Functionally distinct Botrytis cinerea Argonaute proteins in plant-microbe interaction

Der nekrotrophe, filamentöse und phytopathogene Pilz Botrytis cinerea (B. cinerea) verursacht weltweit an mindestens 200 Eudikotyledonen und Monokotyledonen eine schwere Grauschimmelkrankheit. Neben verschiedenen Enzymen sezerniert Botrytis auch sRNAs als virulente Faktoren in Wirte, welche die Pflanzenphysiologie und Pflanzenimmunitätt über Cross-Kingdom-RNA-Interferenz (ckRNAi) manipulieren,. Dabei erfolgt die Translokation der sRNA-Effektoren bidirektional zwischen Pathogen und Wirt. Die Rolle der Ago-vermittelten Regulation auf die Pathogenität durch RNA-Interferenz (RNAi) in B. cinerea ist jedoch noch unklar. In dieser Doktorarbeit wurden vier Gene, Ago1, Ago2, Ago3 und Ago4 in B. cinerea identifiziert, die für Ago-Proteine kodieren. Die phylogenetische Analyse zeigte, dass Ago1, Ago3 und Ago4 zur Klade der Quelling gehören und Ago2 in die Gruppe der MSUD in Botrytis cinerea fällt. Das Expressionsmuster zeigte, dass Botrytis die endogene Ago2-Expression als Reaktion auf eine Infektion von Tomaten im Früh- und Spätstadium unterdrückt, was auf eine mögliche Funktion von Ago2 im Cross-Talk der Pilz-Wirt-Interaktion hinweist. Bei der Phänotypisierung homokaryotischer Mangel-Mutanten von Botrytis, denen Ago1, Ago2, Ago3 bzw. Ago4 fehlten, konnte ich beobachten, dass der Verlust der Ago-Gene keinen Einfluss auf die Myzelmorphologie, Konidiengröße und Konidienbildung hatte, was darauf hindeutet, dass Ago-Gene für die Entwicklung und das Wachstum von Botrytis in der vegetativen Phase nicht erforderlich sind. Darüber hinaus zeigten Ago2-Loss-of-Function-Mutanten eine verminderte Virulenz im Vergleich zu WT und die Komplementierung von Ago2 stellte die verminderte Pathogenität wieder her, was zeigt, dass Ago2 zur Pathogenität von Botrytis während der Infektion auf Tomate beiträgt. Die Abundanz von sRNAs war in den Ago2- und Ago3-Mutationsstämmen erhöht, was die Rolle der mRNA-Degradation für Ago2 und Ago3 anstelle der Stabilisierung im post-transcriptional silencing (PTGS) impliziert. In der Zwischenzeit waren die Transkripte des Retroelements LTR2 in der Ago1- oder Ago2-Loss-of-Function-Mutante signifikant erhöht und LTR1 wurde in der Ago3-Loss-of-Function-Mutante herunterreguliert, was darauf hindeutet, dass Ago2 ein dominanter Slicer in B. cinerea sein könnte, während Ago3 wahrscheinlich ein untergeordneter Slicer ist, der mit Ago2 konkurriert, indem er sRNAs und interferierende RNAi bindet, und Ago4 möglicherweise nicht an PTGS beteiligt ist. Bemerkenswert ist, dass aus mRNA-seq von Ago2 Loss-of-Function-Mutanten acht differentiell exprimierte Gene (DEGs), die in zellulären Komponenten angereichert sind, und drei DEGs (BCIN_16g04050, die eine Glutamat-Dehydrogenase kodieren, BCIN_09g04410 kodiert eine Galaktose-Oxidase und BCIN_03g04090 kodiert eine Glycerin-Kinase), die im KEGG-Pfad angereichert sind, identifiziert wurden, die eine Rolle in der Arginin-Biosynthese, dem Galaktose-Stoffwechsel bzw. dem Lipid-Stoffwechsel spielen. Ein sRNA-Profiling aus diesen identifizierten Genen deckte auf, dass 24-28-nt sRNAs, wahrscheinlich microRNA-like RNAs, in Ago2-Mutationsstämmen dramatisch verloren gingen. Zusammengenommen reguliert Ago2 die Pathogenität positiv, möglicherweise über die Regulierung dieser Gene, die in GO- oder KEGG-Pfaden angereichert sind und die die Ziele der 24-28-nt milRNAs sein könnten, die eine positive Rückkopplungsschleife in B. cinerea bilden.A necrotrophic filamentous phytopathogenic fungus Botrytis cinerea causes severe grey mould disease on at least 200 eudicots and monocots worldwide. In addition to various enzymes, Botrytis also secretes sRNAs as virulent factors into hosts manipu-lating plant physiology and immunity via cross-kingdom RNA interference (ckRNAi) and the translocation of sRNA effectors is bidirectional between pathogen and hosts. However, the function of Ago-mediated regulation on pathogenicity by RNA interference (RNAi) in B. cinerea remains blank. In this doctoral project, four Botrytis genes encoding Ago proteins were identi-fied encompassing Ago1, Ago2, Ago3 and Ago4. Phylogenetic analysis showed that Ago1, Ago3 and Ago4 belong to the clade of quelling and Ago2 falls into the group of MSUD in Botrytis cinerea. The expression pattern indicated that Botrytis sup-pressed endogenous Ago2 expression in response to early- and late-stage infection on tomatoes, suggesting a potential function of Ago2 in the cross-talk of fungal-hosts interaction. Through phenotypical analyses with homokaryotic deficient mutants disrupting Botrytis Ago1, Ago2, Ago3 and Ago4, I observed that loss of Ago genes had no impact on mycelial morphology, conidial size and conidiation, implicating that Ago genes are not required for Botrytis development and growth in the vegeta-tive phase. Furthermore, Ago2 loss-of-function mutants displayed impaired virulence compared to WT and complementation of Ago2 restored reduced pathogenicity, demonstrating that Ago2 contributes to pathogenicity in Botrytis during infection on tomato. The abundance of sRNAs were elevated in Ago2 and Ago3 mutation strains, which suggests the role of mRNA degradation for Ago2 and Ago3 instead of stabili-zation in post-transcriptional gene silencing (PTGS). Meanwhile, transcripts of retro-element LTR2 was significantly increased in Ago1 or Ago2 loss-of-function mutant and LTR1 was down-regulated in Ago3 loss-of-function mutant, indicating that Ago2 might be a dominant slicer in B. cinerea, whereas Ago3 is likely a minor slicer com-peting with Ago2 binding with sRNAs and interfering RNAi, and Ago4 may not in-volve in PTGS. Notably, mRNA-seq from Ago2 loss-of-function mutants showed that eight differentially expressed genes (DEGs) enriched in cellular component and three DEGs (BCIN_16g04050 encoding a glutamate dehydrogenase, BCIN_09g04410 en-coding a galactose oxidase and BCIN_03g04090 encoding glycerol kinase) enriched in the KEGG pathway were identified, which played a role in arginine biosynthesis, galactose metabolism and lipid metabolism, respectively. sRNA profiling derived from these identified genes uncovered that 24-28-nt sRNAs, probably microRNA-like RNAs, dramatically lost in Ago2 mutation strains. Taken together, Ago2 posi-tively regulates pathogenicity, possibly via regulation on these genes enriched in GO or KEGG pathway that might be the targets of 24-28-nt milRNAs forming a positive feedback loop in B. cinerea

