Natural variation of immune epitopes reveals intrabacterial antagonism.

Plants and animals detect biomolecules termed Microbe-Associated Molecular Patterns (MAMPs) and induce immunity. Agricultural production is severely impacted by pathogens which can be controlled by transferring immune receptors. However, most studies use a single MAMP epitope and the impact of diverse multi-copy MAMPs on immune induction is unknown. Here we characterized the epitope landscape from five proteinaceous MAMPs across 4,228 plant-associated bacterial genomes. Despite the diversity sampled, natural variation was constrained and experimentally testable. Immune perception in both Arabidopsis and tomato depended on both epitope sequence and copy number variation. For example, Elongation Factor Tu is predominantly single copy and 92% of its epitopes are immunogenic. Conversely, 99.9% of bacterial genomes contain multiple Cold Shock Proteins and 46% carry a non-immunogenic form. We uncovered a new mechanism for immune evasion, intrabacterial antagonism, where a non-immunogenic Cold Shock Protein blocks perception of immunogenic forms encoded in the same genome. These data will lay the foundation for immune receptor deployment and engineering based on natural variation.

Bacterial Pathogen Infection Triggers Magic Spot Nucleotide Signaling in Arabidopsis thaliana Chloroplasts through Specific RelA/SpoT Homologues.

Magic spot nucleotides (p)ppGpp are important signaling molecules in bacteria and plants. In the latter, RelA-SpoT homologue (RSH) enzymes are responsible for (p)ppGpp turnover. Profiling of (p)ppGpp is more difficult in plants than in bacteria due to lower concentrations and more severe matrix effects. Here, we report that capillary electrophoresis mass spectrometry (CE-MS) can be deployed to study (p)ppGpp abundance and identity in Arabidopsis thaliana. This goal is achieved by combining a titanium dioxide extraction protocol and pre-spiking with chemically synthesized stable isotope-labeled internal reference compounds. The high sensitivity and separation efficiency of CE-MS enables monitoring of changes in (p)ppGpp levels in A. thaliana upon infection with the pathogen Pseudomonas syringae pv. tomato (PstDC3000). We observed a significant increase of ppGpp post infection that is also stimulated by the flagellin peptide flg22 only. This increase depends on functional flg22 receptor FLS2 and its interacting kinase BAK1 indicating that pathogen-associated molecular pattern (PAMP) receptor-mediated signaling controls ppGpp levels. Transcript analyses showed an upregulation of RSH2 upon flg22 treatment and both RSH2 and RSH3 after PstDC3000 infection. Arabidopsis mutants deficient in RSH2 and RSH3 activity display no ppGpp accumulation upon infection and flg22 treatment, supporting the involvement of these synthases in PAMP-triggered innate immune responses to pathogens within the chloroplast.

Identification of a damage-associated molecular pattern (DAMP) receptor and its cognate peptide ligand in sweet potato (Ipomoea batatas).

Sweet potato (Ipomoea batatas) is an important tuber crop, but also target of numerous insect pests. Intriguingly, the abundant storage protein in tubers, sporamin, has intrinsic trypsin protease inhibitory activity. In leaves, sporamin is induced by wounding or a volatile homoterpene and enhances insect resistance. While the signalling pathway leading to sporamin synthesis is partially established, the initial event, perception of a stress-related signal is still unknown. Here, we identified an IbLRR-RK1 that is induced upon wounding and herbivory, and related to peptide-elicitor receptors (PEPRs) from tomato and Arabidopsis. We also identified a gene encoding a precursor protein comprising a peptide ligand (IbPep1) for IbLRR-RK1. IbPep1 represents a distinct signal in sweet potato, which might work in a complementary and/or parallel pathway to the previously described hydroxyproline-rich systemin (HypSys) peptides to strengthen insect resistance. Notably, an interfamily compatibility in the Pep/PEPR system from Convolvulaceae and Solanaceae was identified.

Convergent evolution of plant pattern recognition receptors sensing cysteine-rich patterns from three microbial kingdoms.

