The production of cereal crops is essential to secure a future that feeds the continuously growing population. Rust fungi reduce host fitness by feeding on their living tissue and interfere with the global production of crops. Cereal rusts, like Puccinia hordei (the causal agent of barley leaf rust) and Puccinia triticina (the causal agent of wheat leaf rust), have a narrow host range and colonize only one particular species. The most durable type of resistance, non-host resistance (NHR), is the immunity of an entire plant species to all strains of a pathogen species. Exploring the genetics of NHR has proven to be challenging because most interspecific hybrids are infertile. Previously, barley Rphq2 and Rph22, which encode orthologous lectin receptor-like kinases (LecRKs), were transformed into an experimental barley line, Golden SusPtrit, and showed resistance against adapted and non-adapted leaf rust species. We used these transgenic barley lines in the current project to explore the effect of the LecRKs on four wheat leaf rust (P. triticina) isolates. We used the settling tower method to inoculate four isolates of P. triticina on Rphq2 and Rph22 transgenic families. We found that most transgenic families showed an increase in resistance compared to the non-transgenic control 750-E1. By measuring the infection frequency of the infections, we identified that P. triticina isolates 93012 and 95012 had opposite virulence effects on two barley families, Rphq2-E5 and Rph22-E2A. Although the expression levels of Rphq2 and Rph22 followed an induction trend, we did not find significant differences between the isolates. We conclude that resistance mediated by Rphq2 and Rph22 against P. triticina isolates does not involve an isolate-specific component. Thus, we propose investigating differences between rust species to further explore the molecular aspect of non-host resistance.

Mitogen-activated protein kinases (MAPKs) are a family of highly conserved serine/threonine protein kinases which link upstream receptors to their downstream targets. These targets can be localized in the cytoplasm or the nucleus. Pathogens produce pathogen-associated molecular patterns (PAMPs) that are known to trigger the activation of MAPK cascades. In plants, MAPK signaling cascades regulate development and cellular processes such as stress responses, immunity, and apoptosis by means of the phosphorylation of specific targets. Phosphoproteomics analysis of PAMP-induced Arabidopsis plants led to the identification of several putative MAPK targets. USP1 (Universal Stress Protein A) (At1g11360) and WLIM2A (At2g39900) are two potential phosphorylation targets of MAPKs, and are the focus of this thesis. So far, little is known about their role in plant immunity. CRISPR-Cas9 generated knockout usp1 mutant lines enhanced resistance to infection by Pst DC3000, usp1 mutant showed a reduced level of apoplast reactive oxygen species accumulation and upregulation of defense marker genes such as WRKY29 and FRK1. Transcriptome analyses revealed that immune hormone signaling genes such as salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) are differentially regulated. These hormones are responsible for primary defense responses against biotrophic and necrotrophic pathogens. Although the physiological role of USP1 has been established, the biochemical and molecular functions are unknown. We discovered a new role for USP1, demonstrating that it functions as a molecular chaperone and is involved in thermal priming. Overall, these data show that phosphoprotein USP1 plays an important role in orchestrating plant immunity. CRISPR-Cas9 generated knockout wlim2a mutant showed susceptibility to infection by Pst DC3000. wlim2a mutants showed a reduced level of apoplast reactive oxygen species accumulation and upregulation of defense marker genes such as WRKY29 and FRK1. Transcriptome analyses revealed that immune hormone signaling genes such as salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) are differentially regulated in wlim2a mutants. These hormones are responsible for primary defense responses against biotrophic and necrotrophic pathogens. wlim2a mutants show enhanced fungal infection by Botrytis cinerea. Overall, the data shows that WLIM2A phosphorylation is important during plant immunity.

