Pep1, a Secreted Effector Protein of Ustilago maydis, Is Required for Successful Invasion of Plant Cells

The basidiomycete Ustilago maydis causes smut disease in maize. Colonization of the host plant is initiated by direct penetration of cuticle and cell wall of maize epidermis cells. The invading hyphae are surrounded by the plant plasma membrane and proliferate within the plant tissue. We identified a novel secreted protein, termed Pep1, that is essential for penetration. Disruption mutants of pep1 are not affected in saprophytic growth and develop normal infection structures. However, Δpep1 mutants arrest during penetration of the epidermal cell and elicit a strong plant defense response. Using Affymetrix maize arrays, we identified 116 plant genes which are differentially regulated in Δpep1 compared to wild type infections. Most of these genes are related to plant defense. By in vivo immunolocalization, live-cell imaging and plasmolysis approaches, we detected Pep1 in the apoplastic space as well as its accumulation at sites of cell-to-cell passages. Site-directed mutagenesis identified two of the four cysteine residues in Pep1 as essential for function, suggesting that the formation of disulfide bridges is crucial for proper protein folding. The barley covered smut fungus Ustilago hordei contains an ortholog of pep1 which is needed for penetration of barley and which is able to complement the U. maydis Δpep1 mutant. Based on these results, we conclude that Pep1 has a conserved function essential for establishing compatibility that is not restricted to the U. maydis / maize interaction

Balancing Selection at the Tomato RCR3 Guardee Gene Family Maintains Variation in Strength of Pathogen Defense

Coevolution between hosts and pathogens is thought to occur between interacting molecules of both species. This results in the maintenance of genetic diversity at pathogen antigens (or so-called effectors) and host resistance genes such as the major histocompatibility complex (MHC) in mammals or resistance (R) genes in plants. In plant-pathogen interactions, the current paradigm posits that a specific defense response is activated upon recognition of pathogen effectors via interaction with their corresponding R proteins. According to the''Guard-Hypothesis,'' R proteins (the ``guards'') can sense modification of target molecules in the host (the ``guardees'') by pathogen effectors and subsequently trigger the defense response. Multiple studies have reported high genetic diversity at R genes maintained by balancing selection. In contrast, little is known about the evolutionary mechanisms shaping the guardee, which may be subject to contrasting evolutionary forces. Here we show that the evolution of the guardee RCR3 is characterized by gene duplication, frequent gene conversion, and balancing selection in the wild tomato species Solanum peruvianum. Investigating the functional characteristics of 54 natural variants through in vitro and in planta assays, we detected differences in recognition of the pathogen effector through interaction with the guardee, as well as substantial variation in the strength of the defense response. This variation is maintained by balancing selection at each copy of the RCR3 gene. Our analyses pinpoint three amino acid polymorphisms with key functional consequences for the coevolution between the guardee (RCR3) and its guard (Cf-2). We conclude that, in addition to coevolution at the ``guardee-effector'' interface for pathogen recognition, natural selection acts on the ``guard-guardee'' interface. Guardee evolution may be governed by a counterbalance between improved activation in the presence and prevention of auto-immune responses in the absence of the corresponding pathogen

Proteomic Analysis of the Secretory Response of Aspergillus niger to D-Maltose and D-Xylose

Fungi utilize polysaccharide substrates through extracellular digestion catalyzed by secreted enzymes. Thus far, protein secretion by the filamentous fungus Aspergillus niger has mainly been studied at the level of individual proteins and by genome and transcriptome analyses. To extend these studies, a complementary proteomics approach was applied with the aim to investigate the changes in secretome and microsomal protein composition resulting from a shift to a high level secretion condition. During growth of A. niger on d-sorbitol, small amounts of d-maltose or d-xylose were used as inducers of the extracellular amylolytic and xylanolytic enzymes. Upon induction, protein compositions in the extracellular broth as well as in enriched secretory organelle (microsomal) fractions were analyzed using a shotgun proteomics approach. In total 102 secreted proteins and 1,126 microsomal proteins were identified in this study. Induction by d-maltose or d-xylose resulted in the increase in specific extracellular enzymes, such as glucoamylase A on d-maltose and β-xylosidase D on d-xylose, as well as of microsomal proteins. This reflects the differential expression of selected genes coding for dedicated extracellular enzymes. As expected, the addition of extra d-sorbitol had no effect on the expression of carbohydrate-active enzymes, compared to addition of d-xylose or d-maltose. Furthermore, d-maltose induction caused an increase in microsomal proteins related to translation (e.g., Rpl15) and vesicular transport (e.g., the endosomal-cargo receptor Erv14). Millimolar amounts of the inducers d-maltose and d-xylose are sufficient to cause a direct response in specific protein expression levels. Also, after induction by d-maltose or d-xylose, the induced enzymes were found in microsomes and extracellular. In agreement with our previous findings for d-xylose induction, d-maltose induction leads to recruitment of proteins involved in proteasome-mediated degradation

