Plant innate immunity is activated upon pathogen attack by recognizing their avirulent (avr) genes by Resistant (R) genes leading to R-gene resistance or host resistance. Another form of innate immunity is non-host resistance that is exhibited by a given plant species to most strains of a microbial species. R-gene resistance activates salicylic acid (SA) that is synthesized from methyl salicylic acid (MeSA) by Salicylic Acid Binding Protein 2 (SABP2). It was hypothesized that SABP2 plays the similar role in non-host resistance also. Growth experiments and non-host related gene analysis experiments were conducted on tobacco plants using P.s tabaci and P.s. phaseolicola that are host and non-host pathogens on tobacco respectively. Tobacco control plant C3 that expresses SABP2 and 1-2 that is RNAi silenced in SABP2 expression were used in this study. Results suggest that SABP2 may not have any significant role in tobacco non-host resistance.

No description available.

<p>Systemic Acquired Resistance (SAR) is a plant defense response induced by an initial infection in one part of the plant that leads to broad-spectrum resistance to normally virulent pathogens in distant naïve leaves. As part of the Cameron research team, I contributed to demonstrating that the lipid transfer protein, DIR1 is required for SAR long distance signaling in <em>Arabidopsis</em> and travels from induced to distant tissues during SAR. A highly similar<em> Arabidopsis</em> protein DIR1-like was identified and is thought to be responsible for the occasional SAR-competent phenotype observed in the <em>dir1-1</em> mutant. This work provides evidence for the idea that DIR1 and DIR1-like are paralogs created by a recent duplication event and that similar to DIR1, DIR1-like may travel to distant tissues during SAR. To better understand DIR1 and DIR1-like contribution during SAR, <em>dir1-1dir1-like</em> double mutant transgenic plants were created as well as transgenic plants expressing epitope- (HA and FLAG) and fluorescent- (iLOV and phiLOV) tagged DIR1 and DIR1-like to facilitate visualization of movement during SAR. Several putative DIR1 orthologs were identified in crop plants and cucumber CucDIR1 was shown to be functionally equivalent to AtDIR1 in <em>dir1-1</em> complementation studies providing further evidence that DIR1 plays an important role in SAR across plant species. By analyzing conservation between DIR1, DIR1-like and the putative DIR1 orthologs, several protein residues were identified that may be important for DIR1 function during SAR. DIR1 proteins were modified at these sites and the importance of these residues was supported by the reduced binding of the TNS hydrophobic probe in these DIR1 variants. Taken together, this thesis suggests that DIR1 and DIR1-like both participate in SAR in <em>Arabidopsis</em>, that DIR1 crop orthologs are also important for the SAR response and that DIR1 possesses several sites that are critical for its function in long distance SAR signaling.</p> / Doctor of Philosophy (PhD)

<p><em>Eutrema salsugineum</em> is an extremophile plant native to the Yukon Territory and coastal China. As an extremophile, Yukon <em>Eutrema</em> is tolerant to highly saline, drought conditions and cold temperatures while Shandong <em>Eutrema</em> can survive in highly saline conditions (Griffith et al., 2007; Guevara et al., 2012; Inan et al., 2004). The disease resistance responses of the Yukon and Shandong accessions of <em>Eutrema</em> were investigated to understand how an abiotic stress-tolerant plant responds to biotic stress. A pathosystem was developed using <em>Pseudomonas</em> <em>syringae</em> pv. <em>tomato</em> DC3000 (<em>Pst</em>) to examine <em>Eutrema</em> defense responses. Compared to <em>Arabidopsis </em>(Col-0), both <em>Eutrema</em> accessions exhibited resistance to <em>Pst,</em> with Shandong <em>Eutrema</em> displaying greater resistance than Yukon <em>Eutrema</em>. Resistance to <em>P. syringae</em> pv. <em>maculicola</em> (<em>Psm</em>) was also observed in both accessions. Furthermore, both <em>Eutrema</em> accessions displayed a differential capacity for effector-triggered immunity (ETI). RNA-Seq data of uninoculated Shandong vs. Yukon <em>Eutrema</em> revealed an overrepresentation of defense genes including <em>PR1</em> (<em>pathogenesis-related1</em>; Champigny et al., 2013). Expression of the <em>Eutrema</em> <em>PR1</em> ortholog in uninoculated Shandong leaves combined with enhanced resistance to <em>Pst</em> compared to Yukon <em>Eutrema</em> or Col-0 <em>Arabidopsis</em> suggests that Shandong plants exist in a defense-primed state. Resistance to other pathogens such as <em>Pectobacterium</em> <em>carotovorum</em> ssp. <em>wasabiae</em> (<em>Pcw</em>) further supported the hypothesis that Shandong <em>Eutrema</em> is primed for pathogen tolerance. The <em>Eutrema</em>-<em>P. syringae</em> pathosystem will facilitate future studies to understand how <em>Eutrema</em> deals with multiple or concurrent stresses and this knowledge will contribute to efforts to improve tolerance to both abiotic and biotic stress in crop plants.</p> / Master of Science (MSc)

