S genes is hampered by their recessive nature. Here we explore the use of pathogen-derived effectors as molecular probes to identify S genes. Effectors manipulate specific host processes thereby contributing to disease. Effector targets might therefore represent S genes. Indeed, the Pseudomonas syringae effector HopZ2 was found to target MLO2, an Arabidopsis thaliana homologue of the barley S gene Mlo. Unfortunately, most effector targets identified so far are not applicable as S genes due to detrimental effects they have on other traits. However, some effector targets such as Mlo are successfully used, and with the increase in numbers of effector targets being identified, the numbers of S genes that can be used in resistance breeding will rise as well.

Many plant disease resistance (R) proteins belong to the family of nucleotide-binding-leucine rich repeat (NB-LRR) proteins. NB-LRRs mediate recognition of pathogen-derived effector molecules and subsequently activate host defence. Their multi-domain structure allows these pathogen detectors to simultaneously act as sensor, switch and response factor. Structure-function analyses and the recent elucidation of the 3D structures of subdomains have provided new insight in how these different functions are combined and what the contribution is of the individual subdomains. Besides interdomain contacts, interactions with chaperones, the proteasome and effector baits are required to keep NB-LRRs in a signalling-competent, yet auto-inhibited state. In this review we explore operational models of NB-LRR functioning based on recent advances in understanding their structure.

Plant resistance (R) proteins mediate race-specific immunity and initiate host defenses that are often accompanied by a localized cell-death response. Most R proteins belong to the nucleotide binding-leucine-rich repeat (NB-LRR) protein family, as they carry a central NB-ARC domain fused to an LRR domain. The coiled-coil (CC) domain at the N terminus of some solanaceous NB-LRR proteins is extended with a solanaceae domain (SD). Tomato Mi-1.2, which confers resistance against nematodes, white flies, psyllids, and aphids, encodes a typical SD-CNL protein. Here, we analyzed the role of the extended N terminus for Mi-1.2 activation. Removal of the first part of the N terminus (Nt1) induced Mi-1.2-mediated cell death that could be suppressed by overexpression of the second half of the N-terminal region. Yet, autoactivating NB-ARC-LRR mutants require in trans coexpression of the N-terminal region to induce cell death, indicating that the N terminus functions both as a negative and as a positive regulator. Based on secondary structure predictions, we could link both activities to three distinct subdomains, a typical CC domain and two novel, structurally-conserved helical subdomains called SD1 and SD2. A negative regulatory function could be assigned to the SD1, whereas SD2 and the CC together function as positive regulators of Mi-1.2-mediated cell death.

The plant receptor-like kinase somatic embryogenesis receptor kinase 3 (SERK3)/brassinosteroid insensitive 1-associated kinase 1 (BAK1) is required for pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). Here we show that a distinct member of the SERK family, SERK1, is required for the full functioning of Mi-1, a nucleotide binding leucine-rich repeat (NB-LRR) resistance protein. Mi-1 confers resistance to Meloidogyne spp. (root-knot nematodes, RKNs) and three phloem-feeding insects, including Macrosiphum euphorbiae (potato aphid). SERK1 was identified in a tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) screen in Nicotiana benthamiana. The screen was based on the suppression of a pest-independent hypersensitive response triggered by a constitutively active form of Mi-1, Mi-DS4. To assess the role of SERK1 in Mi-1-mediated resistance, Solanum lycopersicum (tomato) SlSERK genes were cloned. Three SlSERK members were identified with homologies to Arabidopsis AtSERK1 or AtSERK3/BAK1, and were named SlSERK1, SlSERK3A and SlSERK3B. SlSERK1 is ubiquitously expressed in tomato. Reducing SlSERK1 transcript levels in resistant plants, using gene-specific TRV-SERK1 VIGS, revealed a role for SlSERK1 in Mi-1-mediated resistance to potato aphids, but not to RKNs. In addition, Mi-1-dependent SlWRKY72 gene regulation was compromised in SlSERK1-silenced plants, placing SlSERK1 in the Mi-1 signaling pathway. Silencing SlSERK1 in a susceptible tomato background did not reduce the susceptibility to aphids, indicating that SlSERK1 is unlikely to be an essential virulence target. SlSERK1 is an active kinase, mainly localized at the plasma membrane. This work identifies a critical early component of Mi-1 signaling, and demonstrates a role for SlSERK1 in NB-LRR-mediated immunity.

SummaryPlants and animals have evolved structurally related innate immune sensors, designated NLRs, to detect intracellular nonself molecules. NLRs are modular, consisting of N-terminal coiled-coil (CC) or TOLL/interleukin-1 receptor (TIR) domains, a central nucleotide-binding (NB) domain, and C-terminal leucine-rich repeats (LRRs). The polymorphic barley mildew A (MLA) locus encodes CC-containing allelic immune receptors recognizing effectors of the pathogenic powdery mildew fungus. We report the crystal structure of an MLA receptor's invariant CC domain, which reveals a rod-shaped homodimer. MLA receptors also self-associate in vivo, but self-association appears to be independent of effector-triggered receptor activation. MLA CC mutants that fail to self-interact impair in planta cell death activity triggered by the CC domain alone and by an autoactive full-length MLA receptor that mimics its ATP-bound state. Thus, CC domain-dependent dimerization of the immune sensor defines a minimal functional unit and implies a role for the dimeric CC module in downstream immune signaling.

XSP10 is an abundant 10 kDa protein found in the xylem sap of tomato. The protein displays structural similarity to plant lipid transfer proteins (LTPs). LTPs are involved in various physiological processes, including disease resistance, and some are able to bind and transfer diverse lipid molecules. XSP10 abundance in xylem sap declines upon infection with Fusarium oxysporum f. sp. lycopersici (Fol), implying involvement of XSP10 in the plant-pathogen interaction. Here, the biochemical characterization of XSP10 with respect to fatty acid-binding properties is reported; a weak but significant binding to saturated fatty acids was found. Furthermore, XSP10-silenced tomato plants were engineered and it was found that these plants exhibited reduced disease symptom development upon infection with a virulent strain of Fol. Interestingly, the reduced symptoms observed did not correlate with an altered expression profile for known reporter genes of plant defence (PR-1 and WIPI). This work demonstrates that XSP10 has lipid-binding properties and is required for full susceptibility of tomato to Fusarium wilt.