Rice Pti1a negatively regulates RAR1-dependent defense responses

Tomato (Solanum lycopersicum) Pto encodes a protein kinase that confers resistance to bacterial speck disease. A second protein kinase, Pti1, physically interacts with Pto and is involved in Pto-mediated defense signaling. Pti1-related sequences are highly conserved among diverse plant species, including rice (Oryza sativa), but their functions are largely unknown. Here, we report the identification of a null mutant for the Pti1 homolog in rice and the functional characterization of Os Pti1a. The rice pti1a mutant was characterized by spontaneous necrotic lesions on leaves, which was accompanied by a series of defense responses and resistance against a compatible race of Magnaporthe grisea. Overexpression of Pti1a in rice reduced resistance against an incompatible race of the fungus recognized by a resistance (R) protein, Pish. Plants overexpressing Pti1a were also more susceptible to a compatible race of the bacterial pathogen Xanthomonas oryzae pv oryzae. These results suggest that Os Pti1a negatively regulates defense signaling for both R gene-mediated and basal resistance. We also demonstrated that repression of the rice RAR1 gene suppressed defense responses induced in the pti1a mutant, indicating that Pti1a negatively regulates RAR1-dependent defense responses. Expression of a tomato Pti1 cDNA in the rice pti1a mutant suppressed the mutant phenotypes. This contrasts strikingly with the previous finding that Sl Pti1 enhances Pto-mediated hypersensitive response (HR) induction when expressed in tobacco (Nicotiana tabacum), suggesting that the molecular switch controlling HR downstream of pathogen recognition has evolved differently in rice and tomato.     

Adi3 is a Pdk1-interacting AGC kinase that negatively regulates plant cell death

Bacterial speck disease in tomato is caused by Pseudomonas syringae pv. tomato. Resistance to this disease is conferred by the host Pto kinase, which recognizes P. s. pv. tomato strains that express the effector AvrPto. We report here that an AvrPto-dependent Pto-interacting protein 3 (Adi3) is a member of the AGC family of protein kinases. In mammals, AGC kinases are regulated by 3-phosphoinositide-dependent protein kinase-1 (Pdk1). We characterized tomato Pdk1 and showed that Pdk1 and Pto phosphorylate Adi3. Gene silencing of Adi3 in tomato causes MAPKKKalpha-dependent formation of necrotic lesions. Use of a chemical inhibitor of Pdk1, OSU-03012, also implicates Pdk1 and Adi3 in plant cell death regulation. Adi3 thus appears to function analogously to the mammalian AGC kinase protein kinase B/Akt by negatively regulating cell death via Pdk1 phosphorylation. We speculate that the negative regulatory function of Adi3 might be subverted by interaction with Pto/AvrPto, leading to host cell death that is associated with pathogen attack.     

The T-loop Extension of the Tomato Protein Kinase AvrPto-dependent Pto-interacting Protein 3 (Adi3) Directs Nuclear Localization for Suppression of Plant Cell Death

In tomato (Solanum lycopersicum), resistance to Pseudomonas syringae pv. tomato is elicited by the interaction of the host Pto kinase with the pathogen effector protein AvrPto, which leads to various immune responses including localized cell death termed the hypersensitive response. The AGC kinase Adi3 functions to suppress host cell death and interacts with Pto only in the presence of AvrPto. The cell death suppression (CDS) activity of Adi3 requires phosphorylation by 3-phosphoinositide-dependent protein kinase 1 (Pdk1) and loss of Adi3 function is associated with the hypersensitive response cell death initiated by the Pto/AvrPto interaction. Here we studied the relationship between Adi3 cellular localization and its CDS activity. Adi3 is a nuclear-localized protein, and this localization is dictated by a nuclear localization signal found in the Adi3 T-loop extension, an ∼80 amino acid insertion into the T-loop, or activation loop, which is phosphorylated for kinase activation. Nuclear localization of Adi3 is required for its CDS activity and loss of nuclear localization causes elimination of Adi3 CDS activity and induction of cell death. This nuclear localization of Adi3 is dependent on Ser-539 phosphorylation by Pdk1 and non-nuclear Adi3 is found in punctate structures throughout the cell. Our data support a model in which Pdk1 phosphorylation of Adi3 directs nuclear localization for CDS and that disruption of Adi3 nuclear localization may be a mechanism for induction of cell death such as that during the Pto/AvrPto interaction.     

