PRGL: A cell wall proline-rich protein containning GASA domain in Gerbera hybrida

PRPs (proline-rich proteins) are a group of cell wall proteins characterized by their proline and hy- droproline-rich repetitive peptides. The expression of PRPs in plants is stimulated by wounding and environmental stress. GASA (gibberellic acid stimulated in Arabidopsis) proteins are small peptides sharing a 60 amino acid conserved C-terminal domain containing twelve invariant cysteine residues. Most of GASAs reported are localized to apoplasm or cell wall and their expression was regulated by gibberellins (GAs). It has been reported that, in French bean, these two proteins encoding by two distinct genes formed a two-component chitin-receptor involved in plant-pathogen interactions when plant was infected. We cloned a full-length cDNA of PRGL (proline-rich GASA-like) gene which encodes a protein containing both PRP and GASA-like domains. It is demonstrated that PRGL is a new protein with characteristics of PRP and GASA by analyzing its protein structure and gene expression.     

Characterization of PhPRP1, a histidine domain arabinogalactan protein from Petunia hybrida pistils

An arabinogalactan protein, PhPRP1, was purified from Petunia hybrida pistils and shown to be orthologous to TTS-1 and TTS-2 from Nicotiana tabacum and NaTTS from Nicotiana alata. Sequence comparisons among these proteins, and CaPRP1 from Capsicum annuum, reveal a conserved histidine-rich domain and two hypervariable domains. Immunoblots show that TTS-1 and PhPRP1 are also expressed in vegetative tissues of tobacco and petunia respectively. In contrast to the molecular mass heterogeneity displayed by the pistil proteins, the different isoforms found in seedlings, roots, and leaves each has a discrete size (37, 80, 160, and 200 kDa) on SDS-PAGE gels. On the basis of their chemistry, distinctive domain architecture, and the unique pattern of expression, we have named this group of proteins HD-AGPs (histidine domain-arabinogalactan proteins).     

蛋白磷酸化修饰在植物-病原微生物互作中的作用研究进展

蛋白磷酸化修饰是植物细胞信号调控的普遍机制。植物-病原微生物互作过程中, 关键调控蛋白的磷酸化状态影响免疫信号的激活。多种病原微生物通过干扰宿主蛋白的磷酸化状态攻击免疫系统, 以提高致病性。该文对植物免疫调控过程中关键元件的磷酸化修饰及其在免疫信号中的调控作用进行了综述。研究植物-病原菌互作过程中关键蛋白的磷酸化修饰, 有助于深入探讨植物-病原微生物互作的分子机理。该文将为寻找广谱抗病的新途径提供理论依据。     

Plant-induced cell death in the oomycete pathogen Phytophthora parasitica

The activation of programmed cell death in the host during plant-pathogen interactions is an important component of the plant disease resistance mechanism. In this study we show that activation of programmed cell death in microorganisms also regulates plant-pathogen interactions. We found that a form of vacuolar cell death is induced in the oomycete Phytophthora parasitica--the agent that causes black shank disease in Nicotiana tabacum--by extracellular stimuli from resistant tobacco. The single-celled zoospores underwent cell death characterized by dynamic membrane rearrangements, cell shrinkage, formation of numerous large vacuoles in the cytoplasm and degradation of cytoplasmic components before plasma membrane disruption. Phytophthora cell death required protein synthesis but not caspase activation, and was associated with the production of intracellular reactive oxygen species. This characterization of plant-mediated cell death signalling in pathogens will enhance our understanding of the biological processes regulating plant-pathogen interactions, and improve our ability to control crop diseases.     

