Direct delivery of bacterial avirulence proteins into resistant Arabidopsis protoplasts leads to hypersensitive cell death

Many bacterial avirulence (Avr) proteins, including the Pseudomonas syringae proteins, AvrRpt2 and AvrB, appear to be recognized inside the host plant cell by resistance mechanisms mediated by the cognate resistance (R) genes. It is thought that Avr proteins are either delivered directly into the host cell via the bacterial type III secretion system (TTSS) or taken up by the plant cell following secretion into the apoplast through the TTSS. Recently, it was shown that the Xanthomonas campestris AvrBs2 protein can be delivered directly into the host plant cell by the TTSS. However, it is not known whether other type III effectors of phytopathogens behave similarly. Here, using a novel protein transfection method, we demonstrate that AvrRpt2 and AvrB must enter the plant cell to be recognized by R gene-mediated mechanisms. First, we established a hypersensitive cell death assay for protoplasts using the membrane-impermeable, nuclear-staining dye, YO-PRO-1, and transgenic Arabidopsis plants that carry an inducible avrRpt2 gene. Second, we transfected E. coli-produced AvrRpt2 or AvrB proteins into Arabidopsis protoplasts using a protein transfection kit based on the carrier peptide Pep-1, and demonstrated that hypersensitive cell death occurs in a gene-for-gene-specific manner. In contrast, these Avr proteins failed to elicit hypersensitive cell death when they were applied to protoplasts without the carrier peptide. We conclude that our preparations of E. coli-produced AvrRpt2 and AvrB are active, that AvrRpt2 and AvrB must be delivered into the plant cell to be recognized, and that a method based on a carrier peptide can be used to introduce proteins into plant cells.     

Functional analysis of the type III effectors AvrRpt2 and AvrRpm1 of Pseudomonas syringae with the use of a single-copy genomic integration system

Gram-negative phytopathogenic bacteria require a type III secretion apparatus for pathogenesis, presumably to deliver Avr effector proteins directly into plant cells. To extend previous studies of Avr effectors that employed plasmids encoding Avr proteins, we developed a system that permits the integration of any gene into the Pseudomonas syringae genome in single copy. With this system, we confirmed earlier findings showing that P. syringae pv. maculicola strain PsmES4326 expressing the AvrRpt2 effector induces a resistance response in plants with the cognate R gene, RPS2. Chromosomally located avrRpt2, however, provoked a stronger resistance response than that observed with plasmid-expressed AvrRpt2 in RPS2+ plants. Additionally, chromosomal expression of AvrRpt2 conferred a fitness advantage on P. syringae grown in rps2- plants, aiding in growth within leaves and escape to leaf surfaces that was difficult to detect with plasmid-borne avrRpt2. Finally, with the use of the genomic integration system, we found that a chimeric protein composed of the N terminus of the heterologous AvrRpml effector and the C-terminal effector region of AvrRpt2 was delivered to plant cells. Because the C terminus of AvrRpt2 cannot translocate into plant cells on its own, this indicates that the N-terminal region can direct secretion and translocation during an infection, which supports the view that Avr proteins have a modular design. This work establishes a readily manipulatable system to study type III effectors in a biologically realistic context.     

Phospholipase-dependent signalling during the AvrRpm1- and AvrRpt2-induced disease resistance responses in Arabidopsis thaliana

