Identification of Symptomatic Fetuses Infected with Cytomegalovirus Using Amniotic Fluid Peptide Biomarkers

Cytomegalovirus (CMV) is the most common cause of congenital infection, and is a major cause of sensorineural hearing loss and neurological disabilities. Evaluating the risk for a CMV infected fetus to develop severe clinical symptoms after birth is crucial to provide appropriate guidance to pregnant women who might have to consider termination of pregnancy or experimental prenatal medical therapies. However, establishing the prognosis before birth remains a challenge. This evaluation is currently based upon fetal imaging and fetal biological parameters, but the positive and negative predictive values of these parameters are not optimal, leaving room for the development of new prognostic factors. Here, we compared the amniotic fluid peptidome between asymptomatic fetuses who were born as asymptomatic neonates and symptomatic fetuses who were either terminated in view of severe cerebral lesions or born as severely symptomatic neonates. This comparison allowed us to identify a 34-peptide classifier in a discovery cohort of 13 symptomatic and 13 asymptomatic neonates. This classifier further yielded 89% sensitivity, 75% specificity and an area under the curve of 0.90 to segregate 9 severely symptomatic from 12 asymptomatic neonates in a validation cohort, showing an overall better performance than that of classical fetal laboratory parameters. Pathway analysis of the 34 peptides underlined the role of viral entry in fetuses with severe brain disease as well as the potential importance of both beta-2-microglobulin and adiponectin to protect the injured fetal brain infected with CMV. The results also suggested the mechanistic implication of the T calcium channel alpha-1G (CACNA1G) protein in the development of seizures in severely CMV infected children. These results open a new field for potential therapeutic options. In conclusion, this study demonstrates that amniotic fluid peptidome analysis can effectively predict the severity of congenital CMV infection. This peptidomic classifier may therefore be used in clinical settings during pregnancy to improve prenatal counseling.     

A High-Throughput Forward Genetic Screen Identifies Genes Required for Virulence of Pseudomonas syringae pv. maculicola ES4326 on Arabidopsis

Successful pathogenesis requires a number of coordinated processes whose genetic bases remain to be fully characterized. We utilized a high-throughput, liquid media-based assay to screen transposon disruptants of the phytopathogen Pseudomonas syringae pv. maculicola ES4326 to identify genes required for virulence on Arabidopsis. Many genes identified through this screen were involved in processes such as type III secretion, periplasmic glucan biosynthesis, flagellar motility, and amino acid biosynthesis. A small set of genes did not fall into any of these functional groups, and their disruption resulted in context-specific effects on in planta bacterial growth.     

Forward chemical genetic screens in Arabidopsis identify genes that influence sensitivity to the phytotoxic compound sulfamethoxazole

The rise of high mountain chains is widely seen as one of the factors driving rapid diversification of land plants and the formation of biodiversity hotspots. Supporting evidence was reported for the impact of the rapid rise of the Andean mountains but this hypothesis has so far been less explored for the impact of the “roof of the world”. The formation of the Himalaya, and especially the rise of the Qinghai–Tibetan Plateau in the recent 20 million years, altered the monsoon regimes that dominate the current climates of South East Asia. Here, we infer the hypothesis that the rise of Himalaya had a strong impact on the plant diversity in the biodiversity hotspot of the Southwest Chinese Mountains. Our analyses of the diversification pattern of the derived fern genus Lepisorus recovered evidence for changes in plant diversity that correlated with the strengthening of South East Asian monsoon. Southwest China or Southwest China and Japan was recovered as the putative area of origin of Lepisorus and enhancing monsoon regime were found to shape the early diversification of the genus as well as subsequent radiations during the late Miocene and Pliocene. We report new evidence for a coincidence of plant diversification and changes of the climate caused by the uplift of the Himalaya. These results are discussed in the context of the impact of incomplete taxon sampling, uncertainty of divergence time estimates, and limitations of current methods used to assess diversification rates.     

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.     