The Arabidopsis thaliana Receptor-Like Protein RLP30 contributes to immunity against the fungal pathogen Sclerotinia sclerotiorum. Here we identify the RLP30-ligand as a small cysteine-rich protein (SCP) that occurs in many fungi and oomycetes and is also recognized by the Nicotiana benthamiana RLP RE02. However, RLP30 and RE02 share little sequence similarity and respond to different parts of the native/folded protein. Moreover, some Brassicaceae other than Arabidopsis also respond to a linear SCP peptide instead of the folded protein, suggesting that SCP is an eminent immune target that led to the convergent evolution of distinct immune receptors in plants. Surprisingly, RLP30 shows a second ligand specificity for a SCP-nonhomologous protein secreted by bacterial Pseudomonads. RLP30 expression in N. tabacum results in quantitatively lower susceptibility to bacterial, fungal and oomycete pathogens, thus demonstrating that detection of immunogenic patterns by Arabidopsis RLP30 is involved in defense against pathogens from three microbial kingdoms.

Engineered Agrobacterium improves transformation by mitigating plant immunity detection.

Agrobacterium tumefaciens microbe-associated molecular pattern elongation factor Tu (EF-Tu) is perceived by orthologs of the Arabidopsis immune receptor EFR activating pattern-triggered immunity (PTI) that causes reduced T-DNA-mediated transient expression. We altered EF-Tu in A. tumefaciens to reduce PTI and improved transformation efficiency. A robust computational pipeline was established to detect EF-Tu protein variation in a large set of plant bacterial species and identified EF-Tu variants from bacterial pathogen Pseudomonas syringae pv. tomato DC3000 that allow the pathogen to escape EFR perception. Agrobacterium tumefaciens strains were engineered to substitute EF-Tu with DC3000 variants and examined their transformation efficiency in plants. Elongation factor Tu variants with rarely occurred amino acid residues were identified within DC3000 EF-Tu that mitigates recognition by EFR. Agrobacterium tumefaciens strains were engineered by expressing DC3000 EF-Tu instead of native agrobacterial EF-Tu and resulted in decreased plant immunity detection. These engineered A. tumefaciens strains displayed an increased efficiency in transient expression in both Arabidopsis thaliana and Camelina sativa. The results support the potential application of these strains as improved vehicles to introduce transgenic alleles into members of the Brassicaceae 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.

The CEP5 Peptide Promotes Abiotic Stress Tolerance, As Revealed by Quantitative Proteomics, and Attenuates the AUX/IAA Equilibrium in Arabidopsis.

Peptides derived from non-functional precursors play important roles in various developmental processes, but also in (a)biotic stress signaling. Our (phospho)proteome-wide analyses of C-TERMINALLY ENCODED PEPTIDE 5 (CEP5)-mediated changes revealed an impact on abiotic stress-related processes. Drought has a dramatic impact on plant growth, development and reproduction, and the plant hormone auxin plays a role in drought responses. Our genetic, physiological, biochemical, and pharmacological results demonstrated that CEP5-mediated signaling is relevant for osmotic and drought stress tolerance in Arabidopsis, and that CEP5 specifically counteracts auxin effects. Specifically, we found that CEP5 signaling stabilizes AUX/IAA transcriptional repressors, suggesting the existence of a novel peptide-dependent control mechanism that tunes auxin signaling. These observations align with the recently described role of AUX/IAAs in stress tolerance and provide a novel role for CEP5 in osmotic and drought stress tolerance.

Perception of Agrobacterium tumefaciens flagellin by FLS2XL confers resistance to crown gall disease.

Bacterial flagella are perceived by the innate immune systems of plants1 and animals2 alike, triggering resistance. Common to higher plants is the immunoreceptor FLAGELLIN-SENSING 2 (FLS2)3, which detects flagellin via its most conserved epitope, flg22. Agrobacterium tumefaciens, which causes crown gall disease in many crop plants, has a highly diverged flg22 epitope and evades immunodetection by plants so far studied. We asked whether, as a next step in this game of 'hide and seek', there are plant species that have evolved immunoreceptors with specificity for the camouflaged flg22Atum of A. tumefaciens. In the wild grape species Vitis riparia, we discovered FLS2XL, a previously unknown form of FLS2, that provides exquisite sensitivity to typical flg22 and to flg22Atum. As exemplified by ectopic expression in tobacco, FLS2XL can limit crown gall disease caused by A. tumefaciens.