OsCERK1 and OsRLCK176 play important roles in peptidoglycan and chitin signaling in rice innate immunity

Microbe‐associated molecular pattern (MAMP)‐triggered immunity plays critical roles in the basal resistance defense response in plants. Chitin and peptidoglycan (PGN) are major molecular patterns for fungi and bacteria, respectively. Two rice (Oryza sativa) lysin motif‐containing proteins, OsLYP4 and OsLYP6, function as receptors that sense bacterial PGN and fungal chitin. These membrane receptors, which lack intracellular kinase domains, likely contain another component for transmembrane immune signal transduction. Here, we demonstrate that the rice LysM receptor‐like kinase OsCERK1, a key component of the chitin elicitor signaling pathway, also plays an important role in PGN‐triggered immunity in rice. Silencing of OsCERK1 suppressed PGN‐induced (and chitin‐induced) immunity responses, including reactive oxygen species generation, defense gene expression, and callose deposition, indicating that OsCERK1 is essential for both PGN and chitin signaling initiated by OsLYP4 and OsLYP6. OsLYP4 associated with OsLYP6 and the rice chitin receptor chitin oligosaccharide elicitor‐binding protein (CEBiP) in the absence of PGN or chitin, and treatment with PGN or chitin led to their disassociation in vivo. OsCERK1 associated with OsLYP4 or OsLYP6 when induced by PGN but it associated with OsLYP4, OsLYP6, or CEBiP under chitin treatment, suggesting the presence of different patterns of ligand‐induced heterooligomeric receptor complexes. Furthermore, the receptor‐like cytoplasmic kinase OsRLCK176 functions downstream of OsCERK1 in the PGN and chitin signaling pathways, suggesting that these MAMPs share overlapping intracellular signaling components. Therefore, OsCERK1 plays dual roles in PGN and chitin signaling in rice innate immunity and as an adaptor involved in signal transduction at the plasma membrane in conjunction with OsLYP4 and OsLYP6.     

OsSERK1 regulates rice development but not immunity to Xanthomonas oryzae pv. oryzae or Magnaporthe oryzae

Somatic embryogenesis receptor kinase (SERK) proteins play pivotal roles in regulation of plant development and immunity. The rice genome contains two SERK genes, OsSerk1 and OsSerk2. We previously demonstrated that OsSerk2 is required for rice Xa21-mediated resistance to Xanthomonas oryzae pv. oryzae (Xoo) and for normal development. Here we report the molecular characterization of OsSerk1. Overexpression of OsSerk1 results in a semi-dwarf phenotype whereas silencing of OsSerk1 results in a reduced angle of the lamina joint. OsSerk1 is not required for rice resistance to Xoo or Magnaporthe oryzae. Overexpression of OsSerk1 in OsSerk2-silenced lines complements phenotypes associated with brassinosteroid (BR) signaling defects, but not the disease resistance phenotype mediated by Xa21. In yeast, OsSERK1 interacts with itself forming homodimers, and also interacts with the kinase domains of OsSERK2 and BRI1, respectively. OsSERK1 is a functional protein kinase capable of auto-phosphorylation in vitro. We conclude that, whereas OsSERK2 regulates both rice development and immunity, OsSERK1 functions in rice development but not immunity to Xoo and M. oryzae.     

