Nine things to know about elicitins

Elicitins are structurally conserved extracellular proteins in Phytophthora and Pythium oomycete pathogen species. They were first described in the late 1980s as abundant proteins in Phytophthora culture filtrates that have the capacity to elicit hypersensitive (HR) cell death and disease resistance in tobacco. Later, they became well-established as having features of microbe-associated molecular patterns (MAMPs) and to elicit defences in a variety of plant species. Research on elicitins culminated in the recent cloning of the elicitin response (ELR) cell surface receptor-like protein, from the wild potato Solanum microdontum, which mediates response to a broad range of elicitins. In this review, we provide an overview on elicitins and the plant responses they elicit. We summarize the state of the art by describing what we consider to be the nine most important features of elicitin biology

Unravelling the elicitin perception pathway against Phytophthora in potato

Potato (Solanum tuberosum) is the most important non-cereal crop for human consumption and its starch and fibers are also used to produce several industrial products. Potato suffers from many pests and diseases. The most threatening and re-emerging disease of potato worldwide is late blight, which is caused by the notorious oomycete Phytophthora infestans (Chapter 1). This pathogen is a hemi-biotrophic organism that secretes a huge arsenal of apoplastic and host-translocated cytoplasmic effectors in order to colonize the host. Interestingly, wild potato plants have evolved receptors that recognize some of those effectors and trigger defense responses. These wild plants are a major source of resistance genes that can be transferred to the cultivated potato. Resistant cultivars obtained by breeding are highly desirable because the costs for chemical control of the disease are very high and there is also a need to reduce their use in order to preserve the environment. Moreover, the pathogen has developed resistance to some of those chemicals. Receptors that recognize cytoplasmic effectors often belong to the nucleotide-binding leucine-rich repeat (NLR) family of resistance genes, (R genes), and they have been used for several years in resistance breeding. Despite the effectivity of NLRs in providing resistance, so far, most single NLR genes introduced into cultivated potato have been defeated by P. infestans rather quickly. To effectively control the pathogen in the long term, resistance gene stacking approaches in combination with new layers of defense have to be considered. Like NLRs, cell surface-residing receptors, or pattern recognition receptors (PRRs) trigger defense responses and contribute to basal or non-host resistance against pathogens. They do so by recognizing apoplastic effectors or microbe-associated molecular patterns (MAMPs). Usually PRR-triggered resistance is quantitative and not as robust as NLR-based resistance, however it is believed to be more durable. In potato, PRR-based immunity that is triggered by the recognition of MAMPs/effectors has remained unexplored. The first receptor against oomycetes was recently identified from a wild potato species, S. microdontum. The receptor, named ELICITIN RESPONSE (ELR), was found to recognize elicitins, a family of conserved apoplastic effectors with MAMP features, found specifically in Phytophthora and Pythium (Chapter 1). The research described in this thesis focused on studying PRR-triggered immunity in potato against the devastating pathogen P. infestans, by using ELR as a model (Chapter 1). Effector-assisted breeding has proven to be a great tool for identifying resistances against pathogens and was pioneered from research on potato late blight. In a process also known as effector genomics (effectoromics), candidate effectors are predicted from the genome of pathogens such as P. infestans. Candidate effectors are cloned in plant expression vectors and are screened in a wild resistant germplasm for occurrence of specific responses. In Chapter 2 we show how effectoromics can be used to identify PRRs in Solanum spp. Simplified protocols are described for performing the effector screens, selecting plants for crosses and genetically mapping the responses. For performing effector screens, besides the routinely used potato virus X (PVX) agroinfection and agroinfiltration, we describe the use of recombinant apoplastic effector proteins. This strategy can complement the results obtained by the other routinely applied Agrobacterium-based methods or enable screening of Agrobacterium- or PVX-recalcitrant plants. We provide protocols for heterologous apoplastic effector expression in the yeast Pichia pastoris. This includes recombinant effector design, cloning, high throughput P. pastoris clone selection and small scale protein production. We also provide an example with the production of six different P. infestans effectors using this system. ELR is a receptor-like protein (RLP) and as such, it lacks a cytoplasmic signaling