A Role in Immunity for Arabidopsis Cysteine Protease RD21, the Ortholog of the Tomato Immune Protease C14

Secreted papain-like Cys proteases are important players in plant immunity. We previously reported that the C14 protease of tomato is targeted by cystatin-like EPIC proteins that are secreted by the oomycete pathogen Phytophthora infestans (Pinf) during infection. C14 has been under diversifying selection in wild potato species coevolving with Pinf and reduced C14 levels result in enhanced susceptibility for Pinf. Here, we investigated the role C14-EPIC-like interactions in the natural pathosystem of Arabidopsis with the oomycete pathogen Hyaloperonospora arabidopsidis (Hpa). In contrast to the Pinf-solanaceae pathosystem, the C14 orthologous protease of Arabidopsis, RD21, does not evolve under diversifying selection in Arabidopsis, and rd21 null mutants do not show phenotypes upon compatible and incompatible Hpa interactions, despite the evident lack of a major leaf protease. Hpa isolates express highly conserved EPIC-like proteins during infections, but it is unknown if these HpaEPICs can inhibit RD21 and one of these HpaEPICs even lacks the canonical cystatin motifs. The rd21 mutants are unaffected in compatible and incompatible interactions with Pseudomonas syringae pv. tomato, but are significantly more susceptible for the necrotrophic fungal pathogen Botrytis cinerea, demonstrating that RD21 provides immunity to a necrotrophic pathogen

Presence of celiac disease epitopes in modern and old hexaploid wheat varieties: wheat breeding may have contributed to increased prevalence of celiac disease

Gluten proteins from wheat can induce celiac disease (CD) in genetically susceptible individuals. Specific gluten peptides can be presented by antigen presenting cells to gluten-sensitive T-cell lymphocytes leading to CD. During the last decades, a significant increase has been observed in the prevalence of CD. This may partly be attributed to an increase in awareness and to improved diagnostic techniques, but increased wheat and gluten consumption is also considered a major cause. To analyze whether wheat breeding contributed to the increase of the prevalence of CD, we have compared the genetic diversity of gluten proteins for the presence of two CD epitopes (Glia-α9 and Glia-α20) in 36 modern European wheat varieties and in 50 landraces representing the wheat varieties grown up to around a century ago. Glia-α9 is a major (immunodominant) epitope that is recognized by the majority of CD patients. The minor Glia-α20 was included as a technical reference. Overall, the presence of the Glia-α9 epitope was higher in the modern varieties, whereas the presence of the Glia-α20 epitope was lower, as compared to the landraces. This suggests that modern wheat breeding practices may have led to an increased exposure to CD epitopes. On the other hand, some modern varieties and landraces have been identified that have relatively low contents of both epitopes. Such selected lines may serve as a start to breed wheat for the introduction of ‘low CD toxic’ as a new breeding trait. Large-scale culture and consumption of such varieties would considerably aid in decreasing the prevalence of CD

The Lectin Receptor Kinase LecRK-I.9 Is a Novel Phytophthora Resistance Component and a Potential Host Target for a RXLR Effector

In plants, an active defense against biotrophic pathogens is dependent on a functional continuum between the cell wall (CW) and the plasma membrane (PM). It is thus anticipated that proteins maintaining this continuum also function in defense. The legume-like lectin receptor kinase LecRK-I.9 is a putative mediator of CW-PM adhesions in Arabidopsis and is known to bind in vitro to the Phytophthora infestans RXLR-dEER effector IPI-O via a RGD cell attachment motif present in IPI-O. Here we show that LecRK-I.9 is associated with the plasma membrane, and that two T-DNA insertions lines deficient in LecRK-I.9 (lecrk-I.9) have a ‘gain-of-susceptibility’ phenotype specifically towards the oomycete Phytophthora brassicae. Accordingly, overexpression of LecRK-I.9 leads to enhanced resistance to P. brassicae. A similar ‘gain-of-susceptibility’ phenotype was observed in transgenic Arabidopsis lines expressing ipiO (35S-ipiO1). This phenocopy behavior was also observed with respect to other defense-related functions; lecrk-I.9 and 35S-ipiO1 were both disturbed in pathogen- and MAMP-triggered callose deposition. By site-directed mutagenesis, we demonstrated that the RGD cell attachment motif in IPI-O is not only essential for disrupting the CW-PM adhesions, but also for disease suppression. These results suggest that destabilizing the CW-PM continuum is one of the tactics used by Phytophthora to promote infection. As countermeasure the host may want to strengthen CW-PM adhesions and the novel Phytophthora resistance component LecRK-I.9 seems to function in this process