Bacterial pathogens deliver type III effector proteins into plant cells during infection. On susceptible host plants, type III effectors contribute to virulence, but on resistant hosts they betray the pathogen to the plant's immune system and are functionally termed avirulence (Avr) proteins. Recognition induces a complex suite of cellular and molecular events comprising the plant's inducible defence response. As recognition of type III effector proteins occurs inside host cells, defence responses can be elicited by in planta expression of bacterial type III effectors. We demonstrate that recognition of either of two type III effectors, AvrRpm1 or AvrRpt2 from Pseudomonas syringae , induced biphasic accumulation of phosphatidic acid (PA). The first wave of PA accumulation correlated with disappearance of monophosphatidylinosotol (PIP) and is thus tentatively attributed to activation of a PIP specific phospholipase C (PLC) in concert with diacylglycerol kinase (DAGK) activity. Subsequent activation of phospholipase D (PLD) produced large amounts of PA from structural phospholipids. This later wave of PA accumulation was several orders of magnitude higher than the PLC-dependent first wave. Inhibition of phospholipases blocked the response, and feeding PA directly to leaf tissue caused cell death and defence-gene activation. Inhibitor studies ordered these events relative to other known signalling events during the plant defence response. Influx of extracellular Ca²⁺ occurred downstream of PIP-degradation, but upstream of PLD activation. Production of reactive oxygen species occurred downstream of the phospholipases. The data presented indicate that PA is a positive regulator of RPM1- or RPS2-mediated disease resistance signalling, and that the biphasic PA production may be a conserved feature of signalling induced by the coiled-coil nucleotide binding domain leucine-rich repeat class of resistance proteins.     

Fungal effector proteins: past, present and future

The pioneering research of Harold Flor on flax and the flax rust fungus culminated in his gene-for-gene hypothesis. It took nearly 50 years before the first fungal avirulence ( Avr ) gene in support of his hypothesis was cloned. Initially, fungal Avr genes were identified by reverse genetics and map-based cloning from model organisms, but, currently, the availability of many sequenced fungal genomes allows their cloning from additional fungi by a combination of comparative and functional genomics. It is believed that most Avr genes encode effectors that facilitate virulence by suppressing pathogen-associated molecular pattern-triggered immunity and induce effector-triggered immunity in plants containing cognate resistance proteins. In resistant plants, effectors are directly or indirectly recognized by cognate resistance proteins that reside either on the plasma membrane or inside the plant cell. Indirect recognition of an effector (also known as the guard model) implies that the virulence target of an effector in the host (the guardee) is guarded by the resistance protein (the guard) that senses manipulation of the guardee, leading to activation of effector-triggered immunity. In this article, we review the literature on fungal effectors and some pathogen-associated molecular patterns, including those of some fungi for which no gene-for-gene relationship has been established.     

The molecular genetics of virulence of Xanthomonas campestris

Bacteria belonging to the genus Xanthomonas are important pathogens of many plants, and their virulence appears to be due primarily to secreted and surface compounds that could increase host nutrient loss, or avoid or suppress unfavorable conditions in the host. Type II and III secretory pathways are essential for virulence. Some individual extracellular enzymes (type II-secretion dependent) affect final bacterial population levels, whereas some avirulence gene products (type III-secretion dependent) affect virulence by altering host metabolism. Avr proteins, probably secreted via a pilus, can also be recognized by host resistance gene products. Virulence is also associated with bacterial surface polysaccharides, which may help to avoid host defense responses, and regulatory gene systems, which can control virulence gene expression.     

Arabidopsis RIN4 negatively regulates disease resistance mediated by RPS2 and RPM1 downstream or independent of the NDR1 signal modulator and is not required for the virulence functions of bacterial type III effectors AvrRpt2 or AvrRpm1

Bacterial pathogens deliver type III effector proteins into the plant cell during infection. On susceptible (r) hosts, type III effectors can contribute to virulence. Some trigger the action of specific disease resistance (R) gene products. The activation of R proteins can occur indirectly via modification of a host target. Thus, at least some type III effectors are recognized at site(s) where they may act as virulence factors. These data indicate that a type III effector's host target might be required for both initiation of R function in resistant plants and pathogen virulence in susceptible plants. In Arabidopsis thaliana, RPM1-interacting protein 4 (RIN4) associates with both the Resistance to Pseudomonas syringae pv maculicola 1 (RPM1) and Resistance to P. syringae 2 (RPS2) disease resistance proteins. RIN4 is posttranslationally modified after delivery of the P. syringae type III effectors AvrRpm1, AvrB, or AvrRpt2 to plant cells. Thus, RIN4 may be a target for virulence functions of these type III effectors. We demonstrate that RIN4 is not the only host target for AvrRpm1 and AvrRpt2 in susceptible plants because its elimination does not diminish their virulence functions. In fact, RIN4 negatively regulates AvrRpt2 virulence function. RIN4 also negatively regulates inappropriate activation of both RPM1 and RPS2. Inappropriate activation of RPS2 is nonspecific disease resistance 1 (NDR1) independent, in contrast with the established requirement for NDR1 during AvrRpt2-dependent RPS2 activation. Thus, RIN4 acts either cooperatively, downstream, or independently of NDR1 to negatively regulate RPS2 in the absence of pathogen. We propose that many P. syringae type III effectors have more than one target in the host cell. We suggest that a limited set of these targets, perhaps only one, are associated with R proteins. Thus, whereas any pathogen virulence factor may have multiple targets, the perturbation of only one is necessary and sufficient for R activation.     

