NbCSPR underlies age-dependent immune responses to bacterial cold shock protein in Nicotiana benthamiana

Isabel M. L. Saur; Yasuhiro Kadota; Jan Sklenar; Nicholas J. Holton; Elwira Smakowska; Youssef Belkhadir; Cyril Zipfel; John P. Rathjen

doi:10.1073/pnas.1511847113

New Research In

Edited by Frederick M. Ausubel, Harvard Medical School and Massachusetts General Hospital, Boston, MA, and approved January 26, 2016 (received for review June 17, 2015)

Significance

Plants detect pathogens by surface-localized receptors. Few such receptors are known. The coreceptor BRI1-ASSOCIATED KINASE 1 (BAK1) is a frequent member of activated receptor complexes. The proteomics strategy described here uses BAK1 as molecular bait to identify potential receptors that are specifically activated by pathogen components. We demonstrate this approach by identifying Nicotiana benthamiana RECEPTOR-LIKE PROTEIN REQUIRED FOR CSP22 RESPONSIVENESS (NbCSPR). We show that NbCSPR is required for immune responses initiated by the bacterial cold shock protein, confers age-dependent immunity against bacteria, and restricts the transformation of N. benthamiana cells by Agrobacterium. Manipulation of this gene will provide new options for disease control and genetic transformation of crop species.

Abstract

Plants use receptor kinases (RKs) and receptor-like proteins (RLPs) as pattern recognition receptors (PRRs) to sense pathogen-associated molecular patterns (PAMPs) that are typical of whole classes of microbes. After ligand perception, many leucine-rich repeat (LRR)-containing PRRs interact with the LRR-RK BRI1-ASSOCIATED KINASE 1 (BAK1). BAK1 is thus expected to interact with unknown PRRs. Here, we used BAK1 as molecular bait to identify a previously unknown LRR-RLP required for the recognition of the csp22 peptide derived from bacterial cold shock protein. We established a method to identify proteins that interact with BAK1 only after csp22 treatment. BAK1 was expressed transiently in Nicotiana benthamiana and immunopurified after treatment with csp22. BAK1-associated proteins were identified by mass spectrometry. We identified several proteins including known BAK1 interactors and a previously uncharacterized LRR-RLP that we termed RECEPTOR-LIKE PROTEIN REQUIRED FOR CSP22 RESPONSIVENESS (NbCSPR). This RLP associates with BAK1 upon csp22 treatment, and NbCSPR-silenced plants are impaired in csp22-induced defense responses. NbCSPR confers resistance to bacteria in an age-dependent and flagellin-induced manner. As such, it limits bacterial growth and Agrobacterium-mediated transformation of flowering N. benthamiana plants. Transgenic expression of NbCSPR into Arabidopsis thaliana conferred responsiveness to csp22 and antibacterial resistance. Our method may be used to identify LRR-type RKs and RLPs required for PAMP perception/responsiveness, even when the active purified PAMP has not been defined.

Plants and animals sense microbes by detecting a range of pathogen-associated molecular patterns (PAMPs). PAMPs are recognized directly by pattern recognition receptors (PRRs) located on the cell surface. In plants, PRRs usually belong to the receptor kinase (RK) or receptor-like protein (RLP) classes and often contain leucine-rich repeat (LRR) or carbohydrate-binding LysM extracellular domains (1). Perhaps the best-studied PRR is the LRR-RK FLAGELLIN SENSING 2 (FLS2) that recognizes bacterial flagellin or its peptide derivative flg22 (2 ⇓–4). FLS2 and several other LRR-type receptors require the LRR-RK BRI1-ASSOCIATED KINASE 1 (BAK1) for signal transduction. BAK1 (SERK3) is part of the SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) family in Arabidopsis thaliana. BAK1 is sometimes functionally redundant with SERK4/BAK1-LIKE 1 (BKK1) (5). In many cases, BAK1 interacts with receptors in a ligand-induced manner (4 ⇓⇓⇓–8). The BAK1-INTERACTING RKs 1 and 2 (BIR1 and BIR2) negatively regulate BAK1 (9, 10). BIR2 was identified by BAK1 pull-down and is released from the BAK1-FLS2 complex during flg22 perception, whereas BIR1 negatively regulates BAK1-mediated cell death before complex activation. The bir1-1 cell death phenotype is rescued by a mutation in SUPPRESSOR OF BIR1-1 (SOBIR1), sobir1-1. SOBIR1 is a LRR-RK that interacts with RLPs, including the tomato LRR-RLPs Cf-4 and Ve1 (11) and the RLPs ReMAX (12) and RESPONSIVENESS TO BOTRYTIS POLYGALACTURONASES1 (RBPG1/RLP42) from Arabidopsis thaliana (13). SOBIR1 is also required for responses to an elicitor-containing fraction from the necrotrophic fungus Sclerotinia sclerotiorum mediated by RLP30 (14) and forms a constitutive, ligand-independent complex with RLP23 in A. thaliana, which recruits BAK1 upon perception of the PAMP NECROSIS AND ETHYLENE-INDUCING PEPTIDE 1-LIKE PROTEIN 20 (nlp20) (15). Nicotiana benthamiana contains two SOBIR1 homologs, NbSOBIR1 and NbSOBIR1-like (11).

Activation of PRRs leads to PAMP-triggered immunity (PTI) (16). PTI is associated with cellular phenomena such as extracellular alkalinization, influx of apoplastic Ca²⁺, production of reactive oxygen species (ROS), activation of mitogen-activated protein kinases (MAPKs), and reprogramming of host gene expression (17). Adapted bacterial pathogens evade PTI by altering PAMPs to avoid recognition or by secreting virulence effector proteins into the host cytoplasm to inhibit PTI (18). Reduced PTI is associated with disease (18), but is also essential for Agrobacterium-mediated plant transformation and interactions with symbiotic bacteria (19). Bacteria that are not recognized by FLS2 elicit PTI through the perception of alternative PAMPs, and several PAMPs are recognized only by certain plant families (20). For example, A. thaliana recognizes the bacterial PAMP elongation factor-Tu through the LRR-RK ELONGATION FACTOR-TU RECEPTOR (EFR) (21). EFR recruits BAK1 after perception of the EF-Tu–derived peptide elf18, illustrating the capacity of BAK1 to interact with different receptors (8). Likewise, the cold shock protein (CSP) was identified from the bacterium Staphylococcus aureus as a PAMP that is perceived specifically by members of the plant family Solanaceae (22). CSP contains a conserved cold-shock domain (CSD), and the N-terminal 22-amino-acid sequence of the CSP consensus sequence (csp22) elicits immune responses in a BAK1-dependent manner (7, 22). However, a receptor required for CSP-mediated immunity has not yet been identified, despite identification of this PAMP over 10 y ago. Here, we describe a proteomics approach to identify RKs or RLPs required for PTI in response to csp22 using BAK1 as molecular bait. We confirm its utility by identifying a LRR-RLP required for CSP-induced PTI in N. benthamiana (N. benthamiana RLP REQUIRED FOR CSP22 RESPONSIVENESS). NbCSPR induced immune responses after csp22 treatment in an NbBAK1-dependent manner and restricted the growth of adapted and nonadapted bacteria. We further show that perception of CSP from Agrobacterium tumefaciens limits transformation of N. benthamiana and that interfamily transfer of NbCSPR can be a useful strategy to enhance bacterial disease resistance in non-Solanaceaeous plants.

Results

csp22 Responses Are Age-Dependent in N. benthamiana.

Four- to 5-wk-old N. benthamiana plants before the onset of flowering are commonly used to measure immunity and for transient Agrobacterium-mediated transformation (23). Unlike flg22-induced events, csp22-dependent responses are weak and inconsistent in plants of this age. We found that csp22-induced responses were higher in flowering N. benthamiana plants. Under the growth conditions used here, plants were 6 wk old when they flowered. We measured PTI responses including ROS production, Ca²⁺ influx, activation of MAPKs, and up-regulation of PAMP-induced gene (PIGs) expression. All responses triggered by csp22 were greater in 6-wk- than in 4-wk-old plants, but this effect was not seen for flg22 (SI Appendix, Figs. S1 and S2). Therefore, plants at this developmental stage were used to identify proteins required for csp22 responsiveness in N. benthamiana and for all subsequent experiments unless otherwise indicated.