Identification of Hyaloperonospora arabidopsidis Transcript Sequences Expressed during Infection Reveals Isolate-Specific Effectors

Biotrophic plant pathogens secrete effector proteins that are important for infection of the host. The aim of this study was to identify effectors of the downy mildew pathogen Hyaloperonospora arabidopsidis (Hpa) that are expressed during infection of its natural host Arabidopsis thaliana. Infection-related transcripts were identified from Expressed Sequence Tags (ESTs) derived from leaves of the susceptible Arabidopsis Ws eds1-1 mutant inoculated with the highly virulent Hpa isolate Waco9. Assembly of 6364 ESTs yielded 3729 unigenes, of which 2164 were Hpa-derived. From the translated Hpa unigenes, 198 predicted secreted proteins were identified. Of these, 75 were found to be Hpa-specific and six isolate Waco9-specific. Among 42 putative effectors identified there were three Elicitin-like proteins, 16 Cysteine-rich proteins and 18 host-translocated RXLR effectors. Sequencing of alleles in different Hpa isolates revealed that five RXLR genes show signatures of diversifying selection. Thus, EST analysis of Hpa-infected Arabidopsis is proving to be a powerful method for identifying pathogen effector candidates expressed during infection. Delivery of the Waco9-specific protein RXLR29 in planta revealed that this effector can suppress PAMP-triggered immunity and enhance disease susceptibility. We propose that differences in host colonization can be conditioned by isolate-specific effectors

An effector from the Huanglongbing-associated pathogen targets citrus proteases

The citrus industry is facing an unprecedented challenge from Huanglongbing (HLB). All cultivars can be affected by the HLB-associated bacterium ‘Candidatus Liberibacter asiaticus’ (CLas) and there is no known resistance. Insight into HLB pathogenesis is urgently needed in order to develop effective management strategies. Here, we use Sec-delivered effector 1 (SDE1), which is conserved in all CLas isolates, as a molecular probe to understand CLas virulence. We show that SDE1 directly interacts with citrus papain-like cysteine proteases (PLCPs) and inhibits protease activity. PLCPs are defense-inducible and exhibit increased protein accumulation in CLas-infected trees, suggesting a role in citrus defense responses. We analyzed PLCP activity in field samples, revealing specific members that increase in abundance but remain unchanged in activity during infection. SDE1-expressing transgenic citrus also exhibit reduced PLCP activity. These data demonstrate that SDE1 inhibits citrus PLCPs, which are immune-related proteases that enhance defense responses in plants

The Ustilago maydis Effector Pep1 Suppresses Plant Immunity by Inhibition of Host Peroxidase Activity

The corn smut Ustilago maydis establishes a biotrophic interaction with its host plant maize. This interaction requires efficient suppression of plant immune responses, which is attributed to secreted effector proteins. Previously we identified Pep1 (Protein essential during penetration-1) as a secreted effector with an essential role for U. maydis virulence. pep1 deletion mutants induce strong defense responses leading to an early block in pathogenic development of the fungus. Using cytological and functional assays we show that Pep1 functions as an inhibitor of plant peroxidases. At sites of Δpep1 mutant penetrations, H2O2 strongly accumulated in the cell walls, coinciding with a transcriptional induction of the secreted maize peroxidase POX12. Pep1 protein effectively inhibited the peroxidase driven oxidative burst and thereby suppresses the early immune responses of maize. Moreover, Pep1 directly inhibits peroxidases in vitro in a concentration-dependent manner. Using fluorescence complementation assays, we observed a direct interaction of Pep1 and the maize peroxidase POX12 in vivo. Functional relevance of this interaction was demonstrated by partial complementation of the Δpep1 mutant defect by virus induced gene silencing of maize POX12. We conclude that Pep1 acts as a potent suppressor of early plant defenses by inhibition of peroxidase activity. Thus, it represents a novel strategy for establishing a biotrophic interaction

Regulation of proteinaceous effector expression in phytopathogenic fungi

Effectors are molecules used by microbial pathogens to facilitate infection via effector-triggered susceptibility or tissue necrosis in their host. Much research has been focussed on the identification and elucidating the function of fungal effectors during plant pathogenesis. By comparison, knowledge of how phytopathogenic fungi regulate the expression of effector genes has been lagging. Several recent studies have illustrated the role of various transcription factors, chromosome-based control, effector epistasis, and mobilisation of endosomes within the fungal hyphae in regulating effector expression and virulence on the host plant. Improved knowledge of effector regulation is likely to assist in improving novel crop protection strategies