Plant Proteomics
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New secrets of the plant kingdom uncovered after over a century in storage. University of Cambridge

New secrets of the plant kingdom uncovered after over a century in storage. University of Cambridge | Plant Proteomics | Scoop.it

You never know what will turn up when you move house - the University of Cambridge recently moved its extensive Herbarium to the new Sainsbury lab, and found some forgotten treasures (video).

 

They've also got a very nice interactive exhbition of plants collected by Charles Darwin on his Beagle voyage  (http://www.darwinsbeagleplants.org/Darwin/Home.aspx). You can send your students on a digital field trip!

 


Via Mary Williams, Ruth Bastow
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A Small Molecule Inhibitor Partitions Two Distinct Pathways for Trafficking of Tonoplast Intrinsic Proteins in Arabidopsis

A Small Molecule Inhibitor Partitions Two Distinct Pathways for Trafficking of Tonoplast Intrinsic Proteins in Arabidopsis | Plant Proteomics | Scoop.it

Efrain E. Rivera-Serrano, Maria F. Rodriguez-Welsh, Glenn R. Hicks, Marcela Rojas-Pierce

 

Abstract

Tonoplast intrinsic proteins (TIPs) facilitate the membrane transport of water and other small molecules across the plant vacuolar membrane, and members of this family are expressed in specific developmental stages and tissue types. Delivery of TIP proteins to the tonoplast is thought to occur by vesicle–mediated traffic from the endoplasmic reticulum to the vacuole, and at least two pathways have been proposed, one that is Golgi-dependent and another that is Golgi-independent. However, the mechanisms for trafficking of vacuolar membrane proteins to the tonoplast remain poorly understood. Here we describe a chemical genetic approach to unravel the mechanisms of TIP protein targeting to the vacuole in Arabidopsis seedlings. We show that members of the TIP family are targeted to the vacuole via at least two distinct pathways, and we characterize the bioactivity of a novel inhibitor that can differentiate between them. We demonstrate that, unlike for TIP1;1, trafficking of markers for TIP3;1 and TIP2;1 is insensitive to Brefeldin A in Arabidopsis hypocotyls. Using a chemical inhibitor that may target this BFA-insensitive pathway for membrane proteins, we show that inhibition of this pathway results in impaired root hair growth and enhanced vacuolar targeting of the auxin efflux carrier PIN2 in the dark. Our results indicate that the vacuolar targeting of PIN2 and the BFA-insensitive pathway for tonoplast proteins may be mediated in part by common mechanisms.

 

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Synchronization of secretory protein traffic in populations of cells : Nature Methods : Nature Publishing Group

Synchronization of secretory protein traffic in populations of cells : Nature Methods : Nature Publishing Group | Plant Proteomics | Scoop.it
The biotin-reversible interaction between a 'hook' protein localized to a particular cellular compartment and a reporter protein of interest is exploited in a simple system to synchronize protein traffic through the secretory pathway.

 

Gaelle Boncompain,Severine Divoux,Nelly Gareil,Helene de Forges,Aurianne Lescure,Lynda Latreche,Valentina Mercanti,Florence Jollivet,Graça Raposo& Franck Perez

 

Nature Methods9,493–498(2012)doi:10.1038/nmeth.1928

 

Abstract:

To dissect secretory traffic, we developed the retention using selective hooks (RUSH) system. RUSH is a two-state assay based on the reversible interaction of a hook protein fused to core streptavidin and stably anchored in the donor compartment with a reporter protein of interest fused to streptavidin-binding peptide (SBP). Biotin addition causes a synchronous release of the reporter from the hook. Using the RUSH system, we analyzed different transport characteristics of various Golgi and plasma membrane reporters at physiological temperature in living cells. Using dual-color simultaneous live-cell imaging of two cargos, we observed intra- and post-Golgi segregation of cargo traffic, consistent with observation in other systems. We show preliminarily that the RUSH system is usable for automated screening. The system should help increase the understanding of the mechanisms of trafficking and enable screens for molecules that perturb pathological protein transport.

 

 

 

Not a plant paper but certainly an interesting approach.

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Protein phosphorylation is a prerequisite for the Ca2+-dependent activation of Arabidopsis NADPH oxidases...