Many economically important crop systems exhibit an Age-Related Resistance (ARR) response whereby mature plants become resistant to pathogens they were susceptible to when younger. The signaling pathways and mechanisms of ARR have not been well studied. Arabidopsis displays ARR in response to P. syringae pv tomato (Pst). Several studies provide evidence that intercellular salicylic acid (SA) accumulation is required for ARR and SA acts as a direct antimicrobial agent to limit bacterial growth and biofilm-like aggregate formation. SA accumulation mutants are ARR defective; however, a modest level of resistance is occasionally observed leading to the hypothesis that other compounds contribute to ARR as antimicrobial agents. Previous studies demonstrated that CYP71A13 (a key enzyme in indolic biosynthesis) is expressed during the ARR response. I demonstrated that CYP71A12 functionally compensated for CYP71A13 during ARR, as cyp71a12/cyp71a13-1 mutants were consistently ARR-defective compared to their respective single mutants. I demonstrated that dihydrocamalexic acid (DHCA) accumulated in intercellular washing fluids (IWFs) collected from plants during the ARR response using high resolution mass spectrometry-based profiling. DHCA was detected in IWFs collected from wild-type ARR-competent plants and, was absent in IWFs from ARR-incompetent cyp71a12/cyp71a13 mutants. In vitro DHCA antimicrobial activity against P. syringae was not observed, but exogenous infiltration of DHCA into the leaf intercellular space restored ARR in cyp71a12/cyp71a13 mutants Unlike SA which exhibits direct antimicrobial activity against P. syringae, DHCA does not and instead may affect pathogen virulence in other ways. My research provides evidence that intercellular DHCA contributes to ARR in response to P. syringae in Arabidopsis. Understanding the genes and metabolites contributing to ARR will provide useful information for future crop breeding and genetic modification that will mitigate agricultural losses due to disease. / Thesis / Master of Science (MSc) / During Age-Related Resistance (ARR), mature plants including some crop plants become resistant to pathogens they were susceptible to when younger. How ARR works is poorly understood. My objective was to identify potential antimicrobial metabolites contributing to ARR in Arabidopsis against the bacterial pathogen Pseudomonas syringae. Genetic analyses combined with mass-spectrometry based metabolite profiling demonstrates that two cytochromes P450, CYP71A12 and CYP71A13 contribute to ARR. My research provides evidence that DHCA accumulates in the leaf intercellular space in ARR-competent plants, where it may act to inhibit the bacterial infection process. DHCA has low antimicrobial activity against P. syringae suggesting its mechanism of action is not directly antimicrobial. Importantly, application of DHCA to the leaf intercellular space of cyp71a12/cyp71a13 restored ARR, confirming that DHCA contributes to ARR in Arabidopsis. Understanding ARR will provide useful information for future crop breeding and genetic modification that will mitigate agricultural losses due to disease.

