The tomato I gene for Fusarium wilt resistance encodes an atypical leucine‐rich repeat receptor‐like protein whose function is nevertheless dependent on SOBIR1 and SERK3/BAK1

We have identified the tomato I gene for resistance to the Fusarium wilt fungus Fusarium oxysporum f. sp. lycopersici (Fol) and show that it encodes a membrane‐anchored leucine‐rich repeat receptor‐like protein (LRR‐RLP). Unlike most other LRR‐RLP genes involved in plant defence, the I gene is not a member of a gene cluster and contains introns in its coding sequence. The I gene encodes a loopout domain larger than those in most other LRR‐RLPs, with a distinct composition rich in serine and threonine residues. The I protein also lacks a basic cytosolic domain. Instead, this domain is rich in aromatic residues that could form a second transmembrane domain. The I protein recognises the Fol Avr1 effector protein, but, unlike many other LRR‐RLPs, recognition specificity is determined in the C‐terminal half of the protein by polymorphic amino acid residues in the LRRs just preceding the loopout domain and in the loopout domain itself. Despite these differences, we show that I/Avr1‐dependent necrosis in Nicotiana benthamiana depends on the LRR receptor‐like kinases (RLKs) SERK3/BAK1 and SOBIR1. Sequence comparisons revealed that the I protein and other LRR‐RLPs involved in plant defence all carry residues in their last LRR and C‐terminal LRR capping domain that are conserved with SERK3/BAK1‐interacting residues in the same relative positions in the LRR‐RLKs BRI1 and PSKR1. Tyrosine mutations of two of these conserved residues, Q922 and T925, abolished I/Avr1‐dependent necrosis in N. benthamiana, consistent with similar mutations in BRI1 and PSKR1 preventing their interaction with SERK3/BAK1.     

The Brassica napus receptor‐like protein RLM2 is encoded by a second allele of the LepR3/Rlm2 blackleg resistance locus

Leucine‐rich repeat receptor‐like proteins (LRR‐RLPs) are highly adaptable parts of the signalling apparatus for extracellular detection of plant pathogens. Resistance to blackleg disease of Brassica spp. caused by Leptosphaeria maculans is largely governed by host race‐specific R‐genes, including the LRR‐RLP gene LepR3. The blackleg resistance gene Rlm2 was previously mapped to the same genetic interval as LepR3. In this study, the LepR3 locus of the Rlm2 Brassica napus line ‘Glacier DH24287’ was cloned, and B. napus transformants were analysed for recovery of the Rlm2 phenotype. Multiple B. napus, B. rapa and B. juncea lines were assessed for sequence variation at the locus. Rlm2 was found to be an allelic variant of the LepR3 LRR‐RLP locus, conveying race‐specific resistance to L. maculans isolates harbouring AvrLm2. Several defence‐related LRR‐RLPs have previously been shown to associate with the RLK SOBIR1 to facilitate defence signalling. Bimolecular fluorescence complementation (BiFC) and co‐immunoprecipitation of RLM2‐SOBIR1 studies revealed that RLM2 interacts with SOBIR1 of Arabidopsis thaliana when co‐expressed in Nicotiana benthamiana. The interaction of RLM2 with AtSOBIR1 is suggestive of a conserved defence signalling pathway between B. napus and its close relative A. thaliana.     

A pair of G-type lectin receptor-like kinases modulates nlp20-mediated immune responses by coupling to the RLP23 receptor complex

