Candidate Effectors From Uromyces appendiculatus, the Causal Agent of Rust on Common Bean, Can Be Discriminated Based on Suppression of Immune Responses

Rust fungi are devastating pathogens for several important crop plants. The biotrophic lifestyle of rust fungi requires that they influence their host plants to create a favorable environment for growth and reproduction. Rust fungi secrete a variety of effector proteins that manipulate host target proteins to alter plant metabolism and suppress defense responses. Because of the obligate biotrophic lifestyle of rust fungi, direct evidence for effector function is difficult to obtain, and so suites of experiments utilizing expression in heterologous systems are necessary. Here, we present results from a yeast cell death suppression assay and assays for suppression of PAMP-triggered immunity (PTI) and effector triggered immunity (ETI) based on delivery of effectors through the bacterial type III secretion system. In addition, subcellular localization was tested using transient expression of GFP fusion proteins in Nicotiana benthamiana through Agrobacterium infiltration. We tested 31 representative effector candidates from the devastating common bean rust pathogen Uromyces appendiculatus. These effector candidates were selected based on features of their gene families, most important lineage specificity. We show that several of our effector candidates suppress plant defense. Some of them also belong to families of effector candidates that are present in multiple rust species where their homologs probably also have effector functions. In our analysis of candidate effector mRNA expression, some of those effector candidates that gave positive results in the other assays were not up-regulated during plant infection, indicating that either these proteins have functions at multiple life stages or that strong up-regulation of RNA level in planta may not be as important a criterion for identifying effectors as previously thought. Overall, our pipeline for selecting effector candidates based on sequence features followed by screening assays using heterologous expression systems was successful in discriminating effector candidates. This work lays the foundation for functional characterization of U. appendiculatus effectors, the identification of effector targets, and identification of novel sources for resistance in common bean

Suppression or Activation of Immune Responses by Predicted Secreted Proteins of the Soybean Rust Pathogen Phakopsora pachyrhizi

Rust fungi, such as the soybean rust pathogen Phakopsora pachyrhizi, are major threats to crop production. They form specialized haustoria that are hyphal structures intimately associated with host-plant cell membranes. These haustoria have roles in acquiring nutrients and secreting effector proteins that manipulate host immune systems. Functional characterization of effector proteins of rust fungi is important for understanding mechanisms that underlie their virulence and pathogenicity. Hundreds of candidate effector proteins have been predicted for rust pathogens, but it is not clear how to prioritize these effector candidates for further characterization. There is a need for high-throughput approaches for screening effector candidates to obtain experimental evidence for effector-like functions, such as the manipulation of host immune systems. We have focused on identifying effector candidates with immune-related functions in the soybean rust fungus P. pachyrhizi. To facilitate the screening of many P. pachyrhizi effector candidates (named PpECs), we used heterologous expression systems, including the bacterial type III secretion system, Agrobacterium infiltration, a plant virus, and a yeast strain, to establish an experimental pipeline for identifying PpECs with immune-related functions and establishing their subcellular localizations. Several PpECs were identified that could suppress or activate immune responses in nonhost Nicotiana benthamiana, N. tabacum, Arabidopsis, tomato, or pepper plants

Transport of Beads by Several Kinesin Motors

The movements of beads pulled by several kinesin-1 (conventional kinesin) motors are studied both theoretically and experimentally. While the velocity is approximately independent of the number of motors pulling the beads, the walking distance or run-length is strongly increased when more motors are involved. Run-length distributions are measured for a wide range of motor concentrations and matched to theoretically calculated distributions using only two global fit parameters. In this way, the maximal number of motors pulling the beads is estimated to vary between two and seven motors for total kinesin concentrations between 0.1 and 2.5 μg/ml or between 0.27 and 6.7 nM. In the same concentration regime, the average number of pulling motors is found to lie between 1.1 and 3.2 motors

