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Both plants and animals utilize nucleotide-binding leucine-rich repeat immune receptors (NLRs) to perceive the presence of pathogen-derived molecules and induce immune responses. NLR genes are far more abundant and diverse in vascular plants than in animals. Truncated NLRs, which lack one or more of the canonical domains, are also commonly encoded in plant genomes. However, little is known about their functions, especially the N-terminally truncated ones. Here, we show that the Arabidopsis thaliana N-terminally truncated helper NLR (hNLR) gene N REQUIREMENT GENE1 (NRG1C) is highly induced upon pathogen infection and in autoimmune mutants. The immune response and cell death conferred by some Toll/interleukin-1 receptor-type NLRs (TNLs) were compromised in Arabidopsis NRG1C overexpression lines. Detailed genetic analysis revealed that NRG1C antagonizes the immunity mediated by its full-length neighbors NRG1A and NRG1B. Biochemical tests suggested that NRG1C might interfere with the EDS1–SAG101 complex, which functions in immunity signaling together with NRG1A/1B. Interestingly, Brassicaceae NRG1Cs are functionally exchangeable and that the Nicotiana benthamiana N-terminally truncated hNLR NRG2 also antagonizes NRG1 activity. Together, our study uncovers an unexpected negative role of N-terminally truncated hNLRs in immunity in different plant species.

TIR signaling promotes the interactions between lipase-like proteins EDS1/PAD4 and ADR1-L1 immune receptor, and oligomerization of ADR1-L1.

Salicylic acid (SA) is an essential plant defense hormone that promotes immunity against biotrophic and semibiotrophic pathogens. It plays crucial roles in basal defense and the amplification of local immune responses, as well as the establishment of systemic acquired resistance. During the past three decades, immense progress has been made in understanding the biosynthesis, homeostasis, perception, and functions of SA. This review summarizes the current knowledge regarding SA in plant immunity and other biological processes. We highlight recent breakthroughs that substantially advanced our understanding of how SA is biosynthesized from isochorismate, how it is perceived, and how SA receptors regulate different aspects of plant immunity. Some key questions in SA biosynthesis and signaling, such as how SA is produced via another intermediate, benzoic acid, and how SA affects the activities of its receptors in the transcriptional regulation of defense genes, remain to be addressed.

The plant defense hormone salicylic acid (SA) is perceived by two classes of receptors, NPR1 and NPR3/NPR4. They function in two parallel pathways to regulate SA-induced defense gene expression. To better understand the roles of the SA receptors in plant defense, we systematically analyzed their contributions to different aspects of Arabidopsis (Arabidopsis thaliana) plant immunity using the SA-insensitive npr1-1 npr4-4D double mutant. We found that perception of SA by NPR1 and NPR4 is required for activation of N-hydroxypipecolic acid biosynthesis, which is essential for inducing systemic acquired resistance. In addition, both pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) are severely compromised in the npr1-1 npr4-4D double mutant. Interestingly, the PTI and ETI attenuation in npr1-1 npr4-4D is more dramatic compared with the SA-induction deficient2-1 (sid2-1) mutant, suggesting that the perception of residual levels of SA in sid2-1 also contributes to immunity. Furthermore, NPR1 and NPR4 are involved in positive feedback amplification of SA biosynthesis and regulation of SA homeostasis through modifications including 5-hydroxylation and glycosylation. Thus, the SA receptors NPR1 and NPR4 play broad roles in plant immunity.

Disruption of the MEKK1-MKK1/MKK2-MPK4 kinase cascade leads to activation of immunity mediated by the nucleotide-binding leucine-rich repeat (NLR) immune receptor SUMM2, which monitors the phosphorylation status of CRCK3. Here we report that two receptor-like kinases (RLKs), MDS1, and MDS2, function redundantly to promote SUMM2-mediated immunity. Activation of SUMM2-mediated immunity is dependent on MDS1, and to a less extent on MDS2. MDS1 associates with CRCK3 in planta and can phosphorylate CRCK3 in vitro, suggesting that it may target CRCK3 to positively regulate SUMM2-mediated signaling. Our finding highlights a new defense mechanism where RLKs promote NLR-mediated immunity.

Salicylic acid (SA) has emerged as a key plant defense hormone with critical roles in different aspects of plant immunity. Analysis of Arabidopsis mutants revealed complex regulation of pathogen-induced SA biosynthesis. Studies on SA-insensitive mutants led to the identification of the SA receptors and how SA regulates defense gene expression. Consistent with its critical roles in plant immunity, SA is required for the assembly of a normal root microbiome and various pathogen effectors have evolved to target components of SA biosynthesis or signaling. This review discusses recent advances in SA biology, focusing in particular on the regulation of SA biosynthesis and SA perception.

Arabidopsis SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1 (SARD1) and CALMODULIN-BINDING PROTEIN 60g (CBP60g) are two master transcription factors that regulate many defense-related genes in plant immunity. They are required for immunity downstream of the receptor-like protein SUPPRESSOR OF NPR1-1, CONSTITUTIVE 2 (SNC2). Constitutive defense responses in the gain-of-function autoimmune snc2-1D mutant are modestly affected in either sard1 or cbp60g single mutants but completely suppressed in the sard1 cbp60g double mutant. Here we report that CBP60b, another member of the CBP60 family, also functions as a positive regulator of SNC2-mediated immunity. Loss-of-function mutations of CBP60b suppress the constitutive expression of SARD1 and enhanced disease resistance in cbp60g-1 snc2-1D, whereas overexpression of CBP60b leads to elevated SARD1 expression and constitutive defense responses. In addition, transient expression of CBP60b in Nicotiana benthamiana activates the expression of the pSARD1::luciferase reporter gene. Chromatin immunoprecipitation assays further showed that CBP60b is recruited to the promoter region of SARD1, suggesting that it directly regulates SARD1 expression. Interestingly, knocking out CBP60b in the wild-type background leads to ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1)-dependent autoimmunity, suggesting that CBP60b is required for the expression of a guardee/decoy or a negative regulator of immunity mediated by receptors carrying an N-terminal Toll-interleukin-1 receptor-like domain., Arabidopsis SARD1 serves as a master transcription factor in plant immunity. This study shows that CBP60b positively regulates SARD1 expression, and Toll/interleukin-1 receptor signaling is activated when CBP60b is inactivated.