Protein phosphorylation is a prerequisite for the Ca2+-dependent activation of Arabidopsis NADPH oxidases... | Plant Proteomics | Scoop.it

Fascinating to see a possible mechanism for linking kinase-related signalling (phosphorylation) with calcium mediated signalling. The order of various early defense-related signalling events is still a big question. Also interesting to see plant proteins expressed in a heterologous system of human embryonic kidney cells (HEK293T). Use of a heterologous system can be a powerful tool to determine protein functions/mechanisms but - of course - should always be tempered by an awareness that differences between the native and expressed system. In this case, different kinases could influence results (plants lack some common mammalian kinases and have some unique kinases groups) so that we must be careful with assumptions of equivalence. In this paper the mammalian kinases that phosphorylate the expressed plant RbohF/D were not identified (this is reasonable). But this provides some interesting leads to follow up in planta.

 

 

[Sachie Kimura,Hidetaka Kaya,Tomoko Kawarazaki,Goro Hiraoka,Eriko Senzaki,Masataka Michikawa,Kazuyuki Kuchitsu. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

Vol 1823, (2),  398–405 ]

 

Abstract

 

Reactive oxygen species (ROS) produced by NADPH oxidases play critical roles in signalling and development. Given the high toxicity of ROS, their production is tightly regulated. In Arabidopsis, respiratory burst oxidase homologue F (AtrbohF) encodes NADPH oxidase. Here we characterised the activation of AtRbohF using a heterologous expression system. AtRbohF exhibited ROS-producing activity that was synergistically activated by protein phosphorylation and Ca2+. The two EF-hand motifs of AtRbohF in the N-terminal cytosolic region were crucial for its Ca2+-dependent activation. AtrbohD and AtrbohF are involved in stress responses. Although the activation mechanisms for AtRbohD and AtRbohF were similar, AtRbohD had significantly greater ROS-producing activity than AtRbohF, which may reflect their functional diversity, at least in part. We further characterised the interrelationship between Ca2+ and phosphorylation regarding activation and found that protein phosphorylation-induced activation was independent of Ca2+. In contrast, K-252a, a protein kinase inhibitor, inhibited the Ca2+-dependent ROS-producing activity of AtRbohD and AtRbohF in a dose-dependent manner, suggesting that protein phosphorylation is a prerequisite for the Ca2+-dependent activation of Rboh. Positive feedback regulation of Ca2+ and ROS through AtRbohC has been proposed to play a critical role in root hair tip growth. Our findings suggest that Rboh phosphorylation is the initial trigger for the plant Ca2+-ROS signalling network.

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The Ph1 Locus Suppresses Cdk2-Type Activity during Premeiosis and Meiosis in Wheat

The Ph1 Locus Suppresses Cdk2-Type Activity during Premeiosis and Meiosis in Wheat | Plant Proteomics | Scoop.it

I am really pleased to have contributed to this great paper by Peter Shaw and Graham Moore's groups at JIC. Many aspects of the biology are fascinating: the regulation of chromosome pairing and the counter-intuitive increase in histone phosphorylation when one kinase-containing locus is knocked out. However, the aspect of this work that I refer to most frequently is very practical – the selection and dissection of the starting material. Azahara, Ali and Isabelle did a fantastic job of very patiently and repeatedly making careful sample preparations and that is simply one of the best things you can do when starting a proteomic experiment. No matter how sensitive your mass spec is and how well one can profile complex mixtures, choosing a very specific cell type to start with makes all the rest of the work possible.

 

Abstract

Emma Greer, Azahara C. Martín, Ali Pendle, Isabelle Colas, Alexandra M.E. Jones, Graham Moore, and Peter Shaw

 

Despite possessing multiple sets of related (homoeologous) chromosomes, hexaploid wheat (Triticum aestivum) restricts pairing to just true homologs at meiosis. Deletion of a single major locus, Pairing homoeologous1 (Ph1), allows pairing of homoeologs. How can the same chromosomes be processed as homologs instead of being treated as nonhomologs? Ph1 was recently defined to a cluster of defective cyclin-dependent kinase (Cdk)-like genes showing some similarity to mammalian Cdk2. We reasoned that the cluster might suppress Cdk2-type activity and therefore affect replication and histone H1 phosphorylation. Our study does indeed reveal such effects, suggesting that Cdk2-type phosphorylation has a major role in determining chromosome specificity during meiosis.

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Cell - A Mechanism for the Evolution of Phosphorylation Sites

Cell - A Mechanism for the Evolution of Phosphorylation Sites | Plant Proteomics | Scoop.it

Sometimes you read two papers and wish that all of the authors had got together for a collaborative study. This is one of these times. I like the idea that phosphorylation of Ser and Thr residues could substitute for salt bridges between Asp-Glu and I would have loved to see how these fit in - or influenced – work by Nishi et al that I scooped in December (Phosphorylation in protein:protein binding). In particular I would have liked to see if there is a difference in the interface binding between Ser/Thr versus Tyr phosphorylation sites.

