Structural and functional analysis of the cerato-platanin-like protein Cpl1 suggests diverging functions in smut fungi.

Plant-pathogenic fungi are causative agents of the majority of plant diseases and can lead to severe crop loss in infected populations. Fungal colonization is achieved by combining different strategies, such as avoiding and counteracting the plant immune system and manipulating the host metabolome. Of major importance are virulence factors secreted by fungi, which fulfil diverse functions to support the infection process. Most of these proteins are highly specialized, with structural and biochemical information often absent. Here, we present the atomic structures of the cerato-platanin-like protein Cpl1 from Ustilago maydis and its homologue Uvi2 from Ustilago hordei. Both proteins adopt a double-Ψß-barrel architecture reminiscent of cerato-platanin proteins, a class so far not described in smut fungi. Our structure-function analysis shows that Cpl1 binds to soluble chitin fragments via two extended grooves at the dimer interface of the two monomer molecules. This carbohydrate-binding mode has not been observed previously and expands the repertoire of chitin-binding proteins. Cpl1 localizes to the cell wall of U. maydis and might synergize with cell wall-degrading and decorating proteins during maize infection. The architecture of Cpl1 harbouring four surface-exposed loop regions supports the idea that it might play a role in the spatial coordination of these proteins. While deletion of cpl1 has only mild effects on the virulence of U. maydis, a recent study showed that deletion of uvi2 strongly impairs U. hordei virulence. Our structural comparison between Cpl1 and Uvi2 reveals sequence variations in the loop regions that might explain a diverging function.

Fortuitously compatible protein surfaces primed allosteric control in cyanobacterial photoprotection.

Highly specific interactions between proteins are a fundamental prerequisite for life, but how they evolve remains an unsolved problem. In particular, interactions between initially unrelated proteins require that they evolve matching surfaces. It is unclear whether such surface compatibilities can only be built by selection in small incremental steps, or whether they can also emerge fortuitously. Here, we used molecular phylogenetics, ancestral sequence reconstruction and biophysical characterization of resurrected proteins to retrace the evolution of an allosteric interaction between two proteins that act in the cyanobacterial photoprotection system. We show that this interaction between the orange carotenoid protein (OCP) and its unrelated regulator, the fluorescence recovery protein (FRP), evolved when a precursor of FRP was horizontally acquired by cyanobacteria. FRP's precursors could already interact with and regulate OCP even before these proteins first encountered each other in an ancestral cyanobacterium. The OCP-FRP interaction exploits an ancient dimer interface in OCP, which also predates the recruitment of FRP into the photoprotection system. Together, our work shows how evolution can fashion complex regulatory systems easily out of pre-existing components.

MerA functions as a hypothiocyanous acid reductase and defense mechanism in Staphylococcus aureus.

The major pathogen Staphylococcus aureus has to cope with host-derived oxidative stress to cause infections in humans. Here, we report that S. aureus tolerates high concentrations of hypothiocyanous acid (HOSCN), a key antimicrobial oxidant produced in the respiratory tract. We discovered that the flavoprotein disulfide reductase (FDR) MerA protects S. aureus from this oxidant by functioning as a HOSCN reductase, with its deletion sensitizing bacteria to HOSCN. Crystal structures of homodimeric MerA (2.4 Å) with a Cys43 -Cys48 intramolecular disulfide, and reduced MerACys43 S (1.6 Å) showed the FAD cofactor close to the active site, supporting that MerA functions as a group I FDR. MerA is controlled by the redox-sensitive repressor HypR, which we show to be oxidized to intermolecular disulfides under HOSCN stress, resulting in its inactivation and derepression of merA transcription to promote HOSCN tolerance. Our study highlights the HOSCN tolerance of S. aureus and characterizes the structure and function of MerA as a major HOSCN defense mechanism. Crippling the capacity to respond to HOSCN may be a novel strategy for treating S. aureus infections.

Structural insights into the mechanism of archaellar rotational switching.

Signal transduction via phosphorylated CheY towards the flagellum and the archaellum involves a conserved mechanism of CheY phosphorylation and subsequent conformational changes within CheY. This mechanism is conserved among bacteria and archaea, despite substantial differences in the composition and architecture of archaellum and flagellum, respectively. Phosphorylated CheY has higher affinity towards the bacterial C-ring and its binding leads to conformational changes in the flagellar motor and subsequent rotational switching of the flagellum. In archaea, the adaptor protein CheF resides at the cytoplasmic face of the archaeal C-ring formed by the proteins ArlCDE and interacts with phosphorylated CheY. While the mechanism of CheY binding to the C-ring is well-studied in bacteria, the role of CheF in archaea remains enigmatic and mechanistic insights are absent. Here, we have determined the atomic structures of CheF alone and in complex with activated CheY by X-ray crystallography. CheF forms an elongated dimer with a twisted architecture. We show that CheY binds to the C-terminal tail domain of CheF leading to slight conformational changes within CheF. Our structural, biochemical and genetic analyses reveal the mechanistic basis for CheY binding to CheF and allow us to propose a model for rotational switching of the archaellum.