domain that is required for triggering defense responses. It was known that SOMATIC EMBRYOGENESIS RECEPTOR KINASE 3 (SERK3) is a receptor-like kinase (RLK) required for cell death triggered by the elicitin INF1 and that it also biochemically associates with ELR. In Chapter 3 we investigated the association of ELR with another interactor, which has been proposed to be specific for RLPs; the RLK SUPPRESSOR OF BIR1-1 (SOBIR1). Virus-induced gene silencing (VIGS) assays showed that SOBIR1 is required for cell death response triggered by INF1 and for basal resistance to P. infestans in Nicotiana benthamiana. Genetic complementation assays demonstrated that the kinase of SOBIR1 is required for INF1-triggered cell death. Protein co-immunoprecipitation studies showed that ELR is associating with S. microdontum SOBIR1 and its close homolog SOBIR1-like. From our findings it seems that ELR is found in a constitutive complex with SOBIR1, which recruits SERK3 upon INF1 elicitation. True PRRs are known to physically interact with their ligands, however, this was not explored yet since the identification of ELR. In Chapter 4 we studied whether ELR is able to interact with various elicitins by using in planta and in vitro co-immunoprecipitation assays. We showed that ELR is able to physically bind INF1 in both setups, indicating that the interaction is very specific. Moreover, we found that ELR binds with the elicitins ParA1 (P. parasitica) and β-CRY (P. cryptogea). We also found that ELR is able to trigger cell death with additional elicitins, but likely binds them with lower affinity that remained under the detection limit. Interestingly, we observed that one C-terminally tagged version of INF1 was binding to ELR but failed to trigger cell death, in contrast to N-terminally tagged INF1. Similarly, a β-CRY dimer failed to trigger cell death when infiltrated in leaves expressing ELR. We hypothesized that the cause of these phenotypes could be due to altered interaction of ELR with the RLKs SOBIR1 or SERK3. Indeed, with the C-terminally tagged INF1, we found that both SOBIR1 and SERK3 were not in complex with ELR, while the β-CRY dimer was not allowing SOBIR1 to associate. To our knowledge, this is the first time that such a co-receptor inhibition is reported. Our data, therefore, highlight the necessity of performing functional control experiments for tagged proteins and further strengthen our earlier finding that both SOBIR1 and SERK3 are required for INF1-triggered cell death by ELR. These discoveries could prove useful for enabling detailed studies on RLP signaling. When transformed into cultivated potato, ELR was known to enhance resistance against P. infestans, however, it remained unknown what the resistance contribution of this PRR in its native genetic background (i.e. S. microdontum) is. We hypothesized that recent developments of genome editing technologies can be used to perform such studies in non-model plants like (wild) potato. In Chapter 5, we used clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing to target ELR in wild Solanum. We generated a CRISPR/Cas9 construct that was found to be effective in inducing targeted mutations in ELR when tested in a transient setup in planta. Since it was impossible to transform the wild S. microdontum with the construct, we attempted the transformation of five different species that were known to carry ELR homologs. We obtained transformants from S. edinense, S. papita, S. phureja and S. chacoense, which were then screened for altered responses to INF1. Promising transformants with altered responses to INF1 were obtained in the pentaploid S. edinense and tetraploid S. papita. These transformants were subsequently searched for mutations using a mutation enrichment approach, followed by PCR and sequencing. No full ELR knock-outs were obtained, however, partly mutated and partly wildtype alleles could be found in these transformants. Altogether, these findings show that CRISPR/Cas9 approaches are effective for functional characterization of genes in wild Solanum spp. However, they are limited by the transformation efficiency and ploidy level of a said genotype. Finally, in Chapter 6, the findings of this thesis are discussed and placed in a broader perspective. A schematic overview of the perception of elicitins by ELR in wild potato is provided including all of our findings on elicitin binding, interaction with co-receptors SERK3 and SOBIR1, as well as some preliminary findings on AVR3a, an effector that suppresses INF1-triggered cell death as well as receptor endocytosis. In addition we propose two proteomic approaches for PRR identification, taking advantage of the obtained knowledge on effector/PRR or PRR/co-receptor interactions. Overall my research has contributed to the characterization of the first line of induced defense against Phytophthora in potato and could be instrumental for achieving durable resistance against late blight.</p

Recognition of Phytophthora infestans in potato (Solanum tuberosum L.): Scr74 gene as an example