Arabidopsis (Arabidopsis thaliana) MAP KINASE (MPK) proteins can function in multiple MAP kinase cascades and physiological processes. For instance, MPK4 functions in regulating development as well as in plant defense by participating in two independent MAP kinase cascades: the MEKK1-MKK1/MKK2-MPK4 cascade promotes basal resistance against pathogens and is guarded by the NB-LRR protein SUMM2, whereas the ANPs-MKK6-MPK4 cascade plays an essential role in cytokinesis. Here, we report a novel role for MKK6 in regulating plant immune responses. We found that MKK6 functions similarly to MKK1/MKK2 and works together with MEKK1 and MPK4 to prevent autoactivation of SUMM2-mediated defense responses. Interestingly, loss of MKK6 or ANP2/ANP3 results in constitutive activation of plant defense responses. The autoimmune phenotypes of mkk6 and anp2 anp3 mutant plants can be largely suppressed by a constitutively active mpk4 mutant. Further analysis showed that the constitutive defense response in anp2 anp3 is dependent on the defense regulators PAD4 and EDS1, but not on SUMM2, suggesting that the ANP2/ANP3-MKK6-MPK4 cascade may be guarded by a TIR-NB-LRR protein. Our study shows that MKK6 has multiple functions in plant defense responses in addition to cytokinesis.

In plants and animals, nucleotide-binding leucine-rich repeat (NLR) proteins serve as intracellular immune receptors. Defence signalling by NLRs often requires the formation of NLR heteropairs. Our knowledge of the molecular mechanism regulating this process is limited. In a reverse genetic screen to identify the partner of the Arabidopsis typical NLR, SUPRESSOR OF NPR1, CONSTITUTIVE 1 (SNC1), we discovered three NLRs that are redundantly required for SNC1-mediated defence, which were named SIDEKICK SNC1 1 (SIKIC1), SIKIC2 and SIKIC3. Immunoprecipitation–mass spectrometry analyses revealed that SIKIC2 physically associates with SNC1. We also uncovered that the protein level of SIKIC2 is under the control of two previously uncharacterized redundant E3 ubiquitin ligases MUSE1 and MUSE2. As SNC1 accumulation has previously been shown to be regulated by the E3 ubiquitin ligase SCFCPR1, this report provides evidence that the homeostasis of individual components of partnered typical NLRs is subjected to differential regulation via ubiquitin-mediated protein degradation. SNC1 is an intracellular NLR immune receptor controlled by the E3 ubiquitin ligase SCFCPR1. A reverse genetic screen in Arabidopsis identified three clustered NLR partners of SNC1 named SIKICs, which are themselves regulated by novel E3 ubiquitin ligases MUSE1 and MUSE2.

Plant immune responses are mainly activated by two types of receptor. Pattern recognition receptors localized on the plasma membrane perceive extracellular microbial features, and nucleotide-binding leucine-rich repeat receptors (NLRs) recognize intracellular effector proteins from pathogens

In both plants and animals, nucleotide-binding leucine-rich repeat (NLR) immune receptors perceive pathogen-derived molecules to trigger immunity. Global NLR homeostasis must be tightly controlled to ensure sufficient and timely immune output while avoiding aberrant activation, the mechanisms of which are largely unclear. In a previous reverse genetic screen, we identified two novel E3 ligases, SNIPER1 and its homolog SNIPER2, both of which broadly control the levels of NLR immune receptors in Arabidopsis. Protein levels of sensor NLRs (sNLRs) are inversely correlated with SNIPER1 amount and the interactions between SNIPER1 and sNLRs seem to be through the common nucleotide-binding (NB) domains of sNLRs. In support, SNIPER1 can ubiquitinate the NB domains of multiple sNLRs in vitro. Our study thus reveals a novel process of global turnover of sNLRs by two master E3 ligases for immediate attenuation of immune output to effectively avoid autoimmunity. Such unique mechanism can be utilized in the future for engineering broad-spectrum resistance in crops to fend off pathogens that damage our food supply.

The Arabidopsis MEKK1-MKK1/MKK2-MPK4 kinase cascade is monitored by the nucleotide-binding leucine-rich-repeat immune receptor SUMM2. Disruption of this kinase cascade leads to activation of SUMM2-mediated immune responses. MEKK2, a close paralog of MEKK1, is required for defense responses mediated by SUMM2, the molecular mechanism of which is unclear. In this study, we showed that MEKK2 serves as a negative regulator of MPK4. It binds to MPK4 to directly inhibit its phosphorylation by upstream MKKs. Activation of SUMM2-mediated defense responses induces the expression of MEKK2, which in turn blocks MPK4 phosphorylation to further amplify immune responses mediated by SUMM2. Intriguingly, MEKK2 locates in a tandem repeat consisting of MEKK1, MEKK2 and MEKK3, which was generated from a recent gene duplication event, suggesting that MEKK2 evolved from a MAPKKK to become a negative regulator of MAP kinases.

Salicylic acid (SA) has long been known to be essential for basal defense and systemic acquired resistance (SAR). N-Hydroxypipecolic acid (NHP), a recently discovered plant metabolite, also plays a key role in SAR and to a lesser extent in basal resistance. Following pathogen infection, levels of both compounds are dramatically increased. Analysis of SA- or SAR-deficient mutants has uncovered how SA and NHP are biosynthesized. The completion of the SA and NHP biosynthetic pathways in Arabidopsis allowed better understanding of how they are regulated. In this review, we discuss recent progress on SA and NHP biosynthesis and their regulation in plant immunity.

Salicylic acid (SA) and pipecolic acid (Pip) play important roles in plant immunity. Here we analyzed the roles of transcription factors TGACG-BINDING FACTOR 1 (TGA1) and TGA4 in regulating SA and Pip biosynthesis in Arabidopsis thaliana. We quantified the expression levels of SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1 (SARD1) and CALMODULIN-BINDING PROTEIN 60g (CBP60g), which encode two master transcription factors of plant immunity, and the accumulation of SA and Pip in tga1-1 tga4-1 mutant plants. We tested whether SARD1 and CBP60g are direct targets of TGA1 by chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR). In addition to promoting pathogen-induced SA biosynthesis, we found that SARD1 and CBP60g also positively regulated Pip biosynthesis by targeting genes encoding key biosynthesis enzymes of Pip. TGA1/TGA4 were required for full induction of SARD1 and CBP60g in plant defense. ChIP-PCR analysis showed that SARD1 was a direct target of TGA1. In tga1-1 tga4-1 mutant plants, the expression levels of SARD1 and CBP60g along with SA and Pip accumulation following pathogen infection were dramatically reduced compared with those in wild-type plants. Consistent with reduced expression of SARD1 and CBP60g, pathogen-associated molecular pattern (PAMP)-induced pathogen resistance and systemic acquired resistance were compromised in tga1-1 tga4-1. Our study showed that TGA1 and TGA4 regulate Pip and SA biosynthesis by modulating the expression of SARD1 and CBP60g.