 

Summary

Protein phosphorylation provides a mechanism for the rapid, reversible control of protein function. Phosphorylation adds negative charge to amino acid side chains, and negatively charged amino acids (Asp/Glu) can sometimes mimic the phosphorylated state of a protein. Using a comparative genomics approach, we show that nature also employs this trick in reverse by evolving serine, threonine, and tyrosine phosphorylation sites from Asp/Glu residues. Structures of three proteins where phosphosites evolved from acidic residues (DNA topoisomerase II, enolase, and C-Raf) show that the relevant acidic residues are present in salt bridges with conserved basic residues, and that phosphorylation has the potential to conditionally restore the salt bridges. The evolution of phosphorylation sites from glutamate and aspartate provides a rationale for why phosphorylation sometimes activates proteins, and helps explain the origins of this important and complex process.

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Quantitative proteomics reveals dynamic changes in the plasma membrane proteome during Arabidopsis immune signaling

(James Mitch Elmore,Jun Liu,Barrett Smith,Brett Phinney and Gitta Coaker*)

 

Abstract

The plant plasma membrane is a crucial mediator of the interaction between plants and microbes. Understanding how the plasma membrane proteome responds to diverse immune signaling events will lead to a greater understanding of plant immunity and uncover novel targets for crop improvement. Here we report the results from a large-scale quantitative proteomics study of plasma membrane-enriched fractions upon activation of the Arabidopsis thaliana immune receptor RPS2. More than 2300 proteins were identified in total with 1353 proteins reproducibly identified across multiple replications. Label-free spectral counting was employed to quantify relative protein abundance between different treatment samples. Over 20% of upregulated proteins have known roles in plant immune responses. Significantly changing proteins include those involved in calcium and lipid signaling, membrane transport, primary and secondary metabolism, protein phosphorylation, redox homeostasis, and vesicle trafficking. A subset of differentially regulated proteins was independently validated during bacterial infection. This study presents the largest quantitative proteomics dataset of plant immunity to date and provides a framework for understanding global plasma membrane proteome dynamics during plant immune responses.

 

 

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Structural Analysis of Pseudomonas syringae AvrPtoB Bound to Host BAK1 Reveals Two Similar Kinase-Interacting Domains in a Type III Effector

This was a fascinating paper for me. I have a long, if patchy, history with AvrPtoB and BAK1 and it is very satisfying to see how this story is unfolding over time, through the efforts of various research groups. I first because aware of AvrPtoB at Wye College, through the lectures of John Mansfield. Years later I measured the physical binding properties of AvrPtoB and Pto kinase with Bart Feys at Imperial College (unpublished work). As soon as I arrived at the Sainsbury lab I worked with Antje Hesse and John Rathjen and identified BAK1 as interacting with FLS2 (Hesse et al PNAS 2007). Later, with Vardis Ntoukakis and John Rathjen, I worked on the inhibition of Pto (and related kinases) by AvrPtoB (Ntoukakis et al Science 2009). After the publication of the Science paper I had a long discussion with John Mansfield about the apparent paradox between the dispensable ubiquitin ligase activity and our finding that the Pto-like, Fen, kinase is ubiquitinated by AvrPtoB. More recently I have returned to working with BAK1 with Cyril Zipfel’s group (Schwessinger et al PLOS Biology 2011). The duplicated kinase-binding structure of AvrPtoB provides some resolution to the complexities of AvrPtoB’s many interactions with various defence-related kinases.

 

Summary

To infect plants, Pseudomonas syringae pv. tomato delivers ∼30 type III effector proteins into host cells, many of which interfere with PAMP-triggered immunity (PTI). One effector, AvrPtoB, suppresses PTI using a central domain to bind host BAK1, a kinase that acts with several pattern recognition receptors to activate defense signaling. A second AvrPtoB domain binds and suppresses the PTI-associated kinase Bti9 but is conversely recognized by the protein kinase Pto to activate effector-triggered immunity. We report the crystal structure of the AvrPtoB-BAK1 complex, which revealed structural similarity between these two AvrPtoB domains, suggesting that they arose by intragenic duplication. The BAK1 kinase domain is structurally similar to Pto, and a conserved region within both BAK1 and Pto interacts with AvrPtoB. BAK1 kinase activity is inhibited by AvrPtoB, and mutations at the interaction interface disrupt AvrPtoB virulence activity. These results shed light on a structural mechanism underlying host-pathogen coevolution.

