identification-post-translational-modifications-plant-protein

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

University of Warwick

Plant innate immunity relies on a set of pattern recognition receptors (PRRs) that respond to ligands known as pathogen-associated molecular patterns (PAMPs). To overcome such immunity, phytopathogenic bacteria deliver virulence molecules called effector proteins into the plant cell that collectively promote pathogenesis. The vast majority of PRRs controlling PAMP-triggered immunity (PTI) and the mechanisms used by specific effectors to suppress these pathways are mostly unknown. Here, we show that the Arabidopsis LysM receptor kinase CERK1, which is critical for chitin elicitor signaling and resistance to fungal pathogens, plays an essential role in restricting bacterial growth on plants. This is supported by the fact that CERK1 is a target of the bacterial type III effector protein AvrPtoB, which blocks all defense responses through this receptor. AvrPtoB ubiquitinates the CERK1 kinase domain in vitro and targets CERK1 for degradation in vivo. We show that CERK1 is a determinant of bacterial immunity, but its contribution is overcome by bacteria expressing AvrPtoB. Our results reveal a new pathway for plant immunity against bacteria and a role for AvrPtoB E3-ligase activity in suppressing PTI.

AvrPtoB targets the LysM receptor kinase CERK1 to promote bacterial virulence on plants.

Plant pathogenic bacteria secrete effector proteins that attack the host signaling machinery to suppress immunity. Effectors can be recognized by hosts leading to immunity. One such effector is AvrPtoB of Pseudomonas syringae, which degrades host protein kinases, such as tomato Fen, through an E3 ligase domain. Pto kinase, which is highly related to Fen, recognizes AvrPtoB in conjunction with the resistance protein Prf. Here we show that Pto is resistant to AvrPtoB-mediated degradation because it inactivates the E3 ligase domain. AvrPtoB ubiquitinated Fen within the catalytic cleft, leading to its breakdown and loss of the associated Prf protein. Pto avoids this by phosphorylating and inactivating the AvrPtoB E3 domain. Thus, inactivation of a pathogen virulence molecule is one mechanism by which plants resist disease.

Host inhibition of a bacterial virulence effector triggers immunity to infection.

The LysM receptor kinase CERK1 mediates bacterial perception in Arabidopsis.

Cytoplasmic recognition of pathogen virulence effectors by plant NB-LRR proteins leads to strong induction of defence responses termed effector triggered immunity (ETI). In tomato, a protein complex containing the NB-LRR protein Prf and the protein kinase Pto confers recognition of the Pseudomonas syringae effectors AvrPto and AvrPtoB. Although structurally unrelated, AvrPto and AvrPtoB interact with similar residues in the Pto catalytic cleft to activate ETI via an unknown mechanism. Here we show that the Prf complex is oligomeric, containing at least two molecules of Prf. Within the complex, Prf can associate with Pto or one of several Pto family members including Fen, Pth2, Pth3, or Pth5. The dimerization surface for Prf is the novel N-terminal domain, which also coordinates an intramolecular interaction with the remainder of the molecule, and binds Pto kinase or a family member. Thus, association of two Prf N-terminal domains brings the associated kinases into close promixity. Tomato lines containing Prf complexed with Pth proteins but not Pto possessed greater immunity against P. syringae than tomatoes lacking Prf. This demonstrates that incorporation of non-Pto kinases into the Prf complex extends the number of effector proteins that can be recognized.

Prf immune complexes of tomato are oligomeric and contain multiple Pto-like kinases that diversify effector recognition.