Phytophthora infestans is the causal agent of late blight, the most devastating disease of potato worldwide. The P. infestans genome encodes potentially polymorphic genes that evolve continually to evade the recognition of plant R genes, though it has hundreds of predicted and conserved effector proteins recognised by the plant. The gene Scr74 encodes a predicted 74-amino acid secreted cysteine-rich protein belonging to a highly polymorphic gene family within P. infestans. This study screened the recognition of Scr74 genes in wild potato genotypes from August 2013 to January 2014 in the Plant Breeding Laboratory of Wageningen University, the Netherlands. To identify the recognition of the Scr74 gene, we grew potato genotypes in the green house for PVX assays, detached leaf assays and molecular work. Twenty-seven good-quality sequences of the Scr74 gene variant with a length of 74 amino acids were found and more frequent amino acid variation was detected on the mature protein. Seventeen Scr74 constructs were identified as diversified and two effectors were strongly recognised by wild S. verrucosum genotypes via effectoromics from the PVX assay. A strong plant cell death hypersensitive response (HR) was recorded on wild S. verrucosum and S. tuberosum genotypes from the detached leaf assay. This recognition seems to be a useful indicator for the presence of a resistance gene (s) to the polymorphic effectors of P. infestans (as it has seen on Scr74 gene) in the wild potato genotypes

Recognition of Phytophthora infestans in potato (Solanum tuberosum L.): Scr74 gene as an example

Phytophthora infestans is the causal agent of late blight, the most devastating disease of potato worldwide. The P. infestans genome encodes potentially polymorphic genes that evolve continually to evade the recognition of plant R genes, though it has hundreds of predicted and conserved effector proteins recognised by the plant. The gene Scr74 encodes a predicted 74-amino acid secreted cysteine-rich protein belonging to a highly polymorphic gene family within P. infestans. This study screened the recognition of Scr74 genes in wild potato genotypes from August 2013 to January 2014 in the Plant Breeding Laboratory of Wageningen University, the Netherlands. To identify the recognition of the Scr74 gene, we grew potato genotypes in the green house for PVX assays, detached leaf assays and molecular work. Twenty-seven good-quality sequences of the Scr74 gene variant with a length of 74 amino acids were found and more frequent amino acid variation was detected on the mature protein. Seventeen Scr74 constructs were identified as diversified and two effectors were strongly recognised by wild S. verrucosum genotypes via effectoromics from the PVX assay. A strong plant cell death hypersensitive response (HR) was recorded on wild S. verrucosum and S. tuberosum genotypes from the detached leaf assay. This recognition seems to be a useful indicator for the presence of a resistance gene (s) to the polymorphic effectors of P. infestans (as it has seen on Scr74 gene) in the wild potato genotypes.</p

The ELR-SOBIR1 Complex Functions as a Two-Component Receptor-Like Kinase to Mount Defense Against Phytophthora infestans

The ELICITIN RESPONSE protein (ELR) from Solanum microdontum can recognize INF1 elicitin of Phytophthora infestans and trigger defense responses. ELR is a receptor-like protein (RLP) that lacks a cytoplasmic signaling domain and is anticipated to require interaction with a signaling-competent receptor-like kinase. SUPPRESSOR OF BIR1-1 (SOBIR1) has been proposed as a general interactor for RLPs involved in immunity and, as such, is a potential interactor for ELR. Here, we investigate whether SOBIR1 is required for response to INF1 and resistance to P. infestans and whether it associates with ELR. Our results show that virus-induced gene silencing of SOBIR1 in Nicotiana benthamiana leads to loss of INF1-triggered cell death and increased susceptibility to P. infestans. Using genetic complementation, we found that the kinase activity of SOBIR1 is required for INF1-triggered cell death. Coimmunoprecipitation experiments showed that ELR constitutively associates with potato SOBIR1 in planta, forming a bipartite receptor complex. Upon INF1 elicitation, this ELR-SOBIR1 complex recruits SERK3 (SOMATIC EMBRYOGENESIS RECEPTOR KINASE 3) leading to downstream signaling activation. Overall, our study shows that SOBIR1 is required for basal resistance to P. infestans and for INF1-triggered cell death and functions as an adaptor kinase for ELR