Two-component elements mediate interactions between cytokinin and salicylic acid in plant immunity

Recent studies have revealed an important role for hormones in plant immunity. We are now beginning to understand the contribution of crosstalk among different hormone signaling networks to the outcome of plant-pathogen interactions. Cytokinins are plant hormones that regulate development and responses to the environment. Cytokinin signaling involves a phosphorelay circuitry similar to two-component systems used by bacteria and fungi to perceive and react to various environmental stimuli. In this study, we asked whether cytokinin and components of cytokinin signaling contribute to plant immunity. We demonstrate that cytokinin levels in Arabidopsis are important in determining the amplitude of immune responses, ultimately influencing the outcome of plant-pathogen interactions. We show that high concentrations of cytokinin lead to increased defense responses to a virulent oomycete pathogen, through a process that is dependent on salicylic acid (SA) accumulation and activation of defense gene expression. Surprisingly, treatment with lower concentrations of cytokinin results in increased susceptibility. These functions for cytokinin in plant immunity require a host phosphorelay system and are mediated in part by type-A response regulators, which act as negative regulators of basal and pathogen-induced SA-dependent gene expression. Our results support a model in which cytokinin up-regulates plant immunity via an elevation of SA-dependent defense responses and in which SA in turn feedback-inhibits cytokinin signaling. The crosstalk between cytokinin and SA signaling networks may help plants fine-tune defense responses against pathogens.     

Very long chain fatty acid and lipid signaling in the response of plants to pathogens

Recent findings indicate that lipid signaling is essential for plant resistance to pathogens. Besides oxylipins and unsaturated fatty acids known to play important signaling functions during plant-pathogen interactions, the very long chain fatty acid (VLCFA) biosynthesis pathway has been recently associated to plant defense through different aspects. VLCFAs are indeed required for the biosynthesis of the plant cuticle and the generation of sphingolipids. Elucidation of the roles of these lipids in biotic stress responses is the result of the use of genetic approaches together with the identification of the genes/proteins involved in their biosynthesis. This review focuses on recent observations which revealed the complex function of the cuticle and cuticle-derived signals, and the key role of sphingolipids as bioactive molecules involved in signal transduction and cell death regulation during plant-pathogen interactions.Key words: very long chain fatty acids (VLCFAs), plant-pathogen interactions, lipid signaling, sphingolipids, epicuticular waxes, lipid rafts, cuticle, plant defense     

Strangers in the matrix: plant cell walls and pathogen susceptibility

Early in infection, pathogens encounter the outer wall of plant cells. Because pathogen hydrolases targeting the plant cell wall are well-known components of virulence, it has been assumed that wall disassembly by the plant itself also contributes to susceptibility, and now this has been established experimentally. Understanding how plant morphological and developmental remodeling and pathogen cell wall targeted virulence influence infections provides new perspectives about plant-pathogen interactions. The plant cell wall can be an effective physical barrier to pathogens, but also it is a matrix where many proteins involved in pathogen perception are delivered. By breaching the wall, a pathogen potentially reveals itself to the plant and activates responses, setting off events that might halt or limit its advance.     

Searching for potential drug targets in two-component and phosphorelay signal-transduction systems using three-dimensional cluster analysis

Two-component and phosphorelay signal transduction systems are central components in the virulence and antimicrobial resistance responses of a number of bacterial and fungal pathogens; in some cases, these systems are essential for bacterial growth and viability. Herein, we analyze in detail the conserved surface residue clusters in the phosphotransferase domain of histidine kinases and the regulatory domain of response regulators by using complex structure-based three-dimensional cluster analysis. We also investigate the protein-protein interactions that these residue clusters participate in. The Spo0B-Spo0F complex structure was used as the reference structure, and the multiple aligned sequences of phosphotransferases and response regulators were paired correspondingly. The results show that a contiguous conserved residue cluster is formed around the active site, which crosses the interface of histidine kinases and response regulators. The conserved residue clusters of phosphotransferase and the regulatory domains are directly involved in the functional implementation of two-component signal transduction systems and are good targets for the development of novel antimicrobial agents.     

Functional association of essential splicing factor(s) with PRP19 in a protein complex.

