The Bacterial Effector HopX1 Targets JAZ Transcriptional Repressors to Activate Jasmonate Signaling and Promote Infection in Arabidopsis

Pathogenicity of Pseudomonas syringae is dependent on a type III secretion system, which secretes a suite of virulence effector proteins into the host cytoplasm, and the production of a number of toxins such as coronatine (COR), which is a mimic of the plant hormone jasmonate-isoleuce (JA-Ile). Inside the plant cell, effectors target host molecules to subvert the host cell physiology and disrupt defenses. However, despite the fact that elucidating effector action is essential to understanding bacterial pathogenesis, the molecular function and host targets of the vast majority of effectors remain largely unknown. Here, we found that effector HopX1 from Pseudomonas syringae pv. tabaci (Pta) 11528, a strain that does not produce COR, interacts with and promotes the degradation of JAZ proteins, a key family of JA-repressors. We show that hopX1 encodes a cysteine protease, activity that is required for degradation of JAZs by HopX1. HopX1 associates with JAZ proteins through its central ZIM domain and degradation occurs in a COI1-independent manner. Moreover, ectopic expression of HopX1 in Arabidopsis induces the expression of JA-dependent genes, represses salicylic acid (SA)-induced markers, and complements the growth of a COR-deficient P. syringae pv. tomato (Pto) DC3000 strain during natural bacterial infections. Furthermore, HopX1 promoted susceptibility when delivered by the natural type III secretion system, to a similar extent as the addition of COR, and this effect was dependent on its catalytic activity. Altogether, our results indicate that JAZ proteins are direct targets of bacterial effectors to promote activation of JA-induced defenses and susceptibility in Arabidopsis. HopX1 illustrates a paradigm of an alternative evolutionary solution to COR with similar physiological outcome.     

The Phytophthora effector Avh94 manipulates host jasmonic acid signaling to promote infection

The oomycete pathogen Phytophthora sojae is a causal agent of soybean root rot.Upon colonization of soybeans,P.sojae secretes various RXLR effectors to suppress host immune responses,supporting successful infection.Previous research has demonstrated that the RXLR effector Avh94 functions as a virulence effector,but the molecular mechanism underlying its role in virulence remains unknown.Here,we demonstrate that Avh94 overexpression in plants and pathogens promotes Phytophthora infection.Avh94 in...     

Immunomodulation by the Pseudomonas syringae HopZ Type III Effector Family in Arabidopsis

Pseudomonas syringae employs a type III secretion system to inject 20–30 different type III effector (T3SE) proteins into plant host cells. A major role of T3SEs is to suppress plant immune responses and promote bacterial infection. The YopJ/HopZ acetyltransferases are a superfamily of T3SEs found in both plant and animal pathogenic bacteria. In P. syringae, this superfamily includes the evolutionarily diverse HopZ1, HopZ2 and HopZ3 alleles. To investigate the roles of the HopZ family in immunomodulation, we generated dexamethasone-inducible T3SE transgenic lines of Arabidopsis for HopZ family members and characterized them for immune suppression phenotypes. We show that all of the HopZ family members can actively suppress various facets of Arabidopsis immunity in a catalytic residue-dependent manner. HopZ family members can differentially suppress the activation of mitogen-activated protein (MAP) kinase cascades or the production of reactive oxygen species, whereas all members can promote the growth of non-virulent P. syringae. Localization studies show that four of the HopZ family members containing predicted myristoylation sites are localized to the vicinity of the plasma membrane while HopZ3 which lacks the myristoylation site is at least partially nuclear localized, suggesting diversification of immunosuppressive mechanisms. Overall, we demonstrate that despite significant evolutionary diversification, all HopZ family members can suppress immunity in Arabidopsis.     

The phytotoxin coronatine is a multifunctional component of the virulence armament of Pseudomonas syringae

Plant pathogens deploy an array of virulence factors to suppress host defense and promote pathogenicity. Numerous strains of Pseudomonas syringae produce the phytotoxin coronatine (COR). A major aspect of COR function is its ability to mimic a bioactive jasmonic acid (JA) conjugate and thus target the JA-receptor COR-insensitive 1 (COI1). Biological activities of COR include stimulation of JA-signaling and consequent suppression of SA-dependent defense through antagonistic crosstalk, antagonism of stomatal closure to allow bacterial entry into the interior of plant leaves, contribution to chlorotic symptoms in infected plants, and suppression of plant cell wall defense through perturbation of secondary metabolism. Here, we review the virulence function of COR, including updates on these established activities as well as more recent findings revealing COI1-independent activity of COR and shedding light on cooperative or redundant defense suppression between COR and type III effector proteins.     