SARD1 and CBP60g are two master regulators in plant immunity. They are required for the constitutive defense responses in the Arabidopsis snc2-1D mutant, which carries a gain-of-function mutation in a receptor-like protein. Here we report that WRKY54 and WRKY70 are required for activation of SARD1 and CBP60g expression and defense responses in snc2-1D. In addition, the induction of SARD1 and CBP60g by the bacterial pathogen Pseudomonas syringae pv. maculicola is significantly reduced in sid2 wrky54 wrky70 triple mutants compared to the sid2 single mutants, suggesting that WRKY54 and WRKY70 positively regulate the SID2-independent expression of SARD1 and CBP60g during pathogen infection. Our study revealed WRKY54 and WRKY70 as positive regulators of SARD1 and CBP60g expression in plant defense.

Salicylic acid (SA) is a plant hormone critical for pathogen resistance1–3. The NPR proteins have been identified as SA receptors4–10, although how they perceive SA and coordinate hormonal signalling remains elusive. Here we report the mapping of the SA-binding core (SBC) of Arabidopsis thaliana NPR4 and its ligand-bound crystal structure at 2.3 Å resolution. The NPR4 SBC domain, refolded with SA, adopts a unique α-helical fold, which completely buries SA in its hydrophobic core. The lack of ligand entry pathway suggests that SA binding involves a major conformational remodelling of NPR4-SBC, which is validated by HDX-MS analysis of the full-length protein and SA-induced disruption of NPR1-NPR4 interactions. We show that, despite sharing nearly identical hormone-binding residues, NPR1 displays a minimal SA-binding activity compared to NPR4. We further identify two SBC surface residues, whose mutations can alter NPR4’s SA-binding ability and interaction with NPR1. Moreover, we demonstrate that expressing a SA-hypersensitive NPR4 variant could enhance SA-mediated basal immunity without compromising effector-triggered immunity because of its intact ability to re-associate with NPR1 at high SA levels. By unveiling the structural mechanisms of SA perception, our work paves the way for future investigation on the specific roles of the NPR proteins in SA signalling and their potential for engineering plant immunity.

Systemic acquired resistance (SAR) is an immune response induced in the distal parts of plants following defense activation in local tissue. Pipecolic acid (Pip) accumulation orchestrates SAR and local resistance responses. Here, we report the identification and characterization of SAR-DEFICIENT4 ( SARD4 ), which encodes a critical enzyme for Pip biosynthesis in Arabidopsis thaliana . Loss of function of SARD4 leads to reduced Pip levels and accumulation of a Pip precursor, Δ 1 -piperideine-2-carboxylic acid (P2C). In Escherichia coli , expression of the aminotransferase ALD1 leads to production of P2C and addition of SARD4 results in Pip production, suggesting that a Pip biosynthesis pathway can be reconstituted in bacteria by coexpression of ALD1 and SARD4. In vitro experiments showed that ALD1 can use l-lysine as a substrate to produce P2C and P2C is converted to Pip by SARD4. Analysis of sard4 mutant plants showed that SARD4 is required for SAR as well as enhanced pathogen resistance conditioned by overexpression of the SAR regulator FLAVIN-DEPENDENT MONOOXYGENASE1. Compared with the wild type, pathogen-induced Pip accumulation is only modestly reduced in the local tissue of sard4 mutant plants, but it is below detection in distal leaves, suggesting that Pip is synthesized in systemic tissue by SARD4-mediated reduction of P2C and biosynthesis of Pip in systemic tissue contributes to SAR establishment.

Two signal molecules, salicylic acid (SA) and N-hydroxypipecolic acid (NHP), play critical roles in plant immunity. The biosynthetic genes of both compounds are positively regulated by master immune-regulating transcription factors SARD1 and CBP60g. However, the relationship between the SA and NHP pathways is unclear. CALMODULIN-BINDING TRANSCRIPTION FACTOR 1 (CAMTA1), CAMTA2, and CAMTA3 are known redundant negative regulators of plant immunity, but the underlying mechanism also remains largely unknown. In this study, through chromatin immunoprecipitation and electrophoretic mobility shift assays, we uncovered that CBP60g is a direct target of CAMTA3, which also negatively regulates the expression of SARD1, presumably via an indirect effect. The autoimmunity of camta3-1 is suppressed by sard1 cbp60g double mutant as well as ald1 and fmo1, two single mutants defective in NHP biosynthesis. Interestingly, a suppressor screen conducted in the camta1/2/3 triple mutant background yielded various mutants blocking biosynthesis or signaling of either SA or NHP, leading to nearly complete suppression of the extreme autoimmunity of camta1/2/3, suggesting that the SA and NHP pathways can mutually amplify each other. Together, these results reveal that CAMTAs repress the biosynthesis of SA and NHP by modulating the expression of SARD1 and CBP60g, and that the SA and NHP pathways are coordinated to optimize plant immune response.

Spontaneous degradation in hormone synthesis The phytohormone salicylic acid (SA) helps plants respond to biological and physical stresses. Rekhter et al. identified the biosynthetic pathway that produces SA in response to pathogens. A precursor, isochorismic acid, is formed in the chloroplast and then exported to the cytosol. There, enzymatically produced isochorismate-9-glutamate spontaneously decomposes to release SA plus a by-product. The results clarify key steps in the mechanisms involved in synthesizing this important regulator of plant immunity. Science , this issue p. 498

Arabidopsis mitogen-activated protein (MAP) kinase kinase 6 (MKK6) has previously been shown to play important roles in plant immunity and cytokinesis. Here we report that it is also involved in the negative regulation of anthocyanin biosynthesis in Arabidopsis. Elevated anthocyanin levels are observed in mkk6 knock out mutant plants. RNA-sequencing analysis revealed that the expression level of MYB75 (Myb transcription factor 75), which encodes a key positive regulator of anthocyanin biosynthesis, is up-regulated in mkk6 mutant plants, suggesting that increased MYB75 expression contributes to the elevated anthocyanin levels. In addition, suppression of high sucrose-induced anthocyanin production by pathogen associated molecular pattern-triggered immunity is also dependent on MKK6, further supporting that MKK6 is a key negative regulator of anthocyanin biosynthesis in Arabidopsis.

