Botrytis hypersensitive response inducing protein 1 triggers noncanonical PTI to induce plant cell death.

According to their lifestyle, plant pathogens are divided into biotrophic and necrotrophic organisms. Biotrophic pathogens exclusively nourish living host cells, whereas necrotrophic pathogens rapidly kill host cells and nourish cell walls and cell contents. To this end, the necrotrophic fungus Botrytis cinerea secretes large amounts of phytotoxic proteins and cell wall-degrading enzymes. However, the precise role of these proteins during infection is unknown. Here, we report on the identification and characterization of the previously unknown toxic protein hypersensitive response-inducing protein 1 (Hip1), which induces plant cell death. We found the adoption of a structurally conserved folded Alternaria alternata Alt a 1 protein structure to be a prerequisite for Hip1 to exert its necrosis-inducing activity in a host-specific manner. Localization and the induction of typical plant defense responses by Hip1 indicate recognition as a pathogen-associated molecular pattern at the plant plasma membrane. In contrast to other secreted toxic Botrytis proteins, the activity of Hip1 does not depend on the presence of the receptor-associated kinases BRI1-associated kinase 1 and suppressor of BIR1-1. Our results demonstrate that recognition of Hip1, even in the absence of obvious enzymatic or pore-forming activity, induces strong plant defense reactions eventually leading to plant cell death. Botrytis hip1 overexpression strains generated by CRISPR/Cas9 displayed enhanced infection, indicating the virulence-promoting potential of Hip1. Taken together, Hip1 induces a noncanonical defense response which might be a common feature of structurally conserved fungal proteins from the Alt a 1 family.

Nep1-like proteins as a target for plant pathogen control.

The lack of efficient methods to control the major diseases of crops most important to agriculture leads to huge economic losses and seriously threatens global food security. Many of the most important microbial plant pathogens, including bacteria, fungi, and oomycetes, secrete necrosis- and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs), which critically contribute to the virulence and spread of the disease. NLPs are cytotoxic to eudicot plants, as they disturb the plant plasma membrane by binding to specific plant membrane sphingolipid receptors. Their pivotal role in plant infection and broad taxonomic distribution makes NLPs a promising target for the development of novel phytopharmaceutical compounds. To identify compounds that bind to NLPs from the oomycetes Pythium aphanidermatum and Phytophthora parasitica, a library of 587 small molecules, most of which are commercially unavailable, was screened by surface plasmon resonance. Importantly, compounds that exhibited the highest affinity to NLPs were also found to inhibit NLP-mediated necrosis in tobacco leaves and Phytophthora infestans growth on potato leaves. Saturation transfer difference-nuclear magnetic resonance and molecular modelling of the most promising compound, anthranilic acid derivative, confirmed stable binding to the NLP protein, which resulted in decreased necrotic activity and reduced ion leakage from tobacco leaves. We, therefore, confirmed that NLPs are an appealing target for the development of novel phytopharmaceutical agents and strategies, which aim to directly interfere with the function of these major microbial virulence factors. The compounds identified in this study represent lead structures for further optimization and antimicrobial product development.

Disruption of plant plasma membrane by Nep1-like proteins in pathogen-plant interactions.

Lipid membrane destruction by microbial pore-forming toxins (PFTs) is a ubiquitous mechanism of damage to animal cells, but is less prominent in plants. Nep1-like proteins (NLPs) secreted by phytopathogens that cause devastating crop diseases, such as potato late blight, represent the only family of microbial PFTs that effectively damage plant cells by disrupting the integrity of the plant plasma membrane. Recent research has elucidated the molecular mechanism of NLP-mediated membrane damage, which is unique among microbial PFTs and highly adapted to the plant membrane environment. In this review, we cover recent insight into how NLP cytolysins damage plant membranes and cause cell death.

Cytotoxic activity of Nep1-like proteins on monocots.