Recognition of Pep-13/25 MAMPs of Phytophthora localizes to an RLK locus in Solanum microdontum

Pattern-triggered immunity (PTI) in plants is mediated by cell surface-localized pattern recognition receptors (PRRs) upon perception of microbe-associated molecular pattern (MAMPs). MAMPs are conserved molecules across microbe species, or even kingdoms, and PRRs can confer broad-spectrum disease resistance. Pep-13/25 are well-characterized MAMPs in Phytophthora species, which are renowned devastating oomycete pathogens of potato and other plants, and for which genetic resistance is highly wanted. Pep-13/25 are derived from a 42 kDa transglutaminase GP42, but their cognate PRR has remained unknown. Here, we genetically mapped a novel surface immune receptor that recognizes Pep-25. By using effectoromics screening, we characterized the recognition spectrum of Pep-13/25 in diverse Solanaceae species. Response to Pep-13/25 was predominantly found in potato and related wild tuber-bearing Solanum species. Bulk-segregant RNA sequencing (BSR-Seq) and genetic mapping the response to Pep-25 led to a 0.081 cM region on the top of chromosome 3 in the wild potato species Solanum microdontum subsp. gigantophyllum. Some BAC clones in this region were isolated and sequenced, and we found the Pep-25 receptor locates in a complex receptor-like kinase (RLK) locus. This study is an important step toward the identification of the Pep-13/25 receptor, which can potentially lead to broad application in potato and various other hosts of Phytophthora species

Verticillium dahliae LysM effectors differentially contribute to virulence on plant hosts

Chitin-binding LysM effectors contribute to virulence of various plant pathogenic fungi that are causal agents of foliar diseases. Here, we report on LysM effectors of the soil-borne fungal vascular wilt pathogen Verticillium dahliae. Comparative genomics revealed three core LysM effectors that are conserved in a collection of V. dahliae strains. Remarkably, and in contrast to the previously studied LysM effectors of other plant pathogens, no expression of core LysM effectors was monitored in planta in a taxonomically diverse panel of host plants. Moreover, targeted deletion of the individual LysM effector genes in V. dahliae strain JR2 did not compromise virulence in infections on Arabidopsis, tomato or Nicotiana benthamiana. Interestingly, an additional lineage-specific LysM effector is encoded in the genome of V. dahliae strain VdLs17 but not in any other V. dahliae strain sequenced to date. Remarkably, this lineage-specific effector is expressed in planta and contributes to virulence of V. dahliae strain VdLs17 on tomato, but not on Arabidopsis or on N. benthamiana. Functional analysis revealed that this LysM effector binds chitin, is able to suppress chitin-induced immune responses, and protects fungal hyphae against hydrolysis by plant hydrolytic enzymes. Thus, in contrast to the core LysM effectors of V. dahliae, this lineage-specific LysM effector of strain VdLs17 contributes to virulence in planta

Verticillium dahliae LysM effectors differentially contribute to virulence on plant hosts

Chitin-binding lysin motif (LysM) effectors contribute to the virulence of various plant-pathogenic fungi that are causal agents of foliar diseases. Here, we report the LysM effectors of the soil-borne fungal vascular wilt pathogen Verticillium dahliae. Comparative genomics revealed three core LysM effectors that are conserved in a collection of V. dahliae strains. Remarkably, and in contrast with the previously studied LysM effectors of other plant pathogens, no expression of core LysM effectors was monitored in planta in a taxonomically diverse panel of host plants. Moreover, targeted deletion of the individual LysM effector genes in V. dahliae strain JR2 did not compromise virulence in infections on Arabidopsis, tomato or Nicotiana benthamiana. Interestingly, an additional lineage-specific LysM effector is encoded in the genome of V. dahliae strain VdLs17, but not in any other V. dahliae strain sequenced to date. Remarkably, this lineage-specific effector is expressed in planta and contributes to the virulence of V. dahliae strain VdLs17 on tomato, but not on Arabidopsis or N. benthamiana. Functional analysis revealed that this LysM effector binds chitin, is able to suppress chitin-induced immune responses and protects fungal hyphae against hydrolysis by plant hydrolytic enzymes. Thus, in contrast with the core LysM effectors of V. dahliae, this lineage-specific LysM effector of strain VdLs17 contributes to virulence in planta