Functional mapping of harpin HrpZ of Pseudomonas syringae reveals the sites responsible for protein oligomerization, lipid interactions and plant defence induction

Harpin HrpZ is one of the most abundant proteins secreted through the pathogenesis-associated type III secretion system of the plant pathogen Pseudomonas syringae. HrpZ shows membrane-binding and pore-forming activities in vitro, suggesting that it could be targeted to the host cell plasma membrane. We studied the native molecular forms of HrpZ and found that it forms dimers and higher order oligomers. Lipid binding by HrpZ was tested with 15 different membrane lipids, with HrpZ interacting only with phosphatidic acid. Pore formation by HrpZ in artificial lipid vesicles was found to be dependent on the presence of phosphatidic acid. In addition, HrpZ was able to form pores in vesicles prepared from Arabidopsis thaliana plasma membrane, providing evidence for the suggested target of HrpZ in the host. To map the functions associated with HrpZ, we constructed a comprehensive series of deletions in the hrpZ gene derived from P. syringae pv. phaseolicola, and studied the mutant proteins. We found that oligomerization is mainly mediated by a region near the C-terminus of the protein, and that the same region is also essential for membrane pore formation. Phosphatidic acid binding seems to be mediated by two regions separate in the primary structure. Tobacco, a nonhost plant, recognizes, as a defence elicitor, a 24-amino-acid HrpZ fragment which resides in the region indispensable for the oligomerization and pore formation functions of HrpZ.     

Short-term TNFα shedding is independent of cytoplasmic phosphorylation or furin cleavage of ADAM17

Proteolysis of transmembrane molecules is an irreversible post-translational modification enabling autocrine, paracrine and endocrine signaling of many cytokines. The pro-inflammatory activities of membrane bound TNFα (pro-TNFα) strongly depend on ectodomain shedding mediated by the A Disintegrin And Metalloprotease family member ADAM17. Despite the well-documented role of ADAM17 in pro-TNFα cleavage during inflammation, little is known about its regulation. Mitogen-activated protein kinase-induced phosphorylation of the ADAM17 cytoplasmic tail has been described to be required for proper activation. To address, if pro-TNFα shedding depends on cytosolic phosphorylation we analyzed ADAM17 mutants lacking the cytoplasmic domain. ADAM17 mediated shedding of pro-TNFα was induced by PMA, Anisomycin and the phosphatase inhibitors Cantharidin and Calyculin A. Deletion of the entire cytoplasmic portion of ADAM17 abolished furin-dependent proteolytic maturation and pro-TNFα cleavage. Interestingly, we could exclude that resistance to proconvertase processing is the reason for the enzymatic inactivity of ADAM17 lacking the cytoplasmic portion as furin-resistant ADAM17 mutants rescued genetic ADAM17 deficiency after mitogen-activated protein kinase activation. Adding only 6 cytoplasmic amino acids completely restored ADAM17 maturation and shedding of pro-TNFα as well as of both TNF-receptors Finally, we showed that a pro-TNFα mutant lacking the cytoplasmic portion was also shed from the cell surface. We conclude that pro-TNFα cleavage by its major sheddase ADAM17 does not depend on cytosolic phosphorylation and/or interaction. These results have general implications on understanding the activation mechanism controlling the activity of ADAM17.     

Knowledge and valuation of Andean agroforestry species: the role of sex,age, and migration among members of a rural community in Bolivia

Background

Agroforestry is a sustainable land use method with a long tradition in the Bolivian Andes. A better understanding of people’s knowledge and valuation of woody species can help to adjust actor-oriented agroforestry systems. In this case study, carried out in a peasant community of the Bolivian Andes, we aimed at calculating the cultural importance of selected agroforestry species, and at analysing the intracultural variation in the cultural importance and knowledge of plants according to peasants’ sex, age, and migration.

Methods

Data collection was based on semi-structured interviews and freelisting exercises. Two ethnobotanical indices (Composite Salience, Cultural Importance) were used for calculating the cultural importance of plants. Intracultural variation in the cultural importance and knowledge of plants was detected by using linear and generalised linear (mixed) models.