The HopF family of Pseudomonas syringae type III secreted effectors

Pseudomonas syringae is a bacterial phytopathogen that utilizes the type III secretion system to inject effector proteins into plant host cells. Pseudomonas syringae can infect a wide range of plant hosts, including agronomically important crops such as tomatoes and beans. The ability of P. syringae to infect such numerous hosts is caused, in part, by the diversity of effectors employed by this phytopathogen. Over 60 different effector families exist in P. syringae; one such family is HopF, which contains over 100 distinct alleles. Despite this diversity, research has focused on only two members of this family: HopF1 from P. syringae pathovar phaseolicola 1449B and HopF2 from P. syringae pathovar tomato DC3000. In this study, we review the research on HopF family members, including their host targets and molecular mechanisms of immunity suppression, and their enzymatic function. We also provide a phylogenetic analysis of this expanding effector family which provides a basis for a proposed nomenclature to guide future research. The extensive genetic diversity that exists within the HopF family presents a great opportunity to study how functional diversification on an effector family contributes to host specialization.     

Crystal structures of the type III effector protein AvrPphF and its chaperone reveal residues required for plant pathogenesis

The avrPphF locus from Pseudomonas syringae pv. phaseolicola, the causative agent of bean halo-blight disease, encodes proteins which either enhance virulence on susceptible hosts or elicit defense responses on hosts carrying the R1 resistance gene. Here we present the crystal structures of the two proteins from the avrPphF operon. The structure of AvrPphF ORF1 is strikingly reminiscent of type III chaperones from bacterial pathogens of animals, indicating structural conservation of these specialized chaperones, despite high sequence divergence. The AvrPphF ORF2 effector adopts a novel "mushroom"-like structure containing "head" and "stalk" subdomains. The head subdomain possesses limited structural homology to the catalytic domain of bacterial ADP-ribosyltransferases (ADP-RTs), though no ADP-RT activity was detected for AvrPphF ORF2 in standard assays. Nonetheless, this structural similarity identified two clusters of conserved surface-exposed residues important for both virulence mediated by AvrPphF ORF2 and recognition of this effector by bean plants expressing the R1 resistance gene.     

The targeting of plant cellular systems by injected type III effector proteins

The battle between phytopathogenic bacteria and their plant hosts has revealed a diverse suite of strategies and mechanisms employed by the pathogen or the host to gain the higher ground. Pathogens continually evolve tactics to acquire host resources and dampen host defences. Hosts must evolve surveillance and defence systems that are sensitive enough to rapidly respond to a diverse range of pathogens, while reducing costly and damaging inappropriate misexpression. The primary virulence mechanism employed by many bacteria is the type III secretion system, which secretes and translocates effector proteins directly into the cells of their plant hosts. Effectors have diverse enzymatic functions and can target specific components of plant systems. While these effectors should favour bacterial fitness, the host may be able to thwart infection by recognizing the activity or presence of these foreign molecules and initiating retaliatory immune measures. We review the diverse host cellular systems exploited by bacterial effectors, with particular focus on plant proteins directly targeted by effectors. Effector–host interactions reveal different stages of the battle between pathogen and host, as well as the diverse molecular strategies employed by bacterial pathogens to hijack eukaryotic cellular systems.     

Type III effector proteins: doppelgangers of bacterial virulence

Bacterial pathogens have co-evolved with their hosts in their ongoing quest for advantage in the resulting interaction. These intimate associations have resulted in remarkable adaptations of prokaryotic virulence proteins and their eukaryotic molecular targets. An important strategy used by microbial pathogens of animals to manipulate host cellular functions is structural mimicry of eukaryotic proteins. Recent evidence demonstrates that plant pathogens also use structural mimicry of host factors as a virulence strategy. Nearly all virulence proteins from phytopathogenic bacteria have eluded functional annotation on the basis of primary amino-acid sequence. Recent efforts to determine their three-dimensional structures are, however, revealing important clues about the mechanisms of bacterial virulence in plants.