Plant cells recognize microbial patterns with the plasma-membrane-localized pattern-recognition receptors consisting mainly of receptor kinases (RKs) and receptor-like proteins (RLPs). RKs, such as bacterial flagellin receptor FLS2, and their downstream signaling components have been studied extensively. However, newly discovered regulatory components of RLP-mediated immune signaling, such as the nlp20 receptor RLP23, await identification. Unlike RKs, RLPs lack a cytoplasmic kinase domain, instead recruiting the receptor-like kinases (RLKs) BAK1 and SOBIR1. SOBIR1 specifically works as an adapter for RLP-mediated immunity. To identify new regulators of RLP-mediated signaling, we looked for SOBIR1-binding proteins (SBPs) in Arabidopsis thaliana using protein immunoprecipitation and mass spectrometry, identifying two G-type lectin RLKs, SBP1 and SBP2, that physically interacted with SOBIR1. SBP1 and SBP2 showed high sequence similarity, were tandemly repeated on chromosome 4, and also interacted with both RLP23 and BAK1. sbp1 sbp2 double mutants obtained via CRISPR-Cas9 gene editing showed severely impaired nlp20-induced reactive oxygen species burst, mitogen-activated protein kinase (MAPK) activation, and defense gene expression, but normal flg22-induced immune responses. We showed that SBP1 regulated nlp20-induced immunity in a kinase activity-independent manner. Furthermore, the nlp20-induced the RLP23–BAK1 interaction, although not the flg22-induced FLS2–BAK1 interaction, was significantly reduced in sbp1 sbp2. This study identified SBPs as new regulatory components in RLP23 receptor complex that may specifically modulate RLP23-mediated immunity by positively regulating the interaction between the RLP23 receptor and the BAK1 co-receptor.     

The RECEPTOR-LIKE PROTEIN53 immune complex associates with LLG1 to positively regulate plant immunity

Pattern recognition receptors(PRRs) sense ligands in pattern-triggered immunity(PTI). Plant PRRs include numerous receptor-like proteins(RLPs), but many RLPs remain functionally uncharacterized. Here, we examine an Arabidopsis thaliana RLP, RLP53, which positively regulates immune signaling. Our forward genetic screen for suppressors of enhanced disease resistance1(edr1) identified a point mutation in RLP53 that fully suppresses disease resistance and mildewinduced cell death in edr1 mutants. Th...     

A mutational analysis of the cytosolic domain of the tomato Cf‐9 disease‐resistance protein shows that membrane‐proximal residues are important for Avr9‐dependent necrosis

The tomato Cf‐9 gene encodes a membrane‐anchored glycoprotein that imparts race‐specific resistance against the tomato leaf mould fungus Cladosporium fulvum in response to the avirulence protein Avr9. Although the N‐terminal half of the extracellular leucine‐rich repeat (eLRR) domain of the Cf‐9 protein determines its specificity for Avr9, the C‐terminal half, including its small cytosolic domain, is postulated to be involved in signalling. The cytosolic domain of Cf‐9 carries several residues that are potential sites for ubiquitinylation or phosphorylation, or signals for endocytic uptake. A targeted mutagenesis approach was employed to investigate the roles of these residues and cellular processes in Avr9‐dependent necrosis triggered by Cf‐9. Our results indicate that the membrane‐proximal region of the cytosolic domain of Cf‐9 plays an important role in Cf‐9‐mediated necrosis, and two amino acids within this region, a threonine (T835) and a proline (P838), are particularly important for Cf‐9 function. An alanine mutation of T835 had no effect on Cf‐9 function, but an aspartic acid mutation, which mimics phosphorylation, reduced Cf‐9 function. We therefore postulate that phosphorylation/de‐phosphorylation of T835 could act as a molecular switch to determine whether Cf‐9 is in a primed or inactive state. Yeast two‐hybrid analysis was used to show that the cytosolic domain of Cf‐9 interacts with the cytosolic domain of tomato VAP27. This interaction could be disrupted by an alanine mutation of P838, whereas interaction with CITRX remained unaffected. We therefore postulate that a proline‐induced kink in the membrane‐proximal region of the cytosolic domain of Cf‐9 may be important for interaction with VAP27, which may, in turn, be important for Cf‐9 function.     