Results and discussion

The culturally most important woody species were mainly trees and exotic species (e.g. Schinus molle, Prosopis laevigata, Eucalyptus globulus). We found that knowledge and valuation of plants increased with age but that they were lower for migrants; sex, by contrast, played a minor role. The age effects possibly result from decreasing ecological apparency of valuable native species, and their substitution by exotic marketable trees, loss of traditional plant uses or the use of other materials (e.g. plastic) instead of wood. Decreasing dedication to traditional farming may have led to successive abandonment of traditional tool uses, and the overall transformation of woody plant use is possibly related to diminishing medicinal knowledge.

Conclusions

Age and migration affect how people value woody species and what they know about their uses. For this reason, we recommend paying particular attention to the potential of native species, which could open promising perspectives especially for the young migrating peasant generation and draw their interest in agroforestry. These native species should be ecologically sound and selected on their potential to provide subsistence and promising commercial uses. In addition to offering socio-economic and environmental services, agroforestry initiatives using native trees and shrubs can play a crucial role in recovering elements of the lost ancient landscape that still forms part of local people’s collective identity.

     

Trees in the desert: Reproduction and genetic structure of fragmented Ulmus pumila forests in Mongolian drylands

The potential natural distribution of deciduous forests in Central Asia is the subject of ongoing discussions. Ulmus pumila (Siberian elm) is the only tree species occurring in southern and south-eastern Mongolia. In the semi-arid Mongolian Gobi, the species is restricted to ravines and beds of semi-temporary rivers. Compared to zonal occurrences in moister northern Mongolia, elm trees in the Gobi were found to be larger, in spite of their slower growth. Recruitment was very rare in the field although germination studies revealed that seeds were viable, survived osmotic stress, and were tolerant of repeated cycles of moistening and drying. Thus, they should be capable of germination in episodically flooded river beds. Fingerprinting revealed that clonal growth is of negligible importance in the Gobi as almost all U. pumila individuals studied constituted separate genets. Given that many trees were <100 years old and must have become established under current climatic conditions, we infer that the current lack of recruitment is likely to be caused by grazing impact. Our data imply that Ulmus pumila could potentially be much more common in the drylands of southern Mongolia and northern China.     

Similar performance in central and range‐edge populations of a Eurasian steppe grass under different climate and soil pH regimes

The abundant centre hypothesis predicts that changing environmental conditions are detrimental to a species’ abundance and performance towards the periphery of its range. We tested these predictions for the perennial grass Stipa capillata, a species that is commonly found in steppes of Asia but is rare at its north‐western range edge, in central Europe. We compared 21 populations in dry grassland fragments in central Europe and 20 populations in steppe habitats of Kazakhstan. We studied 15 plant performance traits both in situ and under laboratory and common greenhouse conditions, including local density, plant size and biomass production, seed size, weight and viability. To assess environmental conditions, we assembled data on topography, soil properties and climatic parameters. Using variance components analysis and multivariate methods we analyzed whether plant performance and environmental attributes differed more, as predicted, between the core and peripheral regions or whether they differed more among their subregions or populations. Additionally, we tested whether performance was affected by the same set of environmental predictors in each region. Contrary to our expectations, plant performance traits were virtually unchanged between the range periphery and centre. As expected, macroclimatic conditions showed a significant difference between the two regions (annual mean temperature, annual precipitation). The only other measured environmental variable that differed significantly between the two regions was soil pH, which was lower in core‐range populations. Our findings thus do not support the notion of reduced performance at the range edge. Instead, our data lend support to earlier theories of relative habitat constancy, suggesting that peripheral populations can shift to other habitats through plasticity or adaptation.     

Assay conditions in laboratory experiments: is the use of constant rather than fluctuating temperatures justified when investigating temperature-induced plasticity?

Temperature is an important selective agent in nature. Consequently, temperature-induced plasticity which may help buffering detrimental effects of temperature variation has received considerable attention over recent decades. Laboratory studies have almost exclusively used constant temperatures, while in nature, temperature typically shows pronounced daily fluctuations. Using a factorial design with constant versus fluctuating temperatures and a higher versus a lower mean temperature, we here investigate in the butterfly Lycaena tityrus whether the use of constant temperatures is justified. Fluctuating compared to constant temperatures caused shorter development times, increased heat but decreased cold stress resistance, decreased heat-shock protein expression, and increased immunocompetence. Thus, overall, fluctuating temperatures were more beneficial to the butterflies compared to constant ones. However, despite substantial variation across temperature regimes, the ranking of trait values among treatments remained largely unaffected (e.g. lower constant as well as fluctuating temperatures caused increased pupal mass). Thus, we tentatively conclude that there is no general reason for concern about using constant temperatures in studies investigating phenotypic plasticity, which seem to comprise a fair proxy. However, substantial differences in mean values as well as interactive effects suggest that one needs to be cautious. We further demonstrate negative effects of high temperatures on butterfly immune function, which seem to result from a trade-off between the latter and the heat shock response.     