Identification of CSPR from N. benthamiana Using NbBAK1 as Bait.

We exploited the requirement for NbBAK1 in csp22 recognition (7), which suggested a csp22-triggered complex between an unknown receptor protein and NbBAK1. For this approach, we expressed NbBAK1b (referred to here as NbBAK1) (24) from the strong 35S promoter, fused translationally to green fluorescent protein (GFP) at its C terminus (35S:NbBAK1-GFP). Additionally, we created a bak1-5 variant (C508Y) (35S:NbBAK1-5-GFP), as AtBAK1-5 protein shows higher affinity to FLS2 than AtBAK1 (25) and hence might be a better bait in this scheme. We transformed 5-wk-old N. benthamiana leaves with each construct and infiltrated them with csp22 3 d later at the onset of flowering. The putative NbBAK1 protein complexes were purified from leaf extracts using immobilized anti-GFP and isolated proteins digested into peptides before analysis by liquid chromatography-mass spectrometry (LC-MS/MS) (Fig. 1A). Similar numbers of peptides were identified for NbBAK1 and NbBAK1-5 in both mock- and csp22-treated samples. We identified many proteins including an N. benthamiana homolog of BIR1 and two BIR2 homologs (SI Appendix, Table S1, and Fig. 1B) (9, 10). At the protein level, the NbBIR2 variants were 63% identical to AtBIR2. One variant was more abundant in NbBAK1 pull-downs and hence was designated NbBIR2b and the other as NbBIR2a (SI Appendix, Table S2). NbBIR1, NbBIR2a, and NbBIR2b were present in both mock and csp22 treatments. We further identified two LRR-RLPs that were enriched in the csp22-treated samples as CSPR candidates. We termed them receptor candidate 1 (RC1) and 2 (RC2) (Fig. 1B and SI Appendix, Table S1). We cloned the RC1- and RC2-coding regions into binary vectors under the control of the 35S promoter and fused translationally to a C-terminal 5Myc tag. We coexpressed each of these in N. benthamiana leaves with 35S:NbBAK1 fused C-terminally to 3HA and 1FLAG tags (35S:NbBAK1-3HAF) and tested complex formation in the presence of csp22 by coimmunoprecipitation (coIP) experiments. Using anti-FLAG to recover NbBAK1, and probing the complexes by anti-HA and anti-Myc western blots, we found that, in contrast to the MS results, RC1 was constitutively associated with NbBAK1. On the other hand, RC2 copurified with NbBAK1 only after csp22 treatment, and not after treatment with water or flg22 (SI Appendix, Fig. S3A, and Fig. 1C). AtFLS2, RC1, and RC2 associated with NbBAK1-5 independently of csp22 (SI Appendix, Fig. S3B). Similar results were observed for the interaction between AtBAK1-5 and AtFLS2 (25). We concluded that RC2 is likely an RLP required for CSP-mediated PTI in N. benthamiana and from here on refer to it as NbCSPR, for N. benthamiana RLP REQUIRED FOR CSP22 RESPONSIVENESS. The predicted NbCSPR protein contains an N-terminal signal peptide, 28 extracellular tandem LRRs, and a transmembrane domain followed by a short cytoplasmic tail (SI Appendix, Fig. S4). CSP responsivness was identified initially in Nicotiana tabacum suspension cultures (22), and correspondingly we identified a homolog to NbCSPR in N. tabacum (NtCSPR) (SI Appendix, Fig. S5). We also identified NbCSPR sequence homologs in other Solanaceae, including potato (Solanum tuberosum), Solanum commersonii, Nicotiana sylvestris, Nicotiana tomentosiformis, Petunia hybrida, Physalis peruviana, and Withania somnifera (SI Appendix, Fig. S5). Tomato leaves respond to the csp15 peptide lacking the first seven amino acids of csp22 (22), but despite this, we were unable to identify a clear NbCSPR sequence-homolog in tomato (blast.ncbi.nlm.nih.gov/Blast.cgi and https://solgenomics.net/tools/blast/). A. thaliana does not respond to csp22 and, correspondingly, we were unable to identify an NbCSPR homolog in A. thaliana.

Fig. 1.

Identification of NbCSPR using NbBAK1 as molecular bait. (A) Strategy to identify NbCSPR. N. benthamiana leaves were transiently transformed with 35S:NbBAK1-GFP or 35S:NbBAK1-5-GFP (1). Leaves were treated with csp22 (2), leading to complex formation between NbBAK1 and a hypothetical receptor protein (3). The complex was isolated using anti-GFP–conjugated beads (4), and copurifying proteins were identified by LC-MS/MS. (B) Selected LRR-RK and LRR-RLP proteins identified LC-MS/MS after NbBAK1 immunoprecipitation. Each protein and the number of corresponding peptides are identified (from all four experiments). “RC” stands receptor candidate for cold shock protein. (C) NbRC2 forms a complex with NbBAK1 in a csp22-dependent manner. N. benthamiana leaves were cotransformed with 35S:NbBAK1-3HAF or EV and one of pAtFLS2:AtFLS2-3Myc, 35S:NbRC1-5Myc, or 35S:NbRC2-5Myc. Three days postinfiltration, infiltrated leaves were treated with sterile water (mock) or 100 nM csp22 for 15 min before harvesting the tissue. NbBAK1-3HAF was recovered by anti-FLAG pull-down, and immunoprecipitates were probed with anti-Myc and anti-HA western blots after gel electrophoresis. (Left) The input fractions. (Right) Immunoprecipitated fractions (IP).

NbCSPR Forms a Complex with csp22 and Is Required for csp22 Responses.

To test if csp22 and NbCSPR can associate, we purified csp22-GST and flg22-GST from Escherichia coli BL21 cells and purified NbCSPR-3HAF from N. benthamiana leaf extracts by anti-FLAG IP. Both csp22-GST and flg22-GST were biologically active at the concentration (500 nM) used for the association assay, as estimated by their abilities to induce ROS in N. benthamiana (SI Appendix, Fig. S6A). We mixed bead-bound NbCSPR with 500 nM csp22 expressed as a fusion with the GST protein (csp22-GST). After washing the beads, we found that csp22-GST was retained on the NbCSPR-bound beads (SI Appendix, Fig. S6B). NbCSPR did not associate with flg22-GST, nor with csp22-GST when 10 µM free csp22 peptide was added for competition (SI Appendix, Fig. S6C). We cannot, however, exclude the possibility that purification of NbCSPR from N. benthamiana coisolated additional proteins involved in the interaction with csp22. To investigate the requirement for NbCSPR in csp22 responses, we generated gene fragments corresponding to nucleotides 2–299 (TRV:NbCSPRa) and 300–1,001 (TRV:NbCSPRb) of the ORF and cloned them into a tobacco rattle virus (TRV) vector for virus-induced gene silencing (VIGS) (SI Appendix, Table S3) (26). Plants silenced for NbCSPR (TRV:NbCSPRa and TRV:NbCSPRb), but not those silenced for the control gene GFP (TRV:GFP), showed reduced csp22 responses, including diminished ROS production, activation of MAPKs, and up-regulation of PIG expression (Fig. 2 A–C). Silencing of NbCSPR did not affect flg22 responses (SI Appendix, Fig. S6 D–G). We detected the activation of only one MAPK in silenced plants treated with PAMPs, as reported previously (27). Successful silencing was confirmed by reduced NbCSPR mRNA levels (SI Appendix, Fig. S6F) and lack of detectable NbCSPR protein after transient transformation of TRV:NbCSPRa/b plants with 35S:NbCSPR-3HAF (SI Appendix, Fig. S6G). The TRV:NbCSPRa construct was used for all subsequent experiments and is referred to as TRV:NbCSPR from here on.

