Citrus Vascular Proteomics Highlights the Role of Peroxidases and Serine Proteases during Huanglongbing Disease Progression.

Huanglongbing (HLB) is the most devastating and widespread citrus disease. All commercial citrus varieties are susceptible to the HLB-associated bacterium, Candidatus Liberibacter asiaticus (CLas), which resides in the phloem. The phloem is part of the plant vascular system and is involved in sugar transport. To investigate the plant response to CLas, we enriched for proteins surrounding the phloem in an HLB susceptible sweet orange variety, Washington navel (Citrus sinensis (L) Osbeck). Quantitative proteomics revealed global changes in the citrus proteome after CLas inoculation. Plant metabolism and translation were suppressed, whereas defense-related proteins such as peroxidases, proteases and protease inhibitors were induced in the vasculature. Transcript accumulation and enzymatic activity of plant peroxidases in CLas infected sweet orange varieties under greenhouse and field conditions were assessed. Although peroxidase transcript accumulation was induced in CLas infected sweet orange varieties, peroxidase enzymatic activity varied. Specific serine proteases were up-regulated in Washington navel in the presence of CLas based on quantitative proteomics. Subsequent activity-based protein profiling revealed increased activity of two serine proteases, and reduced activity of one protease in two C. sinensis sweet orange varieties under greenhouse and field conditions. The observations in the current study highlight global reprogramming of the citrus vascular proteome and differential regulation of enzyme classes in response to CLas infection. These results open an avenue for further investigation of diverse responses to HLB across different environmental conditions and citrus genotypes.

Quantitative phosphoproteomic analysis reveals common regulatory mechanisms between effector- and PAMP-triggered immunity in plants.

Plant immunity consists of two arms: pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI), induced by surface-localized receptors, and effector-triggered immunity (ETI), induced by intracellular receptors. Despite the little structural similarity, both receptor types activate similar responses with different dynamics. To better understand phosphorylation events during ETI, we employed a phosphoproteomic screen using an inducible expression system of the bacterial effector avrRpt2 in Arabidopsis thaliana, and identified 109 differentially phosphorylated residues of membrane-associated proteins on activation of the intracellular RPS2 receptor. Interestingly, several RPS2-regulated phosphosites overlap with sites that are regulated during PTI, suggesting that these phosphosites may be convergent points of both signaling arms. Moreover, some of these sites are residues of important defense components, including the NADPH oxidase RBOHD, ABC-transporter PEN3, calcium-ATPase ACA8, noncanonical Gα protein XLG2 and H+ -ATPases. In particular, we found that S343 and S347 of RBOHD are common phosphorylation targets during PTI and ETI. Our mutational analyses showed that these sites are required for the production of reactive oxygen species during both PTI and ETI, and immunity against avirulent bacteria and a virulent necrotrophic fungus. We provide, for the first time, large-scale phosphoproteomic data of ETI, thereby suggesting crucial roles of common phosphosites in plant immunity.

An effector from the Huanglongbing-associated pathogen targets citrus proteases.

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

Avr4 promotes Cf-4 receptor-like protein association with the BAK1/SERK3 receptor-like kinase to initiate receptor endocytosis and plant immunity.

The first layer of plant immunity is activated by cell surface receptor-like kinases (RLKs) and proteins (RLPs) that detect infectious pathogens. Constitutive interaction with the SUPPRESSOR OF BIR1 (SOBIR1) RLK contributes to RLP stability and kinase activity. As RLK activation requires transphosphorylation with a second associated RLK, it remains elusive how RLPs initiate downstream signaling. We employed live-cell imaging, gene silencing and coimmunoprecipitation to investigate the requirement of associated kinases for functioning and ligand-induced subcellular trafficking of Cf RLPs that mediate immunity of tomato against Cladosporium fulvum. Our research shows that after elicitation with matching effector ligands Avr4 and Avr9, BRI1-ASSOCIATED KINASE 1/SOMATIC EMBRYOGENESIS RECEPTOR KINASE 3 (BAK1/SERK3) associates with Cf-4 and Cf-9. BAK1/SERK3 is required for the effector-triggered hypersensitive response and resistance of tomato against C. fulvum. Furthermore, Cf-4 interacts with SOBIR1 at the plasma membrane and is recruited to late endosomes upon Avr4 trigger, also depending on BAK1/SERK3. These observations indicate that RLP-mediated resistance and endocytosis require ligand-induced recruitment of BAK1/SERK3, reminiscent of BAK1/SERK3 interaction and subcellular fate of the FLAGELLIN SENSING 2 (FLS2) RLK. This reveals that diverse classes of cell surface immune receptors share common requirements for initiation of resistance and endocytosis.