Kinase activity of SOBIR1 and BAK1 is required for immune signalling

Leucine-rich repeat-receptor-like proteins (LRR-RLPs) and LRR-receptor-like kinases (LRR-RLKs) trigger immune signalling to promote plant resistance against pathogens. LRR-RLPs lack an intracellular kinase domain, and several of these receptors have been shown to constitutively interact with the LRR-RLK Suppressor of BIR1-1/EVERSHED (SOBIR1/EVR) to form signalling-competent receptor complexes. Ligand perception by LRR-RLPs initiates recruitment of the co-receptor BRI1-Associated Kinase 1/Somatic Embryogenesis Receptor Kinase 3 (BAK1/SERK3) to the LRR-RLP/SOBIR1 complex, thereby activating LRR-RLP-mediated immunity. We employed phosphorylation analysis of in planta-produced proteins, live cell imaging, gene silencing and co-immunoprecipitation to investigate the roles of SOBIR1 and BAK1 in immune signalling. We show that Arabidopsis thaliana (At) SOBIR1, which constitutively activates immune responses when overexpressed in planta, is highly phosphorylated. Moreover, in addition to the kinase activity of SOBIR1 itself, kinase-active BAK1 is essential for AtSOBIR1-induced constitutive immunity and for the phosphorylation of AtSOBIR1. Furthermore, the defence response triggered by the tomato LRR-RLP Cf-4 on perception of Avr4 from the extracellular pathogenic fungus Cladosporium fulvum is dependent on kinase-active BAK1. We argue that, in addition to the trans-autophosphorylation of SOBIR1, it is likely that SOBIR1 and BAK1 transphosphorylate, and thereby activate the receptor complex. The signalling-competent cell surface receptor complex subsequently activates downstream cytoplasmic signalling partners to initiate RLP-mediated immunity.     

Biochemical mapping of a ligand-binding domain within Arabidopsis BAM1 reveals diversified ligand recognition mechanisms of plant LRR-RKs

Leucine-rich repeat receptor kinases (LRR-RKs) are the largest sub-family of transmembrane receptor kinases in plants. In several LRR-RKs, a loop-out region called an 'island domain', which intercepts the extracellular tandem LRRs at a position near the transmembrane domain, constitutes the ligand-binding pocket, but the absence of the island domain in numerous LRR-RKs raises questions about which domain recognizes the ligand in non-island domain LRR-RKs. Here, we used photoaffinity labeling followed by chemical and enzymatic digestion to show that BAM1, a CLV1/BAM-family LRR-RK whose extracellular domain comprises 22 consecutive LRRs, directly interacts with the small peptide ligand CLE9 at the LRR6-LRR8 region that is relatively distal from the transmembrane domain. Multiple sequence alignment and homology modeling revealed that the inner concave side of LRR6-LRR8 of CLV1/BAM-family LRR-RKs deviates slightly from the LRR consensus. In support of our findings, the clv1-4 mutant carries a missense mutation at the inner concave side of LRR6 of CLV1, and introduction of the corresponding mutation in BAM1 resulted in complete loss of ligand binding activity. Our results indicate that the ligand recognition mechanisms of plant LRR-RKs are more complex and diverse than anticipated.     

Arabidopsis snc2-1D Activates Receptor-Like Protein-Mediated Immunity Transduced through WRKY70

Plant immune receptors belonging to the receptor-like protein (RLP) family contain extracellular leucine-rich repeats (LRRs) and a short cytoplasmic tail linked by a single transmembrane motif. Here, we report the identification of snc2-1D (for suppressor of npr1-1, constitutive 2), a semidominant Arabidopsis thaliana mutant with constitutively activated defense responses. Map-based cloning of snc2-1D showed that it encodes an RLP. The point mutation in snc2-1D leads to substitution of the second Gly for Arg in the conserved GXXXG motif of the transmembrane helix, suggesting that this residue is important for negative regulation of the protein. Epistasis analysis revealed that the snc2-1D mutant phenotype is not affected by mutations in genes known to be required for the nucleotide binding (NB)-LRR Resistance (R) protein signaling. A suppressor screen of snc2-1D was performed, and map-based cloning of one suppressor revealed that mutations in WRKY70 suppress the constitutive defense responses in snc2-1D, suggesting that WRKY70 functions downstream of snc2-1D. The identification of snc2-1D provides us with a unique system for genetic analysis of resistance pathways downstream of RLPs, which may be distinct from those downstream of NB-LRR type R proteins.     