Fig. 2.

NbCSPR is required for csp22-dependent responses. NbCSPR is required for csp22-dependent responses as determined by VIGS of N. benthamiana plants and measuring (A) ROS production, (B) activation of MAPKs, and (C) up-regulation of PIG expression. Graphed data are ±SEM, *P < 0.05, **P < 0.01, ***P < 0.001 (pairwise Student’s t test comparing TRV:NbCSPR to TRV:GFP plants; n = 8 for ROS; n = 6 for qRT-PCR). Experiments were performed at least three times and representative results are shown.

NbCSPR Does Not Require NbSOBIR1 for csp22 Responses.

The LRR-RK NbSOBIR1 may be generally required for RLP function through direct interaction, perhaps by providing an intracellular signaling component to the complex (15, 28). Indeed, we found that, when overexpressed in N. benthamiana, NbCSPR copurified with NbSOBIR1 in pull-down experiments, but AtFLS2 did not (SI Appendix, Fig. S7 A and B). In agreement, we found that after overexpression in N. benthamiana, NbSOBIR1 can form a complex with NbBAK1 after csp22 treatment (SI Appendix, Fig. S7C). Only a very weak interaction was detected after mock treatment, which may be due to Agrobacterium-mediated transformation. Thus, NbSOBIR1 and NbBAK1 likely associate in a csp22-induced manner. In agreement with the constitutive association of NbCSPR and NbBAK1-5, NbSOBIR1 can form a constitutive complex with NbBAK1-5 (SI Appendix, Fig. S7C). Despite this, cosilencing of NbSOBIR1 and its close homolog NbSOBIR1-like (TRV:NbSOBIR1+SOBIR-like) (11) in N. benthamiana only slightly reduced the accumulation of transiently expressed NbCSPR (SI Appendix, Fig. S7D). TRV:NbSOBIR1+SOBIR-like plants were also not impaired in csp22- or flg22-induced production of ROS, MAPK activation, or PIG up-regulation (SI Appendix, Fig. S7 G and H). In fact, in TRV:NbSOBIR1+SOBIR-like plants, PIGs were induced to a higher extent by csp22 or flg22 treatment by comparison with TRV:GFP plants. Successful silencing was confirmed through reduced NbSOBIR1 and NbSOBIR1-like mRNA levels and the lack of Avr4/Cf4-mediated cell death in TRV:NbSOBIR1+SOBIR-like plants (11) (SI Appendix, Fig. S7 I and J). We thus suggest the existence of an unknown protein(s) that acts redundantly to NbSOBIR1 and NbSOBIR1-like in csp22-triggered immune signaling.

NbCSPR Confers Responsiveness to csp22 in Transgenic A. thaliana Plants Dependent on AtBAK1/AtBKK1.

Next, we tested if interfamily transfer of NbCSPR can confer csp22 recognition to a previously nonresponsive species. We first transformed A. thaliana Col-0 protoplasts with 35S:NbCSPR-3HA to test for csp22-induced MAPK activation. Wild-type Col-0 protoplasts were blind to the PAMP, whereas NbCSPR-expressing protoplasts activated MAPKs in a csp22-dependent manner. Coexpression of NbSOBIR1 intensified the csp22-dependent MAPK activation (SI Appendix, Fig. S8). To further substantiate this, we generated stable transgenic 35S:NbCSPR-5Myc A. thaliana Col-0 plants. We obtained five transgenics, but only one of these, IS-01, expressed NbCSPR-5Myc protein to a detectable level. We measured csp22-dependent responses in this line, including ROS production, seedling growth inhibition (SGI), and MAPK activation. IS-01 developed a weak ROS burst in response to csp22 that was absent in the empty vector line (IS-00). The profile of ROS production was aberrant compared with N. benthamiana leaf discs (Fig. 3 A and B), suggesting that NbCSPR is not properly regulated in A. thaliana, which might be related to the low frequency of productive transformation. In addition, we found that IS-01 plants but not control plants showed weak activation of MAPK after 5 and 15 min (Fig. 3C), a small but significant SGI in response to the elicitor (Fig. 3D) and up-regulation of PATHOGENESIS-RELATED GENE 1 (PR1) expression, a late defense marker also up-regulated by flg22 and elf18 treatment (29, 30) (SI Appendix, Fig. S8E). In agreement with the N. benthamiana data, csp22-dependent MAPK activation in A. thaliana protoplasts expressing NbCSPR was absent in the bak1-5 bkk1-1 double mutant, but present in the sobir1-12 mutant (SI Appendix, Fig. S8). Of note, we always observed that NbCSPR accumulated to lower levels in bak1-5 bkk1-1 protoplasts, which may also partially explain the reduced MAPK activation in response to csp22 in these protoplasts. Flg22 activated MAPKs in Col-0 and sobir1-12 but not bak1-5 bkk1-1 protoplasts expressing NbCSPR in the same experiments. Overall, the data corroborate our findings in N. benthamiana and support a model in which csp22 induces PTI in a manner that depends on protein complexes containing NbCSPR and BAK1 (or BKK1), potentially with SOBIR1 and/or other protein(s) with similar function.

Fig. 3.

NbCSPR confers recognition of csp22 in A. thaliana. Overexpression of NbCSPR in stable transgenic A. thaliana Col-0 plants (IS-01) leads to csp22-dependent responses, including (A and B) production of ROS, (C) MAPK activation, and (D) SGI. Graphed data are ±SEM, **P < 0.01, ***P < 0.001 (pairwise Student’s t test comparing IS-01 to EV plants (IS-00, n = 8). Experiments were performed at least twice and representative results are shown.

NbCSPR Confers Age-Related Resistance to Bacterial Pathogens and Restricts Agrobacterium-Mediated Transformation of N. benthamiana in Flowering Plants.

To test the relevance of NbCSPR for antibacterial immunity, we silenced NbCSPR or NbFLS2 in N. benthamiana using VIGS and infected 4- or 6-wk-old silenced plants with adapted and nonadapted Pseudomonas syringae strains. Both FLS2- and NbCSPR-silenced plants supported more than 1 log growth of the adapted pathogen P. syringae pv. tabaci (Pta) 6605 (Fig. 4A) compared with control plants silenced for GFP. This is consistent with NbCSPR playing an important role in antibacterial immunity. To test this further, we inoculated silenced plants with a mutant strain deficient in the type-III secretion system (Pta 6605 hrcC⁻) (Fig. 4B). Again, bacteria grew significantly more on N. benthamiana plants silenced for NbFLS2 or NbCSPR than on plants silenced for NbGFP. Finally, to test the relative contribution of NbCSPR to bacterial immunity in the absence of flagellin recognition, we inoculated silenced plants with the Pta 6605 fliC⁻ mutant lacking the flagellin gene (31). Accordingly, bacterial growth was not increased on NbFLS2-silenced plants but showed a small but significant increase in 6-wk-old plants silenced for NbCSPR (Fig. 4C). This effect was not seen on 4-wk-old plants (SI Appendix, Fig. S9A). To test a role for NbCSPR against nonadapted pathogens, we inoculated silenced plants with P. syringae pv. phaseolicola 1448A (32) (Fig. 4D). The weak growth of this strain was significantly higher on plants silenced for NbFLS2 or NbCSPR compared with plants silenced for GFP. We also found that NbCSPR contributed to bacterial resistance when transferred into A. thaliana. The stable transgenic lines IS-00 and IS-01 were spray-infected with adapted P. syringae pv. tomato DC3000 bacteria. Plants expressing NbCSPR (IS-01) showed slightly reduced bacterial growth relative to the empty vector (EV) (IS-00) line (Fig. 4E). Taken together, our data show that NbCSPR is an important component of antibacterial immunity. Flowering N. benthamiana plants are recalcitrant to Agrobacterium-mediated transformation (33). As A. tumefaciens contains CSP genes that are likely elicitor-active (SI Appendix, Fig. S9B), we tested if NbCSPR restricts Agrobacterium-mediated transformation. Four-week-old plants silenced for GFP, NbFLS2, or NbCSPR were equally transformable by A. tumefaciens as judged by expression of an intron-GUS marker gene (SI Appendix, Fig. S9C). Older plants were minimally transformable after silencing for GFP or NbFLS2 (Fig. 4F). Strikingly, NbCSPR-silenced plants showed much higher GUS activity comparable to expression in young plants. Similarly, transient expression of an arbitrary gene (N2) encoding the amino acids 1–242 of the Solanum lycopersicum Prf protein (34) (35S:N2-3HAF) in flowering plants revealed greater N2 accumulation in plants silenced for NbCSPR relative to those silenced for GFP (SI Appendix, Fig. S9D). N2 protein levels were unchanged by gene silencing in younger plants (SI Appendix, Fig. S9E). Greater resistance of older plants to Agrobacterium-mediated infiltration may be related to NbCSPR up-regulation of about twofold in 6-wk-old relative to 4-wk-old plants, an effect that was not seen for NbFLS2 (SI Appendix, Fig. S9F). Our data demonstrate a role for NbCSPR in restricting genetic transformation by A. tumefaciens.