Mycoviruses in the Rust Fungus <i>Uromyces fabae</i>

Uromyces fabae, the causal agent of broad bean rust, is a major cause of yield losses in North and East Africa, China, and Australia. It has also served as an important model species for research on rust fungi. Early EST sequencing in U. fabae showed that viruses might be present in this species; however, no follow-up investigations were conducted. In order to identify these viruses, we performed purification of dsRNA followed by Illumina sequencing. We also used ultracentrifugation followed by negative staining electron microscopy to visualize virus particles. We identified 20 viral sequences, which we termed Ufvss. A phylogenetic analysis was performed that grouped Ufvss into totiviruses, polymycoviruses, and virgaviruse; three sequences could not be included in the phylogeny. We also found isometric particles. Our findings contribute to the knowledge of mycoviral diversity in rust fungi and point to the importance of further investigation of these viruses

Similarity-based Influences in Judgment and Decision Making

Psychological similarity—the subjective distance between objects in the world or memory—is a highly influential concept in many areas of cognitive psychology, such as learning, memory, categorization, judgment, and preferential choice. The contributions within this symposium will evaluate the fundamental role that similarity plays in human judgment and decision making. We bring together experts from distinct subdisciplines of psychology, who examine the influence of similarity on categorization, consumer choice, risky choice, social norms, and in memory-based choices. Specifically, the contributions elaborate on three key questions repeatedly pursued within cognitive psychology: 1) how does similarity activate previous experiences and renders them available within a given choice context? 2) how does similarity interact with feature or knowledge abstraction processes? 3) how is similarity represented psychologically? To reach this goal, the contributions within this symposium focus on reinstating similarity-based processes within formal cognitive models and test their predictions experimentally

Report on Notifications Pursuant to §21 German Transfusion Act for 2007

The present report contains the data collected in 2007, pursuant to Section 21 German Transfusion Act, and an analysis of the supply situation over the past 8 years. As in previous years, all blood donation centres located in Germany transmitted data on the collection, manufacture, import and export of blood components for transfusion. According to these data, a total of 6.7 million blood collections were performed in 2007. With 4.7 million, the portion of whole blood donations was at the level of previous years, whereas the number of apheresis donations rose again, to 1.9 million. The portion of autologous blood collections accounts for only 1.1% and thus continues to decline. Since 2003, the number of red blood cell concentrates prepared has been a constant 4.5 million transfusion units. The decay of red blood cell concentrates on the user side in 2000 accounted for 5% while in 2007 it was just above 3%, referred to the total quantity of data reported as transfused and decayed. The manufacture of platelet concentrates rose from 366,000 to 480,000 transfusion units between 2003 and 2007. The production of therapeutic single plasmas, too, markedly increased in 2007 (to 1.2 million transfusion units). In 2007, 2.2 million I of plasma for fractionation were collected in Germany. In addition, 1.0 million I were imported, and 1.8 million I were exported. The quantity available in Germany from a pure arithmetic point of view of 1.4 million I was almost entirely allocated to basic fractionation so that a sufficient plasma supply can be assumed. Due to the fact that manufacturing capacities are still lacking in Germany, recombinant factors need to be imported in their entirety. Since 2003, Germany has by far been the leader in Europe with more than 20 I of fractionation plasma collected per 1,000 inhabitants. Furthermore, regarding the manufacturing figures of red blood cell concentrates, platelet concentrates, and therapeutic single plasma, Germany is in the top third for all these products compared with other European countries. The manufacture of allogeneic stem cell products for haematopoietic reconstitution, obtained by apheresis, has continuously risen to 4,700. A large portion of this (1,810 transplants) could be exported while only a small number (179 preparations) had to be imported. The manufacture of autologous stem cell preparations from cord blood has risen drastically to more than 10,000 in 2007. The interest in the figures collected in compliance with Section 21, German Transfusion Act remains high both in Germany and at an international level. Meanwhile reliable data are available

