Structure-function analysis of plant fructosyltransferases

Fructans are an important class of plant carbohydrates that consist of linear or branched chains of fructosyl moieties. Their synthesis requires fructosyltransferases (FTs) that catalyze the transfer of fructosyl units from a donor substrate (sucrose or fructan) to an acceptor substrate (sucrose or fructan). The fructosyltransferases involved in fructan metabolism are related to acid invertases, enzymes that cleave sucrose into glucose and fructose. An invertase can be considered a fructosyltransferase which transfers the fructose moiety to water. The aim of the present work was to elucidate what determines the different catalytic activities of this enzyme group, by use of molecular methods. In order to study such structure-function relationships we artificially introduced mutational changes and constructed chimeric FTs (enzymes with exchanged regions). The goal was to detect the determining regions or single amino acids. For this purpose we optimized the expression of FTs in the methylotrophic yeast Pichia pastoris and developed the methodology to create the chimeric constructs. Conventional cloning using conveniently located restriction sites and the method of overlapping PCR was used. In a first part domain exchanges between two closely related FTs from cereals were analyzed by expressing the corresponding constructs in Pichia (Chapter 2). The two subunits of FTs (N-terminal large subunit and C-terminal small subunit) were exchanged between Festuca arundinacea (re-classified as Schedonorus arundinaceus) sucrose:sucrose 1-fructosyltransferases (1-SST) and Hordeum vulgare sucrose:sucrose 6- fructosyltransferase (6-SFT). The study revealed that it is the large subunit that carries the structural features responsible for enzyme specificity. In a second part we focused on the conserved motifs (S/N)DPNG, RDP and EC, located on the large subunit, that are presumably essential in the active site of plant FTs. For this purpose two other SST-SFT-chimeras with exchanged N-termini encompassing these motifs, as well as Festuca 1-SST carrying single amino acid substitutions in the RDP- and EC-motif were analyzed (Chapter 3). This study revealed the importance of the three hypothesized active site motifs for the transfructosylation reaction. All three of them were shown to be important for enzyme activity and/or for specificity. In a third part, we addressed the question what structural components determine the relative transferase and hydrolase activities of FTs and vacuolar invertases via a targeted mutational analysis based on sequence comparisons between vacuolar invertases and 1-SSTs, the latter an example of a sucrose-using FT (Chapter 4). We chose Allium cepa invertase and Festuca arundinacea 1-SST for our analysis. Nine amino acids dispersed along the sequence could be identified correlating with either invertase or 1-SST activities. The selected amino acids of onion invertase were mutated to the corresponding amino acids in Festuca 1-SST and vice versa. For both enzymes, the mutations were analyzed independently. Functional expression in Pichia revealed shifts in the catalytic specificity and activity, demonstrating the importance of these amino acids outside the three highly conserved motifs (S/N)DPNG, RDP, and EC for the enzymatic reaction (Chapter 4). This work helped to narrow down the region potentially responsible for enzyme specificity in plant FTs. We could pinpoint the importance of the regions with the highly conserved motifs, and of some additional characteristic single amino acids dispersed along the sequence, for enzyme activity and specificity

An acceptor-substrate binding site determining glycosyl transfer emerges from mutant analysis of a plant vacuolar invertase and a fructosyltransferase

Glycoside hydrolase family 32 (GH32) harbors hydrolyzing and transglycosylating enzymes that are highly homologous in their primary structure. Eight amino acids dispersed along the sequence correlated with either hydrolase or glycosyltransferase activity. These were mutated in onion vacuolar invertase (acINV) according to the residue in festuca sucrose:sucrose 1-fructosyltransferase (saSST) and vice versa. acINV(W440Y) doubles transferase capacity. Reciprocally, saSST(C223N) and saSST(F362Y) double hydrolysis. SaSST(N425S) shows a hydrolyzing activity three to four times its transferase activity. Interestingly, modeling acINV and saSST according to the 3D structure of crystallized GH32 enzymes indicates that mutations saSST(N425S), acINV(W440Y), and the previously reported acINV(W161Y) reside very close together at the surface in the entrance of the active-site pocket. Residues in- and outside the sucrose-binding box determine hydrolase and transferase capabilities of GH32 enzymes. Modeling suggests that residues dispersed along the sequence identify a location for acceptor-substrate binding in the 3D structure of fructosyltransferase

Evaluación preliminar de virus asociados a sistemas productivos de uchuva, gulupa y rosa