The Arabidopsis CERK1‐associated kinase PBL27 connects chitin perception to MAPK activation

Perception of microbe‐associated molecular patterns by host cell surface pattern recognition receptors (PRRs) triggers the intracellular activation of mitogen‐activated protein kinase (MAPK) cascades. However, it is not known how PRRs transmit immune signals to MAPK cascades in plants. Here, we identify a complete phospho‐signaling transduction pathway from PRR‐mediated pathogen recognition to MAPK activation in plants. We found that the receptor‐like cytoplasmic kinase PBL27 connects the chitin receptor complex CERK1‐LYK5 and a MAPK cascade. PBL27 interacts with both CERK1 and the MAPK kinase kinase MAPKKK5 at the plasma membrane. Knockout mutants of MAPKKK5 compromise chitin‐induced MAPK activation and disease resistance to Alternaria brassicicola. PBL27 phosphorylates MAPKKK5 in vitro, which is enhanced by phosphorylation of PBL27 by CERK1. The chitin perception induces disassociation between PBL27 and MAPKKK5 in vivo. Furthermore, genetic evidence suggests that phosphorylation of MAPKKK5 by PBL27 is essential for chitin‐induced MAPK activation in plants. These data indicate that PBL27 is the MAPKKK kinase that provides the missing link between the cell surface chitin receptor and the intracellular MAPK cascade in plants.     

Two Pdk1 phosphorylation sites on the plant cell death suppressor Adi3 contribute to substrate phosphorylation

The tomato AGC kinase Adi3 is phosphorylated by Pdk1 for activation of its cell death suppression activity. The Pdk1 phosphorylation site for activation of Adi3 is at Ser539. However, there is at least one additional Pdk1 phosphorylation site on Adi3 that has an unknown function. Here we identify an Arabidopsis thaliana sequence homologue of Adi3 termed AGC1-3. Two Pdk1 phosphorylation sites were identified on AGC1-3, activation site Ser596 and Ser269, and by homology Ser212 on Adi3 was identified as a second Pdk1 phosphorylation site. While Ser212 is not required for Adi3 autophosphorylation, Ser212 was shown to be required for full phosphorylation of the Adi3 substrate Gal83.     

Expression of the chimeric receptor between the chitin elicitor receptor CEBiP and the receptor-like protein kinase Pi-d2 leads to enhanced responses to the chitin elicitor and disease resistance against Magnaporthe oryzae in rice

We previously reported that rice plants expressing the chimeric receptor consisting of rice chitin oligosaccharides binding protein (CEBiP) and the intracellular protein kinase region of Xa21, which confers resistance to rice bacterial blight, showed enhanced cellular responses to a chitin elicitor N-acetylchitoheptaose and increased resistance to the rice blast fungus Magnaporthe oryzae. Here, we investigated whether CEBiP fused with another type of receptor-like protein kinase (RLK) also functions as a chimeric receptor. Fusion proteins CRPis consist of CEBiP and the intracellular protein kinase region of a true resistance gene Pi-d2. Transgenic rice expressing a CRPi showed enhanced cellular responses specifically to N-acetylchitoheptaose in cultured cells and increased levels of disease resistance against M. oryzae in plants. These responses depended on the amino acid sequences predicted to be essential for the protein kinase activity of CRPi. The structure of the transmembrane domain in CRPi affected the protein accumulation, cellular responses, and disease resistance in transgenic rice. These results suggest that the chimeric receptor consisting of CEBiP and Pi-d2 functions as a receptor for chitin oligosaccharides and CEBiP-based chimeric receptors fused with other RLKs may also act as functional receptors.     

Effector-mediated suppression of chitin-triggered immunity by magnaporthe oryzae is necessary for rice blast disease

Plants use pattern recognition receptors to defend themselves from microbial pathogens. These receptors recognize pathogen-associated molecular patterns (PAMPs) and activate signaling pathways that lead to immunity. In rice (Oryza sativa), the chitin elicitor binding protein (CEBiP) recognizes chitin oligosaccharides released from the cell walls of fungal pathogens. Here, we show that the rice blast fungus Magnaporthe oryzae overcomes this first line of plant defense by secreting an effector protein, Secreted LysM Protein1 (Slp1), during invasion of new rice cells. We demonstrate that Slp1 accumulates at the interface between the fungal cell wall and the rice plasma membrane, can bind to chitin, and is able to suppress chitin-induced plant immune responses, including generation of reactive oxygen species and plant defense gene expression. Furthermore, we show that Slp1 competes with CEBiP for binding of chitin oligosaccharides. Slp1 is required by M. oryzae for full virulence and exerts a significant effect on tissue invasion and disease lesion expansion. By contrast, gene silencing of CEBiP in rice allows M. oryzae to cause rice blast disease in the absence of Slp1. We propose that Slp1 sequesters chitin oligosaccharides to prevent PAMP-triggered immunity in rice, thereby facilitating rapid spread of the fungus within host tissue.     