Identification and Characterization of Two Transmembrane Proteins Required for Virulence of Ustilago maydis.

Smut fungi comprise a large group of biotrophic phytopathogens infecting important crops such as wheat and corn. Through the secretion of effector proteins, the fungus actively suppresses plant immune reactions and modulates its host's metabolism. Consequently, how soluble effector proteins contribute to virulence is already characterized in a range of phytopathogens. However, membrane-associated virulence factors have been much less studied to date. Here, we investigated six transmembrane (TM) proteins that show elevated gene expression during biotrophic development of the maize pathogen Ustilago maydis. We show that two of the six proteins, named Vmp1 and Vmp2 (virulence-associated membrane protein), are essential for the full virulence of U. maydis. The deletion of the corresponding genes leads to a substantial attenuation in the virulence of U. maydis. Furthermore, both are conserved in various related smuts and contain no domains of known function. Our biochemical analysis clearly shows that Vmp1 and Vmp2 are membrane-associated proteins, potentially localizing to the U. maydis plasma membrane. Mass photometry and light scattering suggest that Vmp1 mainly occurs as a monomer, while Vmp2 is dimeric. Notably, the large and partially unstructured C-terminal domain of Vmp2 is crucial for virulence while not contributing to dimerization. Taken together, we here provide an initial characterization of two membrane proteins as virulence factors of U. maydis.

The two paralogous kiwellin proteins KWL1 and KWL1-b from maize are structurally related and have overlapping functions in plant defense.

Many plant-pathogenic bacteria and fungi deploy effector proteins that down-regulate plant defense responses and reprogram plant metabolism for colonization and survival in planta Kiwellin (KWL) proteins are a widespread family of plant-defense proteins that target these microbial effectors. The KWL1 protein from maize (corn, Zea mays) specifically inhibits the enzymatic activity of the secreted chorismate mutase Cmu1, a virulence-promoting effector of the smut fungus Ustilago maydis. In addition to KWL1, 19 additional KWL paralogs have been identified in maize. Here, we investigated the structure and mechanism of the closest KWL1 homolog, KWL1-b (ZEAMA_GRMZM2G305329). We solved the Cmu1-KWL1-b complex to 2.75 Å resolution, revealing a highly symmetric Cmu1-KWL1-b heterotetramer in which each KWL1-b monomer interacts with a monomer of the Cmu1 homodimer. The structure also revealed that the overall architecture of the heterotetramer is highly similar to that of the previously reported Cmu1-KWL1 complex. We found that upon U. maydis infection of Z. mays, KWL1-b is expressed at significantly lower levels than KWL1 and exhibits differential tissue-specific expression patterns. We also show that KWL1-b inhibits Cmu1 activity similarly to KWL1. We conclude that KWL1 and KWL1-b are part of a redundant defense system that tissue-specifically targets Cmu1. This notion was supported by the observation that both KWL proteins are carbohydrate-binding proteins with distinct and likely tissue-related specificities. Moreover, binding by Cmu1 modulated the carbohydrate-binding properties of both KWLs. These findings indicate that KWL proteins are part of a spatiotemporally coordinated, plant-wide defense response comprising proteins with overlapping activities.

Independent Scientific Review under the Endangered Species Act.

The directive from Congress in the Endangered Species Act obliging the US Fish and Wildlife Service and National Marine Fisheries Service along with other federal agencies to use the best available scientific information in their determinations-and calls from stakeholder communities to show that they have done so-have led the federal wildlife agencies to seek external, expert review of their determinations with increasing frequency over time. In the present article, we survey the agency determinations that may be subject to independent science review and the technical tasks embedded in those determinations that can benefit from such review. We go on to identify common failures in scientific review that compromise the quality and reliability of agency determinations and then describe the attributes of independent scientific reviews that enable the agencies to discharge their statutory duties while seeking to conserve threatened and endangered species and the ecosystems on which they depend.

Guidance on the Use of Best Available Science under the U.S. Endangered Species Act.