Necrosis- and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) are found throughout several plant-associated microbial taxa and are typically considered to possess cytolytic activity exclusively on dicot plant species. However, cytolytic NLPs are also produced by pathogens of monocot plants such as the onion (Allium cepa) pathogen Botrytis squamosa. We determined the cytotoxic activity of B. squamosa BsNep1, as well as other previously characterized NLPs, on various monocot plant species and assessed the plant plasma membrane components required for NLP sensitivity. Leaf infiltration of NLPs showed that onion cultivars are differentially sensitive to NLPs, and analysis of their sphingolipid content revealed that the GIPC series A : series B ratio did not correlate to NLP sensitivity. A tri-hybrid population derived from a cross between onion and two wild relatives showed variation in NLP sensitivity within the population. We identified a quantitative trait locus (QTL) for NLP insensitivity that colocalized with a previously identified QTL for B. squamosa resistance and the segregating trait of NLP insensitivity correlated with the sphingolipid content. Our results demonstrate the cytotoxic activity of NLPs on several monocot plant species and legitimize their presence in monocot-specific plant pathogens.

Molecular basis for functional diversity among microbial Nep1-like proteins.

Necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) are secreted by several phytopathogenic microorganisms. They trigger necrosis in various eudicot plants upon binding to plant sphingolipid glycosylinositol phosphorylceramides (GIPC). Interestingly, HaNLP3 from the obligate biotroph oomycete Hyaloperonospora arabidopsidis does not induce necrosis. We determined the crystal structure of HaNLP3 and showed that it adopts the NLP fold. However, the conformations of the loops surrounding the GIPC headgroup-binding cavity differ from those of cytotoxic Pythium aphanidermatum NLPPya. Essential dynamics extracted from µs-long molecular dynamics (MD) simulations reveals a limited conformational plasticity of the GIPC-binding cavity in HaNLP3 relative to toxic NLPs. This likely precludes HaNLP3 binding to GIPCs, which is the underlying reason for the lack of toxicity. This study reveals that mutations at key protein regions cause a switch between non-toxic and toxic phenotypes within the same protein scaffold. Altogether, these data provide evidence that protein flexibility is a distinguishing trait of toxic NLPs and highlight structural determinants for a potential functional diversification of non-toxic NLPs utilized by biotrophic plant pathogens.

Structure-Function Analysis of Immune Receptor AtRLP23 with Its Ligand nlp20 and Coreceptors AtSOBIR1 and AtBAK1.

Pattern-triggered immunity is an inherent feature of the plant immune system. Recognition of either microbe-derived surface structures (patterns) or of plant materials released due to the deleterious impact of microbial infection is brought about by plasma membrane pattern recognition receptors (PRRs). PRRs composed of leucine-rich repeat (LRR) ectodomains are thought to mediate sensing of proteinaceous patterns and to initiate signaling cascades culminating in the activation of generic plant defenses. In contrast to LRR receptor kinases, LRR receptor proteins (LRR-RPs) lack a cytoplasmic kinase domain for initiation of downstream signal transduction. LRR-RPs form heteromeric constitutive, ligand-independent complexes with coreceptor SOBIR1. Upon ligand binding to LRR-RPs, recruitment of coreceptor SERK3/BAK1 results in formation of a ternary PRR complex. Structure-function analysis of LRR-RP-type PRRs is missing. AtRLP23 constitutes an LRR-RP PRR that mediates recognition of a peptide motif (nlp20) found in numerous bacterial, fungal, and oomycete necrosis and ethylene-inducing peptide 1-like proteins (NLPs). We here report the use of a series of AtRLP23 variants to decipher subdomains required for ligand binding and interaction with coreceptors AtSOBIR1 and AtBAK1, respectively. Deletion of LRR1 or LRR3 subdomains efficiently abrogated the ability of AtRLP23 receptor variants to confer nlp20 pattern sensitivity, to bind nlp20, and to recruit AtBAK1 into a ternary PRR complex. This suggests that the very N-terminal part of the AtRLP23 ectodomain is crucial for receptor function. Deletion of the intracellular 17-amino-acid tail of AtRLP23 reduced but did not abolish receptor function, suggesting an auxiliary role of this domain in receptor function. We further found that interaction of AtRLP23 and other LRR-RP-type PRRs with AtSOBIR1 does not require the AtRLP23 LRR ectodomain but is brought about by a GxxxG protein dimerization motif in the transmembrane domain and a stretch of negatively charged glutamic acid residues in the outer juxtamembrane domain of the receptor. Further, AtRLP23 levels were found to be unaltered in Atsobir1-1 mutant genotypes, suggesting that AtSOBIR1 does not act as a protein scaffold in stabilizing LRR-RP-type PRRs in Arabidopsis.

Nep1-like proteins from three kingdoms of life act as a microbe-associated molecular pattern in Arabidopsis.

Necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) are secreted by a wide range of plant-associated microorganisms. They are best known for their cytotoxicity in dicot plants that leads to the induction of rapid tissue necrosis and plant immune responses. The biotrophic downy mildew pathogen Hyaloperonospora arabidopsidis encodes 10 different noncytotoxic NLPs (HaNLPs) that do not cause necrosis. We discovered that these noncytotoxic NLPs, however, act as potent activators of the plant immune system in Arabidopsis thaliana. Ectopic expression of HaNLP3 in Arabidopsis triggered resistance to H. arabidopsidis, activated the expression of a large set of defense-related genes, and caused a reduction of plant growth that is typically associated with strongly enhanced immunity. N- and C-terminal deletions of HaNLP3, as well as amino acid substitutions, pinpointed to a small central region of the protein that is required to trigger immunity, indicating the protein acts as a microbe-associated molecular pattern (MAMP). This was confirmed in experiments with a synthetic peptide of 24 aa, derived from the central part of HaNLP3 and corresponding to a conserved region in type 1 NLPs that induces ethylene production, a well-known MAMP response. Strikingly, corresponding 24-aa peptides of fungal and bacterial type 1 NLPs were also able to trigger immunity in Arabidopsis. The widespread phylogenetic distribution of type 1 NLPs makes this protein family (to our knowledge) the first proteinaceous MAMP identified in three different kingdoms of life.

A conserved peptide pattern from a widespread microbial virulence factor triggers pattern-induced immunity in Arabidopsis.

Microbe- or host damage-derived patterns mediate activation of pattern-triggered immunity (PTI) in plants. Microbial virulence factor (effector)-triggered immunity (ETI) constitutes a second layer of plant protection against microbial attack. Various necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) produced by bacterial, oomycete and fungal microbes are phytotoxic virulence factors that exert immunogenic activities through phytotoxin-induced host cell damage. We here show that multiple cytotoxic NLPs also carry a pattern of 20 amino acid residues (nlp20) that triggers immunity-associated plant defenses and immunity to microbial infection in Arabidopsis thaliana and related plant species with similar characteristics as the prototype pattern, bacterial flagellin. Characteristic differences in flagellin and nlp20 plant responses exist however, as nlp20s fail to trigger extracellular alkalinization in Arabidopsis cell suspensions and seedling growth inhibition. Immunogenic nlp20 peptide motifs are frequently found in bacterial, oomycete and fungal NLPs. Such an unusually broad taxonomic distribution within three phylogenetic kingdoms is unprecedented among microbe-derived triggers of immune responses in either metazoans or plants. Our findings suggest that cytotoxic NLPs carrying immunogenic nlp20 motifs trigger PTI in two ways as typical patterns and by inflicting host cell damage. We further propose that conserved structures within a microbial virulence factor might have driven the emergence of a plant pattern recognition system mediating PTI. As this is reminiscent of the evolution of immune receptors mediating ETI, our findings support the idea that there is a continuum between PTI and ETI.

Evidence for functional diversification within a fungal NEP1-like protein family.

In this study, we functionally analyzed the gene family encoding necrosis- and ethylene-inducing-like proteins (NLP) of the vascular wilt pathogen Verticillium dahliae. We show that the composition of the NLP gene family varies little among V. dahliae isolates. The cytotoxic activity of NLP family members of a tomato-pathogenic V. dahliae strain was determined, demonstrating that only two of the seven NLP induced plant cell death. The genes encoding these cytotoxic NLP were found to be induced in V. dahliae upon colonization of tomato. Interestingly, targeted deletion of either of the two genes in V. dahliae significantly compromised virulence on tomato as well as on Arabidopsis plants, whereas deletion of only one of the two genes affected virulence on Nicotiana benthamiana. This could be attributed to differential induction of the two NLP genes in V. dahliae upon N. benthamiana colonization, revealing that the in planta induction of NLP genes varies between plant hosts. Intriguingly, one of the NLP genes appears to also affect vegetative growth and conidiospore production, because the corresponding deletion strain produced significantly fewer conidiospores and developed extensive aerial mycelium. In conclusion, we demonstrate that the expanded V. dahliae NLP family shows functional diversification, revealing not only differential cytotoxicity between family members but also that the cytotoxic NLP play a role in vegetative growth and asexual reproduction in addition to their contribution to virulence.