ExoS controls the cell contact-mediated switch to effector secretion in Pseudomonas aeruginosa

Type III secretion is used by many gram-negative bacterial pathogens to directly deliver protein toxins (effectors) into targeted host cells. In all cases, secretion of effectors is triggered by host cell contact, although the mechanism is unclear. In Pseudomonas aeruginosa, expression of all type III secretion-related genes is up-regulated when secretion is triggered. We were able to visualize this process using a green fluorescent protein reporter system and to use it to monitor the ability of bacteria to trigger effector secretion on cell contact. Surprisingly, the action of one of the major type III secreted effectors, ExoS, prevented triggering of type III secretion by bacteria that subsequently attached to cells, suggesting that triggering of secretion is feedback regulated. Evidence is presented that translocation (secretion of effectors across the host cell plasma membrane) of ExoS is indeed self-regulated and that this inhibition of translocation can be achieved by either of its two enzymatic activities. The translocator proteins PopB, PopD, and PcrV are secreted via the type III secretion system and are required for pore formation and translocation of effectors across the host cell plasma membrane. Here we present data that secretion of translocators is in fact not controlled by calcium, implying that triggering of effector secretion on cell contact represents a switch in secretion specificity, rather than a triggering of secretion per se. The requirement for a host cell cofactor to control effector secretion may help explain the recently observed phenomenon of target cell specificity in both the Yersinia and P. aeruginosa type III secretion systems.     

青枯劳尔氏菌多基因家族III型效应蛋白在植物病害发展及防御系统中的作用

青枯劳尔氏菌是导致多种重要经济作物毁灭性枯萎（bacterial wilt）的一种土传病害,严重危害热带和亚热带地区食品安全。该细菌通过III型分泌系统（T3SS）向寄主细胞注射大量效应蛋白（T3Es）。效应蛋白是把双刃剑,既可诱导植物感病,又能激活植物防御系统。具有特殊重复结构的效应蛋白被归类成多基因家族,各家族成员协同致病,但其分子机制尚不清楚。本文围绕近年来有关多基因家族效应蛋白结构、功能和致病性等方面最新进展进行综述,为青枯菌致病机理和病害防治提供新思路。     

Identifying type III effectors of plant pathogens and analyzing their interaction with plant cells

Many bacterial pathogens cause disease by injecting virulence proteins (effectors) into host cells via the specialized type III secretion system. Recently, exceptional progress in identifying effectors was made in the phytopathogen Pseudomonas syringae using a novel genetic screen and bioinformatic approach. These studies, along with localization experiments, suggest that most P. syringae effectors function by targeting the plasma membrane, chloroplasts or mitochondria of host cells. The type III secretome of P. syringae is highly variable and dynamic, a lesson gleaned from a comparative genomic analysis. Variation in the effector repertoire is likely to facilitate the adaptation of P. syringae to different hosts.     