Methyl Salicylate Production and Jasmonate Signaling Are Not Essential for Systemic Acquired Resistance in Arabidopsis

Systemic acquired resistance (SAR) develops in response to local microbial leaf inoculation and renders the whole plant more resistant to subsequent pathogen infection. Accumulation of salicylic acid (SA) in noninfected plant parts is required for SAR, and methyl salicylate (MeSA) and jasmonate (JA) are proposed to have critical roles during SAR long-distance signaling from inoculated to distant leaves. Here, we address the significance of MeSA and JA during SAR development in Arabidopsis thaliana. MeSA production increases in leaves inoculated with the SAR-inducing bacterial pathogen Pseudomonas syringae; however, most MeSA is emitted into the atmosphere, and only small amounts are retained. We show that in several Arabidopsis defense mutants, the abilities to produce MeSA and to establish SAR do not coincide. T-DNA insertion lines defective in expression of a pathogen-responsive SA methyltransferase gene are completely devoid of induced MeSA production but increase systemic SA levels and develop SAR upon local P. syringae inoculation. Therefore, MeSA is dispensable for SAR in Arabidopsis, and SA accumulation in distant leaves appears to occur by de novo synthesis via isochorismate synthase. We show that MeSA production induced by P. syringae depends on the JA pathway but that JA biosynthesis or downstream signaling is not required for SAR. In compatible interactions, MeSA production depends on the P. syringae virulence factor coronatine, suggesting that the phytopathogen uses coronatine-mediated volatilization of MeSA from leaves to attenuate the SA-based defense pathway.     

Allele-Specific Virulence Attenuation of the Pseudomonas syringae HopZ1a Type III Effector via the Arabidopsis ZAR1 Resistance Protein

Plant resistance (R) proteins provide a robust surveillance system to defend against potential pathogens. Despite their importance in plant innate immunity, relatively few of the ∼170 R proteins in Arabidopsis have well-characterized resistance specificity. In order to identify the R protein responsible for recognition of the Pseudomonas syringae type III secreted effector (T3SE) HopZ1a, we assembled an Arabidopsis R gene T–DNA Insertion Collection (ARTIC) from publicly available Arabidopsis thaliana insertion lines and screened it for plants lacking HopZ1a-induced immunity. This reverse genetic screen revealed that the Arabidopsis R protein HOPZ-ACTIVATED RESISTANCE 1 (ZAR1; At3g50950) is required for recognition of HopZ1a in Arabidopsis. ZAR1 belongs to the coiled-coil (CC) class of nucleotide binding site and leucine-rich repeat (NBS–LRR) containing R proteins; however, the ZAR1 CC domain phylogenetically clusters in a clade distinct from other related Arabidopsis R proteins. ZAR1–mediated immunity is independent of several genes required by other R protein signaling pathways, including NDR1 and RAR1, suggesting that ZAR1 possesses distinct signaling requirements. The closely-related T3SE protein, HopZ1b, is still recognized by zar1 Arabidopsis plants indicating that Arabidopsis has evolved at least two independent R proteins to recognize the HopZ T3SE family. Also, in Arabidopsis zar1 plants HopZ1a promotes P. syringae growth indicative of an ancestral virulence function for this T3SE prior to the evolution of recognition by the host resistance protein ZAR1. Our results demonstrate that the Arabidopsis resistance protein ZAR1 confers allele-specific recognition and virulence attenuation of the Pseudomonas syringae T3SE protein HopZ1a.     

A Bacterial Acetyltransferase Destroys Plant Microtubule Networks and Blocks Secretion

The eukaryotic cytoskeleton is essential for structural support and intracellular transport, and is therefore a common target of animal pathogens. However, no phytopathogenic effector has yet been demonstrated to specifically target the plant cytoskeleton. Here we show that the Pseudomonas syringae type III secreted effector HopZ1a interacts with tubulin and polymerized microtubules. We demonstrate that HopZ1a is an acetyltransferase activated by the eukaryotic co-factor phytic acid. Activated HopZ1a acetylates itself and tubulin. The conserved autoacetylation site of the YopJ / HopZ superfamily, K289, plays a critical role in both the avirulence and virulence function of HopZ1a. Furthermore, HopZ1a requires its acetyltransferase activity to cause a dramatic decrease in Arabidopsis thaliana microtubule networks, disrupt the plant secretory pathway and suppress cell wall-mediated defense. Together, this study supports the hypothesis that HopZ1a promotes virulence through cytoskeletal and secretory disruption.     