The Endangered Species Act's best available science mandate has been widely emulated and reflects a Congressional directive to ensure that decisions made under the Act are informed by reliable knowledge applied using a structured approach. We build on a standing literature by describing the role of the best science directive in the Act's implementation and best practices that can be employed to realize the directive. Next we describe recurring impediments to realizing determinations by the federal wildlife agencies that are based on the best available science. We then identify the types of data, analyses, and modeling efforts that can serve as best science. Finally, we consider the role and application of best available science in effects analysis and adaptive management. We contend that more rigorous adherence by the wildlife agencies to the best available science directive and more assiduous judicial oversight of agency determinations and actions is essential for effective implementation of the Act, particularly where it has substantial ramifications for listed species, stakeholder segments of society, or both.

A critical assessment of the use of surrogate species in conservation planning in the Sacramento-San Joaquin Delta, California (U.S.A.).

Conservation biology has provided wildlife managers with a wealth of concepts and tools for use in conservation planning; among them is the surrogate species concept. Over the past 20 years, a growing body of empirical literature has demonstrated the limited effectiveness of surrogates as management tools, unless it is first established that the target species and surrogate will respond similarly to a given set of environmental conditions. Wildlife managers and policy makers have adopted the surrogate species concept, reflecting the limited information available on most species at risk of extirpation or extinction and constraints on resources available to support conservation efforts. We examined the use of surrogate species, in the form of cross-taxon response-indicator species (that is, one species from which data are used to guide management planning for another, distinct species) in the Sacramento-San Joaquin Delta, California (U.S.A.). In that system there has been increasing reliance on surrogates in conservation planning for species listed under federal or state endangered species acts, although the agencies applying the surrogate species concept did not first validate that the surrogate and target species respond similarly to relevant environmental conditions. During the same period, conservation biologists demonstrated that the surrogate concept is generally unsupported by ecological theory and empirical evidence. Recently developed validation procedures may allow for the productive use of surrogates in conservation planning, but, used without validation, the surrogate species concept is not a reliable planning tool.

The route to best science in implementation of the Endangered Species Act's consultation mandate: the benefits of structured effects analysis.

The Endangered Species Act is intended to conserve at-risk species and the ecosystems upon which they depend, and it is premised on the notion that if the wildlife agencies that are charged with implementing the statute use the best available scientific information, they can successfully carry out this intention. We assess effects analysis as a tool for using best science to guide agency decisions under the Act. After introducing effects analysis, we propose a framework that facilitates identification and use of the best available information in the development of agency determinations. The framework includes three essential steps--the collection of reliable scientific information, the critical assessment and synthesis of available data and analyses derived from those data, and the analysis of the effects of actions on listed species and their habitats. We warn of likely obstacles to rigorous, structured effect analyses and describe the extent to which independent scientific review may assist in overcoming these obstacles. We conclude by describing eight essential elements that are required for a successful effects analysis.

Further evidence for individual differences in placebo responding: an interactionist perspective.

OBJECTIVE: A prior investigation found that individuals low in optimism are more likely to follow a negative placebo (nocebo) expectation. The present study tested the hypothesis that individuals high in optimism are more likely to follow a positive placebo expectation. METHODS: Individuals (N=56) varying in their level of optimism were randomly assigned to one of three conditions. In the first condition, participants were given the expectation that a placebo sleep treatment would improve their sleep quality (placebo expectation condition). In the second condition, participants engaged in the same sleep treatment activity but were not given the positive placebo expectation (treatment control condition). Finally, a third group did not receive the positive placebo expectation and also did not engage in the placebo sleep treatment (no-placebo control condition). RESULTS: Optimism was positively associated with better sleep quality in the placebo expectation condition (r=.48, P<.05). Optimism scores were not associated with better sleep quality in either the treatment control condition (r=-.17, P=.46) or the no-placebo control condition (r=-.24, P=.35). CONCLUSION: Dispositional optimism relates to placebo responding. This relationship, however, is not manifested in a simple increase or decrease in all types of placebo responding. Rather, it appears that, as optimism increases, response to the positive placebo expectation increases, whereas response to nocebo expectation decreases. It is recommended that future research on personality and placebo effects consider the interaction between situational and dispositional variables.

Expectations and placebo response: a laboratory investigation into the role of somatic focus.

It has been theorized that expectations are an important causal determinant of the placebo effect. Placebo expectations, however, do not always yield placebo effects. In a laboratory study, we tested the hypothesis that one's level of somatic focus moderates the effect of placebo expectations on placebo responding. We also varied whether participants were told the placebo was a drug, could either be a drug or placebo, or was a placebo. The results revealed that individuals who thought they were taking a drug (i.e., unconditional expectations) reported more placebo symptoms when they closely focused on their symptoms. Individuals told they may or may not be receiving a drug (i.e., conditional expectations) did not differ from control participants regardless of how closely they attended to their symptoms. The findings have theoretical implications for expectancy models of the placebo effect as well as for practical research comparing the type of expectations held by individuals in clinical trials and clinical practice.