Agrobacterium-mediated transformation methods have allowed the stable introduction of target genes into the nuclear genomes of recipient plants. Among them, the floral dip approach represents the simplest due to its straightforwardness and high transformation efficiency. In a standard floral dip protocol that most researchers follow, Agrobacterium cells are grown to stationary phase (OD 600≈2.0) in large cultures and resuspended in inoculation medium to OD 600≥0.8. Here, we tested the effects of low Agrobacterium inoculum on transformation rate. Our data revealed that the floral dip method still guarantees a relatively high transformation rate in the Arabidopsis thaliana Col-0 ecotype even with very low Agrobacterium inoculum (OD 600=0.002). Our finding thus simplifies the floral dipping protocol further, which allows transformation with small bacterial culture and enables high-throughput transformation of large numbers of constructs in parallel.

The Arabidopsis receptor-like kinase (RLK) BIR1 (BAK1-INTERACTING RECEPTOR-LIKE KINASE 1) functions as a negative regulator of plant immunity. Previous work showed that loss-of-function of BIR1 leads to constitutive activation of cell death and defense responses. These autoimmune phenotypes are partially dependent on another RLK, SOBIR1. In order to identify additional components involved in the BIR1-regulated plant defense signaling pathway, a suppressor screen was carried out in the bir1-1 pad4-1 mutant background. Mutations in the suppressor mutants were identified by genetic mapping and re-sequencing of the mutant genomes. A number of suppressor mutants were found to carry mutations in an additional RLK, BAK1, indicating that BAK1 is required for activation of cell death and defense responses in bir1-1. Co-immunoprecipitation analysis revealed that BAK1 and SOBIR1 associate with each other in planta when the function of BIR1 is compromised. Although BAK1 was previously characterized as a negative regulator of cell death, our study highlights a novel role of BAK1 in promoting cell death and defense responses in conjunction with SOBIR1.

TGACG-BINDING FACTORs (TGAs) control the developmental or defense-related processes. In Arabidopsis thaliana, the functions of at least TGA2 and PERIANTHIA (PAN) can be repressed by interacting with CC-type glutaredoxins, which have the potential to control the redox state of target proteins. As TGA1 can be redox modulated in planta, we analyzed whether some of the 21 CC-type glutaredoxins (ROXYs) encoded in the Arabidopsis genome can influence TGA1 activity in planta and whether the redox active cysteines of TGA1 are functionally important. We show that the tga1 tga4 mutant and plants ectopically expressing ROXY8 or ROXY9 are impaired in hyponastic growth. As expression of ROXY8 and ROXY9 is activated upon transfer of plants from hyponasty-inducing low light to normal light, they might interfere with the growth-promoting function of TGA1/TGA4 to facilitate reversal of hyponastic growth. The redox-sensitive cysteines of TGA1 are not required for induction or reversal of hyponastic growth. TGA1 and TGA4 interact with ROXYs 8, 9, 18, and 19/GRX480, but ectopically expressed ROXY18 and ROXY19/GRX480 do not interfere with hyponastic growth. Our results therefore demonstrate functional specificities of individual ROXYs for distinct TGAs despite promiscuous protein-protein interactions and point to different repression mechanisms, depending on the TGA/ROXY combination.

Mitogen‐activated protein kinase (MAPK) signaling plays important roles in diverse biological processes. In Arabidopsis, MPK3/MPK6, MKK4/MKK5, and the MAPKKK YODA (YDA) form a MAPK pathway that negatively regulates stomatal development. Brassinosteroid (BR) stimulates this pathway to inhibit stomata production. In addition, MPK3/MPK6 and MKK4/MKK5 also serve as critical signaling components in plant immunity. Here, we report that MAPKKK3/MAPKKK5 form a kinase cascade with MKK4/MKK5 and MPK3/MPK6 to transduce defense signals downstream of multiple plant receptor kinases. Loss of MAPKKK3/MAPKKK5 leads to reduced activation of MPK3/MPK6 in response to different pathogen‐associated molecular patterns (PAMPs) and increased susceptibility to pathogens. Surprisingly, developmental defects caused by silencing of YDA are suppressed in the mapkkk3 mapkkk5 double mutant. On the other hand, loss of YDA or blocking BR signaling leads to increased PAMP‐induced activation of MPK3/MPK6. These results reveal antagonistic interactions between a developmental MAPK pathway and an immune signaling MAPK pathway.

Salicylic acid (SA) is a key signaling molecule in plant immunity. Two types of SA receptors, NPR1 and NPR3/NPR4, were reported to be involved in the perception of SA in Arabidopsis. SA is also synthesized in the non-vascular moss Physcomitrella patens following pathogen infection. Sequence analysis revealed that there is only one NPR1/NPR3/NPR4-like protein in P. patens. This agrees with the phylogenetic study that showed the divergence of NPR1 and NPR3/NPR4 from the same ancestor during the evolution of higher plants. Intriguingly, expression of the P. patens NPR1/NPR3/NPR4-like gene in Arabidopsis does not complement the constitutive defense phenotype of the npr3 npr4 double mutant, but can partially rescue the mutant phenotypes of npr1-1, suggesting that it functions as an NPR1-like positive regulator of SA-mediated immunity and P. patens does not have an SA receptor functioning similarly as NPR3/NPR4. Future characterization of the P. patens NPR1-like protein and analysis of its functions through knockout and biochemical approaches will help us better understand how SA is perceived and what its functions are in P. patens.

BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) plays critical roles in plant developmental and immune signaling pathways. BAK1 and a large number of leucine-rich repeat receptor-like kinases (LRR-RLKs) harbor a mysterious carboxyl-terminal tail (CT) beyond their kinase domain. In this study we analyzed the biological significance of this CT region using a unique bak1 mutant allele which causes deletion of the CT region. We showed that BAK1 CT promotes its kinase activity and is required for pathogen-associated molecular pattern (PAMP)-triggered immunity, but it is dispensable for brassinosteroid responses and BAK1/BKK1-inhibited cell death signaling. Therefore the BAK1 C-terminal tail is differentially required for its functions in development and immunity.

SUMMARY Two conserved splicing factors, SUA and RSN2, were identified from a suppressor screen of snc4-1D. Both are required for alternative splicing of receptor-like kinase (RLK)-encoding SNC4 and CERK1 and their functions, suggesting that pre-mRNA splicing plays important roles in the regulation of plant immunity mediated by the RLKs SNC4 and CERK1., Plant immune receptors belonging to the receptor-like kinase (RLK) family play important roles in the recognition of microbial pathogens and activation of downstream defense responses. The Arabidopsis mutant snc4-1D contains a gain-of-function mutation in the RLK SNC4 (SUPPRESSOR OF NPR1-1, CONSTITUTIVE4), which leads to constitutive activation of defense responses. Analysis of suppressor mutants of snc4-1D identified two conserved splicing factors, SUA (SUPPRESSOR OF ABI3-5) and RSN2 (REQUIRED FOR SNC4-1D 2), that are required for the constitutive defense responses in snc4-1D. In sua and rsn2 mutants, SNC4 splicing is altered and the amount of SNC4 transcripts is reduced. Further analysis showed that SUA and RSN2 are also required for the proper splicing of CERK1 (CHITIN ELICITOR RECEPTOR KINASE1), which encodes another RLK that functions as a receptor for chitin. In sua and rsn2 mutants, induction of reactive oxygen species by chitin is reduced and the non-pathogenic bacteria Pseudomonas syringae pv. tomato DC3000hrcC grows to higher titers than in wild-type plants. Our study suggests that pre-mRNA splicing plays important roles in the regulation of plant immunity mediated by the RLKs SNC4 and CERK1.

In plants, mitogen-activated protein kinase (MAPK) cascades are involved in regulating many biological processes including immunity. They relay signals from membrane-residing immune receptors to downstream components for defense activation. Arabidopsis MPK3/6 and MPK4 are activated in two parallel MAPK cascades during PAMP-triggered immunity. MPK3/6 have been implicated in the activation of various immune responses and their inactivation leads to compromised defense against pathogens. On the other hand, the MEKK1-MKK1/2-MPK4 cascade plays critical roles in basal resistance. Disruption of this MAPK cascade results in constitutive defense responses mediated by the NB-LRR protein SUMM2. Interestingly, SUMM2 guards the MEKK1-MKK1/2-MPK4 cascade activity indirectly through monitoring the phosphorylation status of CRCK3, which is a substrate of MPK4. From the pathogens’ side, a number of effectors are shown to target various components of MAPK cascades in plants. Inactivation of MPK4 by the Pseudomonas effector HopAI1 triggers SUMM2-mediated immunity. Together, these findings suggest intricate interplays between PAMP-triggered immunity and effector-triggered immunity via MAPK signaling.

Plants lack the adaptive immune system possessed by mammals. Instead they rely on innate immunity to defend against pathogen attacks. Genomes of higher plants encode a large number of plant immune receptors belonging to different protein families, which are involved in the detection of pathogens and activation of downstream defense pathways. Plant immunity is tightly controlled to avoid activation of defense responses in the absence of pathogens, as failure to do so can lead to autoimmunity that compromises plant growth and development. Many autoimmune mutants have been reported, most of which are associated with dwarfism and often spontaneous cell death. In this review, we summarize previously reported Arabidopsis autoimmune mutants, categorizing them based on their functional groups. We also discuss how their obvious morphological phenotypes make them ideal tools for epistatic analysis and suppressor screens, and summarize genetic screens that have been carried out in various autoimmune mutant backgrounds.

In fungi and metazoans, extracellular signals are often perceived by G-protein-coupled receptors (GPCRs) and transduced through heterotrimeric G-protein complexes to downstream targets. Plant heterotrimeric G proteins are also involved in diverse biological processes, but little is known about their upstream receptors. Moreover, the presence of bona fide GPCRs in plants is yet to be established. In Arabidopsis (Arabidopsis thaliana), heterotrimeric G protein consists of one Gα subunit (G PROTEIN α-SUBUNIT1), one Gβ subunit (ARABIDOPSIS G PROTEIN β-SUBUNIT1 [AGB1]), and three Gγs subunits (ARABIDOPSIS G PROTEIN γ-SUBUNIT1 [AGG1], AGG2, and AGG3). We identified AGB1 from a suppressor screen of BAK1-interacting receptor-like kinase1-1 (bir1-1), a mutant that activates cell death and defense responses mediated by the receptor-like kinase (RLK) SUPPRESSOR OF BIR1-1. Mutations in AGB1 suppress the cell death and defense responses in bir1-1 and transgenic plants overexpressing SUPPRESSOR OF BIR1-1. In addition, agb1 mutant plants were severely compromised in immunity mediated by three other RLKs, FLAGELLIN-SENSITIVE2 (FLS2), Elongation Factor-TU RECEPTOR (EFR), and CHITIN ELICITOR RECEPTOR KINASE1 (CERK1), respectively. By contrast, G PROTEIN α-SUBUNIT1 is not required for either cell death in bir1-1 or pathogen-associated molecular pattern-triggered immunity mediated by FLS2, EFR, and CERK1. Further analysis of agg1 and agg2 mutant plants indicates that AGG1 and AGG2 are also required for pathogen-associated molecular pattern-triggered immune responses mediated by FLS2, EFR, and CERK1, as well as cell death and defense responses in bir1-1. We hypothesize that the Arabidopsis heterotrimeric G proteins function as a converging point of plant defense signaling by mediating responses initiated by multiple RLKs, which may fulfill equivalent roles to GPCRs in fungi and animals.