In vitro translocation experiments with RxLR-reporter fusion proteins of Avr1b from Phytophthora sojae and AVR3a from Phytophthora infestans fail to demonstrate specific autonomous uptake in plant and animal cells.

Plant-pathogenic oomycetes have a large set of secreted effectors that can be translocated into their host cells during infection. One group of these effectors are the RxLR effectors for which it has been shown, in a few cases, that the RxLR motif is important for their translocation. It has been suggested that the RxLR-leader sequences alone are enough to translocate the respective effectors into eukaryotic cells through binding to surface-exposed phosphoinositol-3-phosphate. These conclusions were primary based on translocation experiments conducted with recombinant fusion proteins whereby the RxLR leader of RxLR effectors (i.e., Avr1b from Phytophthora sojae) were fused to the green fluorescent protein reporter-protein. However, we failed to observe specific cellular uptake for a comparable fusion protein where the RxLR leader of the P. infestans AVR3a was fused to monomeric red fluorescent protein. Therefore, we reexamined the ability of the reported P. sojae AVR1b RxLR leader to enter eukaryotic cells. Different relevant experiments were performed in three independent laboratories, using fluorescent reporter fusion constructs of AVR3a and Avr1b proteins in a side-by-side comparative study on plant tissue and human and animal cells. We report that we were unable to obtain conclusive evidence for specific RxLR-mediated translocation.

Genotyping-by-sequencing-based identification of Arabidopsis pattern recognition receptor RLP32 recognizing proteobacterial translation initiation factor IF1.

Activation of plant pattern-triggered immunity (PTI) relies on the recognition of microbe-derived structures, termed patterns, through plant-encoded surface-resident pattern recognition receptors (PRRs). We show that proteobacterial translation initiation factor 1 (IF1) triggers PTI in Arabidopsis thaliana and related Brassicaceae species. Unlike for most other immunogenic patterns, IF1 elicitor activity cannot be assigned to a small peptide epitope, suggesting that tertiary fold features are required for IF1 receptor activation. We have deployed natural variation in IF1 sensitivity to identify Arabidopsis leucine-rich repeat (LRR) receptor-like protein 32 (RLP32) as IF1 receptor using a restriction site-associated DNA sequencing approach. RLP32 confers IF1 sensitivity to rlp32 mutants, IF1-insensitive Arabidopsis accessions and IF1-insensitive Nicotiana benthamiana, binds IF1 specifically and forms complexes with LRR receptor kinases SOBIR1 and BAK1 to mediate signaling. Similar to other PRRs, RLP32 confers resistance to Pseudomonas syringae, highlighting an unexpectedly complex array of bacterial pattern sensors within a single plant species.

An oomycete NLP cytolysin forms transient small pores in lipid membranes.

Microbial plant pathogens secrete a range of effector proteins that damage host plants and consequently constrain global food production. Necrosis and ethylene-inducing peptide 1-like proteins (NLPs) are produced by numerous phytopathogenic microbes that cause important crop diseases. Many NLPs are cytolytic, causing cell death and tissue necrosis by disrupting the plant plasma membrane. Here, we reveal the unique molecular mechanism underlying the membrane damage induced by the cytotoxic model NLP. This membrane disruption is a multistep process that includes electrostatic-driven, plant-specific lipid recognition, shallow membrane binding, protein aggregation, and transient pore formation. The NLP-induced damage is not caused by membrane reorganization or large-scale defects but by small membrane ruptures. This distinct mechanism of lipid membrane disruption is highly adapted to effectively damage plant cells.

Distinct immune sensor systems for fungal endopolygalacturonases in closely related Brassicaceae.

Plant pattern recognition receptors (PRRs) facilitate recognition of microbial patterns and mediate activation of plant immunity. Arabidopsis thaliana RLP42 senses fungal endopolygalacturonases (PGs) and triggers plant defence through complex formation with SOBIR1 and SERK co-receptors. Here, we show that a conserved 9-amino-acid fragment pg9(At) within PGs is sufficient to activate RLP42-dependent plant immunity. Structure-function analysis reveals essential roles of amino acid residues within the RLP42 leucine-rich repeat and island domains for ligand binding and PRR complex assembly. Sensitivity to pg9(At), which is restricted to A. thaliana and exhibits scattered accession specificity, is unusual for known PRRs. Arabidopsis arenosa and Brassica rapa, two Brassicaceae species closely related to A. thaliana, respectively perceive immunogenic PG fragments pg20(Aa) and pg36(Bra), which are structurally distinct from pg9(At). Our study provides evidence for rapid evolution of polymorphic PG sensors with distinct pattern specificities within a single plant family.