<p>Plants have evolved the circadian clock to anticipate environmental changes and coordinate internal biological processes. Recent studies unveiled the circadian regulation on plant immune responses as well as a reciprocal effect of immune activation on the clock activity. However, it is still largely unknown how the circadian clock interacts with specific immune signals. Plant hormone salicylic acid (SA) is a key immune signal. Its accumulation is sufficient to trigger immune responses and establish broad-spectrum resistance, known as systemic acquired resistance (SAR). My dissertation work studied whether SA could interact with the circadian clock and what potential mechanisms and the biological significance are.</p><p>I first found that SA could reinforce the circadian clock through the modulation of redox state in an NONEXPRESSER OF PR 1 (NPR1)-dependent manner. The basal redox state manifested by the NADPH abundance is shown to display a circadian rhythm. Perturbation in this cellular redox rhythm caused by the immune signal SA is sensed by the master immune regulator NPR1. NPR1 then triggers defense genes expression to generate SAR as well as transcriptionally activates several clock genes to reinforce the circadian clock. Since the basal redox state, which reflects the cellular metabolic activities, is under the circadian control, the reinforced circadian clock may negate the SA-triggered redox perturbation to restore the normal redox rhythm. One of NPR1-regulated clock components is TIMMING OF CAB2 EXPRESSION 1 (TOC1). SA/NPR1-mediated increase in TOC1 expression alone could lead to dampening of SAR through direct transcriptional repression on defense genes. Since maintenance of the immune responses is an energy-costly process, the strength and duration of SAR, a preventative defense strategy, need to be fine-tuned to reduce unnecessary energy expenditure. Therefore, both SA-dependent circadian clock reinforcement and the specific clock component TOC1 induction help to ensure a proper immune induction and a balanced energy allocation between defense and normal metabolic activities.</p><p>Besides the SA effects on the circadian clock, the circadian clock is found to reciprocally regulate SA biosynthesis. The clock gene, CCA1 HIKING EXPEDITION (CHE), and the major SA synthesis gene, ISOCHORISMATE SYNTHASE 1 (ICS1), show in-phase oscillatory rhythms, indicating that CHE may contribute to generation of the circadian rhythm of the basal SA level. I found that CHE, as a transcription factor, directly binds to the promoter of ICS1 to positively regulate its expression. After pathogen infection, CHE promotes endogenous SA biosynthesis and acts as a positive regulator of SAR. The function of the clock component CHE in activating ICS1 not only reveals a novel transcriptional regulatory mechanism of SA accumulation but also provides a new molecular link between the circadian clock and plant immunity.</p><p>In summary, my dissertation studies identified previously unknown molecular mechanisms of how the circadian clock mediates SA biosynthesis and SA-triggered immune responses. The interplay between the circadian clock and SA achieves a balance between activation of immune responses and maintenance of normal metabolic activities. Further studies may explore how other plant immune signals affect the circadian clock as well as how different clock components coordinately regulate the plant immunity. These future directions will broaden our understanding about the clock-immunity crosstalk.</p> / Dissertation

The plant phenylpropanoid pathway is initiated from deamination of phenylalanine to form cinnamic acid followed by hydroxylation and methylation of the aromatic ring to generate a variety of phenolic compounds including lignin monomers, flavonoid compounds and sinapate esters. The incorporation of phenylpropanoid metabolism served as a key step in the early land-colonization of plants from aqueous environment since phenolic compounds play important roles in plant development and abiotic/biotic stress responses. Lignin is a heteropolymer of hydroxycinnamyl alcohols that are derived from the major branch of plant phenylpropanoid pathway. The main function of lignin is to enhance the strength of plant cell wall and waterproof the vascular system for long-distance transportation of water and solutes. In addition, lignin is also involved in protecting plants against pathogen attack. My Ph.D. research is to investigate how lignin biosynthesis contributes to plant immunity. The results showed that the expression of major lignin biosynthetic genes was induced upon host fungal pathogen infection. Moreover, a mutant disrupted in the lignin gene F5H1 showed enhanced susceptibility when challenged with several fungal pathogens. F5H1 encodes a ferulic acid 5-hydroxylase that is uniquely present in angiosperm plants, leading to the biosynthesis of syringyl lignin monomer, which is not present in gymnosperm plants. Subsequent research demonstrated that f5h1 mutation impaired the penetration (pre-invasion) resistance but did not impact post-invasion resistance. Furthermore, the pathogen-induced expression of lignin genes was independent of well-characterized defensive signaling pathways, and regulated by a novel regulating mechanism. F5H1 contributes to pmr2-mediated resistance but acts independently of other molecular components of penetration resistance including PEN1, PEN2, and PEN3. In contrast to f5h1, a knockout mutant of flavonoid pathway gene chalcone isomerase (CHI/TT5) showed enhanced resistance to host anthracnose pathogen Colletotrichum higginsianum in a salicylic acid (SA)-dependent manner. Taken together, our results for the first time provide genetic evidence demonstrating that lignin biosynthetic gene F5H1 plays critical roles in plant penetration resistance and that an uncharted pathway in flavonoid metabolism confers an SA-dependent resistance pathway in Arabidopsis.

