Parasitic Molecular Strategies to Influence Host Plant Signaling and Gene Transcription

Parasitic plants are a constraint on agriculture worldwide. Plants of the genus Cuscuta are obligate holoparasites with a broad host spectrum for nearly all dicotyledonous plants. As leaf- and rootless plants, Cuscuta spp. wind around stems of host plants and penetrate host tissue with haustoria. They directly connect to the vasculature and exhaust water, nutrients and carbohydrates. Thus, the haustorium development and the establishment of a connection to the host represent essential steps in the parasite’s life cycle. To date, little is known concerning the development of such host-parasite connections on molecular level. The aim of this work was to gain knowledge about specific molecular signals of Cuscuta spp. that get sensed by host plants and manipulate hosts towards susceptibility. On the host plant side, the major focus was to identify Cuscuta-derived transcription factor targets or receptors that recognize parasitic molecules and further induce cellular signaling programs related to susceptibility or development. To shed light on the transcriptomic reprogramming during the early stages of infection with Cuscuta spp., a transcriptome analysis by RNA sequencing after infiltration of Cuscuta Extract into N. benthamiana leaves has been performed. Additionally, an intense literature search with a focus on RNA sequencing data providing regulated transcripts of host and parasite genes was initiated. To investigate the molecular cues that might be necessary to switch-on host responses, intracellular processes and the connection to the host vascular system, promoters of upregulated host genes at the Cuscuta spp. infection site have been used to control the expression of the luciferase reporter gene. To establish a promoter:luciferase based bioassay to screen for inducing Cuscuta-derived molecular cues, a promoter was needed to control luciferase expression leading to significantly increased light emission after treatment with Cuscuta Extract. The promoter:luciferase construct of USUALLY MULTIPLE ACIDS MOVE IN AND OUT TRANSPORTER 25 (pUMAMIT25:luc) that showed a specific reaction to Cuscuta Extract was used to purify the Cuscuta-derived molecular cue. The purification and identification of the Cuscuta-derived molecule is part of an ongoing project. The molecule seems to be difficult to isolate since it did not bind to most of the tested chromatography columns. A characterization showed that the molecular cue had no charge, was hydrophilic and was not of proteidogenous nature. An in silico analysis of the host UMAMIT25 which seems manipulated by C. reflexa revealed its involvement in amino acid transport, that can be relevant for Cuscuta spp. nutrition and growth. Additional beneficial transporter genes like sucrose transporters were analyzed and the corresponding promoter:luciferase constructs showed increased light emission upon Cuscuta Extract treatment. The experiments of this work leave it open whether the molecular cue might be a sucrose gradient, a Cuscuta-derived CLE peptide, a Transcription activator-like (TAL) effector-like or a still elusive molecular trigger

Eine experimentelle Methode zur Untersuchung von Raumluftströmungen in einem stark verkleinerten Modellraum: Entwicklung und Anwendung

In der vorliegenden Arbeit wurde ein Verfahren zur experimentellen Untersuchung nicht-isothermer Raumluftströmungen in einem stark verkleinerten Modellraum unter Berücksichtigung aller Ähnlichkeitskennzahlen entwickelt. Mit diesem Verfahren konnte erstmals eine sehr hohe Genauigkeit bezüglich der Ähnlichkeitskennzahlen Reynolds-Zahl Re, Rayleigh-Zahl Ra, Prandtl-Zahl Pr sowie der Archimedes-Zahl Ar erreicht werden. Die Anpassung der Randbedingungen an den Modellmaßstab erfolgt bei dieser Methode über die Anpassung der Materialeigenschaften eines beliebigen Arbeitsgases durch Erhöhen des Drucks p. Dieses Verfahren konnte in der weltweit einzigartigen Experimentieranlage SCALEX (SCALEX = scaled convective airflow laboratory experiment) umgesetzt und auf zwei Beispielproblemstellungen der Raumluftströmung (isotherme sowie gemischte Konvektion) erfolgreich angewendet werden. Dazu wurden die Geschwindigkeitsverteilungen der groß-skaligen Strömungsstrukturen in einem im Maßstab von 1:10 verkleinerten Modellraum hinreichend komplexer Geometrie für den Fall isothermer sowie gemischter Konvektion untersucht. Die Geschwindigkeitsmessungen wurden mit einem 2D-laser Doppler velocimetry System durchgeführt. Als Arbeitsgas wurde Schwefelhexafluorid (SF6) bei einem Druck von p = 4.517bar verwendet. Die Ergebnisse der vorliegenden Arbeit sowie die neu entstandene Anlage tragen zu einem umfangreichen Verständnis sowie einem vereinfachten experimentellen Zugang zum komplexen Themengebiet der Raumluftströmungen bei.In the present work a new experimental method for the investigation of non-isothermal indoor airflows in a reduced scale model room with respect to the full set of dimensionless numbers has been developed. The method provides a high accuracy with respect to the dimensionless numbers Reynolds number Re, Rayleigh number Ra, Prandtl number Pr and Archimedes number Ar. The adjustment of the boundary conditions to the chosen scale is achieved by adjusting the material properties of a working gas by increasing its pressure. The technical realization of this theoretical approach could be demonstrated by setting up the worldwide unique experimental setup, the SCALEX-facility (SCALEX = scaled convective airflow laboratory experiment) and applying it to two exemplary problems (isothermal and mixed convection) of indoor airflow research. For this purpose the velocity distributions of the large-scale flow structures have been investigated in a 1:10 scale model room with sufficient high complex geometry. The velocity measurements were performed using a 2D laser Doppler velocimetry system. Sulfur hexafluoride (SF6) at a pressure of p = 4.517bar have been used as working gas. The results of the present work as well as the set up SCALEX-facility account for a comprehensive understanding and an easy experimental access to the complex subject of indoor airflow research