Child and adolescent abuse and subsequent victimization: a prospective study.

OBJECTIVE: We investigated the possible reciprocal relationship between victimization experiences and psychological functioning by assessing abuse experiences in childhood, adolescence, and during a 2-month follow-up period. METHOD: At the beginning of the study (Time 1), abuse histories, trauma and depressive symptoms, and interpersonal functioning were assessed in 551 college women. Subsequent victimization experiences and psychological outcomes were assessed at the follow-up (Time 2). RESULTS: Path analyses indicated that verbal abuse by the mother and father were predictive of various psychological outcomes as measured at Time 1 and emerged as the only significant predictors of adolescent dating violence. Adolescent dating violence subsequently predicted the experience of dating violence during the 2-month follow-up period. Paternal physical abuse predicted adolescent sexual victimization which subsequently predicted all symptom measures at Time 1. Conversely, the experience of adolescent physical dating violence was not predictive of any of the symptom measures at Time 1. For those women who experienced dating violence during the follow-up, however, the severity of their abusive experiences was related to both depression and interpersonal problems assessed at Time 2. In comparison, for those women who experienced sexual victimization during the follow-up period, the severity of their abusive experiences was related to trauma symptoms. Interpersonal problems emerged as both an aftereffect of adolescent sexual victimization experience and a predictor of a subsequent sexual victimization experience during the follow-up. CONCLUSIONS: Given that emotional abuse emerged as a predictor of adolescent dating violence and psychological outcomes, researchers and clinicians need to continue to explore this problem. Further, it is important to assess how interpersonal problems contribute to the risk of subsequent sexual victimization and to try to break the cycle between adolescent abuse experiences and subsequent physical and sexual assaults.

Goal activation, expectations, and the placebo effect.

Motivational factors receive little attention in current theories of the placebo effect. Reasons for this position are reviewed, and an argument is made for reconsidering the influence of motivation on the placebo effect. The authors hypothesize that nonconscious goals alter reactions to a placebo expectation. Specifically, the authors predict that the placebo effect is most likely to occur when individuals have a goal that can be fulfilled by confirmation of the placebo expectation. The authors tested this notion in 5 experiments. The results demonstrate the role of motivation in the placebo effect across a variety of symptom domains and via 4 different goal activation techniques. Moreover, this moderating effect occurred for both positive and negative placebo expectations, across different placebo effect measures, and in brief laboratory experiments as well as in lengthier studies. It is argued that theories regarding the placebo effect should incorporate motivational factors.

Reconsidering the role of personality in placebo effects: dispositional optimism, situational expectations, and the placebo response.

OBJECTIVE: Prior investigations have failed to find reliable personality differences in placebo responding. The present study tests the hypothesis that personality and situational variables interact to determine placebo responding. METHODS: Optimists and pessimists were randomly assigned to one of three conditions. In the first condition, the participants were told that they were to ingest a pill that would make them feel unpleasant (deceptive-expectation group). In the second condition, the participants were told that they were to ingest a pill that would make them feel either unpleasant or was an inactive substance (conditional-expectation group). Finally, a third group was told they were to ingest a pill that was inactive (control group). RESULTS: Pessimists were more likely than optimists to follow a negative-placebo expectation when given a deceptive expectation, but not when given a conditional expectation. CONCLUSION: The personality variable optimism-pessimism relates to placebo responding when individuals are given a deceptive but not a conditional expectation. This suggests that personality and situational variables interact to determine placebo responding.

Videotaped interrogations and confessions: a simple change in camera perspective alters verdicts in simulated trials.

Prior research has indicated that altering the perspective from which a videotaped confession is recorded influences assessments of the confession's voluntariness. The authors examined whether this camera perspective bias persists in more ecologically valid contexts. In Study 1, neither a realistic videotaped trial simulation nor potentially corrective judicial instruction was sufficient to mitigate the prejudicial effect of camera perspective on mock jurors' assessments of voluntariness or on their all-important final verdicts. Study 2 suggests that perhaps the best camera perspective to use is one that focuses trial fact finders' attention on the interrogator, as this particular vantage point may facilitate decision makers' capacity to detect coercive influences, which in turn could, in some cases, improve assessments of the confession's reliability.