Fig. 4.

NbCSPR contributes to antibacterial immunity. N. benthamiana plants were silenced for GFP, NbFLS2, or NbCSPR before infection by dipping into P. syringae suspensions. Silenced plants were infected with (A) P. syringae pv. tabaci (Pta) 6605, (B) Pta 6605 hrcC⁻, (C) Pta 6605 fliC⁻, and (D) P. syringae pv. phaseolicola 1448A (Pph). Graphed data are ±SEM, *P < 0.05, **P < 0.01 (pairwise Student’s t test comparing TRV:NbFLS2 or TRV:NbCSPR to TRV:GFP plants; n = 6). (E) Stable transgenic Col-0 plants transformed with 35S:EV-5Myc (IS-00) or 35S:NbCSPR-5Myc (IS-01) were spray-infected with P. syringae pv. tomato DC3000 bacteria. Plants were dip- and spray-infected using a bacterial suspension of 5 × 10⁷ cfu/mL, and samples were taken after 3 d. (F) Transformation of 6-wk-old N. benthamiana plants is restricted by NbCSPR. N. benthamiana plants were silenced for GFP, NbFLS2, or NbCSPR before infiltration with A. tumefaciens GV3101 pMp90 carrying a 35S:intron-GUS construct (21). Leaves were harvested 2 d postinfiltration, and GUS activity was detected by GUS staining. Blue color indicates transformation with the GUS gene. All experiments were performed at least twice, and representative results are shown.

Potentiation of csp22 Responses by flg22 Pretreatment.

In contrast to wild-type Pta 6605, NbCSPR restricted growth of Pta 6605 fliC⁻ only in 6-wk-old plants. We thus investigated the role of flagellin perception on csp22-mediated immune responses. We found that prior flg22 treatment caused higher csp22-dependent production of ROS, PIG up-regulation, and MAPK activation including activation of a second MAPK (Fig. 5 A–C). Interestingly, both csp22-induced ROS and MAPK assays showed decreases after csp22 pretreatment, which may be a similar phenomenon to the refractory period of diminished FLS2-mediated responses after initial flg22 perception (35). Treatment of N. benthamiana leaves with 100 nM csp22 or the unrelated PAMP chitin at 100 µg/mL significantly up-regulated NbCSPR expression, but this effect was far higher upon treatment with 100 nM flg22. Conversely, flg22 treatment up-regulated NbFLS2 to only a small extent, whereas its induction by csp22 was negligible (SI Appendix, Fig. S10 A and B). PTI responses induced by flg22 were not increased by prior csp22 treatment (SI Appendix, Fig. S10 C–E). Similarly, prior flg22 treatment caused higher elf18-dependent production of ROS in A. thaliana, but elf18 pretreatment did not result in increased flg22-mediated ROS production (SI Appendix, Fig. S10 E and F). Overall, prior flg22 treatment increased csp22 responses in N. benthamiana and elf18 responses in A. thaliana but not vice versa, perhaps consistent with the fact that flagellin is an external PAMP, and CSP and EF-Tu are internal.

Fig. 5.

flg22 perception potentiates csp22 responsiveness in 4-wk old N. benthamiana plants. Increase in csp22-dependent (A) ROS production, (B) expression of PIG relative to mock-treated controls, and (C) MAPK activation in N. benthamiana leaves after flg22 pretreatment. flg22 was removed and replaced with sterile water before treatment with csp22. Graphed data are ±SEM, **P < 0.01, ***P < 0.001 (pairwise Student’s t test comparing flg22 or csp22 pretreated plants to mock-pretreated plants; n = 8 for ROS; n = 6 for qRT-PCR). Experiments were performed at least twice, and representative results are shown.

Discussion

We report here identification of a LRR-RLP required for csp22 responses using a previously undescribed biochemical approach. NbCSPR encodes a previously undescribed LRR-RLP that can form a constitutive complex with SOBIR1. NbCSPR associates with NbBAK1 after elicitation and is required for immunity to bacterial pathogens. It is active in 6-wk-old plants where it restricts the growth of adapted and nonadapted pathogens and transient transformation by A. tumefaciens. Interestingly, our results suggest a mechanism in which PAMP perception is coordinated temporally as prior flagellin perception potentiates NbCSPR-mediated immunity in 4-wk-old plants.

We used a proteomics approach to identify LRR-RKs or LRR-RLPs that depend on common complex components such as BAK1. In contrast to previous pull-down experiments, our method aims to identify LRR-RKs and LRR-RLPs that form a complex with BAK1 in response to a specific ligand. It is well established that BAK1 is a central regulator of immunity through interaction with LRR-RKs or RLPs after PAMP perception (6, 7). We showed previously that csp22-dependent ROS production is NbBAK1-dependent and as such predicted a csp22-induced interaction between NbBAK1 and an unknown LRR-RK or RLP. Through purifying NbBAK1-GFP (or NbBAK1-5-GFP) after csp22 treatment, we identified known interactors of BAK1 including N. benthamiana homologs of AtBIR1 and AtBIR2 (9, 10). Notably, we did not detect a release of either NbBIR2 variant from NbBAK1 after csp22 treatment as has been reported for AtBIR2 (10). This may reflect a biological difference or was perhaps due to NbBAK1 overexpression. Most importantly, we identified two proteins that were enriched in csp22-treated samples. Subsequent coIP analysis confirmed our LC-MS/MS results and showed the csp22-dependent association of one of these proteins with NbBAK1. Overall, the approach was successful and offers a general strategy to identify BAK1-associated proteins that play specific roles in PAMP perception/responsiveness. Genetic tests showed that NbCSPR is required for csp22-dependent responses and antibacterial immunity. Plants silenced for NbCSPR were deficient in csp22-triggered ROS production, MAPK activation, and up-regulation of PIGs. Consistent with this, the silenced plants were more susceptible to infection by adapted and nonadapted P. syringae pathogens. Silencing of NbCSPR allowed a similar increase in bacterial growth as silencing NbFLS2. Moreover, plants silenced for NbCSPR were transformed more efficiently by A. tumefaciens than TRV:GFP plants, but this effect was not seen for NbFLS2. This result reflects the fact that A. tumefaciens possesses a conserved CSP protein containing the csp22 motif (SI Appendix, Fig. S9B), but its variant flagellin is not recognized (19). Recognition of A. tumefaciens CSP may suggest why NbCSPR peptides were recovered from NbBAK-GFP preparations before csp22 treatment. Restriction of Agrobacterium-mediated transformation by NbCSPR is not unexpected because EFR also limits transformation in A. thaliana and transgenic N. benthamiana (21).