Plants use several strategies to defend themselves against microbial pathogens. These include basal resistance, which is induced in response to pathogen encoded effector proteins, and resistance (R) protein-mediated resistance that is activated upon direct or indirect recognition of pathogen encoded avirulence protein(s). The activation of Rmediated signaling is often associated with generation of a signal, which, upon its translocation to the distal uninfected parts, confers broad-spectrum immunity against related or unrelated pathogens. This phenomenon known as systemic acquired resistance (SAR) is one of the well-established forms of induced defense response. However, the molecular mechanism underlying SAR remains largely unknown. Induction of plant defense is often associated with a fitness cost, likely because it involves reprogramming of the energy-providing metabolic pathways. Glycerol metabolism is one such pathway that feeds into primary metabolism, including lipid biosynthesis. In this study, I evaluated the role of glycerol-3-phosphate (G3P) in host-pathogen interaction. Inoculation with the hemibiotrophic fungal pathogen Colletotrichum higginsianum led to increased accumulation of G3P in wild-type plants. Mutants impaired in biosynthesis of G3P showed enhanced susceptibility, suggesting a correlation between G3P levels and basal defense. Conversely, increased biosynthesis of G3P correlated with enhanced resistance. The Arabidopsis genome encodes one copy of glycerol kinase (GK), which catalyzes phosphorylation of glycerol to G3P, and five copies of G3P dehydrogenase (G3Pdh), which catalyze reduction of dihydroxyacetone phosphate to G3P. Analysis of plants mutated in various G3Pdh's showed that plastidal lipid biosynthesis was only dependent on the GLY1 isoform but the pathogen induced G3P pool required the function of GLY1 and two other G3Pdh isoforms. Interestingly, compromised G3P biosynthesis in GK and G3Pdh mutants also compromised SAR, which was restored when G3P was provided exogenously. Detailed biochemical analysis showed that G3P was transported to distal tissues and that this process was dependent on a lipid transfer protein, DIR1. Together, these results show that G3P plays an important role in both basal- and induced-defense responses.

La méthylation et déméthylation de l'ADN jouent un rôle majeur dans la stabilité des génomes, l'empreinte génomique, la paramutation et le développement. En revanche, le rôle de cette régulation épigénétique a été peu étudiée dans les interactions hôtes-pathogènes. Dans ce projet de thèse, nous avons tout d'abord montré que la méthylation de l'ADN régule négativement la résistance d'Arabidopsis thaliana à une souche de Pseudomonas syringae pathogène. Nous avons également identifié un grand nombre de gènes de l'immunité ciblés directement par la méthylation de l'ADN dirigée par petits ARN dans leurs régions promotrices. Nous proposons que cette régulation génique permettrait de maintenir une faible expression basale de ces gènes et d'éviter ainsi des effets délétères qui seraient causés par une expression constitutive de la réponse immunitaire. De plus, nous montrons que la déméthylase active REPRESSOR OF SILENCING 1 (ROS1) facilite l'activation transcriptionnelle de gènes de l'immunité en laissant potentiellement des éléments de régulation en cis accessibles à des facteurs de transcription. Nous avons également démontré que ce facteur contribue à la résistance à P. syringae chez Arabidopsis, caractérisant ainsi la première déméthylase eucaryote dans la résistance antibactérienne. Sur la base de ces résultats, nous proposons que la méthylation de l'ADN maintient une faible expression basale de gènes de l'immunité en absence de pathogène, tandis que la déméthylation active assure une induction rapide de ces gènes au cours de la réponse immunitaire en favorisant potentiellement le recrutement de facteurs de transcription sur la chromatine. / DNA methylation and demethylation are regulatory processes involved in genome stability, genomic imprinting, paramutation and development. Until recently, very little was known about the role of these epigenetic processes in plant disease resistance and in the transcriptional control of immune-responsive genes. Here we provide evidence that DNA methylation negatively regulates antibacterial resistance against a virulent Pseudomonas syringae strain in Arabidopsis. Accordingly, we have identified a subset of defense genes that are targeted and repressed by RNA-directed DNA methylation (RdDM), presumably to prevent trade-off effects that would be caused by their constitutive expression and/or sustained induction. In addition, we found that the active DNA demethylase facilitates the transcriptional activation of some of these defense genes by pruning DNA methylation at their promoter regions and leaving cis-elements accessible for transcription factor binding. In addition, we show that the active demethylase REPRESSOR OF SILENCING 1 (ROS1) positively regulates late immune responses including Pathogen Associated Molecular Pattern (PAMP)-triggered callose deposition and salicylic acid (SA)-dependent defense response. We also demonstrate that ROS1 restricts Pto DC3000 propagation in Arabidopsis leaf secondary veins, providing the first example for a role of an active DNA demethylase in antibacterial resistance. Based on these findings we propose that DNA methylation maintains a low basal expression of some immune-responsive genes in normal growth condition, while active DNA demethylation ensures a rapid and pervasive induction of these genes upon bacterial pathogen detection.

