Wenbo Ma

Type III Effector Diversification via Both Pathoadaptation and Horizontal Transfer in Response to a Coevolutionary Arms Race

The concept of the coevolutionary arms race holds a central position in our understanding of pathogen–host interactions. Here we identify the molecular mechanisms and follow the stepwise progression of an arms race in a natural system. We show how the evolution and function of the HopZ family of type III secreted effector proteins carried by the plant pathogen Pseudomonas syringae are influenced by a coevolutionary arms race between pathogen and host. We surveyed 96 isolates of P. syringae and identified three homologs (HopZ1, HopZ2, and HopZ3) distributed among ∼45% of the strains. All alleles were sequenced and their expression was confirmed. Evolutionary analyses determined that the diverse HopZ1 homologs are ancestral to P. syringae, and have diverged via pathoadaptive mutational changes into three functional and two degenerate forms, while HopZ2 and HopZ3 have been brought into P. syringae via horizontal transfer from other ecologically similar bacteria. A PAML selection analysis revealed that the C terminus of HopZ1 is under strong positive selection. Despite the extensive genetic variation observed in this family, all three homologs have cysteine–protease activity, although their substrate specificity may vary. The introduction of the ancestral hopZ1 allele into strains harboring alternate alleles results in a resistance protein-mediated defense response in their respective hosts, which is not observed with the endogenous allele. These data indicate that the P. syringae HopZ family has undergone allelic diversification via both pathoadaptive mutational changes and horizontal transfer in response to selection imposed by the host defense system. This genetic diversity permits the pathogen to avoid host defenses while still maintaining a virulence-associated protease, thereby allowing it to thrive on its current host, while simultaneously impacting its host range.

Oomycete pathogens encode RNA silencing suppressors

Effectors are essential virulence proteins produced by a broad range of parasites, including viruses, bacteria, fungi, oomycetes, protozoa, insects and nematodes. Upon entry into host cells, pathogen effectors manipulate specific physiological processes or signaling pathways to subvert host immunity. Most effectors, especially those of eukaryotic pathogens, remain functionally uncharacterized. Here, we show that two effectors from the oomycete plant pathogen Phytophthora sojae suppress RNA silencing in plants by inhibiting the biogenesis of small RNAs. Ectopic expression of these Phytophthora suppressors of RNA silencing enhances plant susceptibility to both a virus and Phytophthora, showing that some eukaryotic pathogens have evolved virulence proteins that target host RNA silencing processes to promote infection. These findings identify RNA silencing suppression as a common strategy used by pathogens across kingdoms to cause disease and are consistent with RNA silencing having key roles in host defense.

Bacterial effector activates jasmonate signaling by directly targeting JAZ transcriptional repressors.

Gram-negative bacterial pathogens deliver a variety of virulence proteins through the type III secretion system (T3SS) directly into the host cytoplasm. These type III secreted effectors (T3SEs) play an essential role in bacterial infection, mainly by targeting host immunity. However, the molecular basis of their functionalities remains largely enigmatic. Here, we show that the Pseudomonas syringae T3SE HopZ1a, a member of the widely distributed YopJ effector family, directly interacts with jasmonate ZIM-domain (JAZ) proteins through the conserved Jas domain in plant hosts. JAZs are transcription repressors of jasmonate (JA)-responsive genes and major components of the jasmonate receptor complex. Upon interaction, JAZs can be acetylated by HopZ1a through a putative acetyltransferase activity. Importantly, P. syringae producing the wild-type, but not a catalytic mutant of HopZ1a, promotes the degradation of HopZ1-interacting JAZs and activates JA signaling during bacterial infection. Furthermore, HopZ1a could partially rescue the virulence defect of a P. syringae mutant that lacks the production of coronatine, a JA-mimicking phytotoxin produced by a few P. syringae strains. These results highlight a novel example by which a bacterial effector directly manipulates the core regulators of phytohormone signaling to facilitate infection. The targeting of JAZ repressors by both coronatine toxin and HopZ1 effector suggests that the JA receptor complex is potentially a major hub of host targets for bacterial pathogens.

Terminal reassortment drives the quantum evolution of type III effectors in bacterial pathogens.