We have previously shown that the yeast PRP19 protein is a spliceosomal component, but is not tightly associated with small nuclear RNAs. It appears to associate with the spliceosome concomitant with or just after dissociation of the U4 small nuclear RNA during spliceosome assembly. We have found that PRP19 is associated with a protein complex in the splicing extract and that at least one of the associated components is essential for splicing. Taking advantage of the epitope tagging technique, we have isolated the PRP19-associated complex by affinity chromatography. The isolated complex is functional for complementation for the heat-inactivated prp19 mutant extract, and consists of at least seven polypeptides in addition to PRP19. At least three of these can interact directly with the PRP19 protein. We also show that the PRP19 protein itself is in an oligomeric form, which might be a prerequisite for its interaction with these proteins.     

Is myelin basic protein crystallizable?

Myelin basic protein (MBP) is the predominant extrinsic protein in both central and peripheral nervous system myelins. It is thought to be involved in the stabilizing interactions between myelin membranes, and it may play an important role in demyelinating diseases such as multiple sclerosis. In spite of the fact that this abundant protein has been known for almost three decades, its three-dimensional crystal structure has not yet been determined. In this study we report on our extensive attempts to crystallize the major 18.5 kDa isoform of MBP. We used MBP having different degrees of purity, ranging from crude MBP (that was acid or salt extracted from isolated myelin), to highest purity single isoform. We used conventional strategies in our search for a suitable composition or a crystallization medium. We applied both full and incomplete factorial searches for crystallization conditions. We analyzed the available data on proteins which have previously resisted crystallization, and applied this information to our own experiments. Nevertheless, despite our efforts which included 4600 different conditions, we were unable to induce crystallization of MBP. Previous work on MBP indicates that when it is removed from its native environment in the myelin membrane and put in crystallization media, the protein adopts a random coil conformation and persists as a population of structurally non-identical molecules. This thermodynamically preferred state presumably hinders crystallization, because the most fundamental factor of protein crystallization-homogeneity of tertiary structure-is lacking. We conclude that as long as its random coil flexibility is not suppressed, 18.5 kDa MBP and possibly also its isoforms will remain preeminent examples of proteins that cannot be crystallized.     

Silica deposition by a strongly cationic proline-rich protein from systemically resistant cucumber plants

Infection of one leaf of cucumber (Cucumis sativa) plants can render other leaves resistant to various pathogens. This so-called systemic acquired resistance (SAR) can be functionally mimicked by certain chemicals. All these treatments enhanced expression of a gene encoding a novel proline-rich protein (PRP1) which has C-terminal repetitive sequences containing an unusually high amount of lysine and arginine residues. Antibodies raised against a synthetic peptide derived from four of the repetitive sequences cross-reacted mainly with a cell wall polypeptide of an apparent MW of 8 kDa. The protein accumulated upon SAR induction, though it does not appear to take part in oxidative protein cross-linking, at least in the hypocotyl tissue. The synthetic peptide derived from the repetitive sequences was able to polymerize orthosilicic acid to insoluble silica, a property not resulting directly from the primary protein sequence, but rather from the high positive charge density. Our results suggest that during induction of SAR, the synthesis of a strongly cationic PRP prepares the cell walls for enhanced silica deposition which is known to participate in cell wall reinforcement, localized at the site of attempted penetration of fungi into epidermal cells. Constitutive accumulation of related PRPs may function in silica deposition during certain developmental stages, a process important for various physiological functions of green plants.     

植物与病原菌互作的蛋白质组学研究进展

深入认识植物与病原菌的识别方式、亲和性或非亲和性的互作模式,对于揭示植物-病原菌互作机制研究具有重要意义.利用蛋白质组学方法研究病原菌侵染植物过程,分析相关的基因和蛋白,有助于从分子水平上探究植物-病原菌相互作用机制.本文概述了植物-病原菌的互作机制,系统介绍了差异蛋白质组学分析方法在植物-病原真菌、植物-病原细菌两类互作系统中的应用,分析了植物与病原菌互作过程中可能涉及的差异表达功能蛋白,并对当前蛋白质组学技术在植物与病原菌互作研究中存在的诸多问题进行了探讨.     