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The International Proteomics Tutorial Programme

The International Proteomics Tutorial Programme | Plant Proteomics | Scoop.it

An Initiative to create an Open-Access Teaching Resource.

 

This is worth following and as the slides are open access I hope this will develop and be maintaned as a great teaching resource.

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Proteome Science | Full text | Identification of a novel Plasmopara halstedii elicitor protein combining de novo peptide sequencing algorithms and RACE-PCR

Often high-quality MS/MS spectra of tryptic peptides do not match to any database entry because of only partially sequenced genomes and therefore, protein identification requires de novo peptide sequencing.
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Receptor Kinase Signaling Pathways in Plant-Microbe Interactions - Annual Review of Phytopathology, 50(1):451

Receptor Kinase Signaling Pathways in Plant-Microbe Interactions - Annual Review of Phytopathology, 50(1):451 | Plant Proteomics | Scoop.it

A very enjoyable review: Although the FLS2 CLV3 debate is not raised, the wide overview of structural studies and pathogenic and symboitic RLKS is nice.

 

Meritxell Antolín-Llovera, Martina K. Ried, Andreas Binder, and Martin Parniske

 

ABSTRACT

Plant receptor-like kinases (RLKs) function in diverse signaling pathways, including the responses to microbial signals in symbiosis and defense. This versatility is achieved with a common overall structure: an extracytoplasmic domain (ectodomain) and an intracellular protein kinase domain involved in downstream signal transduction. Various surfaces of the leucine-rich repeat (LRR) ectodomain superstructure are utilized for interaction with the cognate ligand in both plant and animal receptors. RLKs with lysin-motif (LysM) ectodomains confer recognitional specificity toward N-acetylglucosamine-containing signaling molecules, such as chitin, peptidoglycan (PGN), and rhizobial nodulation factor (NF), that induce immune or symbiotic responses. Signaling downstream of RLKs does not follow a single pattern; instead, the detailed analysis of brassinosteroid (BR) signaling, innate immunity, and symbiosis revealed at least three largely nonoverlapping pathways. In this review, we focus on RLKs involved in plant-microbe interactions and contrast the signaling pathways leading to symbiosis and defense.

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Commentaries and Letters to the Editor of The Plant Cell on recognition specificity of the FLS2 receptor (2012)

Does the Arabidopsis receptor FLS2 respond to CLV3 and Ax21 peptides? "The editors of The Plant Cell feel that they have taken responsibility for opening up the scientific debate on FLS2 and innate immune signaling, and they have gone as far as possible to ensure a fair and balanced discussion of issues of particular interest to its readership":

 

Cathie Martin. Commentaries and Letters to the Editor of The Plant Cell http://tinyurl.com/d6mgbmf

 

Katharina Mueller, Delphine Chinchilla, Markus Albert, Anna K. Jehle, Hubert Kalbacher, Thomas Boller, and Georg Felix. Contamination Risks in Work with Synthetic Peptides: flg22 as an Example of a Pirate in Commercial Peptide Preparations http://tinyurl.com/c2u3pr6

 

Cécile Segonzac, Zachary L. Nimchuk, Martina Beck, Paul T. Tarr, Silke Robatzek, Elliot M. Meyerowitz, and Cyril Zipfel. The Shoot Apical Meristem Regulatory Peptide CLV3 Does Not Activate Innate Immunity http://tinyurl.com/bslnq4o

 

Horim Lee, Ashok Khatri, Julia M. Plotnikov, Xue-Cheng Zhang, and Jen Sheen. Complexity in Differential Peptide–Receptor Signaling: Response to Segonzac et al. and Mueller et al. Commentaries http://tinyurl.com/c2csqm3

 

Cristian H. Danna, Xue-Cheng Zhang, Ashok Khatri, Andrew F. Bent, Pamela C. Ronald, and Frederick M. Ausubel. FLS2-Mediated Responses to Ax21-Derived Peptides: Response to the Mueller et al. Commentary http://tinyurl.com/d2a36cr

 

Mueller et al. also used the CLV3 peptide as negative control in this Plant cell paper: http://tinyurl.com/bszkm24

 

These are the two contested papers:

 