SOBIR1 requires the GxxxG dimerization motif in its transmembrane domain to form constitutive complexes with receptor-like proteins.

Receptor-like proteins (RLPs), forming an important group of transmembrane receptors in plants, play roles in development and immunity. RLPs contain extracellular leucine-rich repeats (LRRs) and, in contrast with receptor-like kinases (RLKs), lack a cytoplasmic kinase required for the initiation of downstream signalling. Recent studies have revealed that the RLK SOBIR1/EVR (SUPPRESSOR OF BIR1-1/EVERSHED) specifically interacts with RLPs. SOBIR1 stabilizes RLPs and is required for their function. However, the mechanism by which SOBIR1 associates with RLPs and regulates RLP function remains unknown. The Cf immune receptors of tomato (Solanum lycopersicum), mediating resistance to the fungus Cladosporium fulvum, are RLPs that also interact with SOBIR1. Here, we show that both the LRR and kinase domain of SOBIR1 are dispensable for association with the RLP Cf-4, whereas the highly conserved GxxxGxxxG motif present in the transmembrane domain of SOBIR1 is essential for its interaction with Cf-4 and additional RLPs. Complementation assays in Nicotiana benthamiana, in which endogenous SOBIR1 levels were knocked down by virus-induced gene silencing, showed that the LRR domain as well as the kinase activity of SOBIR1 are required for the Cf-4/Avr4-triggered hypersensitive response (HR). In contrast, the LRRs and kinase activity of SOBIR1 are not required for facilitation of Cf-4 accumulation. Together, these results suggest that, in addition to being a stabilizing scaffold for RLPs, SOBIR1 is also required for the initiation of downstream signalling through its kinase domain.

PBL13 Is a Serine/Threonine Protein Kinase That Negatively Regulates Arabidopsis Immune Responses.

Receptor-like cytoplasmic kinases (RLCKs) are a subset of plant receptor-like kinases lacking both extracellular and transmembrane domains. Some of the 46 members in the Arabidopsis (Arabidopsis thaliana) RLCK subfamily VII have been linked to plant innate immunity; however, most remain uncharacterized. Thus, multiple subfamily VII members are expected to be involved in plant immune signaling. Here, we investigate the role of AvrPphB SUSCEPTIBLE1-LIKE13 (PBL13), a subfamily VII RLCK with unique domain architecture. Unlike other characterized RLCKs, PBL13 transfer DNA insertion lines exhibit enhanced disease resistance after inoculation with virulent Pseudomonas syringae. The pbl13-2 knockout also exhibits elevated basal-level expression of the PATHOGENESIS-RELATED GENE1 defense marker gene, enhanced reactive oxygen species (ROS) burst in response to perception of bacterial microbial patterns, and accelerated flagellin-induced activation of mitogen-activated protein kinases. Recombinant PBL13 is an active kinase, and its primary autophosphorylated sites map to a 15-amino acid repeat motif unique to PBL13. Complementation of pbl13-2 with PBL13-3xFLAG converts the enhanced resistance and elevated ROS phenotypes back to wild-type levels. In contrast, kinase-dead PBL13(K111A)-3xFLAG was unable to rescue pbl13-2 disease phenotypes. Consistent with the enhanced ROS burst in the pbl13-2 knockout, PBL13 is able to associate with the nicotinamide adenine dinucleotide phosphate, reduced oxidase RESPIRATORY BURST OXIDASE HOMOLOG PROTEIN D (RBOHD) by split-luciferase complementation assay, and this association is disrupted by flagellin treatment. We conclude that the PBL13 kinase negatively regulates plant innate immunity to pathogenic bacteria and can associate with RBOHD before pathogen perception. These data are consistent with the hypothesis that PBL13 acts to prevent inappropriate activation of defense responses in the absence of pathogen challenge.

Elicitin recognition confers enhanced resistance to Phytophthora infestans in potato.