An autophosphorylation site database for leucine‐rich repeat receptor‐like kinases in Arabidopsis thaliana

Leucine‐rich repeat receptor‐like kinases (LRR RLKs) form a large family of plant signaling proteins consisting of an extracellular domain connected by a single‐pass transmembrane sequence to a cytoplasmic kinase domain. Autophosphorylation on specific Ser and/or Thr residues in the cytoplasmic domain is often critical for the activation of several LRR RLK family members with proven functional roles in plant growth regulation, morphogenesis, disease resistance, and stress responses. While identification and functional characterization of in vivo phosphorylation sites is ultimately required for a full understanding of LRR RLK biology and function, bacterial expression of recombinant LRR RLK cytoplasmic catalytic domains for identification of in vitro autophosphorylation sites provides a useful resource for further targeted identification and functional analysis of in vivo sites. In this study we employed high‐throughput cloning and a variety of mass spectrometry approaches to generate an autophosphorylation site database representative of more than 30% of the approximately 223 LRR RLKs in Arabidopsis thaliana. We used His‐tagged constructs of complete cytoplasmic domains to identify a total of 592 phosphorylation events across 73 LRR RLKs, with 497 sites uniquely assigned to specific Ser (268 sites) or Thr (229 sites) residues in 68 LRR RLKs. Multiple autophosphorylation sites per LRR RLK were the norm, with an average of seven sites per cytoplasmic domain, while some proteins showed more than 20 unique autophosphorylation sites. The database was used to analyze trends in the localization of phosphorylation sites across cytoplasmic kinase subdomains and to derive a statistically significant sequence motif for phospho‐Ser autophosphorylation.     

Endosomal signaling of the tomato leucine-rich repeat receptor-like protein LeEix2

Extracellular leucine-rich repeat (LRR) receptor-like proteins (RLPs) represent a unique class of cell-surface receptors, as they lack a functional cytoplasmic domain. Our knowledge of how RLPs that do not contain a kinase or Toll domain function is very limited. The tomato RLP receptor LeEix2 signals to induce defense responses mediated by the fungal protein ethylene-inducing xylanase (EIX). The movement of FYVE-positive endosomes before and after EIX application was examined using spinning disc confocal microscopy. We found that while FYVE-positive endosomes generally observe a random movement pattern, following EIX application a subpopulation of FYVE-positive endosomes follow a directional movement pattern. Further, cellular endosomes travel greater distances at higher speeds following EIX application. Time-course experiments conducted with specific inhibitors demonstrate the involvement of endosomal signaling in EIX-triggered defense responses. Abolishing the existence of endosomes or the endocytic event prevented EIX-induced signaling. Endocytosis/endosome inhibitors, such as Dynasore or 1-butanol, inhibit EIX-induced signaling. Moreover, treatment with Endosidin1, which inhibits an early step in plasma membrane/endosome trafficking, enhances the induction of defense responses by EIX. Our data indicate a distinct endosomal signaling mechanism for induction of defense responses in this RLP system.     