Danna, C.H., Millet, Y.A., Koller, T., Han, S.W., Bent, A.F., Ronald, P.C., and Ausubel, F.M. (2011). The Arabidopsis flagellin receptor FLS2 mediates the perception of Xanthomonas Ax21 secreted peptides. Proc. Natl. Acad. Sci. USA 108: 9286–9291. http://tinyurl.com/brwkb4w

 

Lee, H., Chah, O.-K., and Sheen, J. (2011). Stem-cell-triggered immunity through CLV3p-FLS2 signalling. Nature 473: 376–379. http://tinyurl.com/3thgqut


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ER-Quality Control Chaperones Facilitate the Biogenesis of Cf Receptor-Like Proteins Involved in Pathogen Resistance of Tomato

ER-Quality Control Chaperones Facilitate the Biogenesis of Cf Receptor-Like Proteins Involved in Pathogen Resistance of Tomato | Plant Proteomics | Scoop.it

Cf proteins are receptor-like proteins (RLPs) that mediate resistance of tomato (Solanum lycopersicum) to the foliar pathogen Cladosporium fulvum. These transmembrane immune receptors, which carry extracellular leucine-rich repeats (LRRs) that are subjected to post-translational glycosylation, perceive effectors of the pathogen and trigger a defense response that results in plant resistance. To identify proteins required for the functionality of these RLPs, we performed immunopurification of a functional Cf-4-eGFP fusion protein transiently expressed in Nicotiana benthamiana, followed by mass spectrometry. The endoplasmic reticulum (ER) HSP70 binding proteins (BiPs) and lectin-type calreticulins (CRTs), which are chaperones involved in ER-Quality Control (ER-QC), were co-purifying with Cf-4-eGFP. The tomato and N. benthamiana genomes encode four BiP homologs and silencing experiments revealed that these BiPs are important for overall plant viability. For the three tomato CRTs, VIGS targeting the plant-specific CRT3a gene resulted in a significantly compromised Cf-4-mediated defense response and loss of full resistance to C. fulvum. We show that upon knock-down of CRT3a the Cf-4 protein accumulated, but the pool of Cf-4 protein carrying complex-type N-linked glycans was largely reduced. Together, our study on proteins required for Cf function reveals a unique role for the calreticulins in the biogenesis and functionality of this type of RLPs involved in plant defense.

 

Thomas W.H. Liebrand, Patrick Smit, Ahmed Abd-El-Haliem, Ronnie de Jonge, Jan H.G. Cordewener, Antoine H.P. America, Jan Sklenar, Alexandra M.E. Jones, Silke Robatzek, Bart P.H.J. Thomma, Wladimir I.L. Tameling and Matthieu H.A.J. Joosten


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Nature Methods correspondence on phosphorylation site stiochiometry

In Nature Methods correspondence this month [Vol:9 (2012)DOI:doi:10.1038/nmeth.1941] there is an exchange between Chris Soon Heng Tan, Gary Bader and Steve Gygi et al. I always enjoy such public exchanges; they feel very much like observing science in action. Here Tan and Bader pick up on a point from a paper by Wu et al (Nat Meth, 2011, 8, 677-83) that described the stiochiometries of 5000 yeast phosphorylation sites. Wu et al mentioned that lower occupancy phosphorylation sites appeared to be more highly conserved. I guess this struck Tan and Bader as counterintuitive as they did a more detailed analysis of the absolute abundances of the proteins and concluded that phosphorylation sites of higher stoichiometry are more conserved.

 

I am glad to see Tan and Bader also mention that the limit of detection by mass spectrometry may be a reason why low occupancy phosphorylation sites on lower abundance proteins are under reported. I think this is true and remains a technical challenge. Of course our instruments have limits of detection and this is all too often forgotten by those discussing absence. It is great to see this level of measurement and discussion and at my own small scale I have frequently argued against the idea that ‘phosphorylation correlates with activation’; these may be true for certain enzymes but this lazy line of thinking was particularly popular in papers using 2D gel electrophoresis.

 

But I am left with a feeling of bewilderment, not so much from these papers but from the ideas that follow. Certainly for building mathematical models stoichiometry is important, and conceptually it is very appealing too. But I wonder how a cell measures such things*? If 1% phosphorylation of an abundant protein and 90% phosphorylation of a rarer protein, both equal about 100 phosphosrylated molecules -  is that functionally equivalent? Of course that is naive too; what is the function of the protein, what does it interact with and how are these altered by x% phosphorylation? I suspect that a cell operates on a fuzzy ‘just about enough’ system that is very robust. Anyway as good papers should do, this correspondence has stimulated thought and wonder.