1 recurso en línea (páginas 390-396).Plant viruses may pose a threat to crops in Colombia. To evaluate the potential risk of yield losses due to plant virus infection, a literature analysis followed by a first field study was carried out focusing on purple passion fruit (Passiflora edulis Sims), cape gooseberry (Physalis peruviana L.), and ornamental rose (Rosa sp.), which are important Colombian exports. Over the past three years, plant material was collected from 21 farms in Cundinamarca and Boyacá, Colombia, two regions that are in close proximity to El Dorado International Airport, the country’s largest air freight terminal. Plants were visually inspected and subsequently tested by bioassay and serological methods. Overall, in the samples investigated by the two diagnostic methods, plant viruses were detected. Detected viruses belong to the genus Poty-, Tobamo-, Nepo-, Ilar-, and Tospovirus. The extent of the distribution and occurrence of these viruses in each crop has to be determined in a representative field study. Such a monitoring program could be supported by a standardized farmer interview. The development of suitable plant virus diagnostic and managements tools is the focus of a cooperation project between German and Colombian universities, the Colombian Agricultural Institute (ICA), the Colombian Corporation of Agricultural Investigation (AGROSAVIA) and the International Center for Tropical Agriculture (CIAT).Los virus representan una amenaza para las plantas cultivadas en Colombia, y algunos estudios indican que las pérdidas económicas causadas por estos problemas fitosanitarios podrían evitarse mediante procedimientos preventivos estándar. Como objeto de estudio, se seleccionaron tres importantes productos de Colombia, como lo son: rosa ornamental (Rosa sp.), uchuva (Physalis peruviana L.) y gulupa (Passiflora edulis Sims). En el presente artículo, se muestran los primeros desarrollos resultantes del monitoreo de virus en estas plantas, para determinar el estado de las virosis en fincas colombianas. Se realizaron análisis serológicos de 21 fincas en Cundinamarca y Boyacá, Colombia, en 2016-18. El objetivo de esta investigación fue el desarrollo de un protocolo piloto para el diagnóstico rutinario, que se pueda aplicar en un programa de certificación de material vegetal para la determinación de la presencia de virus, en varios productos hortícolas colombianos. Con base en este protocolo, se pueden determinar los riesgos que representan ciertos virus y se puede considerar la necesidad de certificar el material de siembra evaluado según la presencia de virus. Se están desarrollando herramientas de diagnóstico confiables y prácticas, para la detección de los virus más relevantes, en un proyecto conjunto entre universidades alemanas y colombianas, el Instituto Colombiano de Agropecuario (ICA), la Corporación Colombiana de Investigación Agropecuaria (AGROSAVIA) y el Centro Internacional de Agricultura Tropical (CIAT).Bibliografía: páginas 395-39

An acceptor-substrate binding site determining glycosyl transfer emerges from mutant analysis of a plant vacuolar invertase and a fructosyltransferase

Glycoside hydrolase family 32 (GH32) harbors hydrolyzing and transglycosylating enzymes that are highly homologous in their primary structure. Eight amino acids dispersed along the sequence correlated with either hydrolase or glycosyltransferase activity. These were mutated in onion vacuolar invertase (acINV) according to the residue in festuca sucrose:sucrose 1-fructosyltransferase (saSST) and vice versa. acINV(W440Y) doubles transferase capacity. Reciprocally, saSST(C223N) and saSST(F362Y) double hydrolysis. SaSST(N425S) shows a hydrolyzing activity three to four times its transferase activity. Interestingly, modeling acINV and saSST according to the 3D structure of crystallized GH32 enzymes indicates that mutations saSST(N425S), acINV(W440Y), and the previously reported acINV(W161Y) reside very close together at the surface in the entrance of the active-site pocket. Residues in- and outside the sucrose-binding box determine hydrolase and transferase capabilities of GH32 enzymes. Modeling suggests that residues dispersed along the sequence identify a location for acceptor-substrate binding in the 3D structure of fructosyltransferases

The Arabidopsis protein phosphatase PP2C38 negatively regulates the central immune kinase BIK1

Plants recognize pathogen-associated molecular patterns (PAMPs) via cell surface-localized pattern recognition receptors (PRRs), leading to PRR-triggered immunity (PTI). The Arabidopsis cytoplasmic kinase BIK1 is a downstream substrate of several PRR complexes. How plant PTI is negatively regulated is not fully understood. Here, we identify the protein phosphatase PP2C38 as a negative regulator of BIK1 activity and BIK1-mediated immunity. PP2C38 dynamically associates with BIK1, as well as with the PRRs FLS2 and EFR, but not with the co-receptor BAK1. PP2C38 regulates PAMP-induced BIK1 phosphorylation and impairs the phosphorylation of the NADPH oxidase RBOHD by BIK1, leading to reduced oxidative burst and stomatal immunity. Upon PAMP perception, PP2C38 is phosphorylated on serine 77 and dissociates from the FLS2/EFR-BIK1 complexes, enabling full BIK1 activation. Together with our recent work on the control of BIK1 turnover, this study reveals another important regulatory mechanism of this central immune component

What did we achieve with VALITEST an EU project on validation in plant pest diagnostics?