Anion channel SLAH3 is a regulatory target of chitin receptor-associated kinase PBL27 in microbial stomatal closure.

In plants, antimicrobial immune responses involve the cellular release of anions and are responsible for the closure of stomatal pores. Detection of microbe-associated molecular patterns (MAMPs) by pattern recognition receptors (PRRs) induces currents mediated via slow-type (S-type) anion channels by a yet not understood mechanism. Here, we show that stomatal closure to fungal chitin is conferred by the major PRRs for chitin recognition, LYK5 and CERK1, the receptor-like cytoplasmic kinase PBL27, and the SLAH3 anion channel. PBL27 has the capacity to phosphorylate SLAH3, of which S127 and S189 are required to activate SLAH3. Full activation of the channel entails CERK1, depending on PBL27. Importantly, both S127 and S189 residues of SLAH3 are required for chitin-induced stomatal closure and anti-fungal immunity at the whole leaf level. Our results demonstrate a short signal transduction module from MAMP recognition to anion channel activation, and independent of ABA-induced SLAH3 activation.

Peptide Feeding and Mechanical Wounding for Tomato Seedlings.

Plants need to respond appropriately to wounding and herbivorous insects. Peptide signals have been implicated in local and systemic induction of appropriate plant defense responses. To study these peptide signals and their perception in host plants, it is important to have reproducible bioassays. Several assays, such as treatment of peptide solution via pressure infiltration, have been developed. Here, we provide detailed protocols for peptide feeding and mechanical wounding for tomato seedlings. To directly introduce peptides into tomato seedlings, peptide solution is fed through the excised stem via the transpiration stream. To mimic the wounding caused by insect feeding, leaflets of tomato seedlings are mechanically damaged with a hemostat; and wounded and systemic unwounded leaves are harvested and analyzed separately. Samples from both assays may be further assessed by examining the transcript level of marker genes by quantitative real-time PCR (qRT-PCR).

The dynamics of root cap sloughing in Arabidopsis is regulated by peptide signalling.

The root cap protects the stem cell niche of angiosperm roots from damage. In Arabidopsis, lateral root cap (LRC) cells covering the meristematic zone are regularly lost through programmed cell death, while the outermost layer of the root cap covering the tip is repeatedly sloughed. Efficient coordination with stem cells producing new layers is needed to maintain a constant size of the cap. We present a signalling pair, the peptide IDA-LIKE1 (IDL1) and its receptor HAESA-LIKE2 (HSL2), mediating such communication. Live imaging over several days characterized this process from initial fractures in LRC cell files to full separation of a layer. Enhanced expression of IDL1 in the separating root cap layers resulted in increased frequency of sloughing, balanced with generation of new layers in a HSL2-dependent manner. Transcriptome analyses linked IDL1-HSL2 signalling to the transcription factors BEARSKIN1/2 and genes associated with programmed cell death. Mutations in either IDL1 or HSL2 slowed down cell division, maturation and separation. Thus, IDL1-HSL2 signalling potentiates dynamic regulation of the homeostatic balance between stem cell division and sloughing activity.

The systemin receptor SYR1 enhances resistance of tomato against herbivorous insects.