Enhancement of MAMP signaling by chimeric receptors improves disease resistance in plants

Plants activate defense responses through the recognition of microbe-associated molecular patterns (MAMPs). Recently, several pattern-recognition receptors (PRRs) have been identified in plants, paving the way for manipulating MAMP signaling. CEBiP is a receptor for the chitin elicitor (CE) identified in the rice plasma membrane and XA21 is a member of the receptor-like protein kinase (RLK) family that confers disease resistance to rice bacterial leaf blight expressing the sulfated protein Ax21. To improve resistance to rice blast, the most serious fungal disease of rice, we aimed to create a defense system that combines high affinity of CEBiP for CE and the ability of XA21 to confer disease resistance. Cultured rice cells expressing the chimeric receptor CRXA, which consists of CEBiP and the intracellular region of XA21, induced cell death accompanied by an increased production of reactive oxygen and nitrogen species after exposure to CE. Rice plants expressing the chimeric receptor exhibited more resistance to rice blast. Engineering PRRs may be a new strategy in molecular breeding for achieving disease resistance.Key words: chimeric receptor, chitin signal, disease resistance, HR cell death, MAMP-induced resistance, rice blast fungus     

Rice NRR, a negative regulator of disease resistance, interacts with Arabidopsis NPR1 and rice NH1

Arabidopsis NPR1/NIM1 is a key regulator of systemic acquired resistance (SAR), which confers lasting broad-spectrum resistance. Over-expression of Arabidopsis NPR1 or the NPR1 homolog 1 (NH1) in rice results in enhanced resistance to the pathogen Xanthomonasoryzae pv. oryzae (Xoo), suggesting the presence of a related defense pathway in rice. We investigated this pathway in rice by identifying proteins that interact with NH1. Here we report the isolation and characterization of a rice cDNA encoding a novel protein, named NRR (for negative regulator of resistance). NRR interacts with NPR1 in the NPR1-interacting domain (NI25) consisting of 25 amino acids. NRR also interacts with NH1; however, NI25 was not sufficient for a strong interaction, indicating a difference between the rice and the Arabidopsis proteins. Silencing of NRR in rice had little effect on resistance to Xoo. When constitutively over-expressed in rice, NRR affected basal resistance, age-related resistance and Xa21-mediated resistance, causing enhanced susceptibility to Xoo. This phenotype was correlated with elevated NRR mRNA and protein levels and increased Xoo growth. Over-expression of NRR suppressed the induction of defense-related genes. NRR:GFP (green fluorescent protein) protein was localized to the nucleus, indicating that NRR may act directly to suppress the activation of defense genes. The fact that NRR compromises Xa21-mediated resistance indicates cross-talk or overlap between NH1- and Xa21-mediated pathways.     

Presence of LYM2 dependent but CERK1 independent disease resistance in Arabidopsis

Plants have the ability to detect invading fungi through the perception of chitin fragments released from the fungal cell walls. Plant chitin receptor consists of two types of plasma membrane proteins, CEBiP and CERK1. However, the contribution of these proteins to chitin signaling is different between Arabidopsis and rice. In Arabidopsis, it seems CERK1 receptor kinase is enough for both ligand perception and signaling, whereas both CEBiP and OsCERK1 are required for chitin signaling in rice. Here we report that Arabidopsis CEBiP homolog, LYM2, is not involved in chitin signaling but contributes to resistance against a fungal pathogen, Alternaria brassicicola, indicating the presence of a novel disease resistance mechanism in Arabidopsis.     