peer reviewedEnsuring the reliability of diagnostic activities is an essential cornerstone of Plant Health strategies to reduce the risk of entry and spread of plant pests in a region and ultimately their impacts. Diagnostic tests should be validated to ensure that they are fit for purpose. Validation is usually done by diagnostic laboratories although companies commercializing diagnostic kits also produce validation data for their products. Due to the high number of pest , matrix and method combinations and given the significant resources required to validate tests, it is essential that validation data are shared with the entire diagnostic community and produced in a harmonized way to facilitate their use by different stakeholders. Indeed, the selection of tests to be used in specific contexts is not the sole responsibility of diagnostic laboratories and also involve National Plant Protection Organizations. The VALITEST EU project (2018-2021) was established to tackle all these issues. New validation data for tests targeting important pests for the EPPO region were produced. Guidelines to improve and harmonize the validation framework were developed. Sharing of validation data and experience was ensured through the development of new or existing databases, the organization of training courses and the dissemination of the project outputs in scientific publications and Standards. Finally, the involvement of researchers, diagnosticians, policy makers, inspectors, industries etc. and the establishment of the European Plant Diagnostic Industry Association were important actions to strengthen the interactions between Plant Health stakeholders

Fruit infestation of Drosophila suzukii by fast immunological detection

Spotted Wing Drosophila SWD (Drosophila suzukii) is a serious pest in Europe attacking many softskinned crops such as several berry species and grapevine. SWD is an invasive species arriving Europe in 2008. In contrast to most other Drosophila species, SWD deposits its eggs with the serrated ovipositor into mature, intact, and undamaged fruits. Larvae develop within the fruit that collapse within days. Before collapse the infestation is hardly seen. This may lead to infested fruits that collapse in the shops or after being sold to consumers. Today, farmers may test for infestation by incubation for 48h of the fruits followed by a treatment with saltwater. Larvae escape the fruits and can be counted on the water surface. A more efficient and accurate detection of fruit infestation is currently not available. We aim at developing an antibody-based detection system allowing to rapidly analyze a high number of fruit samples for potential infestation with eggs and larvae of SWD. Some 7 000 potential target proteins have been selected from Casas-Vila (2018) for in silico analysis of the expression profiles. The proteins most stably expressed during embryo, larvae, and pupae stages have been selected and used as targets for ELISA and lateral flow device

Sensitivity of field tests, serological and molecular techniques for Plum Pox Virus detection in various tissues

Sensitivity of field tests (AgriStrip  and Immunochromato), DAS-ELISA, two step RT-PCR and real-time RT-PCR for <em>Plum pox virus</em> (PPV) detection was tested in various tissues of apricot, peach, plum and damson plum trees infected with isolates belonging to PPV-D, PPV-M or PPV-Rec, the three strains present in Slovenia. Flowers of apricot and plum in full bloom proved to be a very good source for detection of PPV. PPV could be detected with all tested techniques in symptomatic parts of leaves in May and with one exception even in the beginning of August, but it was not detected in asymptomatic leaves using field tests, DAS-ELISA and partly also molecular techniques. PPV was detected only in some of the samples of asymptomatic parts of the leaves with symptoms and of stalks by field tests and DAS-ELISA. Infections were not detected in buds in August using field tests or DAS-ELISA. Field tests are useful for confirmation of the PPV infection in symptomatic leaves, but in tissues without symptoms DAS-ELISA should be combined or replaced by molecular techniques

Учебная программа для специальности: 1-86 0101 Социальная работа (социо-медико-психологическая деятельность)

Potato blackleg is a seedborne disease that can cause significant economic losses for growers. Disease development depends mainly on two drivers, namely seed inoculum and local climatic conditions. To better establish the relationship between these two drivers, blackleg development was monitored in Swiss field trials at multiple locations from 2010 to 2013 involving three sets of naturally infected seed lots planted in each of three locations. The seed lot itself was thereby the most important factor explaining differences in disease development, rather than environmental factors. In a further on‐farm project conducted at various locations in Switzerland and southern Germany from 2013 to 2015, the implementation of a seed‐testing procedure was investigated. A total of 177 seed lots were tested for natural latent infection with soft rot Pectobacteriaceae and the corresponding blackleg incidence was tracked in 242 fields. The reliability of the relationship between latent infection and field incidence was found to be strongly linked to the bacterial species. Dickeya spp. field infection could be predicted with an acceptable reliability, whereas Pectobacterium carotovorum subsp. brasiliense, even when detected as latent tuber infection, was not consistently expressed as visual blackleg. Moreover, commonly found mixed latent infections with several bacterial species made it even harder to predict which bacteria would cause blackleg symptoms. Finally, variability in the reliability of seed testing may also be explained by differences in local farming practices. These trials over several years with naturally infected potato seed highlight the usefulness and limits of seed testing to manage blackleg