The nonresponsive species A. thaliana initiated csp22-dependent production of ROS, MAPK activation, and SGI after transformation with 35S:NbCSPR-5Myc. The transfer of NbCSPR to protoplasts of A. thaliana allowed csp22-dependent MAPK activation in the transformed cells, whereas wild-type Col-0 protoplasts were blind to the PAMP. Importantly, NbCSPR-mediated signaling in A. thaliana protoplasts required AtBAK1 and/or its close paralogue AtBKK1. Finally, we showed that NbCSPR expressed in N. benthamiana tissue associated with csp22-GST and that this interaction was abrogated when excess free csp22 peptide was used in competition for binding. We therefore conclude that NbCSPR is required for csp22 responses in N. benthamiana and may be the csp22 receptor.

NbSOBIR1 is required for accumulation and functionality of multiple RLPs, perhaps by stabilizing the respective receptor or by providing transmembrane signaling capability (11 ⇓⇓⇓–15, 28). Although NbSOBIR1 associated with NbCSPR and also with NbBAK1 after csp22 treatment, silencing of NbSOBIR1 and its close homolog NbSOBIR1-like only weakly affected accumulation of transiently expressed NbCSPR. This may explain why neither NbSOBIR1 nor its close homolog NbSOBIR1-like were required for csp22-induced responses. We used the TRV:NbSOBIR1+NbSOBIR-like silencing construct that targets both genes (11). Cosilencing of NbSOBIR1 and NbSOBIR1-like was confirmed by qRT-PCR and the lack of Avr4/Cf4-induced hypersensitive response, as shown previously (11). The same plants exhibited all csp22-induced responses. We further found that SOBIR1 was dispensable for NbCSPR-dependent csp22 responsiveness in the A. thaliana sobir1-12 protoplasts. This may be due to the strong NbCSPR protein levels detected during these experiments. Overall, our data suggest that SOBIR1 is involved in NbCSPR function, but that additional proteins may act redundantly to NbSOBIR1 in csp22-mediated immunity.

CSP responses were far greater in plants that were transitioning to flowering than in younger plants. This may be due to an increase in NbCSPR expression or several other untested regulatory mechanisms. The difference is biologically significant because older plants were more resistant to Pta bacteria lacking flagellin and were recalcitrant to transformation by A. tumefaciens. Both effects were reversed by NbCSPR silencing. Despite the fact that csp22 generally exhibited weaker PTI responses than flg22 (7, 19, 22), plants silenced for NbCSPR showed strikingly similar levels of bacterial growth compared with NbFLS2-silenced plants. This was true for adapted and nonadapted P. syringae. However, we cannot exclude differential silencing levels of each gene. The Pta fliC⁻ strain that cannot activate FLS2 showed similar growth on NbFLS2-silenced plants to TRV:GFP plants, as expected. Growth of this strain was slightly but significantly higher in NbCSPR-silenced plants, again demonstrating a role for NbCSPR in antibacterial immunity. Likewise, the efficiency of Agrobacterium-mediated transformation in plants silenced for GFP or NbFLS2 was similar, whereas NbCSPR-silenced plants showed both strongly enhanced GUS activity and accumulation of the N2 protein after transient transformation. Similarly, resistance to Xanthomonas oryzae pv. oryzae mediated by the rice LRR-RKs Xa21 and Xa3/Xa26 is developmentally regulated (36, 37). Our data further show that younger plants can compensate for their deficiency in csp22 perception by up-regulating NbCSPR expression in response to flg22. This potentiated all csp22-induced responses tested here and may explain why NbCSPR does not restrict the growth of the flagellin-deficient strain Pta 6605 fliC⁻ in 4-wk-old plants. This is an important observation because one potential interpretation is that flagellin and CSP perception occur sequentially. This would accord with the fact that flagellin is an external PAMP that is immediately visible to the infected plant, whereas CSP and EF-Tu are internal and must be released before host perception. This model implies that FLS2 identifies the invading microbe as bacterial. Consistent with this view, both eukaryotic pathogens and N. benthamiana itself express proteins with conserved CSDs, and a protein with a CSD from Nicotiana sylvestris elicited a defense response on N. tabacum cells (22). Hence, an additional level of regulation may be necessary for appropriate deployment of CSP recognition, perhaps also to avoid an auto-immune response. We further speculate that the importance of developmental regulation of NbCSPR might be related to the difficulty in recovering 35S:NbCSPR A. thaliana transgenics.

In summary, we have used a proteomics procedure to purify and identify a previously undescribed LRR-RLP, CSPR, involved in antibacterial immunity. NbCSPR transfer to A. thaliana confers responsiveness to the bacterial PAMP csp22, which suggests that NbCSPR directly recognizes csp22. We cannot exclude completely that NbCSPR acts as a coreceptor for the csp22-binding determinant. Our data suggest that transfer of NbCSPR to plant species otherwise blind to CSP may be useful for conferring resistance to bacterial diseases in agriculture. In addition, knocking out or silencing the expression of the NbCSPR will improve transient Agrobacterium-mediated transformation of N. benthamiana for industrial and experimental uses.

Materials and Methods

De novo identification of NbBAK1-associated proteins by LC-MS/MS following anti-GFP immunoprecipitation: NbBAK1-GFP was overexpressed in N. benthamiana. Proteins were extracted and coIP performed as described using anti-GFP (ChromoTek) (38). LC-MS/MS, software processing, and peptide identification were performed as described (39) with the difference that a combined Sol genomics/TGAC N. benthamiana predicted protein database was used for protein identification.

All other materials and methods can be found in SI Appendix, SI Materials and Methods.

Acknowledgments

We thank Dr. B. Schwessinger for preliminary data describing the use of AtBAK1 as molecular bait; Dr. S. Robatzek and Dr. M. Joosten for 35S:Avr4, 35S:Cf4-GFP, and TRV:NbSOBIR1+NbSOBIR-like constructs; and Dr. A. Gust for the sobir1-12 seeds. This work was supported by Future Fellowship FT0992129 and Discovery Program DP110103322 from the Australian Research Council (to J.P.R.) and by the Gatsby Charitable Foundation, the European Research Council (PHOSPHinnATE), and the 2Blades Foundation (C.Z.). I.M.L.S. was supported by a European Molecular Biology Organization Short-Term Fellowship (ASTF 441-2012) and Australian National University Scholarships. Y.K. was supported by a RIKEN Special Postdoctoral Research Fellowship, Excellent Young Researcher Overseas Visit Program of Japan Society for the Promotion of Science, and the Uehara Memorial Foundation. Y.B. was supported by funds from the Austrian Academy of Science through the Gregor Mendel Institute.

Footnotes

↵¹Present address: Department of Plant Microbe Interactions, Max Planck Institute for Plant Breeding Research, Carl-von-Linné-Weg 10, 50829 Cologne, Germany.
↵²Present address: RIKEN Center for Sustainable Resource Science, Suehiro-cho 1-7-22 Tsurumi-ku, Yokohama 230-0045, Japan.
↵³To whom correspondence may be addressed. Email: john.rathjen{at}anu.edu.au or cyril.zipfel{at}tsl.ac.uk.