The discovery in tomato of systemin, the first plant peptide hormone1,2, was a fundamental change for the concept of plant hormones. Numerous other peptides have since been shown to play regulatory roles in many aspects of the plant life, including growth, development, fertilization and interactions with symbiotic organisms3-6. Systemin, an 18 amino acid peptide derived from a larger precursor protein 7 , was proposed to act as the spreading signal that triggers systemic defence responses observed in plants after wounding or attack by herbivores1,7,8. Further work culminated in the identification of a leucine-rich repeat receptor kinase (LRR-RK) as the systemin receptor 160 (SR160)9,10. SR160 is a tomato homologue of Brassinosteroid Insensitive 1 (BRI1), which mediates the regulation of growth and development in response to the steroid hormone brassinolide11-13. However, a role of SR160/BRI1 as systemin receptor could not be corroborated by others14-16. Here, we demonstrate that perception of systemin depends on a pair of distinct LRR-RKs termed SYR1 and SYR2. SYR1 acts as a genuine systemin receptor that binds systemin with high affinity and specificity. Further, we show that presence of SYR1, although not decisive for local and systemic wound responses, is important for defence against insect herbivory.

Author Correction: The pattern-recognition receptor CORE of Solanaceae detects bacterial cold-shock protein.

In the version of this Article originally published, Fig. 6b, which is composed of individual pictures of six plants, inadvertently and erroneously displayed the same image of one Col-0 wt plant twice. This has been corrected so that Fig. 6b now shows two different representative plants for the Col-0 wt control.

The pattern-recognition receptor CORE of Solanaceae detects bacterial cold-shock protein.

Plants and animals recognize microbial invaders by detecting microbe-associated molecular patterns (MAMPs) by cell surface receptors. Many plant species of the Solanaceae family detect the highly conserved nucleic acid binding motif RNP-1 of bacterial cold-shock proteins (CSPs), represented by the peptide csp22, as a MAMP. Here, we exploited the natural variation in csp22 perception observed between cultivated tomato (Solanum lycopersicum) and Solanum pennellii to map and identify the leucine-rich repeat (LRR) receptor kinase CORE (cold shock protein receptor) of tomato as the specific, high-affinity receptor site for csp22. Corroborating its function as a genuine receptor, heterologous expression of CORE in Arabidopsis thaliana conferred full sensitivity to csp22 and, importantly, it also rendered these plants more resistant to infection by the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Our study also confirms the biotechnological potential of enhancing plant immunity by interspecies transfer of highly effective pattern-recognition receptors such as CORE to different plant families.

A fungal pathogen secretes plant alkalinizing peptides to increase infection.

Plant infections caused by fungi are often associated with an increase in the pH of the surrounding host tissue(1). Extracellular alkalinization is thought to contribute to fungal pathogenesis, but the underlying mechanisms are poorly understood. Here, we show that the root-infecting fungus Fusarium oxysporum uses a functional homologue of the plant regulatory peptide RALF (rapid alkalinization factor)(2,3) to induce alkalinization and cause disease in plants. An upshift in extracellular pH promotes infectious growth of Fusarium by stimulating phosphorylation of a conserved mitogen-activated protein kinase essential for pathogenicity(4,5). Fungal mutants lacking a functional Fusarium (F)-RALF peptide failed to induce host alkalinization and showed markedly reduced virulence in tomato plants, while eliciting a strong host immune response. Arabidopsis plants lacking the receptor-like kinase FERONIA, which mediates the RALF-triggered alkalinization response(6), displayed enhanced resistance against Fusarium. RALF homologues are found across a number of phylogenetically distant groups of fungi, many of which infect plants. We propose that fungal pathogens use functional homologues of alkalinizing peptides found in their host plants to increase their infectious potential and suppress host immunity.

Detection of the plant parasite Cuscuta reflexa by a tomato cell surface receptor.

Parasitic plants are a constraint on agriculture worldwide. Cuscuta reflexa is a stem holoparasite that infests most dicotyledonous plants. One exception is tomato, which is resistant to C. reflexa We discovered that tomato responds to a small peptide factor occurring in Cuscuta spp. with immune responses typically activated after perception of microbe-associated molecular patterns. We identified the cell surface receptor-like protein CUSCUTA RECEPTOR 1 (CuRe1) as essential for the perception of this parasite-associated molecular pattern. CuRe1 is sufficient to confer responsiveness to the Cuscuta factor and increased resistance to parasitic C. reflexa when heterologously expressed in otherwise susceptible host plants. Our findings reveal that plants recognize parasitic plants in a manner similar to perception of microbial pathogens.