Pflanzen erkennen potentielle Pathogene anhand von konservierten Mikroben-assoziierten molekularen Mustern (MAMPs) welche sie über membranlokalisierte Rezeptoren wahrnehmen. Der durch diese Rezeptoren aktivierte Signalweg spielt eine wesentliche Rolle in der pflanzlichen angeborenen Immunität. Das Binden eines MAMPs an die oberflächenexponierten Ektodomänen der Rezeptoren führt typischerweise dazu, dass diese homo- oder heteromere Komplexe bilden. Diese Komplexe können aus rezeptorartigen Kinasen (RLKs), rezeptorartigen Proteinen (RLPs) sowie aus rezeptorartigen zytoplasmatischen Kinasen (RLCKs), welche keine extrazelluläre Domäne zur Ligandenbindung besitzen, bestehen. Der Fokus dieser Arbeit liegt auf einem möglichen heteromeren Signalkomplex der unteranderem aus der lysinhaltigen-Motiv (LysM) RLK CERK1 besteht. CERK1 spielt eine Rolle in der durch Chitin induzierten Signaltransduktion und Abwehrantwort in Arabidopsis. In einer vorangegangenen Hefe-Zwei-Hybrid-Analyse wurde die RLCK CLR1 als möglicher Interaktor der CERK1 Kinasedomäne identifiziert. Vergleichende Sequenzanalysen zeigen, dass die Aminosäuresequenz von CLR1 eine hohe Homologie zu den Sequenzen der Kinasedomänen anderer Arabidopsis LysM-RLKs aufweist. Dies könnte möglicherweise für die Funktion des Proteins eine Rolle spielen. Die auf TAIR10 annotierte CLR1 Sequenz scheint falsch annotiert worden zu sein, da das eigentliche Protein laut Analysen in dieser Arbeit wahrscheinlich erst 23 Aminosäuren Richtung C-Terminus beginnt, wodurch dann ein mögliches N-Myristoylierungsmotiv exponiert wird. In vitro wird CLR1 direkt von der CERK1 Kinasedomäne phosphoryliert. CLR1 Fusionsproteine wurden in stabil transgenen Arabidopsis-Pflanzen CERK1-abhängig durch Chitin phosphoryliert. Unabhängig von der möglichen N-terminalen Myristoylierung scheint CLR1 sowohl in vitro also auch in vivo ein Phosphorylierungssubstrat von CERK1 darzustellen. Mikrosomale Fraktionierungen und Analysen zur subzellulären Lokalisation in transgenen Pflanzen zeigten dass die Mehrheit der CLR1 Proteine löslich ist, wobei auch eine kleine Subpopulation von CLR1 membrangebunden in Pflanzenzellen vorliegt. Drei unabhängige T DNA Insertionslinien wurden isoliert und im Hinblick auf die Weiterleitung Chitin-induzierter Signale und Immunität gegen pilzliche und bakterielle Schädlinge getestet. Die clr1 T-DNA Linien wiesen eine verringerte ROS Produktion, MAPK Aktivierung und Expression von Abwehrgenen auf, was eine Rolle für CLR1 im Chitin-induzierten Signalweg bestätigt. Dabei hing die Ausprägung des Phänotyps von der Position der T-DNA ab. clr1 Pflanzen waren nicht in der Resistenz gegen pilzliche Schädlinge beeinträchtigt, wohingegen sie eine leicht erhöhte Anfälligkeit gegenüber bakterieller Infektionen zeigten. Da der CLR1 Promotor erhöhte Aktivität in Hydathoden zeigt, könnte CLR1 darin involviert sein selektiv das Eintreten von Pathogenen über diese konstitutiv geöffneten Öffnungen einzugrenzen.

The circadian clock regulates a wide range of biological processes, allowing plants to be prepared for predictable daily diurnal changes in environmental cues such as light and temperature. Recent studies have suggested that the circadian clock may also control plant immunity. The exact nature of the interaction between the circadian clock and plant pathogens remains unknown. Our focus in this study is on the elucidation of the role of the biological clock in plant immunity against the necrotrophic pathogen to Botrytis cinerea. In order to do this we tested the level of susceptibility to B. cinerea in Arabidopsis thaliana wild type and transgenic plants: toc1, cca1/lhy, cca1/toc1, lhy/toc1, cca1/lhy/toc1, GLK1 OE, GLK2 OE, glk1, glk2, and glk1/glk2. We demonstrated that the time of infection plays a role in susceptibility to B. cinerea. Specifically, we found that plants are more susceptible to infection in the subjective morning. We also found that genetic mutations in core clock components or in GLK genes leads to changes in susceptibility to B. cinerea. Our data suggests that clock genes are not solely responsible for plant immune responses to B. cinerea but rather the ways in which the biological clock system regulates outcome pathways. Furthermore, when we entrain the biological clock by changing the photoperiod (day length) in normal earth conditions LD 24h and SD 24h, we observed that short day plants had higher susceptibility to B. cinerea than long day plants. In addition, when we entrain the biological clock in different photoperiods, the LD 30h photoperiod plants displayed similar responses as those in the SD 24h photoperiod. The data indicates that day length is not responsible for the control of plant immunity; it is the ability of light to entrain the biological clock that is important. Together, the data strongly support the conclusion that the circadian clock plays a role in plant defense regulation.

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