Transcriptional reprogramming during induction of salicylic acid (SA)-mediated defenses is regulated primarily by NPR1 (NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1), likely through interactions with TGA bZIP transcription factors. To ascertain the contributions of clade I TGA factors (TGA1 and TGA4) to defense responses, a tga1-1 tga4-1 double mutant was constructed and challenged with Pseudomonas syringae and Hyaloperonospora arabidopsidis. Although the mutant displayed enhanced susceptibility to virulent P. syringae, it was not compromised in systemic acquired resistance against this pathogen or resistance against avirulent H. arabidopsidis. Microarray analysis of nonelicited and SA-treated plants indicated that clade I TGA factors regulate fewer genes than NPR1. Approximately half of TGA-dependent genes were regulated by NPR1 but, in all cases, the direction of change was opposite in the two mutants. In support of the microarray data, the NPR1-independent disease resistance observed in the autoimmune resistance (R) gene mutant snc1 is partly compromised by tga1-1 tga4-1 mutations, and a triple mutant of clade I TGA factors with npr1-1 is more susceptible than either parent. These results suggest that clade I TGA factors are required for resistance against virulent pathogens and avirulent pathogens mediated by at least some R gene specificities, acting substantially through NPR1-independent pathways.

The Arabidopsis immune receptor FLS2 perceives bacterial flagellin epitope flg22 to activate defenses through the central cytoplasmic kinase BIK1. The heterotrimeric G proteins composed of the non-canonical Gα protein XLG2, the Gβ protein AGB1, and the Gγ proteins AGG1 and AGG2 are required for FLS2-mediated immune responses through an unknown mechanism. Here we show that in the pre-activation state, XLG2 directly interacts with FLS2 and BIK1, and it functions together with AGB1 and AGG1/2 to attenuate proteasome-mediated degradation of BIK1, allowing optimum immune activation. Following the activation by flg22, XLG2 dissociates from AGB1 and is phosphorylated by BIK1 in the N terminus. The phosphorylated XLG2 enhances the production of reactive oxygen species (ROS) likely by modulating the NADPH oxidase RbohD. The study demonstrates that the G proteins are directly coupled to the FLS2 receptor complex and regulate immune signaling through both pre-activation and post-activation mechanisms. DOI: http://dx.doi.org/10.7554/eLife.13568.001, eLife digest Living cells need to be able to detect changes in their environment and respond accordingly. This ability involves signals from outside of the cell triggering changes to the activity inside the cell. Heterotrimeric G proteins are important for this kind of signaling in a wide range of organisms. In animals and fungi, these proteins directly work with a specific class of receptor proteins called G protein-coupled receptors (or GPCRs for short). Plants also have heterotrimeric G proteins, but it remains unclear whether they similiarly work with GPCRs. Plants detect invading microbes by using receptors that are completely different from GPCRs. For example, a receptor called FLS2 from the model plant Arabidopsis senses a telltale protein produced by bacteria, and then passes the signal to another protein called BIK1 to activate the plant’s defenses. Heterotrimeric G proteins are required for this process, but the underlying mechanisms remain unknown. Liang, Ding et al. now show that heterotrimeric G proteins regulate FLS2-controlled defenses by directly interacting with FLS2 and BIK1. Heterotrimeric G proteins also enhance defenses in at least two different ways. Firstly, in the absence of an infection, heterotrimeric G proteins stabilize the BIK1 protein to ensure that it is ready to respond. Secondly, if FLS2 does detect the telltale bacterial protein, BIK1 marks one of the heterotrimeric G proteins with a phosphate group. This then allows the G protein to boost the activity of another plant enzyme that is vital for defense signaling. In the future, it will be important to work out how activation of FLS2 leads to the activation of heterotrimeric G proteins. Furthermore, heterotrimeric G proteins are likely to regulate additional plant proteins when defenses are activated, and further studies are needed to identify these proteins. DOI: http://dx.doi.org/10.7554/eLife.13568.002

The Arabidopsis receptor-like kinase (RLK) BIR1 (BAK1-INTERACTING RECEPTOR-LIKE KINASE 1) functions as a negative regulator of plant immunity. Previous work showed that loss-of-function of BIR1 leads to constitutive activation of cell death and defense responses. These autoimmune phenotypes are partially dependent on another RLK, SOBIR1. In order to identify additional components involved in the BIR1-regulated plant defense signaling pathway, a suppressor screen was carried out in the bir1-1 pad4-1 mutant background. Mutations in the suppressor mutants were identified by genetic mapping and re-sequencing of the mutant genomes. A number of suppressor mutants were found to carry mutations in an additional RLK, BAK1, indicating that BAK1 is required for activation of cell death and defense responses in bir1-1. Co-immunoprecipitation analysis revealed that BAK1 and SOBIR1 associate with each other in planta when the function of BIR1 is compromised. Although BAK1 was previously characterized as a negative regulator of cell death, our study highlights a novel role of BAK1 in promoting cell death and defense responses in conjunction with SOBIR1.

Genetic screens have proven to be a useful tool in the dissection of biological processes in plants. Specifically, suppressor screens have been widely used to study signal transduction pathways. Here we provide a detailed protocol for ethyl methanesulfonate (EMS) mutagenesis used in our suppressor screens in Arabidopsis and discuss the basic principles behind suppressor screen design and downstream analyses.

Plants utilize a large number of immune receptors to recognize pathogens and activate defense responses. A small number of these receptors belong to the receptor-like protein family. Previously, we showed that a gain-of-function mutation in the receptor-like protein SNC2 (for Suppressor of NPR1, Constitutive2) leads to constitutive activation of defense responses in snc2-1D mutant plants. To identify defense signaling components downstream of SNC2, we carried out a suppressor screen in the snc2-1D mutant background of Arabidopsis (Arabidopsis thaliana). Map-based cloning of one of the suppressor genes, BDA1 (for bian da; “becoming big” in Chinese), showed that it encodes a protein with amino-terminal ankyrin repeats and carboxyl-terminal transmembrane domains. Loss-of-function mutations in BDA1 suppress the dwarf morphology and constitutive defense responses in snc2-1D npr1-1 (for nonexpressor of pathogenesis-related genes1,1) and also result in enhanced susceptibility to bacterial pathogens. In contrast, a gain-of-function allele of bda1 isolated from a separate genetic screen to search for mutants with enhanced pathogen resistance was found to constitutively activate cell death and defense responses. These data suggest that BDA1 is a critical signaling component that functions downstream of SNC2 to regulate plant immunity.