Bacterial Outer Membrane Vesicles Induce Plant Immune Responses.

Gram-negative bacteria continuously pinch off portions of their outer membrane, releasing membrane vesicles. These outer membrane vesicles (OMVs) are involved in multiple processes including cell-to-cell communication, biofilm formation, stress tolerance, horizontal gene transfer, and virulence. OMVs are also known modulators of the mammalian immune response. Despite the well-documented role of OMVs in mammalian-bacterial communication, their interaction with plants is not well studied. To examine whether OMVs of plant pathogens modulate the plant immune response, we purified OMVs from four different plant pathogens and used them to treat Arabidopsis thaliana. OMVs rapidly induced a reactive oxygen species burst, medium alkalinization, and defense gene expression in A. thaliana leaf discs, cell cultures, and seedlings, respectively. Western blot analysis revealed that EF-Tu is present in OMVs and that it serves as an elicitor of the plant immune response in this form. Our results further show that the immune coreceptors BAK1 and SOBIR1 mediate OMV perception and response. Taken together, our results demonstrate that plants can detect and respond to OMV-associated molecules by activation of their immune system, revealing a new facet of plant-bacterial interactions.

A new species of Metopocetus (Cetacea, Mysticeti, Cetotheriidae) from the Late Miocene of the Netherlands.

The family Cetotheriidae has played a major role in recent discussions of baleen whale phylogenetics. Within this group, the enigmatic, monotypic Metopocetus durinasus has been interpreted as transitional between herpetocetines and other members of the family, but so far has been restricted to a single, fragmentary cranium of uncertain provenance and age. Here, we expand the genus and shed new light on its phylogenetic affinities and functional morphology by describing Metopocetus hunteri sp. nov. from the Late Miocene of the Netherlands. Unlike the holotype of M. durinasus, the material described here is confidently dated and preserves both the tympanic bulla and additional details of the basicranium. M. hunteri closely resembles M. durinasus, differing primarily in its somewhat less distally expanded compound posterior process of the tympanoperiotic. Both species are characterised by the development of an unusually large fossa on the ventral surface of the paroccipital process, which extends anteriorly on to the compound posterior process and completely floors the facial sulcus. In life, this enlarged fossa may have housed the posterior sinus and/or the articulation of the stylohyal. Like other cetotheriids, Metopocetus also bears a well-developed, posteriorly-pointing dorsal infraorbital foramen near the base of the ascending process of the maxilla, the precise function of which remains unclear.

The rice immune receptor XA21 recognizes a tyrosine-sulfated protein from a Gram-negative bacterium.

Surveillance of the extracellular environment by immune receptors is of central importance to eukaryotic survival. The rice receptor kinase XA21, which confers robust resistance to most strains of the Gram-negative bacterium Xanthomonas oryzae pv. oryzae (Xoo), is representative of a large class of cell surface immune receptors in plants and animals. We report the identification of a previously undescribed Xoo protein, called RaxX, which is required for activation of XA21-mediated immunity. Xoo strains that lack RaxX, or carry mutations in the single RaxX tyrosine residue (Y41), are able to evade XA21-mediated immunity. Y41 of RaxX is sulfated by the prokaryotic tyrosine sulfotransferase RaxST. Sulfated, but not nonsulfated, RaxX triggers hallmarks of the plant immune response in an XA21-dependent manner. A sulfated, 21-amino acid synthetic RaxX peptide (RaxX21-sY) is sufficient for this activity. Xoo field isolates that overcome XA21-mediated immunity encode an alternate raxX allele, suggesting that coevolutionary interactions between host and pathogen contribute to RaxX diversification. RaxX is highly conserved in many plant pathogenic Xanthomonas species. The new insights gained from the discovery and characterization of the sulfated protein, RaxX, can be applied to the development of resistant crop varieties and therapeutic reagents that have the potential to block microbial infection of both plants and animals.