 

(*This question has been bugging me a lot recently along with its corollary; are we taking the correct measurements?)

 

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Suppression of EGFR endocytosis by dynamin delpetion...

Interesting work looking at where signalling occurs. In keeping with my comparing papers, I would very much like to know how TGFb behaves in a similar experiment. The case for TGFb endosomal signalling is strong (I think) so perhaps dynamin delpetion would have quite a different result in that pathway.

 

Suppression of EGFR endocytosis by dynamin depletion reveals that EGFR signaling occurs primarily at the plasma membrane.

 

Sousa et al.

Abstract: The role of endocytosis in the control of EGF receptor (EGFR) activation and cell signaling was explored by using mouse fibroblasts in which dynamin was conditionally depleted. Dynamin is a GTPase shown to play an important role in the control clathrin mediated endocytosis of EGFR and other cell surface receptors. In this report, we demonstrate that EGF binding activity and the display of high and low affinity EGFRs on the cell surface are not affected by dynamin depletion. By contrast, dynamin depletion leads to a strong inhibition of EGFR endocytosis, robust enhancement of EGFR autophosphorylation and ubiquitination, and slower kinetics of EGFR degradation. Surprisingly, MAPK stimulation induced by either low or high EGF concentrations is not affected by dynamin depletion. While a similar initial Akt response is detected in control or dynamin depleted fibroblasts, a somewhat more sustained Akt stimulation is detected in the dynamin depleted cells. These experiments demonstrate that dynamin-mediated endocytosis leads to attenuation of EGFR activation and degradation and that stimulation of the MAPK response and Akt activation are primarily mediated by activated EGFR located in the plasma membrane

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Effector-Mediated Suppression of Chitin-Triggered Immunity by Magnaporthe oryzae Is Necessary for Rice Blast Disease

Abstract

 

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.

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Open science is a research accelerator : Nature Chemistry : Nature Publishing Group

Open science is a research accelerator : Nature Chemistry : Nature Publishing Group | Plant Proteomics | Scoop.it

Data analysis in proteomics is becoming a real bottleneck for us. Many projects identify several hundred proteins (if not thousands) and we are trying various methods to quantify them. The questions each project asks are often different and so we use a wide variety of programs, each with its own learning curve and errors, to address these. 

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Phosphorylation in Protein-Protein Binding: Effect on Stability and Function

Not a plant paper, but it is nice to see some testing of the very long standing and common assumptions about how phosphorylation regulates proteins.

 

 

Summary

Posttranslational modifications offer a dynamic way to regulate protein activity, subcellular localization, and stability. Here we estimate the effect of phosphorylation on protein binding and function for different types of complexes from human proteome. We find that phosphorylation sites tend to be located on binding interfaces in heterooligomeric and weak transient homooligomeric complexes. Analysis of molecular mechanisms of phosphorylation shows that phosphorylation may modulate the strength of interactions directly on interfaces and that binding hotspots tend to be phosphorylated in heterooligomers. Although the majority of complexes do not show significant estimated stability differences upon phosphorylation or dephosphorylation, for about one-third of all complexes it causes relatively large changes in binding energy. We discuss the cases where phosphorylation mediates the complex formation and regulates the function. We show that phosphorylation sites are more likely to be evolutionary conserved than other interfacial residues.

 

 

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PNAS: Phytophthora infestans effector AVRblb2 prevents secretion of a plant immune protease at the haustorial interface

PNAS: Phytophthora infestans effector AVRblb2 prevents secretion of a plant immune protease at the haustorial interface | Plant Proteomics | Scoop.it

In response to pathogen attack, plant cells secrete antimicrobial molecules at the site of infection. However, how plant pathogens interfere with defense-related focal secretion remains poorly known. Here we show that the host-translocated RXLR-type effector protein AVRblb2 of the Irish potato famine pathogen Phytophthora infestans focally accumulates around haustoria, specialized infection structures that form inside plant cells, and promotes virulence by interfering with the execution of host defenses. AVRblb2 significantly enhances susceptibility of host plants to P. infestans by targeting the host papain-like cysteine protease C14 and specifically preventing its secretion into the apoplast. Plants altered in C14 expression were significantly affected in susceptibility to P. infestans in a manner consistent with a positive role of C14 in plant immunity. Our findings point to a unique counterdefense strategy that plant pathogens use to neutralize secreted host defense proteases. Effectors, such as AVRblb2, can be used as molecular probes to dissect focal immune responses at pathogen penetration sites.


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