Genetic variation of powdery mildew resistance in Arabidopsis thaliana

In interactions between beneficial or pathogenic microbes and host organisms, particular host proteins, referred to as compatibility factors, are considered to be essential for the establishment of compatibility. Induced resistance of the dicot plant Arabidopsis thaliana to powdery mildew fungi based on the lack of compatibility factors has been previously shown in the pmr mutants in the Col-0 ecotype. With respect to natural resistance of Arabidopsis to powdery mildews, only resistance mediated by the unusual resistance (R) gene RPW8 was identified in different accessions. In this study, an approach based on natural variation was conducted to analyze powdery mildew resistance in A. thaliana with an emphasis on the identification of new compatibility factors. To this end, loci mediating resistance were mapped in accessions, in which resistance to the compatible powdery mildew Golovinomyces orontii was likely due to a defect in a compatibility factor (i.e. these accessions showing monogenic and recessively or semi-dominantly inherited resistance). In six accessions, the resistance locus was mapped to the lower arm of chromosome III. In other ecotypes analyzed, resistance was likely to be of polygenic origin. The occurrence of only few accessions selected for putative dominantly inherited resistance mediated by prototypical R-genes indicated that this type of resistance to powdery mildews is probably very rare if not non-existent in A. thaliana. The results of allelism tests between resistant accessions yielded an unexpectedly high percentage of susceptible plants in the F2 progeny, a contradiction to the expected segregation of either two either identical or closely linked genes. It was suggested that this phenomenon could either reflect epistatic effects or be the consequence of pairing between two homologous epialleles (paramutation). A comparative microscopic analysis of resistance in selected accessions revealed differences in timing and strength of defense responses in the plant; either due to different loci responsible for resistance or owing to different genetic backgrounds. In general, resistance to G. orontii was characterized by a reduced production of fungal conidiophores on the leaf surface, pronounced cell death, callose deposition and hydrogen peroxide accumulation, but only in some accessions by reduced host cell entry and retarded hyphal growth. Fine mapping of resistance in segregating F2 populations via mapping populations and analysis of recombinant inbred lines allowed a restriction of the putative target gene region, within which several candidate genes were analyzed. One of these genes was RPW8, an atypical R-gene. Based on results from dsRNAi-mediated RPW8 transcript depletion, RPW8 was proposed to be responsible for resistance to G. orontii in accessions Bu-0, Co-3, Do-0, Ei-4, Ei-5, Kas-1, Nok-3, Ob-0 and Sha. The observed recessive or semi-dominant inheritance of resistance indicated a dosage-dependent effect of RPW8-mediated resistance. Sequence analysis of the conceptual RPW8 protein in several accessions revealed a region of high variability between the predicted transmembrane and coiled-coil domains. It is suggested that powdery mildew resistance in A. thaliana appears to be either of polygenic origin or due to RPW8

Functional and biochemical characterization of viral and host factors involved in the Zika virus replication cycle

Arboviruses are one of the greatest threats to human health since they cause millions of infections each year. The mosquito-borne flavivirus Zika virus (ZIKV), which is listed as a priority disease, has been declared a public health emergency of global concern by the World Health Organization (WHO) in 2016. Even though most infections are asymptomatic or manifest only moderate symptoms, infections have been related to neurological disorders such as the Guillain-Barré syndrome and microcephaly. Despite years of intensive research, there are presently no effective antiviral therapies or vaccines. Their development requires further studies of the fundamental principles of the ZIKV life cycle to provide the necessary knowledge for the development of effective therapeutic measures. Upon ZIKV infection, the cell’s endoplasmic reticulum (ER) is significantly remodeled resulting in the formation of defined ER membrane invaginations termed vesicle packets (VPs), which are thought to be the sites where viral RNA replication takes place. Since their formation is coupled to viral replication, it has been challenging to retrieve mechanistic information on VP biogenesis thus far, as perturbations that reduce viral replication inevitably impact VP formation. To overcome this hurdle, my first project was to develop an expression system supporting the formation of ZIKV VPs to enable research focusing entirely on their biogenesis and the factors involved. I was able to demonstrate that ZIKV VPs are forming upon expression of the viral replicase (NS1-NS5) via a construct comprising in addition the 5’ and 3’ untranslated region (UTRs). Ultrastructural characterization of VPs in transfected cells revealed that the VPs are morphologically identical to those produced in infected cells. This newly created system was given the name pIRO (plasmid-induced replication organelle). A thorough deletion mutagenesis study focusing on RNA elements contained in the 5’ and 3’UTR revealed that for VP formation the 5’UTR is largely dispensable. In contrast, the 3’UTR was required as deletion of distinct RNA elements contained therein decreased the efficiency of VP induction. In conclusion, I was able to show that ZIKV VP formation occurs independent of RNA elements with the size and morphology of VPs being independent of the length of the viral RNA. The second part of my PhD thesis was devoted to characterizing the mode of action of a novel ZIKV NS4A inhibitor. This topic was a perfect fit to the first part of my thesis, given the essential role of NS4A in VP formation. The inhibitor was developed by a collaborator who did a large-scale cell-based high-content screen, resulting in the identification of (2E)-N-benzyl-3-(4-butoxyphenyl) prop-2-enamide (SBI-0090799). This compound exhibits potent and wide- spread antiviral activity against numerous ZIKV strains in vitro. Using a combination of biochemical, virological, and imaging-based techniques, I confirmed that SBI-0090799 inhibits ZIKV replication by blocking the de novo formation of ZIKV VPs. Resistance mutations mapping to NS4A rescued viral RNA replication and restored VP formation in cells treated with high concentrations of the compound. These findings suggest that SBI-0090799 perturbs VP formation by interfering with NS4A, either by preventing it from inducing ER membrane curvature or inhibiting it from binding viral or cellular factors contributing to VP formation. Thus, the mechanism of action of SBI-0090799 is comparable to the one of hepatitis C virus NS5A inhibitors that are in clinical use. The third part of my PhD study aimed to unravel the role of cholesterol in the viral replication cycle. Although it has already been shown that cholesterol plays a critical role in virus entry, particularly at the stage of viral envelope fusion with the endosome, as well as virion assembly, direct viral protein-cholesterol interactions have not been determined thus far. To fill this gap in knowledge I performed chemo-proteomics in ZIKV-infected human cells, using a photoactivatable and clickable cholesterol probe, to identify cholesterol binding viral proteins. I discovered that both the structural protein prM and its cleavage product, the M protein, can be efficiently cross-linked to cholesterol. Combining bioinformatics analyses and site-directed mutagenesis, alongside with cholesterol binding assays, I was able to show that the M protein has two functional cholesterol binding domains (CRAC motifs) in its transmembrane domains (TMD) 2 and 3 (CRAC2 and CRAC3, respectively). Using reverse genetics studies, I could show that the M protein’s ability to bind cholesterol is not required for the processing of the viral polyprotein, viral RNA replication, and subcellular localization of the uncleaved prM protein. However, mutations affecting the cholesterol binding motif 2 (CRAC2) significantly impaired viruses in their ability to infect cells having low cholesterol levels. Furthermore, I was able to show that complete exchange of the cholesterol binding motif 3 (CRAC3) severely affected virus particle assembly. In line with these results, atomistic molecular dynamics simulations confirmed cholesterol binding to membrane-associated wild type M protein, whereas M proteins containing mutations in CRAC2 and CRAC3 lost cholesterol interactions. In conclusion, I was able to uncover a bifunctional role for the cholesterol interaction of the M protein in the ZIKV life cycle: facilitating virus entry requiring CRAC2 and virus particle assembly requiring CRAC3