Author contributions: I.M.L.S., Y.K., N.J.H., E.S., Y.B., C.Z., and J.P.R. designed research; I.M.L.S., Y.K., J.S., N.J.H., and E.S. performed research; I.M.L.S. contributed new reagents/analytic tools; I.M.L.S., Y.K., J.S., N.J.H., E.S., Y.B., C.Z., and J.P.R. analyzed data; and I.M.L.S., C.Z., and J.P.R. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1511847113/-/DCSupplemental.

http://www.pnas.org/preview_site/misc/userlicense.xhtml

References

↵
1. Cao Y, et al.
(2014) The kinase LYK5 is a major chitin receptor in Arabidopsis and forms a chitin-induced complex with related kinase CERK1. eLife 3, doi:10.7554/eLife.03766
.
OpenUrl Abstract/FREE Full Text
↵
1. Chinchilla D,
2. Bauer Z,
3. Regenass M,
4. Boller T,
5. Felix G
(2006) The Arabidopsis receptor kinase FLS2 binds flg22 and determines the specificity of flagellin perception. Plant Cell 18(2):465–476
.
OpenUrl Abstract/FREE Full Text
↵
1. Gómez-Gómez L,
2. Boller T
(2000) FLS2: An LRR receptor-like kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis. Mol Cell 5(6):1003–1011
.
OpenUrl CrossRef PubMed
↵
1. Sun Y, et al.
(2013) Structural basis for flg22-induced activation of the Arabidopsis FLS2-BAK1 immune complex. Science 342(6158):624–628
.
OpenUrl Abstract/FREE Full Text
↵
1. Roux M, et al.
(2011) The Arabidopsis leucine-rich repeat receptor-like kinases BAK1/SERK3 and BKK1/SERK4 are required for innate immunity to hemibiotrophic and biotrophic pathogens. Plant Cell 23(6):2440–2455
.
OpenUrl Abstract/FREE Full Text
↵
1. Chinchilla D, et al.
(2007) A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defence. Nature 448(7152):497–500
.
OpenUrl CrossRef PubMed
↵
1. Heese A, et al.
(2007) The receptor-like kinase SERK3/BAK1 is a central regulator of innate immunity in plants. Proc Natl Acad Sci USA 104(29):12217–12222
.
OpenUrl Abstract/FREE Full Text
↵
1. Schulze B, et al.
(2010) Rapid heteromerization and phosphorylation of ligand-activated plant transmembrane receptors and their associated kinase BAK1. J Biol Chem 285(13):9444–9451
.
OpenUrl Abstract/FREE Full Text
↵
1. Gao M, et al.
(2009) Regulation of cell death and innate immunity by two receptor-like kinases in Arabidopsis. Cell Host Microbe 6(1):34–44
.
OpenUrl CrossRef PubMed
↵
1. Halter T, et al.
(2014) The leucine-rich repeat receptor kinase BIR2 is a negative regulator of BAK1 in plant immunity. Curr Biol 24(2):134–143
.
OpenUrl CrossRef PubMed
↵
1. Liebrand TW, et al.
(2013) Receptor-like kinase SOBIR1/EVR interacts with receptor-like proteins in plant immunity against fungal infection. Proc Natl Acad Sci USA 110(24):10010–10015
.
OpenUrl Abstract/FREE Full Text
↵
1. Zhang L, et al.
(2014) Fungal endopolygalacturonases are recognized as microbe-associated molecular patterns by the Arabidopsis receptor-like protein RESPONSIVENESS TO BOTRYTIS POLYGALACTURONASES1. Plant Physiol 164(1):352–364
.
OpenUrl Abstract/FREE Full Text
↵
1. Jehle AK,
2. Fürst U,
3. Lipschis M,
4. Albert M,
5. Felix G
(2013) Perception of the novel MAMP eMax from different Xanthomonas species requires the Arabidopsis receptor-like protein ReMAX and the receptor kinase SOBIR. Plant Signal Behav 8(12):e27408
.
OpenUrl CrossRef PubMed
↵
1. Zhang W, et al.
(2013) Arabidopsis receptor-like protein30 and receptor-like kinase suppressor of BIR1-1/EVERSHED mediate innate immunity to necrotrophic fungi. Plant Cell 25(10):4227–4241
.
OpenUrl Abstract/FREE Full Text
↵
1. Albert I, et al.
(2015) An RLP23–SOBIR1–BAK1 complex mediates NLP-triggered immunity. Nature Plants 1:15140
.
OpenUrl CrossRef
↵
1. Monaghan J,
2. Zipfel C
(2012) Plant pattern recognition receptor complexes at the plasma membrane. Curr Opin Plant Biol 15(4):349–357
.
OpenUrl CrossRef PubMed
↵
1. Macho AP,
2. Zipfel C
(2014) Plant PRRs and the activation of innate immune signaling. Mol Cell 54(2):263–272
.
OpenUrl CrossRef PubMed
↵
1. Jones JD,
2. Dangl JL
(2006) The plant immune system. Nature 444(7117):323–329
.
OpenUrl CrossRef PubMed
↵
1. Felix G,
2. Duran JD,
3. Volko S,
4. Boller T
(1999) Plants have a sensitive perception system for the most conserved domain of bacterial flagellin. Plant J 18(3):265–276
.
OpenUrl CrossRef PubMed
↵
1. Boller T,
2. Felix G
(2009) A renaissance of elicitors: Perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors. Annu Rev Plant Biol 60:379–406
.
OpenUrl CrossRef PubMed
↵
1. Zipfel C, et al.
(2006) Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacterium-mediated transformation. Cell 125(4):749–760
.
OpenUrl CrossRef PubMed
↵
1. Felix G,
2. Boller T
(2003) Molecular sensing of bacteria in plants. The highly conserved RNA-binding motif RNP-1 of bacterial cold shock proteins is recognized as an elicitor signal in tobacco. J Biol Chem 278(8):6201–6208
.
OpenUrl Abstract/FREE Full Text
↵
1. Shamloul M,
2. Trusa J,
3. Mett V,
4. Yusibov V
(2014) Optimization and utilization of Agrobacterium-mediated transient protein production in Nicotiana. J Vis Exp doi:10.3791/51204
.
OpenUrl CrossRef
↵
1. Chaparro-Garcia A, et al.
(2011) The receptor-like kinase SERK3/BAK1 is required for basal resistance against the late blight pathogen phytophthora infestans in Nicotiana benthamiana. PLoS One 6(1):e16608
.
OpenUrl CrossRef PubMed
↵
1. Schwessinger B, et al.
(2011) Phosphorylation-dependent differential regulation of plant growth, cell death, and innate immunity by the regulatory receptor-like kinase BAK1. PLoS Genet 7(4):e1002046
.
OpenUrl CrossRef PubMed
↵
1. Dong Y,
2. Burch-Smith TM,
3. Liu Y,
4. Mamillapalli P,
5. Dinesh-Kumar SP
(2007) A ligation-independent cloning tobacco rattle virus vector for high-throughput virus-induced gene silencing identifies roles for NbMADS4-1 and -2 in floral development. Plant Physiol 145(4):1161–1170
.
OpenUrl Abstract/FREE Full Text
↵
1. Segonzac C, et al.
(2011) Hierarchy and roles of pathogen-associated molecular pattern-induced responses in Nicotiana benthamiana. Plant Physiol 156(2):687–699
.
OpenUrl Abstract/FREE Full Text
↵
1. Gust AA,
2. Felix G
(2014) Receptor like proteins associate with SOBIR1-type of adaptors to form bimolecular receptor kinases. Curr Opin Plant Biol 21:104–111
.
OpenUrl CrossRef PubMed
↵
1. Asai T, et al.
(2002) MAP kinase signalling cascade in Arabidopsis innate immunity. Nature 415(6875):977–983
.
OpenUrl CrossRef PubMed
↵
1. Lu X, et al.
(2009) Uncoupling of sustained MAMP receptor signaling from early outputs in an Arabidopsis endoplasmic reticulum glucosidase II allele. Proc Natl Acad Sci USA 106(52):22522–22527
.
OpenUrl Abstract/FREE Full Text
↵
1. Shimizu R, et al.
(2003) The DeltafliD mutant of Pseudomonas syringae pv. tabaci, which secretes flagellin monomers, induces a strong hypersensitive reaction (HR) in non-host tomato cells. Mol Genet Genomics 269(1):21–30
.
OpenUrl PubMed
↵
1. Arnold DL,
2. Lovell HC,
3. Jackson RW,
4. Mansfield JW
(2011) Pseudomonas syringae pv. phaseolicola: From ‘has bean’ to supermodel. Mol Plant Pathol 12(7):617–627
.
OpenUrl CrossRef PubMed
↵
1. Leuzinger K, et al.
(2013) Efficient agroinfiltration of plants for high-level transient expression of recombinant proteins. J Vis Exp 77, doi:10.3791/50521
.
OpenUrl CrossRef
↵
1. Saur IM,
2. Conlan BF,
3. Rathjen JP
(2015) The N-terminal domain of the tomato immune protein Prf contains multiple homotypic and Pto kinase interaction sites. J Biol Chem 290(18):11258–11267
.
OpenUrl Abstract/FREE Full Text
↵
1. Smith JM,
2. Salamango DJ,
3. Leslie ME,
4. Collins CA,
5. Heese A
(2014) Sensitivity to Flg22 is modulated by ligand-induced degradation and de novo synthesis of the endogenous flagellin-receptor FLAGELLIN-SENSING2. Plant Physiol 164(1):440–454
.
OpenUrl Abstract/FREE Full Text
↵
1. Century KS, et al.
(1999) Short communication: Developmental control of Xa21-mediated disease resistance in rice. Plant J 20(2):231–236
.
OpenUrl CrossRef PubMed
↵
1. Cao Y, et al.
(2007) The expression pattern of a rice disease resistance gene xa3/xa26 is differentially regulated by the genetic backgrounds and developmental stages that influence its function. Genetics 177(1):523–533
.
OpenUrl Abstract/FREE Full Text
↵
1. Kadota Y, et al.
(2014) Direct regulation of the NADPH oxidase RBOHD by the PRR-associated kinase BIK1 during plant immunity. Mol Cell 54(1):43–55
.
OpenUrl CrossRef PubMed
↵
1. Caillaud MC, et al.
(2014) The plasmodesmal protein PDLP1 localises to haustoria-associated membranes during downy mildew infection and regulates callose deposition. PLoS Pathog 10(10):e1004496
.
OpenUrl CrossRef PubMed