Pseudomonas syringae type III effector HopZ1 targets a host enzyme to suppress isoflavone biosynthesis and promote infection in soybean

Type III secreted effectors (T3SEs), such as Pseudomonas syringae HopZ1, are essential bacterial virulence proteins injected into the host cytosol to facilitate infection. However, few direct targets of T3SEs are known. Investigating the target(s) of HopZ1 in soybean, a natural P. syringae host, we find that HopZ1 physically interacts with the isoflavone biosynthesis enzyme, 2-hydroxyisoflavanone dehydratase (GmHID1). P. syringae infection induces gmhid1 expression and production of daidzein,?a major soybean isoflavone. Silencing gmhid1 increases susceptibility to P. syringae infection, supporting a role for GmHID1 in innate immunity. P.?syringae expressing active but not the catalytic mutant of HopZ1 inhibits daidzein induction and promotes bacterial multiplication in soybean. HopZ1-enhanced P. syringae multiplication is at least partially dependent on GmHID1. Thus, GmHID1 is a virulence target of HopZ1 to promote P. syringae infection of soybean. This work highlights the isoflavonoid biosynthesis pathway as an antibacterial defense mechanism and a direct T3SE target.     

Catalytic domain of the diversified Pseudomonas syringae type III effector HopZ1 determines the allelic specificity in plant hosts

The type III secretion systems (T3SS) and secreted effectors (T3SEs) are essential virulence factors in Gram‐negative bacteria. During the arms race, plants have evolved resistance (R) genes to detect specific T3SEs and activate defence responses. However, this immunity can be efficiently defeated by the pathogens through effector evolution. HopZ1 of the plant pathogen Pseudomonas syringae is a member of the widely distributed YopJ T3SE family. Three alleles are known to be present in P. syringae, with HopZ1a most resembling the ancestral allelic form. In this study, molecular mechanisms underlying the sequence diversification‐enabled HopZ1 allelic specificity is investigated. Using domain shuffling experiments, we present evidence showing that a central domain upstream of the conserved catalytic cysteine residue determines HopZ1 recognition specificity. Random and targeted mutagenesis identified three amino acids involved in HopZ1 allelic specificity. Particularly, the exchange of cysteine141 in HopZ1a with lysine137 at the corresponding position in HopZ1b abolished HopZ1a recognition in soybean. This position is under strong positive selection, suggesting that the cysteine/lysine mutation might be a key step driving the evolution of HopZ1. Our data support a model in which sequence diversification imposed by the plant R gene‐associated immunity has driven HopZ1 evolution by allowing allele‐specific substrate‐binding.     

HopW1 from Pseudomonas syringae Disrupts the Actin Cytoskeleton to Promote Virulence in Arabidopsis

A central mechanism of virulence of extracellular bacterial pathogens is the injection into host cells of effector proteins that modify host cellular functions. HopW1 is an effector injected by the type III secretion system that increases the growth of the plant pathogen Pseudomonas syringae on the Columbia accession of Arabidopsis. When delivered by P. syringae into plant cells, HopW1 causes a reduction in the filamentous actin (F-actin) network and the inhibition of endocytosis, a known actin-dependent process. When directly produced in plants, HopW1 forms complexes with actin, disrupts the actin cytoskeleton and inhibits endocytosis as well as the trafficking of certain proteins to vacuoles. The C-terminal region of HopW1 can reduce the length of actin filaments and therefore solubilize F-actin in vitro. Thus, HopW1 acts by disrupting the actin cytoskeleton and the cell biological processes that depend on actin, which in turn are needed for restricting P. syringae growth in Arabidopsis.     

The YopJ superfamily in plant-associated bacteria

Bacterial pathogens employ the type III secretion system to secrete and translocate effector proteins into their hosts. The primary function of these effector proteins is believed to be the suppression of host defence responses or innate immunity. However, some effector proteins may be recognized by the host and consequently trigger a targeted immune response. The YopJ/HopZ/AvrRxv family of bacterial effector proteins is a widely distributed and evolutionarily diverse family, found in both animal and plant pathogens, as well as plant symbionts. How can an effector family effectively promote the virulence of pathogens on hosts from two separate kingdoms? Our understanding of the evolutionary relationships among the YopJ superfamily members provides an excellent opportunity to address this question and to investigate the functions and virulence strategies of a diverse type III effector family in animal and plant hosts. In this work, we briefly review the literature on YopJ, the archetypal member from Yersinia pestis, and discuss members of the superfamily in species of Pseudomonas, Xanthomonas, Ralstonia and Rhizobium. We review the molecular and cellular functions, if known, of the YopJ homologues in plants, and highlight the diversity of responses in different plant species, with a particular focus on the Pseudomonas syringae HopZ family. The YopJ superfamily provides an excellent foundation for the study of effector diversification in the context of wide‐ranging, co‐evolutionary interactions.