Guillaume de Machaut: Notation and the Compositional Process

In recent scholarship it has been increasingly emphasized that Machaut, particularly in his later secular chansons, developed a new ideal of vertical sound, namely one which rested on a three- rather than a two-voice basis. The present article links this innovation to the composer's layered concept of notation and to the linear individuality of each of the three voices as evidenced by his use of repeated rhythmic motifs in the ballades of the late narrative poem. Le voir dit.En recientes estudios se ha enfatizado cada vez más que Machaut, particularmente en sus últimas chansons profanas, desarrolló un nuevo ideal de verticalidad musical, basado concretamente en tres, en lugar de dos voces. El presente artículo relaciona esta innovación con el concepto de notación estratificada del compositor y con la individualidad lineal de cada una de las tres voces, como se pone de manifiesto por su uso de motivos rítmicos repetidos en las baladas del poema narrativo tardío, Le voir dit

Mode and change of mode in the 13th-century motet

The many different versions in which motets were notated in various manuscripts from the 13th century testify to the great flexibility of this genre. The present paper pursues those few clues in practical and theoretical sources which indicate that the rhythmic mode itself was often subject to change within a particular group of motets. Evidence of this practice is found in the early group of the manuscripts and then, again, in tlie later, more liturgically oriented sources, including Las Huelgas. In the former, first and second modes could be interchanged to correspond to Latin or French texts, respectively, and in later times the notated combination of third and fifth modes, now considered out of date, could simply be performed as sixth and second modes. In both cases the change of mode appears to represent a quasi-improvisatory practice which could be performed without altering the written version, i.e. it was probably much more common than the sources themselves indicate.La mayoría de las diferentes versiones en las que eran anotados los motetes en varios manuscritos desde el siglo XIII dan fe de la gran flexibilidad de este género. El presente artículo se centra en las pocas claves existentes en las fuentes prácticas y teóricas que indican que el modo rítmico en sí mismo estaba, a menudo, sujeto a cambios dentro de un grupo concreto de motetes. La evidencia de esta práctica se halla en un grupo temprano de manuscritos y después, de nuevo, en los posteriores, que son fuentes más orientadas hacia la liturgia, incluyendo Las Huelgas. Al principio, el primero y segundo modos podían ser intercambiados para corresponderse con textos en latín o en francés, respectivamente, y en posteriores ocasiones la combinación anotada de tercer y quinto modos, ahora considerada anticuada, podía ser interpretada simplemente como sexto y segundo modos. En ambos casos el cambio de modo aparece para representar una práctica casi improvisatoria la cual podía ser interpretada sin alterar la versión escrita, o sea, era una práctica posiblemente más común que lo que indican las propias fuentes

Force delivery from a transpalatal arch for the correction of unilateral first molar cross-bite

The forces and moments delivered by prefabricated transpalatal arches of the makes Unitek, GAC, and Ormco were studied in laboratory experiments. The Unitek and GAC arches were made of steel, and the Ormco arch of beta-titanium alloy (TMA). Three types of activation were investigated: for bilateral expansion in a statically indeterminate system and for unilateral expansion in a statically determinate system with and without torque activation. In addition to the arch design, composition, and mode of activation, the influence of arch size and degree of activation were studied. It was found that activation for unilateral expansion with the inclusion of torque in the statically determinate system produced forces and moments suitable for the correction of a unilateral cross-bit