Article Alerts

Email Article

Citation Tools

Request Permissions

Proceedings of the National Academy of Sciences: 118 (8)

Current Issue

Submit

[1] ↵
Cao Y, et al.
(2014) The kinase LYK5 is a major chitin receptor in Arabidopsis and forms a chitin-induced complex with related kinase CERK1. eLife 3, doi:10.7554/eLife.03766
.
OpenUrl Abstract/FREE Full Text

[2] Cao Y, et al.

[3] ↵
Chinchilla D,
Bauer Z,
Regenass M,
Boller T,
Felix G
(2006) The Arabidopsis receptor kinase FLS2 binds flg22 and determines the specificity of flagellin perception. Plant Cell 18(2):465–476
.
OpenUrl Abstract/FREE Full Text

[4] Chinchilla D,

[5] Bauer Z,

[6] Regenass M,

[7] Boller T,

[8] Felix G

[9] ↵
Gómez-Gómez L,
Boller T
(2000) FLS2: An LRR receptor-like kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis. Mol Cell 5(6):1003–1011
.
OpenUrl CrossRef PubMed

[10] Gómez-Gómez L,

[11] Boller T

[12] ↵
Sun Y, et al.
(2013) Structural basis for flg22-induced activation of the Arabidopsis FLS2-BAK1 immune complex. Science 342(6158):624–628
.
OpenUrl Abstract/FREE Full Text

[13] Sun Y, et al.

[14] ↵
Roux M, et al.
(2011) The Arabidopsis leucine-rich repeat receptor-like kinases BAK1/SERK3 and BKK1/SERK4 are required for innate immunity to hemibiotrophic and biotrophic pathogens. Plant Cell 23(6):2440–2455
.
OpenUrl Abstract/FREE Full Text

[15] Roux M, et al.

[16] ↵
Chinchilla D, et al.
(2007) A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defence. Nature 448(7152):497–500
.
OpenUrl CrossRef PubMed

[17] Chinchilla D, et al.

[18] ↵
Heese A, et al.
(2007) The receptor-like kinase SERK3/BAK1 is a central regulator of innate immunity in plants. Proc Natl Acad Sci USA 104(29):12217–12222
.
OpenUrl Abstract/FREE Full Text

[19] Heese A, et al.

[20] ↵
Schulze B, et al.
(2010) Rapid heteromerization and phosphorylation of ligand-activated plant transmembrane receptors and their associated kinase BAK1. J Biol Chem 285(13):9444–9451
.
OpenUrl Abstract/FREE Full Text

[21] Schulze B, et al.

[22] ↵
Gao M, et al.
(2009) Regulation of cell death and innate immunity by two receptor-like kinases in Arabidopsis. Cell Host Microbe 6(1):34–44
.
OpenUrl CrossRef PubMed

[23] Gao M, et al.

[24] ↵
Halter T, et al.
(2014) The leucine-rich repeat receptor kinase BIR2 is a negative regulator of BAK1 in plant immunity. Curr Biol 24(2):134–143
.
OpenUrl CrossRef PubMed

[25] Halter T, et al.

[26] ↵
Liebrand TW, et al.
(2013) Receptor-like kinase SOBIR1/EVR interacts with receptor-like proteins in plant immunity against fungal infection. Proc Natl Acad Sci USA 110(24):10010–10015
.
OpenUrl Abstract/FREE Full Text

[27] Liebrand TW, et al.

[28] ↵
Zhang L, et al.
(2014) Fungal endopolygalacturonases are recognized as microbe-associated molecular patterns by the Arabidopsis receptor-like protein RESPONSIVENESS TO BOTRYTIS POLYGALACTURONASES1. Plant Physiol 164(1):352–364
.
OpenUrl Abstract/FREE Full Text

[29] Zhang L, et al.

[30] ↵
Jehle AK,
Fürst U,
Lipschis M,
Albert M,
Felix G
(2013) Perception of the novel MAMP eMax from different Xanthomonas species requires the Arabidopsis receptor-like protein ReMAX and the receptor kinase SOBIR. Plant Signal Behav 8(12):e27408
.
OpenUrl CrossRef PubMed

[31] Jehle AK,

[32] Fürst U,

[33] Lipschis M,

[34] Albert M,

[35] Felix G

[36] ↵
Zhang W, et al.
(2013) Arabidopsis receptor-like protein30 and receptor-like kinase suppressor of BIR1-1/EVERSHED mediate innate immunity to necrotrophic fungi. Plant Cell 25(10):4227–4241
.
OpenUrl Abstract/FREE Full Text

[37] Zhang W, et al.

[38] ↵
Albert I, et al.
(2015) An RLP23–SOBIR1–BAK1 complex mediates NLP-triggered immunity. Nature Plants 1:15140
.
OpenUrl CrossRef

[39] Albert I, et al.

[40] ↵
Monaghan J,
Zipfel C
(2012) Plant pattern recognition receptor complexes at the plasma membrane. Curr Opin Plant Biol 15(4):349–357
.
OpenUrl CrossRef PubMed

[41] Monaghan J,

[42] Zipfel C

[43] ↵
Macho AP,
Zipfel C
(2014) Plant PRRs and the activation of innate immune signaling. Mol Cell 54(2):263–272
.
OpenUrl CrossRef PubMed

[44] Macho AP,

[45] Zipfel C

[46] ↵
Jones JD,
Dangl JL
(2006) The plant immune system. Nature 444(7117):323–329
.
OpenUrl CrossRef PubMed

[47] Jones JD,

[48] Dangl JL

[49] ↵
Felix G,
Duran JD,
Volko S,
Boller T
(1999) Plants have a sensitive perception system for the most conserved domain of bacterial flagellin. Plant J 18(3):265–276
.
OpenUrl CrossRef PubMed

[50] Felix G,

[51] Duran JD,

[52] Volko S,

[53] Boller T

[54] ↵
Boller T,
Felix G
(2009) A renaissance of elicitors: Perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors. Annu Rev Plant Biol 60:379–406
.
OpenUrl CrossRef PubMed

[55] Boller T,

[56] Felix G

[57] ↵
Zipfel C, et al.
(2006) Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacterium-mediated transformation. Cell 125(4):749–760
.
OpenUrl CrossRef PubMed

[58] Zipfel C, et al.