Diverse evolutionary mechanisms shape the type III effector virulence factor repertoire in the plant pathogen Pseudomonas syringae

Many gram-negative pathogenic bacteria directly translocate effector proteins into eukaryotic host cells via type III delivery systems. Type III effector proteins are determinants of virulence on susceptible plant hosts; they are also the proteins that trigger specific disease resistance in resistant plant hosts. Evolution of type III effectors is dominated by competing forces: the likely requirement for conservation of virulence function, the avoidance of host defenses, and possible adaptation to new hosts. To understand the evolutionary history of type III effectors in Pseudomonas syringae, we searched for homologs to 44 known or candidate P. syringae type III effectors and two effector chaperones. We examined 24 gene families for distribution among bacterial species, amino acid sequence diversity, and features indicative of horizontal transfer. We assessed the role of diversifying and purifying selection in the evolution of these gene families. While some P. syringae type III effectors were acquired recently, others have evolved predominantly by descent. The majority of codons in most of these genes were subjected to purifying selection, suggesting selective pressure to maintain presumed virulence function. However, members of 7 families had domains subject to diversifying selection.     

The effector protein Avr2 of the xylem-colonizing fungus Fusarium oxysporum activates the tomato resistance protein I-2 intracellularly

To promote host colonization, many plant pathogens secrete effector proteins that either suppress or counteract host defences. However, when these effectors are recognized by the host's innate immune system, they trigger resistance rather than promoting virulence. Effectors are therefore key molecules in determining disease susceptibility or resistance. We show here that Avr2, secreted by the vascular wilt fungus Fusarium oxysporum f. sp. lycopersici ( Fol ), shows both activities: it is required for full virulence in a susceptible host and also triggers resistance in tomato plants carrying the resistance gene I-2 . Point mutations in AVR2 , causing single amino acid changes, are associated with I-2 -breaking Fol strains. These point mutations prevent recognition by I-2 , both in tomato and when both genes are co-expressed in leaves of Nicotiana benthamiana . Fol strains carrying the Avr2 variants are equally virulent, showing that virulence and avirulence functions can be uncoupled. Although Avr2 is secreted into the xylem sap when Fol colonizes tomato, the Avr2 protein can be recognized intracellularly by I-2, implying uptake by host cells.     

RXLR-mediated entry of Phytophthora sojae effector Avr1b into soybean cells does not require pathogen-encoded machinery

Effector proteins secreted by oomycete and fungal pathogens have been inferred to enter host cells, where they interact with host resistance gene products. Using the effector protein Avr1b of Phytophthora sojae, an oomycete pathogen of soybean (Glycine max), we show that a pair of sequence motifs, RXLR and dEER, plus surrounding sequences, are both necessary and sufficient to deliver the protein into plant cells. Particle bombardment experiments demonstrate that these motifs function in the absence of the pathogen, indicating that no additional pathogen-encoded machinery is required for effector protein entry into host cells. Furthermore, fusion of the Avr1b RXLR-dEER domain to green fluorescent protein (GFP) allows GFP to enter soybean root cells autonomously. The conclusion that RXLR and dEER serve to transduce oomycete effectors into host cells indicates that the >370 RXLR-dEER-containing proteins encoded in the genome sequence of P. sojae are candidate effectors. We further show that the RXLR and dEER motifs can be replaced by the closely related erythrocyte targeting signals found in effector proteins of Plasmodium, the protozoan that causes malaria in humans. Mutational analysis of the RXLR motif shows that the required residues are very similar in the motifs of Plasmodium and Phytophthora. Thus, the machinery of the hosts (soybean and human) targeted by the effectors may be very ancient.     

Internalization of Flax Rust Avirulence Proteins into Flax and Tobacco Cells Can Occur in the Absence of the Pathogen

Translocation of pathogen effector proteins into the host cell cytoplasm is a key determinant for the pathogenicity of many bacterial and oomycete plant pathogens. A number of secreted fungal avirulence (Avr) proteins are also inferred to be delivered into host cells, based on their intracellular recognition by host resistance proteins, including those of flax rust (Melampsora lini). Here, we show by immunolocalization that the flax rust AvrM protein is secreted from haustoria during infection and accumulates in the haustorial wall. Five days after inoculation, the AvrM protein was also detected within the cytoplasm of a proportion of plant cells containing haustoria, confirming its delivery into host cells during infection. Transient expression of secreted AvrL567 and AvrM proteins fused to cerulean fluorescent protein in tobacco (Nicotiana tabacum) and flax cells resulted in intracellular accumulation of the fusion proteins. The rust Avr protein signal peptides were functional in plants and efficiently directed fused cerulean into the secretory pathway. Thus, these secreted effectors are internalized into the plant cell cytosol in the absence of the pathogen, suggesting that they do not require a pathogen-encoded transport mechanism. Uptake of these proteins is dependent on signals in their N-terminal regions, but the primary sequence features of these uptake regions are not conserved between different rust effectors.     

