A bacterial E3 ubiquitin ligase targets a host protein kinase to disrupt plant immunity

Many bacterial pathogens of plants and animals use a type III secretion system to deliver diverse virulence-associated 'effector' proteins into the host cell. The mechanisms by which these effectors act are mostly unknown; however, they often promote disease by suppressing host immunity. One type III effector, AvrPtoB, expressed by the plant pathogen Pseudomonas syringae pv. tomato, has a carboxy-terminal domain that is an E3 ubiquitin ligase. Deletion of this domain allows an amino-terminal region of AvrPtoB (AvrPtoB(1-387)) to be detected by certain tomato varieties leading to immunity-associated programmed cell death. Here we show that a host kinase, Fen, physically interacts with AvrPtoB(1-387 )and is responsible for activating the plant immune response. The AvrPtoB E3 ligase specifically ubiquitinates Fen and promotes its degradation in a proteasome-dependent manner. This degradation leads to disease susceptibility in Fen-expressing tomato lines. Various wild species of tomato were found to exhibit immunity in response to AvrPtoB(1-387 )and not to full-length AvrPtoB. Thus, by acquiring an E3 ligase domain, AvrPtoB has thwarted a highly conserved host resistance mechanism.     

A Xanthomonas uridine 5'-monophosphate transferase inhibits plant immune kinases

Plant innate immunity is activated on the detection of pathogen-associated molecular patterns (PAMPs) at the cell surface, or of pathogen effector proteins inside the plant cell. Together, PAMP-triggered immunity and effector-triggered immunity constitute powerful defences against various phytopathogens. Pathogenic bacteria inject a variety of effector proteins into the host cell to assist infection or propagation. A number of effector proteins have been shown to inhibit plant immunity, but the biochemical basis remains unknown for the vast majority of these effectors. Here we show that the Xanthomonas campestris pathovar campestris type III effector AvrAC enhances virulence and inhibits plant immunity by specifically targeting Arabidopsis BIK1 and RIPK, two receptor-like cytoplasmic kinases known to mediate immune signalling. AvrAC is a uridylyl transferase that adds uridine 5'-monophosphate to and conceals conserved phosphorylation sites in the activation loop of BIK1 and RIPK, reducing their kinase activity and consequently inhibiting downstream signalling.     

The structural basis for activation of plant immunity by bacterial effector protein AvrPto

Pathogenic microbes use effectors to enhance susceptibility in host plants. However, plants have evolved a sophisticated immune system to detect these effectors using cognate disease resistance proteins, a recognition that is highly specific, often elicits rapid and localized cell death, known as a hypersensitive response, and thus potentially limits pathogen growth. Despite numerous genetic and biochemical studies on the interactions between pathogen effector proteins and plant resistance proteins, the structural bases for such interactions remain elusive. The direct interaction between the tomato protein kinase Pto and the Pseudomonas syringae effector protein AvrPto is known to trigger disease resistance and programmed cell death through the nucleotide-binding site/leucine-rich repeat (NBS-LRR) class of disease resistance protein Prf. Here we present the crystal structure of an AvrPto-Pto complex. Contrary to the widely held hypothesis that AvrPto activates Pto kinase activity, our structural and biochemical analyses demonstrated that AvrPto is an inhibitor of Pto kinase in vitro. The AvrPto-Pto interaction is mediated by the phosphorylation-stabilized P+1 loop and a second loop in Pto, both of which negatively regulate the Prf-mediated defences in the absence of AvrPto in tomato plants. Together, our results show that AvrPto derepresses host defences by interacting with the two defence-inhibition loops of Pto.     

Mediation of pathogen resistance by exudation of antimicrobials from roots

Most plant species are resistant to most potential pathogens. It is not known why most plant-microbe interactions do not lead to disease, although recent work indicates that this basic disease resistance is multi-factorial. Here we show that the exudation of root-derived antimicrobial metabolites by Arabidopsis thaliana confers tissue-specific resistance to a wide range of bacterial pathogens. However, a Pseudomonas syringae strain that is both at least partly resistant to these compounds and capable of blocking their synthesis/exudation is able to infect the roots and cause disease. We also show that the ability of this P. syringae strain to block antimicrobial exudation is dependent on the type III secretory system.     

The assembly of a GTPase-kinase signalling complex by a bacterial catalytic scaffold

The fidelity and specificity of information flow within a cell is controlled by scaffolding proteins that assemble and link enzymes into signalling circuits. These circuits can be inhibited by bacterial effector proteins that post-translationally modify individual pathway components. However, there is emerging evidence that pathogens directly organize higher-order signalling networks through enzyme scaffolding, and the identity of the effectors and their mechanisms of action are poorly understood. Here we identify the enterohaemorrhagic Escherichia coli O157:H7 type III effector EspG as a regulator of endomembrane trafficking using a functional screen, and report ADP-ribosylation factor (ARF) GTPases and p21-activated kinases (PAKs) as its relevant host substrates. The 2.5?? crystal structure of EspG in complex with ARF6 shows how EspG blocks GTPase-activating-protein-assisted GTP hydrolysis, revealing a potent mechanism of GTPase signalling inhibition at organelle membranes. In addition, the 2.8?? crystal structure of EspG in complex with the autoinhibitory Iα3-helix of PAK2 defines a previously unknown catalytic site in EspG and provides an allosteric mechanism of kinase activation by a bacterial effector. Unexpectedly, ARF and PAKs are organized on adjacent surfaces of EspG, indicating its role as a 'catalytic scaffold' that effectively reprograms cellular events through the functional assembly of GTPase-kinase signalling complex.     