[59] ↵
Felix G,
Boller T
(2003) Molecular sensing of bacteria in plants. The highly conserved RNA-binding motif RNP-1 of bacterial cold shock proteins is recognized as an elicitor signal in tobacco. J Biol Chem 278(8):6201–6208
.
OpenUrl Abstract/FREE Full Text

[60] Felix G,

[61] Boller T

[62] ↵
Shamloul M,
Trusa J,
Mett V,
Yusibov V
(2014) Optimization and utilization of Agrobacterium-mediated transient protein production in Nicotiana. J Vis Exp doi:10.3791/51204
.
OpenUrl CrossRef

[63] Shamloul M,

[64] Trusa J,

[65] Mett V,

[66] Yusibov V

[67] ↵
Chaparro-Garcia A, et al.
(2011) The receptor-like kinase SERK3/BAK1 is required for basal resistance against the late blight pathogen phytophthora infestans in Nicotiana benthamiana. PLoS One 6(1):e16608
.
OpenUrl CrossRef PubMed

[68] Chaparro-Garcia A, et al.

[69] ↵
Schwessinger B, et al.
(2011) Phosphorylation-dependent differential regulation of plant growth, cell death, and innate immunity by the regulatory receptor-like kinase BAK1. PLoS Genet 7(4):e1002046
.
OpenUrl CrossRef PubMed

[70] Schwessinger B, et al.

[71] ↵
Dong Y,
Burch-Smith TM,
Liu Y,
Mamillapalli P,
Dinesh-Kumar SP
(2007) A ligation-independent cloning tobacco rattle virus vector for high-throughput virus-induced gene silencing identifies roles for NbMADS4-1 and -2 in floral development. Plant Physiol 145(4):1161–1170
.
OpenUrl Abstract/FREE Full Text

[72] Dong Y,

[73] Burch-Smith TM,

[74] Liu Y,

[75] Mamillapalli P,

[76] Dinesh-Kumar SP

[77] ↵
Segonzac C, et al.
(2011) Hierarchy and roles of pathogen-associated molecular pattern-induced responses in Nicotiana benthamiana. Plant Physiol 156(2):687–699
.
OpenUrl Abstract/FREE Full Text

[78] Segonzac C, et al.

[79] ↵
Gust AA,
Felix G
(2014) Receptor like proteins associate with SOBIR1-type of adaptors to form bimolecular receptor kinases. Curr Opin Plant Biol 21:104–111
.
OpenUrl CrossRef PubMed

[80] Gust AA,

[81] Felix G

[82] ↵
Asai T, et al.
(2002) MAP kinase signalling cascade in Arabidopsis innate immunity. Nature 415(6875):977–983
.
OpenUrl CrossRef PubMed

[83] Asai T, et al.

[84] ↵
Lu X, et al.
(2009) Uncoupling of sustained MAMP receptor signaling from early outputs in an Arabidopsis endoplasmic reticulum glucosidase II allele. Proc Natl Acad Sci USA 106(52):22522–22527
.
OpenUrl Abstract/FREE Full Text

[85] Lu X, et al.

[86] ↵
Shimizu R, et al.
(2003) The DeltafliD mutant of Pseudomonas syringae pv. tabaci, which secretes flagellin monomers, induces a strong hypersensitive reaction (HR) in non-host tomato cells. Mol Genet Genomics 269(1):21–30
.
OpenUrl PubMed

[87] Shimizu R, et al.

[88] ↵
Arnold DL,
Lovell HC,
Jackson RW,
Mansfield JW
(2011) Pseudomonas syringae pv. phaseolicola: From ‘has bean’ to supermodel. Mol Plant Pathol 12(7):617–627
.
OpenUrl CrossRef PubMed

[89] Arnold DL,

[90] Lovell HC,

[91] Jackson RW,

[92] Mansfield JW

[93] ↵
Leuzinger K, et al.
(2013) Efficient agroinfiltration of plants for high-level transient expression of recombinant proteins. J Vis Exp 77, doi:10.3791/50521
.
OpenUrl CrossRef

[94] Leuzinger K, et al.

[95] ↵
Saur IM,
Conlan BF,
Rathjen JP
(2015) The N-terminal domain of the tomato immune protein Prf contains multiple homotypic and Pto kinase interaction sites. J Biol Chem 290(18):11258–11267
.
OpenUrl Abstract/FREE Full Text

[96] Saur IM,

[97] Conlan BF,

[98] Rathjen JP

[99] ↵
Smith JM,
Salamango DJ,
Leslie ME,
Collins CA,
Heese A
(2014) Sensitivity to Flg22 is modulated by ligand-induced degradation and de novo synthesis of the endogenous flagellin-receptor FLAGELLIN-SENSING2. Plant Physiol 164(1):440–454
.
OpenUrl Abstract/FREE Full Text

[100] Smith JM,

[101] Salamango DJ,

[102] Leslie ME,

[103] Collins CA,

[104] Heese A

[105] ↵
Century KS, et al.
(1999) Short communication: Developmental control of Xa21-mediated disease resistance in rice. Plant J 20(2):231–236
.
OpenUrl CrossRef PubMed

[106] Century KS, et al.

[107] ↵
Cao Y, et al.
(2007) The expression pattern of a rice disease resistance gene xa3/xa26 is differentially regulated by the genetic backgrounds and developmental stages that influence its function. Genetics 177(1):523–533
.
OpenUrl Abstract/FREE Full Text

[108] Cao Y, et al.

[109] ↵
Kadota Y, et al.
(2014) Direct regulation of the NADPH oxidase RBOHD by the PRR-associated kinase BIK1 during plant immunity. Mol Cell 54(1):43–55
.
OpenUrl CrossRef PubMed

[110] Kadota Y, et al.

[111] ↵
Caillaud MC, et al.
(2014) The plasmodesmal protein PDLP1 localises to haustoria-associated membranes during downy mildew infection and regulates callose deposition. PLoS Pathog 10(10):e1004496
.
OpenUrl CrossRef PubMed

[112] Caillaud MC, et al.

Main menu

User menu

Search

New Research In

NbCSPR underlies age-dependent immune responses to bacterial cold shock protein in Nicotiana benthamiana

Significance

Abstract

Results

csp22 Responses Are Age-Dependent in N. benthamiana.

Identification of CSPR from N. benthamiana Using NbBAK1 as Bait.

NbCSPR Forms a Complex with csp22 and Is Required for csp22 Responses.

NbCSPR Does Not Require NbSOBIR1 for csp22 Responses.

NbCSPR Confers Responsiveness to csp22 in Transgenic A. thaliana Plants Dependent on AtBAK1/AtBKK1.

NbCSPR Confers Age-Related Resistance to Bacterial Pathogens and Restricts Agrobacterium-Mediated Transformation of N. benthamiana in Flowering Plants.

Potentiation of csp22 Responses by flg22 Pretreatment.

Discussion

Materials and Methods

Acknowledgments

Footnotes

References

Citation Manager Formats

You May Also be Interested in

Similar Articles

Articles

PNAS Portals

Information

Main menu

User menu

Search

New Research In

Physical Sciences

Featured Portals

Articles by Topic

Social Sciences

Featured Portals

Articles by Topic

Biological Sciences

Featured Portals

Articles by Topic

NbCSPR underlies age-dependent immune responses to bacterial cold shock protein in Nicotiana benthamiana

Significance

Abstract

Results

csp22 Responses Are Age-Dependent in N. benthamiana.

Identification of CSPR from N. benthamiana Using NbBAK1 as Bait.

NbCSPR Forms a Complex with csp22 and Is Required for csp22 Responses.

NbCSPR Does Not Require NbSOBIR1 for csp22 Responses.

NbCSPR Confers Responsiveness to csp22 in Transgenic A. thaliana Plants Dependent on AtBAK1/AtBKK1.

NbCSPR Confers Age-Related Resistance to Bacterial Pathogens and Restricts Agrobacterium-Mediated Transformation of N. benthamiana in Flowering Plants.

Potentiation of csp22 Responses by flg22 Pretreatment.

Discussion

Materials and Methods

Acknowledgments

Footnotes

References

Citation Manager Formats

Sign up for Article Alerts

Jump to section

You May Also be Interested in

Similar Articles

Articles

PNAS Portals

Information