Evolutionary dynamics of leucine-rich repeat receptor-like kinases and related genes in plants：A phylogenomic approach

Leucine-rich repeat （LRR） receptor-like kinases （RLKs）, evolutionarily related LRR receptor-like proteins （RLPs） and receptor-like cytoplasmic kinases （RLCKs） have important roles in plant signaling, and their gene subfamilies are large with a complicated history of gene duplication and loss. In three pairs of closely related lineages, including Arabidopsis thaliana and A. lyrata （Arabidopsis）, Lotus japonicus, and Medicago truncatula （Legumes）, Oryza sativa ssp. japonica, and O. sativa ssp. indica （Rice）, we find that LRR RLKs comprise the largest group of these LRR-related subfamilies, while the related RLCKs represent the smal est group. In addition, comparison of orthologs indicates a high frequency of reciprocal gene loss of the LRR RLK/LRR RLP/RLCK subfamilies. Furthermore, pairwise comparisons show that reciprocal gene loss is often associated with lineage-specific duplication（s） in the alternative lineage. Last, analysis of genes in A. thaliana involved in development revealed that most are highly conserved orthologs without species-specific duplication in the two Arabidopsis species and originated from older Arabidopsis-specific or rosid-specific duplications. We discuss＆amp;nbsp;potential pitfal s related to functional prediction for genes that have undergone frequent turnover （duplications, losses, and domain architecture changes）, and conclude that prediction based on phylogenetic relationships wil likely outperform that based on sequence similarity alone.     

MOL1 is required for cambium homeostasis in Arabidopsis

Plants maintain pools of pluripotent stem cells which allow them to constantly produce new tissues and organs. Stem cell homeostasis in shoot and root tips depends on negative regulation by ligand–receptor pairs of the CLE peptide and leucine‐rich repeat receptor‐like kinase (LRR‐RLK) families. However, regulation of the cambium, the stem cell niche required for lateral growth of shoots and roots, is poorly characterized. Here we show that the LRR‐RLK MOL1 is necessary for cambium homeostasis in Arabidopsis thaliana. By employing promoter reporter lines, we reveal that MOL1 is active in a domain that is distinct from the domain of the positively acting CLE41/PXY signaling module. In particular, we show that MOL1 acts in an opposing manner to the CLE41/PXY module and that changing the domain or level of MOL1 expression both result in disturbed cambium organization. Underlining discrete roles of MOL1 and PXY, both LRR‐RLKs are not able to replace each other when their expression domains are interchanged. Furthermore, MOL1 but not PXY is able to rescue CLV1 deficiency in the shoot apical meristem. By identifying genes mis‐expressed in mol1 mutants, we demonstrate that MOL1 represses genes associated with stress‐related ethylene and jasmonic acid hormone signaling pathways which have known roles in coordinating lateral growth of the Arabidopsis stem. Our findings provide evidence that common regulatory mechanisms in different plant stem cell niches are adapted to specific niche anatomies and emphasize the importance of a complex spatial organization of intercellular signaling cascades for a strictly bidirectional tissue production.     

Chaperones of the endoplasmic reticulum are required for Ve1‐mediated resistance to Verticillium

The tomato receptor‐like protein (RLP) Ve1 mediates resistance to the vascular fungal pathogen Verticillium dahliae. To identify the proteins required for Ve1 function, we transiently expressed and immunopurified functional Ve1‐enhanced green fluorescent protein (eGFP) from Nicotiana benthamiana leaves, followed by mass spectrometry. This resulted in the identification of peptides originating from the endoplasmic reticulum (ER)‐resident chaperones HSP70 binding proteins (BiPs) and a lectin‐type calreticulin (CRT). Knock‐down of the different BiPs and CRTs in tomato resulted in compromised Ve1‐mediated resistance to V. dahliae in most cases, showing that these chaperones play an important role in Ve1 functionality. Recently, it has been shown that one particular CRT is required for the biogenesis of the RLP‐type Cladosporium fulvum resistance protein Cf‐4 of tomato, as silencing of CRT3a resulted in a reduced pool of complex glycosylated Cf‐4 protein. In contrast, knock‐down of the various CRTs in N. benthamiana or N. tabacum did not result in reduced accumulation of mature complex glycosylated Ve1 protein. Together, this study shows that the BiP and CRT ER chaperones differentially contribute to Cf‐4‐ and Ve1‐mediated immunity.     