Modulation of an RNA-binding protein by abscisic-acid-activated protein kinase

Protein kinases are involved in stress signalling in both plant and animal systems. The hormone abscisic acid mediates the responses of plants to stresses such as drought, salinity and cold. Abscisic-acid-activated protein kinase (AAPK -- found in guard cells, which control stomatal pores -- has been shown to regulate plasma membrane ion channels. Here we show that AAPK-interacting protein 1 (AKIP1), with sequence homology to heterogeneous nuclear RNA-binding protein A/B, is a substrate of AAPK. AAPK-dependent phosphorylation is required for the interaction of AKIP1 with messenger RNA that encodes dehydrin, a protein implicated in cell protection under stress conditions. AAPK and AKIP1 are present in the guard-cell nucleus, and in vivo treatment of such cells with abscisic acid enhances the partitioning of AKIP1 into subnuclear foci which are reminiscent of nuclear speckles. These results show that phosphorylation-regulated RNA target discrimination by heterogeneous nuclear RNA-binding proteins may be a general phenomenon in eukaryotes, and implicate a plant hormone in the regulation of protein dynamics during rapid subnuclear reorganization.     

Maintenance of an unfolded polypeptide by a cognate chaperone in bacterial type III secretion.

Many bacterial pathogens use a type III protein secretion system to deliver virulence effector proteins directly into the host cell cytosol, where they modulate cellular processes. A requirement for the effective translocation of several such effector proteins is the binding of specific cytosolic chaperones, which typically interact with discrete domains in the virulence factors. We report here the crystal structure at 1.9 A resolution of the chaperone-binding domain of the Salmonella effector protein SptP with its cognate chaperone SicP. The structure reveals that this domain is maintained in an extended, unfolded conformation that is wound around three successive chaperone molecules. Short segments from two different SptP molecules are juxtaposed by the chaperones, where they dimerize across a hydrophobic interface. These results imply that the chaperones associated with the type III secretion system maintain their substrates in a secretion-competent state that is capable of engaging the secretion machinery to travel through the type III apparatus in an unfolded or partially folded manner.     

Structural basis for overhang-specific small interfering RNA recognition by the PAZ domain

Short RNAs mediate gene silencing, a process associated with virus resistance, developmental control and heterochromatin formation in eukaryotes. RNA silencing is initiated through Dicer-mediated processing of double-stranded RNA into small interfering RNA (siRNA). The siRNA guide strand associates with the Argonaute protein in silencing effector complexes, recognizes complementary sequences and targets them for silencing. The PAZ domain is an RNA-binding module found in Argonaute and some Dicer proteins and its structure has been determined in the free state. Here, we report the 2.6 A crystal structure of the PAZ domain from human Argonaute eIF2c1 bound to both ends of a 9-mer siRNA-like duplex. In a sequence-independent manner, PAZ anchors the 2-nucleotide 3' overhang of the siRNA-like duplex within a highly conserved binding pocket, and secures the duplex by binding the 7-nucleotide phosphodiester backbone of the overhang-containing strand and capping the 5'-terminal residue of the complementary strand. On the basis of the structure and on binding assays, we propose that PAZ might serve as an siRNA-end-binding module for siRNA transfer in the RNA silencing pathway, and as an anchoring site for the 3' end of guide RNA within silencing effector complexes.     

Genome sequence of the plant pathogen Ralstonia solanacearum

Ralstonia solanacearum is a devastating, soil-borne plant pathogen with a global distribution and an unusually wide host range. It is a model system for the dissection of molecular determinants governing pathogenicity. We present here the complete genome sequence and its analysis of strain GMI1000. The 5.8-megabase (Mb) genome is organized into two replicons: a 3.7-Mb chromosome and a 2.1-Mb megaplasmid. Both replicons have a mosaic structure providing evidence for the acquisition of genes through horizontal gene transfer. Regions containing genetically mobile elements associated with the percentage of G+C bias may have an important function in genome evolution. The genome encodes many proteins potentially associated with a role in pathogenicity. In particular, many putative attachment factors were identified. The complete repertoire of type III secreted effector proteins can be studied. Over 40 candidates were identified. Comparison with other genomes suggests that bacterial plant pathogens and animal pathogens harbour distinct arrays of specialized type III-dependent effectors.     