SipB-SipC Complex Is Essential for Translocon Formation

The delivery of effector proteins by Salmonella across the host cell membrane requires a subset of effectors secreted by the type III secretion system (TTSS) known as translocators. SipC and SipB are translocator proteins that are inserted into host membranes and presumably form a channel that translocates type III effectors into the host cell. The molecular events of how these translocators insert into the host cell membrane remain unknown. We have previously shown that the SipC C-terminal amino acid region (321–409) is required for the translocation of effectors into host cells. In this study, we demonstrate that the ability to form SipC-SipB complex is essential for their insertion into the host membrane. The SipB-interacting domain of SipC is near its C-terminal amino acid region (340–409). In the absence of SipB, SipC was not detected in the membrane fraction. Furthermore, SipC mutants that no longer interact with SipB are defective in inserting into the host cell membrane. We propose a mechanism whereby SipC binds SipB through its C-terminal region to facilitate membrane-insertion and subsequent translocon formation in the host cell membrane.     

Coiled-coil domains enhance the membrane association of Salmonella type III effectors

Coiled-coil domains in eukaryotic and prokaryotic proteins contribute to diverse structural and regulatory functions. Here we have used in silico analysis to predict which proteins in the proteome of the enteric pathogen, Salmonella enterica serovar Typhimurium, harbour coiled-coil domains. We found that coiled-coil domains are especially prevalent in virulence-associated proteins, including type III effectors. Using SopB as a model coiled-coil domain type III effector, we have investigated the role of this motif in various aspects of effector function including chaperone binding, secretion and translocation, protein stability, localization and biological activity. Compared with wild-type SopB, SopB coiled-coil mutants were unstable, both inside bacteria and after translocation into host cells. In addition, the putative coiled-coil domain was required for the efficient membrane association of SopB in host cells. Since many other Salmonella effectors were predicted to contain coiled-coil domains, we also investigated the role of this motif in their intracellular targeting in mammalian cells. Mutation of the predicted coiled-coil domains in PipB2, SseJ and SopD2 also eliminated their membrane localization in mammalian cells. These findings suggest that coiled-coil domains represent a common membrane-targeting determinant for Salmonella type III effectors.     

SCAMP3 is a component of the Salmonella-induced tubular network and reveals an interaction between bacterial effectors and post-Golgi trafficking

Salmonella enterica are facultative intracellular bacterial pathogens that proliferate within host cells in a membrane-bounded compartment, the Salmonella -containing vacuole (SCV). Intracellular replication of Salmonella is mediated by bacterial effectors translocated on to the cytoplasmic face of the SCV membrane by a type III secretion system. Some of these effectors manipulate the host endocytic pathway, resulting in the formation in epithelial cells of tubules enriched in late endosomal markers, known as Salmonella -induced filaments (SIFs). However, much less is known about possible interference of Salmonella with the secretory pathway. Here, a small-interference RNA screen revealed that secretory carrier membrane proteins (SCAMPs) 2 and 3 contribute to the maintenance of SCVs in the Golgi region of HeLa cells. This is likely to reflect a function of SCAMPs in vacuolar membrane dynamics. Moreover, SCAMP3, which accumulates on the trans -Golgi network in uninfected cells, marked tubules induced by Salmonella effectors that overlapped with SIFs but which also comprised distinct tubules lacking late endosomal proteins. We propose that SCAMP3 tubules reflect a manipulation of specific post-Golgi trafficking that might allow Salmonella to acquire nutrients and membrane, or to control host immune responses.