Botrytis hypersensitive response inducing protein 1 triggers noncanonical PTI to induce plant cell death

According to their lifestyle, plant pathogens are divided into biotrophic and necrotrophic organisms. Biotrophic pathogens exclusively nourish living host cells, whereas necrotrophic pathogens rapidly kill host cells and nourish cell walls and cell contents. To this end, the necrotrophic fungus Botrytis cinerea secretes large amounts of phytotoxic proteins and cell wall-degrading enzymes. However, the precise role of these proteins during infection is unknown. Here, we report on the identification and characterization of the previously unknown toxic protein hypersensitive response-inducing protein 1 (Hip1), which induces plant cell death. We found the adoption of a structurally conserved folded Alternaria alternata Alt a 1 protein structure to be a prerequisite for Hip1 to exert its necrosis-inducing activity in a host-specific manner. Localization and the induction of typical plant defense responses by Hip1 indicate recognition as a pathogen-associated molecular pattern at the plant plasma membrane. In contrast to other secreted toxic Botrytis proteins, the activity of Hip1 does not depend on the presence of the receptor-associated kinases BRI1-associated kinase 1 and suppressor of BIR1-1. Our results demonstrate that recognition of Hip1, even in the absence of obvious enzymatic or pore-forming activity, induces strong plant defense reactions eventually leading to plant cell death. Botrytis hip1 overexpression strains generated by CRISPR/Cas9 displayed enhanced infection, indicating the virulence-promoting potential of Hip1. Taken together, Hip1 induces a noncanonical defense response which might be a common feature of structurally conserved fungal proteins from the Alt a 1 family.

The secreted Botrytis protein hypersensitive response inducing protein 1 activates plant defense to kill plant cells.     

Attention‐deficit/hyperactivity disorder as a risk factor for cardiovascular diseases: a nationwide population‐based cohort study

Accumulating evidence suggests a higher risk for cardiovascular diseases among individuals with mental disorders, but very little is known about the risk for overall and specific groups of cardiovascular diseases in people with attention‐deficit/hyperactivity disorder (ADHD). To fill this knowledge gap, we investigated the prospective associations between ADHD and a wide range of cardiovascular diseases in adults. In a nationwide population‐based cohort study, we identified 5,389,519 adults born between 1941 and 1983, without pre‐existing cardiovascular diseases, from Swedish registers. The study period was from January 1, 2001 to December 31, 2013. Incident cardiovascular disease events were identified according to ICD codes. Hazard ratios (HR) with 95% confidence intervals (CI) were calculated using Cox proportional hazards regression model, with ADHD as a time‐varying exposure. After an average 11.80 years of follow‐up, 38.05% of individuals with ADHD versus 23.57% of those without ADHD had at least one diagnosis of cardiovascular disease (p<0.0001). ADHD was significantly associated with increased risk of any cardiovascular disease (HR=2.05, 95% CI: 1.98‐2.13) after adjusting for sex and year of birth. Further adjustments for education level, birth country, type 2 diabetes mellitus, obesity, dyslipidemia, sleep problems and heavy smoking attenuated the association, which however remained significant (HR=1.84, 95% CI: 1.77‐1.91). Further adjustment for psychiatric comorbidities attenuated but could not fully explain the association (HR=1.65, 95% CI: 1.59‐1.71). The strongest associations were found for cardiac arrest (HR=2.28, 95% CI: 1.81‐2.87), hemorrhagic stroke (HR=2.16, 95% CI: 1.68‐2.77), and peripheral vascular disease/arteriosclerosis (HR=2.05, 95% CI: 1.76‐2.38). Stronger associations were observed in males and younger adults, while comparable associations were found among individuals with or without psychotropic medications and family history of cardiovascular diseases. These data suggest that ADHD is an independent risk factor for a wide range of cardiovascular diseases. They highlight the importance of carefully monitoring cardiovascular health and developing age‐appropriate and individualized strategies to reduce the cardiovascular risk in individuals with ADHD.