Plants rely heavily on receptor-like kinases (RLKs) for perception and integration of external and internal stimuli. The Arabidopsis regulatory leucine-rich repeat RLK (LRR-RLK) BAK1 is involved in steroid hormone responses, innate immunity, and cell death control. Here, we describe the differential regulation of three different BAK1-dependent signaling pathways by a novel allele of BAK1, bak1-5. Innate immune signaling mediated by the BAK1-dependent RKs FLS2 and EFR is severely compromised in bak1-5 mutant plants. However, bak1-5 mutants are not impaired in BR signaling or cell death control. We also show that, in contrast to the RD kinase BRI1, the non-RD kinases FLS2 and EFR have very low kinase activity, and we show that neither was able to trans-phosphorylate BAK1 in vitro. Furthermore, kinase activity for all partners is completely dispensable for the ligand-induced heteromerization of FLS2 or EFR with BAK1 in planta, revealing another pathway specific mechanistic difference. The specific suppression of FLS2- and EFR-dependent signaling in bak1-5 is not due to a differential interaction of BAK1-5 with the respective ligand-binding RK but requires BAK1-5 kinase activity. Overall our results demonstrate a phosphorylation-dependent differential control of plant growth, innate immunity, and cell death by the regulatory RLK BAK1, which may reveal key differences in the molecular mechanisms underlying the regulation of ligand-binding RD and non-RD RKs.

Phosphorylation-dependent differential regulation of plant growth, cell death, and innate immunity by the regulatory receptor-like kinase BAK1.

Detailed phenotypic characterization reveals that several BAK1 fusion proteins with C-terminal tags strongly impair complementation of bak1 null mutants with respect to responsiveness to the bacterial pathogen-associated molecular patterns flagellin and EF-Tu. This raises concerns about the widespread use of such protein variants of this important regulatory Leu-rich repeat receptor-like kinase (RLK) for functional analyses of RLK-based signaling.

Cautionary notes on the use of C-terminal BAK1 fusion proteins for functional studies.

The major virulence strategy of phytopathogenic bacteria is to secrete effector proteins into the host cell to target the immune machinery. AvrPto and AvrPtoB are two such effectors from Pseudomonas syringae, which disable an overlapping range of kinases in Arabidopsis and Tomato. Both effectors target surface-localized receptor-kinases to avoid bacterial recognition. In turn, tomato has evolved an intracellular effector-recognition complex composed of the NB-LRR protein Prf and the Pto kinase. Structural analyses have shown that the most important interaction surface for AvrPto and AvrPtoB is the Pto P+1 loop. AvrPto is an inhibitor of Pto kinase activity, but paradoxically, this kinase activity is a prerequisite for defense activation by AvrPto. Here using biochemical approaches we show that disruption of Pto P+1 loop stimulates phosphorylation in trans, which is possible because the Pto/Prf complex is oligomeric. Both P+1 loop disruption and transphosphorylation are necessary for signalling. Thus, effector perturbation of one kinase molecule in the complex activates another. Hence, the Pto/Prf complex is a sophisticated molecular trap for effectors that target protein kinases, an essential aspect of the pathogen's virulence strategy. The data presented here give a clear view of why bacterial virulence and host recognition mechanisms are so often related and how the slowly evolving host is able to keep pace with the faster-evolving pathogen.

Article	Year
AvrPtoB targets the LysM receptor kinase CERK1 to promote bacterial virulence on plants. Current biology : CB\| PubMed ID: 19249211	2009
Host inhibition of a bacterial virulence effector triggers immunity to infection. Science (New York, N.Y.)\| PubMed ID: 19423826	2009
The LysM receptor kinase CERK1 mediates bacterial perception in Arabidopsis. Plant signaling & behavior\| PubMed ID: 19816132	2009
Prf immune complexes of tomato are oligomeric and contain multiple Pto-like kinases that diversify effector recognition. The Plant journal : for cell and molecular biology\| PubMed ID: 19919571	2010
Phosphorylation-dependent differential regulation of plant growth, cell death, and innate immunity by the regulatory receptor-like kinase BAK1. PLoS genetics\| PubMed ID: 21593986	2011
Cautionary notes on the use of C-terminal BAK1 fusion proteins for functional studies. The Plant cell\| PubMed ID: 22129600	2011
The tomato Prf complex is a molecular trap for bacterial effectors based on Pto transphosphorylation. PLoS pathogens\| PubMed ID: 23382672	2013
Editorial: Mechanisms regulating immunity in plants. Frontiers in plant science\| PubMed ID: 23544032	2013

Vardis Ntoukakis

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