A genome-wide functional investigation into the roles of receptor-like proteins in Arabidopsis

Receptor-like proteins (RLPs) are cell surface receptors that typically consist of an extracellular leucine-rich repeat domain, a transmembrane domain, and a short cytoplasmatic tail. In several plant species, RLPs have been found to play a role in disease resistance, such as the tomato (Solanum lycopersicum) Cf and Ve proteins and the apple (Malus domestica) HcrVf2 protein that mediate resistance against the fungal pathogens Cladosporium fulvum, Verticillium spp., and Venturia inaequalis, respectively. In addition, RLPs play a role in plant development; Arabidopsis (Arabidopsis thaliana) TOO MANY MOUTHS (TMM) regulates stomatal distribution, while Arabidopsis CLAVATA2 (CLV2) and its functional maize (Zea mays) ortholog FASCINATED EAR2 regulate meristem maintenance. In total, 57 RLP genes have been identified in the Arabidopsis genome and a genome-wide collection of T-DNA insertion lines was assembled. This collection was functionally analyzed with respect to plant growth and development and sensitivity to various stress responses, including susceptibility toward pathogens. A number of novel developmental phenotypes were revealed for our CLV2 and TMM insertion mutants. In addition, one AtRLP gene was found to mediate abscisic acid sensitivity and another AtRLP gene was found to influence nonhost resistance toward Pseudomonas syringae pv phaseolicola. This genome-wide collection of Arabidopsis RLP gene T-DNA insertion mutants provides a tool for future investigations into the biological roles of RLPs.     

The Diverse Roles of Extracellular Leucine-rich Repeat-containing Receptor-like Proteins in Plants

Plant cells use various types of cell surface receptor molecules to sense extracellular signals and modulate cell-to-cell communication in many biological processes. Extracellular leucine-rich repeat (eLRR) receptor-like proteins (RLPs) represent an important class of such cell surface receptors. RLPs differ from receptor-like kinases (RLKs), which compose the largest class of cell surface receptors in many plant species, because they lack a cytoplasmic kinase domain. RLPs play roles in both developmental processes and disease resistance. A total of 57 RLP encoding genes has been identified in Arabidopsis. Two of them, CLAVATA2 (CLV2) and Too Many Mouths (TMM) have a function in meristem maintenance and stomatal distribution, respectively, whereas few others act in basal defense against pathogens. Although the function of most RLPs in Arabidopsis remains unclear, considerable progress has been made in understanding RLP functioning and signaling over the years. This review focuses on the function of RLPs in plants. Furthermore, the function of distinct RLP domains and the role of conserved residues important for perception and ligand specificity are discussed. The role of RLP proteins in multimeric complexes to sense biotic and abiotic extracellular signals is also addressed.     

SOBIR1 and AGB1 independently contribute to nonhost resistance to Pyricularia oryzae (syn. Magnaporthe oryzae) in Arabidopsis thaliana

ABSTRACT

Rice blast caused by Pyricularia oryzae (syn. Magnaporthe oryzae) is a disease devastating to rice. We have studied the Arabidopsis-P. oryzae pathosystem as a model system for nonhost resistance (NHR) and found that SOBIR1, but not BAK1, is a positive regulator of NHR to P. oryzae in Arabidopsis. AGB1 is also involved in NHR. However, the genetic interactions between SOBIR1, BAK1, and AGB1 are uncharacterized. In this study, we delineated the genetic interactions between SOBIR1, BAK1, and AGB1 in NHR to P. oryzae in Arabidopsis and found SOBIR1 and AGB1 independently control NHR to P. oryzae in Arabidopsis pen2-1 mutant plants. Furthermore, XLG2, but not TMM, has a positive role in penetration resistance to P. oryzae in Arabidopsis pen2-1 mutant plants. Our study characterized genetic interactions in Arabidopsis NHR.