The C-terminal domain of Fcj1 is required for formation of crista junctions and interacts with the TOB/SAM complex in mitochondria

Crista junctions (CJs) are tubular invaginations of the inner membrane of mitochondria that connect the inner boundary with the cristae membrane. These architectural elements are critical for mitochondrial function. The yeast inner membrane protein Fcj1, called mitofilin in mammals, was reported to be preferentially located at CJs and crucial for their formation. Here we investigate the functional roles of individual domains of Fcj1. The most conserved part of Fcj1, the C-terminal domain, is essential for Fcj1 function. In its absence, formation of CJ is strongly impaired and irregular, and stacked cristae are present. This domain interacts with full-length Fcj1, suggesting a role in oligomer formation. It also interacts with Tob55 of the translocase of outer membrane β-barrel proteins (TOB)/sorting and assembly machinery (SAM) complex, which is required for the insertion of β-barrel proteins into the outer membrane. The association of the TOB/SAM complex with contact sites depends on the presence of Fcj1. The biogenesis of β-barrel proteins is not significantly affected in the absence of Fcj1. However, down-regulation of the TOB/SAM complex leads to altered cristae morphology and a moderate reduction in the number of CJs. We propose that the C-terminal domain of Fcj1 is critical for the interaction of Fcj1 with the TOB/SAM complex and thereby for stabilizing CJs in close proximity to the outer membrane. These results assign novel functions to both the C-terminal domain of Fcj1 and the TOB/SAM complex

The tomato receptor CuRe1 senses a cell wall protein to identify Cuscuta as a pathogen

Parasitic plants of the genus Cuscuta penetrate shoots of host plants with haustoria and build a connection to the host vasculature to exhaust water, solutes and carbohydrates. Such infections usually stay unrecognized by the host and lead to harmful host plant damage. Here, we show a molecular mechanism of how plants can sense parasitic Cuscuta. We isolated an 11 kDa protein of the parasite cell wall and identified it as a glycine-rich protein (GRP). This GRP, as well as its minimal peptide epitope Crip21, serve as a pathogen-associated molecular pattern and specifically bind and activate a membrane-bound immune receptor of tomato, the Cuscuta Receptor 1 (CuRe1), leading to defense responses in resistant hosts. These findings provide the initial steps to understand the resistance mechanisms against parasitic plants and further offer great potential for protecting crops by engineering resistance against parasitic plants

Three Pseudomonas putida FNR Family Proteins with Different Sensitivities to O-2

The Escherichia coli fumarate-nitrate reduction regulator (FNR) protein is the paradigm for bacterial O2-sensing transcription factors. However, unlike E. coli, some bacterial species possess multiple FNR proteins that presumably have evolved to fulfill distinct roles. Here, three FNR proteins (ANR, PP_3233, and PP_3287) from a single bacterial species, Pseudomonas putida KT2440, have been analyzed. Under anaerobic conditions, all three proteins had spectral properties resembling those of [4Fe-4S] proteins. The reactivity of the ANR [4Fe-4S] cluster with O2 was similar to that of E. coli FNR, and during conversion to the apo-protein, via a [2Fe-2S] intermediate, cluster sulfur was retained. Like ANR, reconstituted PP_3233 and PP_3287 were converted to [2Fe-2S] forms when exposed to O2, but their [4Fe-4S] clusters reacted more slowly. Transcription from an FNR-dependent promoter with a consensus FNR-binding site in P. putida and E. coli strains expressing only one FNR protein was consistent with the in vitro responses to O2. Taken together, the experimental results suggest that the local environments of the iron-sulfur clusters in the different P. putida FNR proteins influence their reactivity with O2, such that ANR resembles E. coli FNR and is highly responsive to low concentrations of O2, whereas PP_3233 and PP_3287 have evolved to be less sensitive to O2

A multi-country test of brief reappraisal interventions on emotions during the COVID-19 pandemic.

The COVID-19 pandemic has increased negative emotions and decreased positive emotions globally. Left unchecked, these emotional changes might have a wide array of adverse impacts. To reduce negative emotions and increase positive emotions, we tested the effectiveness of reappraisal, an emotion-regulation strategy that modifies how one thinks about a situation. Participants from 87 countries and regions (n = 21,644) were randomly assigned to one of two brief reappraisal interventions (reconstrual or repurposing) or one of two control conditions (active or passive). Results revealed that both reappraisal interventions (vesus both control conditions) consistently reduced negative emotions and increased positive emotions across different measures. Reconstrual and repurposing interventions had similar effects. Importantly, planned exploratory analyses indicated that reappraisal interventions did not reduce intentions to practice preventive health behaviours. The findings demonstrate the viability of creating scalable, low-cost interventions for use around the world

Parasitic Cuscuta factor(s) and the detection by tomato initiates plant defense

Dodders (Cuscuta spp.) are holoparasitic plants that enwind stems of host plants and penetrate those by haustoria to connect to the vascular bundles. Having a broad host plant spectrum, Cuscuta spp infect nearly all dicot plants – only cultivated tomato as one exception is mounting an active defense specifically against C. reflexa. In a recent work we identified a pattern recognition receptor of tomato, “Cuscuta Receptor 1“ (CuRe1), which is critical to detect a “Cuscuta factor” (CuF) and initiate defense responses such as the production of ethylene or the generation of reactive oxygen species. CuRe1 also contributes to the tomato resistance against C. reflexa. Here we point to the fact that CuRe1 is not the only relevant component for full tomato resistance but it requires additional defense mechanisms, or receptors, respectively, to totally fend off the parasite