Infinitely many solutions for a class of p(x)p(x)-Laplacian equations in RN\mathbb{R}^{N}

In this paper, we study the existence of infinitely many solutions for a class of $p(x)$-Laplacian equations in $\mathbb{R}^{N}$, where the nonlinearity is sublinear. The main tool used here is a variational method combined with the theory of variable exponent Sobolev spaces. Recent results from the literature are extended

Convergence for pseudo monotone semiflows on product ordered topological spaces

AbstractIn this paper, we consider a class of pseudo monotone semiflows, which only enjoy some weak monotonicity properties and are defined on product-ordered topological spaces. Under certain conditions, several convergence principles are established for each precompact orbit of such a class of semiflows to tend to an equilibrium, which improve and extend some corresponding results already known. Some applications to delay differential equations are presented

Periodic solutions for nonlinear nth order differential equations with delays

AbstractBy applying the continuation theorem of coincidence degree theory, we establish the existence of 2π-periodic solutions for a class of nonlinear nth order differential equations with delays

Existence and uniqueness of periodic solutions for a kind of Liénard equation with a deviating argument

AbstractIn this work, we use the coincidence degree theory to establish new results on the existence and uniqueness of T-periodic solutions for a kind of Liénard equation with a deviating argument of the form x″(t)+f(x(t))x′(t)+g(t,x(t−τ(t)))=p(t)

Dynamics of smooth essentially strongly order-preserving semiflows with application to delay differential equations

AbstractIn this paper, we introduce a class of smooth essentially strongly order-preserving semiflows and improve the limit set dichotomy for essentially strongly order-preserving semiflows. Generic convergence and stability properties of this class of smooth essentially strongly order-preserving semiflows are then developed. We also establish the generalized Krein–Rutman Theorem for a compact and eventually essentially strongly positive linear operator. By applying the main results of this paper to essentially cooperative and irreducible systems of delay differential equations, we obtain some results on generic convergence and stability, the linearized stability of an equilibrium and the existence of the most unstable manifold in these systems. The obtained results improve some corresponding ones already known

The Young Environmental Citizens in Nordic Countries : Their Concerns, Values, Engagement, and Intended Future Actions

This chapter presents an analysis of students’ concerns, values, engagement, and intended future participation on environmental issues in relation to their home socioeconomic background, gender, and migrant status. Analyzing IEA International Civic and Citizenship Education Study (ICCS) 2016 data of Nordic countries, we first present descriptions of student responses to all questions related to environmental issues and compare Nordic results with European and international averages. Then, we construct a composite score of student environmental citizenship for investigating its relationship with student background factors such as gender, migrant status, and home socioeconomic status through comparing means between student groups with different background characteristics. Lastly, we apply factorial ANOVA analysis method to examine the effect sizes of student background factors and the interactions between them on youth environmental citizenship in the four countries. The results show that there are both similarities and small variations in elements of student environmental citizenship among the Nordic countries and in comparison with their European and international peers. Nordic students stand out as the concerned environmental citizens while they are somehow lower than their European and international peers in engagement, values, and intended participation of environmental citizenship. We find that student environmental citizenship is socially divided in all Nordic countries as it differs significantly between students from different socioeconomic strata and genders. Although not all differences of student environmental citizenship by migrant status are statistically significant among the Nordic countries, we find some significant influence of migrant status interaction with socioeconomic statuses and genders.Peer reviewe

Construct of Youth Environmental Citizenship among International Large-Scale Educational Studies

Environmental citizenship has become an integral element of civic and citizenship education curricula, both globally and at the European level. Previous analysis provided important evidence that environmental-citizenship education practice in Nordic schools has a significant positive association with heightened attitudes and magnified behaviours among students toward environmental actions now and in the future. This paper starts with critiques on the strengths and limitations of ICCS 2016 data measures of youth environmental citizenship, and then it provides an overview of measures applied in a few other large-scale international studies such as PISA 2015 and PISA 2018. At the end, we present a measurement proposal on environmental citizenship through combining the measures from these studies