Salicylic acid (SA) serves as a critical signaling molecule in plant defense. Two transcription factors, SARD1 and CBP60g, control SA biosynthesis through regulating pathogen-induced expression of Isochorismate Synthase1, which encodes a key enzyme for SA biosynthesis. Here, we report that Pattern-Triggered Immunity Compromised Receptor-like Cytoplasmic Kinase1 (PCRK1) and PCRK2 function as key regulators of SA biosynthesis. In the pcrk1 pcrk2 double mutant, pathogen-induced expression of SARD1, CBP60g, and ICS1 is greatly reduced. The pcrk1 pcrk2 double mutant, but neither of the single mutants, exhibits reduced accumulation of SA and enhanced disease susceptibility to bacterial pathogens. Both PCRK1 and PCRK2 interact with the pattern recognition receptor FLS2, and treatment with pathogen-associated molecular patterns leads to rapid phosphorylation of PCRK2. Our data suggest that PCRK1 and PCRK2 function downstream of pattern recognition receptor in a signal relay leading to the activation of SA biosynthesis.

Genetic screens have proven to be a useful tool in the dissection of biological processes in plants. Specifically, suppressor screens have been widely used to study signal transduction pathways. Here we provide a detailed protocol for ethyl methanesulfonate (EMS) mutagenesis used in our suppressor screens in Arabidopsis and discuss the basic principles behind suppressor screen design and downstream analyses.

Systemic acquired resistance (SAR) is a defense mechanism induced in the distal parts of plants after primary infection. It confers long-lasting protection against a broad spectrum of microbial pathogens. Lack of high-throughput assays has hampered the forward genetic analysis of SAR. Here, we report the development of an easy and efficient assay for SAR and its application in a forward genetic screen for SAR-deficient mutants in Arabidopsis (Arabidopsis thaliana). Using the new assay for SAR, we identified six flavin-dependent monooxygenase1, four AGD2-like defense response protein1, three salicylic acid induction-deficient2, one phytoalexin deficient4, and one avrPphB-susceptible3 alleles as well as a gain-of-function mutant of CALMODULIN-BINDING TRANSCRIPTION ACTIVATOR3 designated camta3-3D. Like transgenic plants overexpressing CAMTA3, camta3-3D mutant plants exhibit compromised SAR and enhanced susceptibility to virulent pathogens, suggesting that CAMTA3 is a critical regulator of both basal resistance and SAR.

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.

In both plants and animals, nucleotide-binding (NB) domain and leucine-rich repeat (LRR)-containing proteins (NLR) function as sensors of pathogen-derived molecules and trigger immune responses. Although NLR resistance (R) proteins were first reported as plant immune receptors more than 15 years ago, how these proteins activate downstream defense responses is still unclear. Here we report that the Toll-like/interleukin-1 receptor (TIR)-NB-LRR R protein, suppressor of npr1-1 , constitutive 1 (SNC1) functions through its associated protein, Topless-related 1 (TPR1). Knocking out TPR1 and its close homologs compromises immunity mediated by SNC1 and several other TIR-NB-LRR–type R proteins, whereas overexpression of TPR1 constitutively activates SNC1-mediated immune responses. TPR1 functions as a transcriptional corepressor and associates with histone deacetylase 19 in vivo. Among the target genes of TPR1 are Defense no Death 1 ( DND1 ) and Defense no Death 2 ( DND2 ), two known negative regulators of immunity that are repressed during pathogen infection, suggesting that TPR1 activates R protein-mediated immune responses through repression of negative regulators.

Salicylic acid (SA) is a plant defense hormone required for immunity. Arabidopsis NPR1 and NPR3/NPR4 were previously shown to bind SA and all three proteins were proposed as SA receptors. NPR1 functions as a transcriptional co-activator, whereas NPR3/NPR4 were suggested to function as E3 ligases that promote NPR1 degradation. Here we report that NPR3/NPR4 function as transcriptional co-repressors and SA inhibits their activities to promote the expression of downstream immune regulators. npr4-4D, a gain-of-function npr4 allele that renders NPR4 unable to bind SA, constitutively represses SA-induced immune responses. In contrast, the equivalent mutation in NPR1 abolishes its ability to bind SA and promote SA-induced defense gene expression. Further analysis revealed that NPR3/NPR4 and NPR1 function independently to regulate SA-induced immune responses. Our study indicates that both NPR1 and NPR3/NPR4 are bona fide SA receptors, but play opposite roles in transcriptional regulation of SA-induced defense gene expression.

SummaryUpon recognition of bacterial flagellin, the plant receptor FLS2 heterodimerizes with brassinosteroid insensitive 1-associated receptor kinase 1 (BAK1) and activates plant defense responses. Because constitutive activation of defense responses is detrimental, plant resistance signaling pathways must be negatively controlled, although the mechanisms involved are unclear. We identified Arabidopsis BIR1 as a BAK1-interacting receptor-like kinase. Knocking out BIR1 leads to extensive cell death, activation of constitutive defense responses, and impairment in the activation of MPK4, a negative regulator of plant resistance (R) protein signaling, by flagellin. sobir1-1, a mutant obtained in a screen for suppressors of the bir1-1 phenotype, rescued cell death observed in bir1-1. SOBIR1 encodes another receptor-like kinase whose overexpression activates cell death and defense responses. Our data suggest that BIR1 negatively regulates multiple plant resistance signaling pathways, one of which is the SOBIR1-dependent pathway identified here.

Mitogen-activated protein kinase (MAPK) cascades play important roles in regulating plant innate immune responses. In a genetic screen to search for mutants with constitutive defense responses, we identified multiple alleles of mpk4 and mekk1 that exhibit cell death and constitutive defense responses. Bimolecular fluorescence complementation (BiFC) analysis showed that both MPK4 and MEKK1 interact with MKK1 and MKK2, two closely related MAPK kinases. mkk1 and mkk2 single mutant plants do not have obvious mutant phenotypes. To test whether MKK1 and MKK2 function redundantly, mkk1 mkk2 double mutants were generated. The mkk1 mkk2 double mutant plants die at seedling stage and the seedling-lethality phenotype is temperature-dependent. Similar to the mpk4 and mekk1 mutants, the mkk1 mkk2 double mutant seedlings accumulate high levels of H2O2, display spontaneous cell death, constitutively express Pathogenesis Related (PR) genes and exhibit pathogen resistance. In addition, activation of MPK4 by flg22 is impaired in the mkk1 mkk2 double mutants, suggesting that MKK1 and MKK2 function together with MPK4 and MEKK1 in a MAP kinase cascade to negatively regulate innate immune responses in plants.