Potato late blight, caused by the destructive Irish famine pathogen Phytophthora infestans, is a major threat to global food security(1,2). All late blight resistance genes identified to date belong to the coiled-coil, nucleotide-binding, leucine-rich repeat class of intracellular immune receptors(3). However, virulent races of the pathogen quickly evolved to evade recognition by these cytoplasmic immune receptors(4). Here we demonstrate that the receptor-like protein ELR (elicitin response) from the wild potato Solanum microdontum mediates extracellular recognition of the elicitin domain, a molecular pattern that is conserved in Phytophthora species. ELR associates with the immune co-receptor BAK1/SERK3 and mediates broad-spectrum recognition of elicitin proteins from several Phytophthora species, including four diverse elicitins from P. infestans. Transfer of ELR into cultivated potato resulted in enhanced resistance to P. infestans. Pyramiding cell surface pattern recognition receptors with intracellular immune receptors could maximize the potential of generating a broader and potentially more durable resistance to this devastating plant pathogen.

Arabidopsis thaliana receptor-like protein AtRLP23 associates with the receptor-like kinase AtSOBIR1.

Plants employ a large number of receptors localizing to the cell surface to sense extracellular signals. Receptor-like proteins (RLPs) form an important group of such trans-membrane receptors, containing an extracellular domain which is involved in signal perception and a short cytoplasmic domain. In contrast to receptor-like kinases (RLKs), RLPs lack a cytoplasmic kinase domain. How intracellular signaling is triggered downstream of RLPs upon perception of an extracellular signal, is therefore still poorly understood. Recently, the RLK SOBIR1 (Suppressor Of BIR1-1) was identified as an essential regulatory RLK of various RLPs involved in plant immunity against fungal pathogens. (1) Given that SOBIR1 appears to be a crucial component of RLP-containing complexes, we aimed to identify additional proteins interacting with SOBIR1. Here, we report on the immunopurification of a functional Arabidopsis thaliana (At)SOBIR1-yellow fluorescent protein (YFP) fusion protein stably expressed in Arabidopsis, followed by mass-spectrometry to identify co-purifying proteins. Interestingly, and in line with various studies showing interaction between RLPs and SOBIR1, we discovered that AtSOBIR1 interacts with AtRLP23, an RLP of which the function is currently unknown.

Functional analysis of the tomato immune receptor Ve1 through domain swaps with its non-functional homolog Ve2.

Resistance in tomato against race 1 strains of the fungal vascular wilt pathogens Verticillium dahliae and V. albo-atrum is mediated by the Ve locus. This locus comprises two closely linked inversely oriented genes, Ve1 and Ve2, which encode cell surface receptors of the extracellular leucine-rich repeat receptor-like protein (eLRR-RLP) type. While Ve1 mediates Verticillium resistance through monitoring the presence of the recently identified V. dahliae Ave1 effector, no functionality for Ve2 has been demonstrated in tomato. Ve1 and Ve2 contain 37 eLRRs and share 84% amino acid identity, facilitating investigation of Ve protein functionality through domain swapping. In this study it is shown that Ve chimeras in which the first thirty eLRRs of Ve1 were replaced by those of Ve2 remain able to induce HR and activate Verticillium resistance, and that deletion of these thirty eLRRs from Ve1 resulted in loss of functionality. Also the region between eLRR30 and eLRR35 is required for Ve1-mediated resistance, and cannot be replaced by the region between eLRR30 and eLRR35 of Ve2. We furthermore show that the cytoplasmic tail of Ve1 is required for functionality, as truncation of this tail results in loss of functionality. Moreover, the C-terminus of Ve2 fails to activate immune signaling as chimeras containing the C-terminus of Ve2 do not provide Verticillium resistance. Furthermore, Ve1 was found to interact through its C-terminus with the eLRR-containing receptor-like kinase (eLRR-RLK) interactor SOBIR1 that was recently identified as an interactor of eLRR-RLP (immune) receptors. Intriguingly, also Ve2 was found to interact with SOBIR1.

Two for all: receptor-associated kinases SOBIR1 and BAK1.