Type VI secretion delivers bacteriolytic effectors to target cells

Peptidoglycan is the major structural constituent of the bacterial cell wall, forming a meshwork outside the cytoplasmic membrane that maintains cell shape and prevents lysis. In Gram-negative bacteria, peptidoglycan is located in the periplasm, where it is protected from exogenous lytic enzymes by the outer membrane. Here we show that the type VI secretion system of Pseudomonas aeruginosa breaches this barrier to deliver two effector proteins, Tse1 and Tse3, to the periplasm of recipient cells. In this compartment, the effectors hydrolyse peptidoglycan, thereby providing a fitness advantage for P. aeruginosa cells in competition with other bacteria. To protect itself from lysis by Tse1 and Tse3, P. aeruginosa uses specific periplasmically localized immunity proteins. The requirement for these immunity proteins depends on intercellular self-intoxication through an active type VI secretion system, indicating a mechanism for export whereby effectors do not access donor cell periplasm in transit.     

The RNA-binding protein FCA is an abscisic acid receptor

The phytohormone abscisic acid (ABA) regulates various physiological processes in plants. The molecular mechanisms by which this is achieved are not fully understood. Genetic approaches have characterized several downstream components of ABA signalling, but a receptor for ABA has remained elusive. Although studies indicate that several ABA response genes encode RNA-binding or RNA-processing proteins, none has been found to be functional in binding ABA. Here we show that FCA, an RNA-binding protein involved in flowering, binds ABA with high affinity in an interaction that is stereospecific and follows receptor kinetics. The interaction between FCA and ABA has molecular effects on downstream events in the autonomous floral pathway and, consequently, on the ability of the plant to undergo transition to flowering. We further show that ABA binding exerts a direct control on the FCA-mediated processing of precursor messenger RNA. Our results indicate that FCA is an ABA receptor involved in RNA metabolism and in controlling flowering time.     

植物细胞壁中富含甘氨酸的蛋白质

在植物的某些组织中存在富含甘氨酸的蛋白质，根据ＧＲＰｓ中甘氨酸的含量和氨基酸的种类、数目及排列顺序的差异，推测植物的ＧＲＰｓ存在３种类型，这些ＧＲＰｓ在植物的生长发育及植物防御过程中起重要作用，已有大量的ＧＲＰ基因被分离鉴定，并对一些ＧＲＰ基因的启动子进行了分析，ＧＲＰｓ基因的表达受植物发育的调节，具有组织或器官特异性，很多ＧＲＰ基因的表达还受环境因素的影响。     

bicoid RNA localization requires specific binding of an endosomal sorting complex

bicoid messenger RNA localizes to the anterior of the Drosophila egg, where it is translated to form a morphogen gradient of Bicoid protein that patterns the head and thorax of the embryo. Although bicoid was the first localized cytoplasmic determinant to be identified, little is known about how the mRNA is coupled to the microtubule-dependent transport pathway that targets it to the anterior, and it has been proposed that the mRNA is recognized by a complex of many redundant proteins, each of which binds to the localization element in the 3' untranslated region (UTR) with little or no specificity. Indeed, the only known RNA-binding protein that co-localizes with bicoid mRNA is Staufen, which binds non-specifically to double-stranded RNA in vitro. Here we show that mutants in all subunits of the ESCRT-II complex (VPS22, VPS25 and VPS36) abolish the final Staufen-dependent step in bicoid mRNA localization. ESCRT-II is a highly conserved component of the pathway that sorts ubiquitinated endosomal proteins into internal vesicles, and functions as a tumour-suppressor by removing activated receptors from the cytoplasm. However, the role of ESCRT-II in bicoid localization seems to be independent of endosomal sorting, because mutations in ESCRT-I and III components do not affect the targeting of bicoid mRNA. Instead, VPS36 functions by binding directly and specifically to stem-loop V of the bicoid 3' UTR through its amino-terminal GLUE domain, making it the first example of a sequence-specific RNA-binding protein that recognizes the bicoid localization signal. Furthermore, VPS36 localizes to the anterior of the oocyte in a bicoid-mRNA-dependent manner, and is required for the subsequent recruitment of Staufen to the bicoid complex. This function of ESCRT-II as an RNA-binding complex is conserved in vertebrates and may clarify some of its roles that are independent of endosomal sorting.     

Energy source of flagellar type III secretion

Bacterial flagella contain a specialized secretion apparatus that functions to deliver the protein subunits that form the filament and other structures to outside the membrane. This apparatus is related to the injectisome used by many gram-negative pathogens and symbionts to transfer effector proteins into host cells; in both systems this export mechanism is termed 'type III' secretion. The flagellar secretion apparatus comprises a membrane-embedded complex of about five proteins, and soluble factors, which include export-dedicated chaperones and an ATPase, FliI, that was thought to provide the energy for export. Here we show that flagellar secretion in Salmonella enterica requires the proton motive force (PMF) and does not require ATP hydrolysis by FliI. The export of several flagellar export substrates was prevented by treatment with the protonophore CCCP, with no accompanying decrease in cellular ATP levels. Weak swarming motility and rare flagella were observed in a mutant deleted for FliI and for the non-flagellar type-III secretion ATPases InvJ and SsaN. These findings show that the flagellar secretion apparatus functions as a proton-driven protein exporter and that ATP hydrolysis is not essential for type III secretion.