The RAR1 and SGT1 proteins function synergistically or antagonistically in plant innate immune responses. Here, we show that the rice orthologs OsRAR1 and OsSGT1 physically interact in vivo and in yeast. They displayed conserved roles in Arabidopsis disease resistance through ectopic expression in the Arabidopsis rar1 and sgt1 mutants. Overexpression of OsRar1 and OsSGT1 in rice significantly increased basal resistance to a virulent bacterial blight Xanthomonas oryzae pv. oryzae PXO99 but not to another virulent strain DY89031, suggesting race-specificlike basal resistance conferred by OsRar1 and OsSGT1. OsRar1-OE and OsSGT1-OE plants also enhanced resistance to all four virulent blast fungal Magnaporthe oryzae races. Overexpression of the OsSGT1-green fluorescent protein (GFP) fusion most likely caused a dominant negative phenotype which led to race-specific-like basal resistance. Transgenic plants overexpressing OsSGT1-GFP show enhanced resistance to DY89031 but decreased resistance to PXO99, implying that OsSGT1 might be the target of a component required for DY89031 virulence or OsSGT1GFP might stabilize weak resistance proteins against DY89031. Consistent with the hypothesis of the dominant negative regulation, we observed the reduced sensitivity to auxin of OsSGT1-GFP plants compared with the wild-type ones, and the curling-root phenotype in OsSGT1-OE plants. These results collectively suggest that OsRar1 and OsSGT1 might be differentially required for rice basal disease resistance. Our current study also provides new insight into the roles of OsSGT1 in basal disease resistance. Additional keywords: development, R-mediated resistance.

Summary Plant defences require a multitude of tightly regulated resistance responses. In Arabidopsis, the unique gain-of-function mutant suppressor of npr1-1 constitutive 1 (snc1) carries a point mutation in a Resistance (R)-gene, resulting in constitutive activation of defence responses without interaction with pathogens. This has allowed us to identify various downstream signalling components essential in multiple defence pathways. One mutant that suppresses snc1-mediated constitutive resistance is modifier of snc1 5 (mos5), which carries a 15-bp deletion in UBA1, one of two ubiquitin-activating enzyme genes in Arabidopsis. A mutation in UBA2 does not suppress snc1, suggesting that these two genes are not equally required in Arabidopsis disease resistance. On the other hand, a mos5 uba2 double mutant is lethal, implying partial redundancy of the two homologues. Apart from affecting snc1-mediated resistance, mos5 also exhibits enhanced disease susceptibility to a virulent pathogen and is impaired in response to infection with avirulent bacteria carrying the protease elicitor AvrRpt2. The mos5 mutation in the C-terminus of UBA1 might affect binding affinity of the downstream ubiquitin-conjugating enzymes, thus perturbing ubiquitination of target proteins. Furthermore, SGT1b and RAR1, which are necessary for resistance conferred by the SNC1-related R-genes RPP4 and RPP5, are dispensable in snc1-mediated resistance. Our data reveal the definite requirement for the ubiquitination pathway in the activation and downstream signalling of several R-proteins.

International audience; Nod-like receptors (NLRs) serve as immune receptors in plants and animals. The stability of NLRs is tightly regulated, though its mechanism is not well understood. Here, we show the crucial impact of N-terminal acetylation on the turnover of one plant NLR, Suppressor of NPR1, Constitutive 1 (SNC1), in Arabidopsis thaliana. Genetic and biochemical analyses of SNC1 uncovered its multilayered regulation by different N-terminal acetyltransferase (Nat) complexes. SNC1 exhibits a few distinct N-terminal isoforms generated through alternative initiation and N-terminal acetylation. Its first Met is acetylated by N-terminal acetyltransferase complex A (NatA), while the second Met is acetylated by N-terminal acetyltransferase complex B (NatB). Unexpectedly, the NatA-mediated acetylation serves as a degradation signal, while NatB-mediated acetylation stabilizes the NLR protein, thus revealing antagonistic N-terminal acetylation of a single protein substrate. Moreover, NatA also contributes to the turnover of another NLR, RESISTANCE TO P. syringae pv maculicola 1. The intricate regulation of protein stability by Nats is speculated to provide flexibility for the target protein in maintaining its homeostasis.

Recognition of pathogens by host plants leads to rapid transcriptional reprogramming and activation of defence responses. The expression of many defence regulators is induced in this process, but the mechanisms of how they are controlled transcriptionally are largely unknown. Here we use chromatin immunoprecipitation sequencing to show that the transcription factors SARD1 and CBP60g bind to the promoter regions of a large number of genes encoding key regulators of plant immunity. Among them are positive regulators of systemic immunity and signalling components for effector-triggered immunity and PAMP-triggered immunity, which is consistent with the critical roles of SARD1 and CBP60g in these processes. In addition, SARD1 and CBP60g target a number of genes encoding negative regulators of plant immunity, suggesting that they are also involved in negative feedback regulation of defence responses. Based on these findings we propose that SARD1 and CBP60g function as master regulators of plant immune responses., SARD1 and CBP60g are two plant transcription factors that regulate salicylic acid biosynthesis in response to pathogens. Here, Sun et al. show that they bind a wide array of loci related to multiple defence signalling pathways suggesting a broader role as regulators of the plant immune response.

Heterotrimeric G proteins (G-proteins) are versatile signaling elements conserved in Eukaryotes. In animals G-proteins relay signals from 7-transmembrane spanning G protein-coupled receptors (GPCRs) to intracellular downstream effectors; however, the existence of GPCRs in plants is controversial. Contrastingly, a surplus of receptor-like kinases (RLKs) provides signal recognition at the plant cell surface. It is established that G proteins are involved in plant defense and suggested that they relay signals from defense-related RLKs. However, it is unclear how the signaling is conducted, as physical interaction between the RLKs and G proteins has not been demonstrated. Using yeast split-ubiquitin system and Bimolecular Fluorescence Complementation assays, we demonstrate physical interaction between the Gα, Gγ1 and Gγ2 subunits, and the defense-related RD-type receptor like kinases CERK1, BAK1 and BIR1. At the same time, no interaction was detected with the non-RD RLK FLS2. We hypothesize that G-proteins mediate signal transduction immediately downstream of the pathogenesis-related RLKs.