Two LysM receptor molecules, CEBiP and OsCERK1, cooperatively regulate chitin elicitor signaling in rice

Chitin is a major molecular pattern for various fungi, and its fragments, chitin oligosaccharides, are known to induce various defense responses in plant cells. A plasma membrane glycoprotein, CEBiP (chitin elicitor binding protein) and a receptor kinase, CERK1 (chitin elicitor receptor kinase) (also known as LysM-RLK1), were identified as critical components for chitin signaling in rice and Arabidopsis, respectively. However, it is not known whether each plant species requires both of these two types of molecules for chitin signaling, nor the relationships between these molecules in membrane signaling. We report here that rice cells require a LysM receptor-like kinase, OsCERK1, in addition to CEBiP, for chitin signaling. Knockdown of OsCERK1 resulted in marked suppression of the defense responses induced by chitin oligosaccharides, indicating that OsCERK1 is essential for chitin signaling in rice. The results of a yeast two-hybrid assay indicated that both CEBiP and OsCERK1 have the potential to form hetero- or homo-oligomers. Immunoprecipitation using a membrane preparation from rice cells treated with chitin oligosaccharides suggested the ligand-induced formation of a receptor complex containing both CEBiP and OsCERK1. Blue native PAGE and chemical cross-linking experiments also suggested that a major portion of CEBiP exists as homo-oligomers even in the absence of chitin oligosaccharides.     

OsRAR1 and OsSGT1 physically interact and function in rice basal disease resistance

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-specific-like 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 OsSGT1-GFP 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.     

Disruption of PAMP-induced MAP kinase cascade by a Pseudomonas syringae effector activates plant immunity mediated by the NB-LRR protein SUMM2

Pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) serves as a primary plant defense response against microbial pathogens, with MEKK1, MKK1/MKK2, and MPK4 functioning as a MAP kinase cascade downstream of PAMP receptors. Plant Resistance (R) proteins sense specific pathogen effectors to initiate a second defense mechanism, termed effector-triggered immunity (ETI). In a screen for suppressors of the mkk1 mkk2 autoimmune phenotype, we identify the nucleotide-binding leucine-rich repeat (NB-LRR) protein SUMM2 and find that the MEKK1-MKK1/MKK2-MPK4 cascade negatively regulates SUMM2-mediated immunity. Further, the MEKK1-MKK1/MKK2-MPK4 cascade positively regulates basal defense targeted by the Pseudomonas syringae pathogenic effector HopAI1, which inhibits MPK4 kinase activity. Inactivation of MPK4 by HopAI1 results in activation of SUMM2-mediated defense responses. Our data suggest that SUMM2 is an R protein that becomes active when the MEKK1-MKK1/MKK2-MPK4 cascade is disrupted by pathogens, supporting the hypothesis that R proteins evolved to protect plants when microbial effectors suppress basal resistance.     

Screening for resistance against Pseudomonas syringae in rice-FOX Arabidopsis lines identified a putative receptor-like cytoplasmic kinase gene that confers resistance to major bacterial and fungal pathogens in Arabidopsis and rice

Approximately 20,000 of the rice-FOX Arabidopsis transgenic lines, which overexpress 13,000 rice full-length cDNAs at random in Arabidopsis, were screened for bacterial disease resistance by dip inoculation with Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). The identities of the overexpressed genes were determined in 72 lines that showed consistent resistance after three independent screens. Pst DC3000 resistance was verified for 19 genes by characterizing other independent Arabidopsis lines for the same genes in the original rice-FOX hunting population or obtained by reintroducing the genes into ecotype Columbia by floral dip transformation. Thirteen lines of these 72 selections were also resistant to the fungal pathogen Colletotrichum higginsianum. Eight genes that conferred resistance to Pst DC3000 in Arabidopsis have been introduced into rice for overexpression, and transformants were evaluated for resistance to the rice bacterial pathogen, Xanthomonas oryzae pv. oryzae. One of the transgenic rice lines was highly resistant to Xanthomonas oryzae pv. oryzae. Interestingly, this line also showed remarkably high resistance to Magnaporthe grisea, the fungal pathogen causing rice blast, which is the most devastating rice disease in many countries. The causal rice gene, encoding a putative receptor-like cytoplasmic kinase, was therefore designated as BROAD-SPECTRUM RESISTANCE 1. Our results demonstrate the utility of the rice-FOX Arabidopsis lines as a tool for the identification of genes involved in plant defence and suggest the presence of a defence mechanism common between monocots and dicots.