Leucine-rich repeat-receptor-like proteins (LRR-RLPs) are ubiquitous cell surface receptors lacking a cytoplasmic signalling domain. For most of these LRR-RLPs, it remained enigmatic how they activate cellular responses upon ligand perception. Recently, the LRR-receptor-like kinase (LRR-RLK) SUPPRESSOR OF BIR1-1 (SOBIR1) was shown to be essential for triggering defence responses by certain LRR-RLPs that act as immune receptors. In addition to SOBIR1, the regulatory LRR-RLK BRI1-ASSOCIATED KINASE-1 (BAK1) is also required for LRR-RLP function. Here, we compare the roles of SOBIR1 and BAK1 as regulatory LRR-RLKs in immunity and development. BAK1 has a general regulatory role in plasma membrane-associated receptor complexes comprising LRR-RLPs and/or LRR-RLKs. By contrast, SOBIR1 appears to be specifically required for the function of receptor complexes containing LRR-RLPs.

Fungal endopolygalacturonases are recognized as microbe-associated molecular patterns by the arabidopsis receptor-like protein RESPONSIVENESS TO BOTRYTIS POLYGALACTURONASES1.

Plants perceive microbial invaders using pattern recognition receptors that recognize microbe-associated molecular patterns. In this study, we identified RESPONSIVENESS TO BOTRYTIS POLYGALACTURONASES1 (RBPG1), an Arabidopsis (Arabidopsis thaliana) leucine-rich repeat receptor-like protein, AtRLP42, that recognizes fungal endopolygalacturonases (PGs) and acts as a novel microbe-associated molecular pattern receptor. RBPG1 recognizes several PGs from the plant pathogen Botrytis cinerea as well as one from the saprotroph Aspergillus niger. Infiltration of B. cinerea PGs into Arabidopsis accession Columbia induced a necrotic response, whereas accession Brno (Br-0) showed no symptoms. A map-based cloning strategy, combined with comparative and functional genomics, led to the identification of the Columbia RBPG1 gene and showed that this gene is essential for the responsiveness of Arabidopsis to the PGs. Transformation of RBPG1 into accession Br-0 resulted in a gain of PG responsiveness. Transgenic Br-0 plants expressing RBPG1 were equally susceptible as the recipient Br-0 to the necrotroph B. cinerea and to the biotroph Hyaloperonospora arabidopsidis. Pretreating leaves of the transgenic plants with a PG resulted in increased resistance to H. arabidopsidis. Coimmunoprecipitation experiments demonstrated that RBPG1 and PG form a complex in Nicotiana benthamiana, which also involves the Arabidopsis leucine-rich repeat receptor-like protein SOBIR1 (for SUPPRESSOR OF BIR1). sobir1 mutant plants did not induce necrosis in response to PGs and were compromised in PG-induced resistance to H. arabidopsidis.

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.

Receptor-like kinase SOBIR1/EVR interacts with receptor-like proteins in plant immunity against fungal infection.

The plant immune system is activated by microbial patterns that are detected as nonself molecules. Such patterns are recognized by immune receptors that are cytoplasmic or localized at the plasma membrane. Cell surface receptors are represented by receptor-like kinases (RLKs) that frequently contain extracellular leucine-rich repeats and an intracellular kinase domain for activation of downstream signaling, as well as receptor-like proteins (RLPs) that lack this signaling domain. It is therefore hypothesized that RLKs are required for RLPs to activate downstream signaling. The RLPs Cf-4 and Ve1 of tomato (Solanum lycopersicum) mediate resistance to the fungal pathogens Cladosporium fulvum and Verticillium dahliae, respectively. Despite their importance, the mechanism by which these immune receptors mediate downstream signaling upon recognition of their matching ligand, Avr4 and Ave1, remained enigmatic. Here we show that the tomato ortholog of the Arabidopsis thaliana RLK Suppressor Of BIR1-1/Evershed (SOBIR1/EVR) and its close homolog S. lycopersicum (Sl)SOBIR1-like interact in planta with both Cf-4 and Ve1 and are required for the Cf-4- and Ve1-mediated hypersensitive response and immunity. Tomato SOBIR1/EVR interacts with most of the tested RLPs, but not with the RLKs FLS2, SERK1, SERK3a, BAK1, and CLV1. SOBIR1/EVR is required for stability of the Cf-4 and Ve1 receptors, supporting our observation that these RLPs are present in a complex with SOBIR1/EVR in planta. We show that SOBIR1/EVR is essential for RLP-mediated immunity and propose that the protein functions as a regulatory RLK of this type of cell-surface receptors.

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.