Many bacterial pathogens employ a type III secretion system to deliver type III secreted effectors (T3SEs) into host cells, where they interact directly with host substrates to modulate defense pathways and promote disease. This interaction creates intense selective pressures on these secreted effectors, necessitating rapid evolution to overcome host surveillance systems and defenses. Using computational and evolutionary approaches, we have identified numerous mosaic and truncated T3SEs among animal and plant pathogens. We propose that these secreted virulence genes have evolved through a shuffling process we have called “terminal reassortment.” In terminal reassortment, existing T3SE termini are mobilized within the genome, creating random genetic fusions that result in chimeric genes. Up to 32% of T3SE families in species with relatively large and well-characterized T3SE repertoires show evidence of terminal reassortment, as compared to only 7% of non-T3SE families. Terminal reassortment may permit the near instantaneous evolution of new T3SEs and appears responsible for major modifications to effector activity and function. Because this process plays a more significant role in the evolution of T3SEs than non-effectors, it provides insight into the evolutionary origins of T3SEs and may also help explain the rapid emergence of new infectious agents.

Evolution of prokaryotic and eukaryotic virulence effectors.

Coevolutionary interactions between plants and their bacterial and eukaryotic pathogens are mediated by virulence effectors. These effectors face the daunting challenge of carrying out virulence functions, while also potentially exposing the pathogen to host defense systems. Very strong selective pressures are imposed by these competing roles, and the subsequent genetic changes leave their footprints in the extant allelic variation. This review examines the evolutionary processes that drive pathogen-host interactions as revealed by the genetic signatures left in virulence effectors, and speculate on the different pressures imposed on bacterial versus eukaryotic pathogens. We find numerous instances of positive selection for new allelic forms, and diversifying selection for genetic variability, which results in altered host-pathogen interactions. We also describe how the genetic structure of both bacterial and eukaryotic virulence effectors may contribute to their rapid generation and turnover.

The HopZ Family of Pseudomonas syringae Type III Effectors Require Myristoylation for Virulence and Avirulence Functions in Arabidopsis thaliana

Pseudomonas syringae utilizes the type III secretion system to translocate effector proteins into plant cells, where they can contribute to the pathogens ability to infect and cause disease. Recognition of these effectors by resistance proteins induces defense responses that typically include a programmed cell death reaction called the hypersensitive response. The YopJ/HopZ family of type III effector proteins is a common family of effector proteins found in animal- and plant-pathogenic bacteria. The HopZ family in P. syringae includes HopZ1a(PsyA2), HopZ1b(PgyUnB647), HopZ1c(PmaE54326), HopZ2(Ppi895A) and HopZ3(PsyB728a). HopZ1a is predicted to be most similar to the ancestral hopZ allele and causes a hypersensitive response in multiple plant species, including Arabidopsis thaliana. Therefore, it has been proposed that host defense responses have driven the diversification of this effector family. In this study, we further characterized the hypersensitive response induced by HopZ1a and demonstrated that it is not dependent on known resistance genes. Further, we identified a novel virulence function for HopZ2 that requires the catalytic cysteine demonstrated to be required for protease activity. Sequence analysis of the HopZ family revealed the presence of a predicted myristoylation sequence in all members except HopZ3. We demonstrated that the myristoylation site is required for membrane localization of this effector family and contributes to the virulence and avirulence activities of HopZ2 and HopZ1a, respectively. This paper provides insight into the selective pressures driving virulence protein evolution by describing a detailed functional characterization of the diverse HopZ family of type III effectors with the model plant Arabidopsis.

Phytophthora effector targets a novel component of small RNA pathway in plants to promote infection