The tomato receptor CuRe1 senses a cell wall protein to identify Cuscuta as a pathogen.

Parasitic plants of the genus Cuscuta penetrate shoots of host plants with haustoria and build a connection to the host vasculature to exhaust water, solutes and carbohydrates. Such infections usually stay unrecognized by the host and lead to harmful host plant damage. Here, we show a molecular mechanism of how plants can sense parasitic Cuscuta. We isolated an 11 kDa protein of the parasite cell wall and identified it as a glycine-rich protein (GRP). This GRP, as well as its minimal peptide epitope Crip21, serve as a pathogen-associated molecular pattern and specifically bind and activate a membrane-bound immune receptor of tomato, the Cuscuta Receptor 1 (CuRe1), leading to defense responses in resistant hosts. These findings provide the initial steps to understand the resistance mechanisms against parasitic plants and further offer great potential for protecting crops by engineering resistance against parasitic plants.

Eudicot plant-specific sphingolipids determine host selectivity of microbial NLP cytolysins.

Necrosis and ethylene-inducing peptide 1-like (NLP) proteins constitute a superfamily of proteins produced by plant pathogenic bacteria, fungi, and oomycetes. Many NLPs are cytotoxins that facilitate microbial infection of eudicot, but not of monocot plants. Here, we report glycosylinositol phosphorylceramide (GIPC) sphingolipids as NLP toxin receptors. Plant mutants with altered GIPC composition were more resistant to NLP toxins. Binding studies and x-ray crystallography showed that NLPs form complexes with terminal monomeric hexose moieties of GIPCs that result in conformational changes within the toxin. Insensitivity to NLP cytolysins of monocot plants may be explained by the length of the GIPC head group and the architecture of the NLP sugar-binding site. We unveil early steps in NLP cytolysin action that determine plant clade-specific toxin selectivity.

An RLP23-SOBIR1-BAK1 complex mediates NLP-triggered immunity.

Plants and animals employ innate immune systems to cope with microbial infection. Pattern-triggered immunity relies on the recognition of microbe-derived patterns by pattern recognition receptors (PRRs). Necrosis and ethylene-inducing peptide 1-like proteins (NLPs) constitute plant immunogenic patterns that are unique, as these proteins are produced by multiple prokaryotic (bacterial) and eukaryotic (fungal, oomycete) species. Here we show that the leucine-rich repeat receptor protein (LRR-RP) RLP23 binds in vivo to a conserved 20-amino-acid fragment found in most NLPs (nlp20), thereby mediating immune activation in Arabidopsis thaliana. RLP23 forms a constitutive, ligand-independent complex with the LRR receptor kinase (LRR-RK) SOBIR1 (Suppressor of Brassinosteroid insensitive 1 (BRI1)-associated kinase (BAK1)-interacting receptor kinase 1), and recruits a second LRR-RK, BAK1, into a tripartite complex upon ligand binding. Stable, ectopic expression of RLP23 in potato (Solanum tuberosum) confers nlp20 pattern recognition and enhanced immunity to destructive oomycete and fungal plant pathogens, such as Phytophthora infestans and Sclerotinia sclerotiorum. PRRs that recognize widespread microbial patterns might be particularly suited for engineering immunity in crop plants.

Immune receptor complexes at the plant cell surface.

Immunity to microbial infection is a common feature of metazoans and plants. Plants employ plasma membrane and cytoplasmic receptor systems for sensing microbe-derived or host-derived patterns and effectors and to trigger inducible immune defenses. Different biochemical types of plasma membrane immune receptors mediate recognition predominantly of peptide and carbohydrate patterns. Current research highlights the role of immune receptor complex formation in plant immunity. In particular, ligand binding by immune receptors generates molecular surfaces that enable either receptor homo-dimerization or co-receptor recruitment for subsequent signal transduction. New insight into negative regulatory principles of immune receptor function further suggests substantial dynamics in protein-protein interactions at the plasma membrane that we are only beginning to understand.