Abbreviations: PRR: pattern recognition receptor, RLK: receptor-like kinase, RLP: receptor-like protein, BAK1: BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED RECEPTOR KINASE 1, BIR1: BAK1-INTERACTING RECEPTOR-LIKE KINASE 1, SOBIR1: SUPPRESSOR OF BIR1-1-1, AGB1: ARABIDOPSIS G PROTEIN ß-SUBUNIT 1, XLG2: EXTRA-LARGE G PROTEIN 2     

Domain swapping and gene shuffling identify sequences required for induction of an Avr-dependent hypersensitive response by the tomato Cf-4 and Cf-9 proteins

The tomato Cf-4 and Cf-9 genes confer resistance to infection by the biotrophic leaf mold pathogen Cladosporium. Their protein products induce a hypersensitive response (HR) upon recognition of the fungus-encoded Avr4 and Avr9 peptides. Cf-4 and Cf-9 share >91% sequence identity and are distinguished by sequences in their N-terminal domains A and B, their N-terminal leucine-rich repeats (LRRs) in domain C1, and their LRR copy number (25 and 27 LRRs, respectively). Analysis of Cf-4/Cf-9 chimeras, using several different bioassays, has identified sequences in Cf-4 and Cf-9 that are required for the Avr-dependent HR in tobacco and tomato. A 10-amino acid deletion within Cf-4 domain B relative to Cf-9 was required for full Avr4-dependent induction of an HR in most chimeras analyzed. Additional sequences required for Cf-4 function are located in LRRs 11 and 12, a region that contains only eight of the 67 amino acids that distinguish it from Cf-9. One chimera, with 25 LRRs that retained LRR 11 of Cf-4, induced an attenuated Avr4-dependent HR. The substitution of Cf-9 N-terminal LRRs 1 to 9 with the corresponding sequences from Cf-4 resulted in attenuation of the Avr9-induced HR, as did substitution of amino acid A433 in LRR 15. The amino acids L457 and K511 in Cf-9 LRRs 16 and 18 are essential for induction of the Avr9-dependent HR. Therefore, important sequence determinants of Cf-9 function are located in LRRs 10 to 18. This region contains 15 of the 67 amino acids that distinguish it from Cf-4, in addition to two extra LRRs. Our results demonstrate that sequence variation within the central LRRs of domain C1 and variation in LRR copy number in Cf-4 and Cf-9 play a major role in determining recognition specificity in these proteins.     

Endoplasmic reticulum-quality control chaperones facilitate the biogenesis of Cf receptor-like proteins involved in pathogen resistance of tomato

Cf proteins are receptor-like proteins (RLPs) that mediate resistance of tomato (Solanum lycopersicum) to the foliar pathogen Cladosporium fulvum. These transmembrane immune receptors, which carry extracellular leucine-rich repeats that are subjected to posttranslational glycosylation, perceive effectors of the pathogen and trigger a defense response that results in plant resistance. To identify proteins required for the functionality of these RLPs, we performed immunopurification of a functional Cf-4-enhanced green fluorescent protein fusion protein transiently expressed in Nicotiana benthamiana, followed by mass spectrometry. The endoplasmic reticulum (ER) heat shock protein70 binding proteins (BiPs) and lectin-type calreticulins (CRTs), which are chaperones involved in ER-quality control, were copurifying with Cf-4-enhanced green fluorescent protein. The tomato and N. benthamiana genomes encode four BiP homologs and silencing experiments revealed that these BiPs are important for overall plant viability. For the three tomato CRTs, virus-induced gene silencing targeting the plant-specific CRT3a gene resulted in a significantly compromised Cf-4-mediated defense response and loss of full resistance to C. fulvum. We show that upon knockdown of CRT3a the Cf-4 protein accumulated, but the pool of Cf-4 protein carrying complex-type N-linked glycans was largely reduced. Together, our study on proteins required for Cf function reveals an important role for the CRT ER chaperone CRT3a in the biogenesis and functionality of this type of RLP involved in plant defense.