Significance Phytophthora is a major threat to agriculture. However, the molecular interaction of these severe pathogens with plant hosts is poorly understood. Here, we report that the Phytophthora Suppressor of RNA Silencing 1 (PSR1) effectively promotes infection in Arabidopsis thaliana by directly targeting an essential protein containing a aspartate–glutamate–alanine–histidine-box RNA helicase domain. This PSR1-Interacting Protein 1 (PINP1) is required for the accumulation of distinct classes of endogenous small RNAs and acts as a positive regulator of plant immunity. Silencing of PINP1 impaired the assembly of microRNA-processing complexes in the nucleus, leading to defects in development and immunity. This study revealed a conserved RNA helicase as a regulator of RNA silencing and provides mechanistic insight into Phytophthora pathogenesis. A broad range of parasites rely on the functions of effector proteins to subvert host immune response and facilitate disease development. The notorious Phytophthora pathogens evolved effectors with RNA silencing suppression activity to promote infection in plant hosts. Here we report that the Phytophthora Suppressor of RNA Silencing 1 (PSR1) can bind to an evolutionarily conserved nuclear protein containing the aspartate–glutamate–alanine–histidine-box RNA helicase domain in plants. This protein, designated PSR1-Interacting Protein 1 (PINP1), regulates the accumulation of both microRNAs and endogenous small interfering RNAs in Arabidopsis. A null mutation of PINP1 causes embryonic lethality, and silencing of PINP1 leads to developmental defects and hypersusceptibility to Phytophthora infection. These phenotypes are reminiscent of transgenic plants expressing PSR1, supporting PINP1 as a direct virulence target of PSR1. We further demonstrate that the localization of the Dicer-like 1 protein complex is impaired in the nucleus of PINP1-silenced or PSR1-expressing cells, indicating that PINP1 may facilitate small RNA processing by affecting the assembly of dicing complexes. A similar function of PINP1 homologous genes in development and immunity was also observed in Nicotiana benthamiana. These findings highlight PINP1 as a previously unidentified component of RNA silencing that regulates distinct classes of small RNAs in plants. Importantly, Phytophthora has evolved effectors to target PINP1 in order to promote infection.

Computational prediction of type III secreted proteins from gram-negative bacteria

BackgroundType III secretion system (T3SS) is a specialized protein delivery system in gram-negative bacteria that injects proteins (called effectors) directly into the eukaryotic host cytosol and facilitates bacterial infection. For many plant and animal pathogens, T3SS is indispensable for disease development. Recently, T3SS has also been found in rhizobia and plays a crucial role in the nodulation process. Although a great deal of efforts have been done to understand type III secretion, the precise mechanism underlying the secretion and translocation process has not been fully understood. In particular, defined secretion and translocation signals enabling the secretion have not been identified from the type III secreted effectors (T3SEs), which makes the identification of these important virulence factors notoriously challenging. The availability of a large number of sequenced genomes for plant and animal-associated bacteria demands the development of efficient and effective prediction methods for the identification of T3SEs using bioinformatics approaches.ResultsWe have developed a machine learning method based on the N-terminal amino acid sequences to predict novel type III effectors in the plant pathogen Pseudomonas syringae and the microsymbiont rhizobia. The extracted features used in the learning model (or classifier) include amino acid composition, secondary structure and solvent accessibility information. The method achieved a precision of over 90% on P. syringae in a cross validation study. In combination with a promoter screen for the type III specific promoters, this classifier trained on the P. syringae data was applied to predict novel T3SEs from the genomic sequences of four rhizobial strains. This application resulted in 57 candidate type III secreted proteins, 17 of which are confirmed effectors.ConclusionOur experimental results demonstrate that the machine learning method based on N-terminal amino acid sequences combined with a promoter screen could prove to be a very effective computational approach for predicting novel type III effectors in gram-negative bacteria. Our method and data are available to the public upon request.

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.

Chromatin configuration as a battlefield in plant-bacteria interactions

Regulation of chromatin dynamics by remodeling complexes is well known to play critical roles in gene expression changes during plant development and stress response. Recent discoveries implicated that chromatin remodelers also regulate plant immunity. Upon pathogen infection, histone modifying enzymes and ATP-dependent chromatin remodelers, in conjunction with other regulatory proteins, are recruited to defense-related gene regions, resulting in transcription reprogramming. The presence of a large number of potential components of chromatin remodeling complexes may contribute to the integration of various signals, and eventually lead to the quantitative and kinetical immune response. Biotrophic and hemibiotrophic pathogens have evolved virulence factors to suppress plant defense, which is essential for disease development. The conserved nature of chromatin remodeling complexes makes them attractive targets of these pathogens. Here, we update our knowledge on the emerging role of chromatin configuration in the tight control of plant defense response against bacterial infection, and discuss the potential functions of bacterial virulence proteins to subvert immunity by modulating chromatin-remodeling processes.