Cooperative binding and activation of fibronectin by a bacterial surface protein

Integrin-dependent cell invasion of some pathogenic bacteria is mediated by surface proteins targeting the extracellular matrix protein fibronectin (FN). Although the structural basis for bacterial FN recognition is well understood, it has been unclear why proteins such as streptococcal SfbI contain several FN-binding sites. We used microcalorimetry to reveal cooperative binding of FN fragments to arrays of binding sites in SfbI. In combination with thermodynamic analyses, functional cell-based assays show that SfbI induces conformational changes in the N-terminal 100-kDa region of FN (FN100kDa), most likely by competition with intramolecular interactions defining an inactive state of FN100kDa. This study provides insights into how long range conformational changes resulting in FN activation may be triggered by bacterial pathogens.     

Host pathogen protein interactions predicted by comparative modeling

Pathogens have evolved numerous strategies to infect their hosts, while hosts have evolved immune responses and other defenses to these foreign challenges. The vast majority of host-pathogen interactions involve protein-protein recognition, yet our current understanding of these interactions is limited. Here, we present and apply a computational whole-genome protocol that generates testable predictions of host-pathogen protein interactions. The protocol first scans the host and pathogen genomes for proteins with similarity to known protein complexes, then assesses these putative interactions, using structure if available, and, finally, filters the remaining interactions using biological context, such as the stage-specific expression of pathogen proteins and tissue expression of host proteins. The technique was applied to 10 pathogens, including species of Mycobacterium, apicomplexa, and kinetoplastida, responsible for "neglected" human diseases. The method was assessed by (1) comparison to a set of known host-pathogen interactions, (2) comparison to gene expression and essentiality data describing host and pathogen genes involved in infection, and (3) analysis of the functional properties of the human proteins predicted to interact with pathogen proteins, demonstrating an enrichment for functionally relevant host-pathogen interactions. We present several specific predictions that warrant experimental follow-up, including interactions from previously characterized mechanisms, such as cytoadhesion and protease inhibition, as well as suspected interactions in hypothesized networks, such as apoptotic pathways. Our computational method provides a means to mine whole-genome data and is complementary to experimental efforts in elucidating networks of host-pathogen protein interactions.     

Towards genomic and proteomic studies of protein phosphorylation in plant-pathogen interactions

Phosphorylation is an effective method of post-translational protein modification but understanding its significance is hindered by its biological complexity. Many protein kinases and phosphatases have been identified that connect signal perception mechanisms to plant defence responses. Recent studies of mitogen-activated protein kinases, calcium-dependent protein kinases and other kinases and phosphatases have revealed some important mechanisms, but have also raised new questions. The regulation of any phosphorylation pathway is complex and dynamic. There are many protein kinases and phosphatases in the plant genome, which makes it hard to delineate the phosphorylation machinery fully. Genomics and proteomics have already identified new components and will continue to influence the study of phosphorylation profoundly in plant-pathogen interactions.     

The PRP31 gene encodes a novel protein required for pre-mRNA splicing in Saccharomyces cerevisiae.

The pre-mRNA splicing factor Prp31p was identified in a screen of temperature-sensitive yeast strains for those exhibiting a splicing defect upon shift to the non- permissive temperature. The wild-type PRP31 gene was cloned and shown to be essential for cell viability. The PRP31 gene is predicted to encode a 60 kDa polypeptide. No similarities with other known splicing factors or motifs indicative of protein-protein or RNA-protein interaction domains are discernible in the predicted amino acid sequence. A PRP31 allele bearing a triple repeat of the hemagglutinin epitope has been generated. The tagged protein is functional in vivo and a single polypeptide species of the predicted size was detected by Western analysis with proteins from yeast cell extracts. Functional Prp31p is required for the processing of pre-mRNA species both in vivo and in vitro, indicating that the protein is directly involved in the splicing pathway.