Similarity-based Influences in Judgment and Decision Making

Psychological similarity—the subjective distance between objects in the world or memory—is a highly influential concept in many areas of cognitive psychology, such as learning, memory, categorization, judgment, and preferential choice. The contributions within this symposium will evaluate the fundamental role that similarity plays in human judgment and decision making. We bring together experts from distinct subdisciplines of psychology, who examine the influence of similarity on categorization, consumer choice, risky choice, social norms, and in memory-based choices. Specifically, the contributions elaborate on three key questions repeatedly pursued within cognitive psychology: 1) how does similarity activate previous experiences and renders them available within a given choice context? 2) how does similarity interact with feature or knowledge abstraction processes? 3) how is similarity represented psychologically? To reach this goal, the contributions within this symposium focus on reinstating similarity-based processes within formal cognitive models and test their predictions experimentally

Diels-Alder ligation of peptides and proteins

The development of the Diels-Alder cycloaddition as a new method for the site-specific chemoselective ligation of peptides and proteins under mild conditions is reported. Peptides equipped with a 2,4-hexadienyl ester and an N-terminal maleimide react in aqueous media to give cycloadducts in high yields and depending on the amino acid sequence with high stereoselectivity. Except for the cysteine SH group the transformation is compatible with all amino acid side chain functional groups. For ligation to proteins the hexadienyl group was attached to avidin and streptavidin noncovalently by means of complex formation with a biotinylated peptide or by covalent attachment of a hexadienyl ester-containing label to lysine side chains incorporated into the proteins. Site-specific attachment of the hexadienyl unit into a Rab protein was achieved by means of expressed protein ligation followed by protection of the generated cysteine SH by means of Ellman's reagent. The protein reacted with different maleimido-modified peptides under mild conditions to give the fully functional cycloadducts in high yield. The results demonstrate that the Diels-Alder ligation offers an advantageous and technically straightforward new opportunity for the site-specific equipment of peptides and proteins with further functional groups and labels. It proceeds under very mild conditions and is compatible with most functional groups found in proteins. Its combination with other ligation methods, in particular expressed protein ligation is feasible

Candidate Effectors From Uromyces appendiculatus, the Causal Agent of Rust on Common Bean, Can Be Discriminated Based on Suppression of Immune Responses

Rust fungi are devastating pathogens for several important crop plants. The biotrophic lifestyle of rust fungi requires that they influence their host plants to create a favorable environment for growth and reproduction. Rust fungi secrete a variety of effector proteins that manipulate host target proteins to alter plant metabolism and suppress defense responses. Because of the obligate biotrophic lifestyle of rust fungi, direct evidence for effector function is difficult to obtain, and so suites of experiments utilizing expression in heterologous systems are necessary. Here, we present results from a yeast cell death suppression assay and assays for suppression of PAMP-triggered immunity (PTI) and effector triggered immunity (ETI) based on delivery of effectors through the bacterial type III secretion system. In addition, subcellular localization was tested using transient expression of GFP fusion proteins in Nicotiana benthamiana through Agrobacterium infiltration. We tested 31 representative effector candidates from the devastating common bean rust pathogen Uromyces appendiculatus. These effector candidates were selected based on features of their gene families, most important lineage specificity. We show that several of our effector candidates suppress plant defense. Some of them also belong to families of effector candidates that are present in multiple rust species where their homologs probably also have effector functions. In our analysis of candidate effector mRNA expression, some of those effector candidates that gave positive results in the other assays were not up-regulated during plant infection, indicating that either these proteins have functions at multiple life stages or that strong up-regulation of RNA level in planta may not be as important a criterion for identifying effectors as previously thought. Overall, our pipeline for selecting effector candidates based on sequence features followed by screening assays using heterologous expression systems was successful in discriminating effector candidates. This work lays the foundation for functional characterization of U. appendiculatus effectors, the identification of effector targets, and identification of novel sources for resistance in common bean.This article is published as Qi M, Mei Y, Grayczyk JP, Darben LM, Rieker MEG, Seitz JM, Voegele RT, Whitham SA and Link TI (2019) Candidate Effectors From Uromyces appendiculatus, the Causal Agent of Rust on Common Bean, Can Be Discriminated Based on Suppression of Immune Responses. Front. Plant Sci. 10:1182. doi: 10.3389/fpls.2019.01182.</p