Greenhouse environments often promote bacterial and fungal infections in important crop plants. Exogenous application of chemical inducers could help reduce the severity of infection, or even prevent infection. Small molecules such as glycerol, azelaic acid and pipecolic acid have been implicated as being important signaling molecules during Systemic Acquired Resistance (SAR). To examine if these small molecules could be used to induce resistance in crop plants, exogenous treatment assays were developed in cucumber. Glycerol spray and azelaic acid infiltration induced modest resistance at locally treated leaves. Pipecolic acid soil treatment induced modest resistance in aerial tissue of cucumber plants, and strong resistance when plants were treated weekly. This knowledge may be useful in promoting the commercialization of SAR-associated compounds to protect important crop plants against disease. Plants possess multiple defense pathways that include an SA signaling component to initiate resistance to microbial pathogens. However, during Age-Related Resistance (ARR) in Arabidopsis, a number of studies support that SA acts as an anti-microbial and anti-biofilm agent against Pseudomonas syringae pv. tomato (Pst) in the plant intercellular space. Little is known about the role of Pst biofilm formation during infection of young plants or if other defense responses act to suppress bacterial biofilm formation. Therefore Pst biofilm formation and the effect of PAMP Triggered Immunity (PTI) on bacterial biofilm formation was examined. PTI was induced with flg22 in wild-type Col-0, fls2, bak1-3 (PTI mutants) and sid2-2 (SA biosynthesis mutant). In vivo bacterial biofilm-like aggregate formation was monitored using Pst DC3000 PDSK-GFPuv and epifluorescence microscopy. Pst aggregate occurrence and size were positively correlated with bacterial success in susceptible plants (wild-type Col-0, fls2, bak1-3, sid2-2), while fewer and smaller bacterial aggregates were observed in Col-0 undergoing PTI. To determine if the extracellular polysaccharide, alginate was a major contributor to biofilm formation, in vivo bacterial aggregate formation was monitored using alginate deficient Pst-GFP. Alginate deficient Pst-GFP and wild-type Pst grew to similar levels in wild-type plants suggesting that the ability to produce alginate was not necessary for Pst pathogenicity and success in planta. Fewer alginate-deficient Pst aggregates were observed compared to wild-type Pst in inoculated plants, suggesting that the ability to produce alginate was modestly important for aggregate formation. These data provide novel insights into how biofilms form in planta, the association between pathogen virulence and biofilm formation, and how plant defense responses such as PTI not only reduce bacterial growth, but also target biofilms. / Thesis / Master of Science (MSc)

Pseudomonas syringae is an important bacterial plant pathogen that, as a species, is known to cause disease on hundreds of different plant species. However, any individual pathovar of P. syringae typically only causes disease on one or a few plant species, which constitute the host range of the pathovar. Plants are generally resistant to most pathogens primarily because the plant innate immune system is capable of recognizing conserved microbial-associated molecular patterns (MAMPs). Adapted pathovars of P. syringae secrete effector proteins through a Type Three Secretion System (T3SS) to suppress the immune response elicited by their MAMPs. However, secretion of effectors can also trigger a strong plant immune response if the plant harbors resistance proteins capable of recognizing the secreted effectors. Successful pathovars, therefore, must secrete a combination of effectors capable of suppressing MAMP/Pattern-Triggered Immunity (PTI) without eliciting Effector-Triggered Immunity. Here we identify several novel strategies employed by P. syringae to overcome the plant immune system and cause disease. First, we demonstrate that, in place of the canonical T3SS used by all known pathogens of P. syringae, several apparently nonpathogenic isolates of P. syringae employ a novel T3SS that is functional but not necessary for colonization of plants. Despite being closely related to pathogenic isolates of P. syringae, the isolates employing the noncanonical T3SS do not cause disease on any tested plants and instead appear to act more as commensal organisms. Second, we advance the understanding of PTI by identifying a second region of bacterial flagellin that triggers PTI in addition to the archetypical MAMP flg22, which is recognized by the archetypical plant receptor FLS2. This new elicitor, termed flgII-28, is also detected by FLS2 and appears to be under selection in very closely related lineages of P. syringae. Alleles of flagellin present in one recently expanded and agriculturally problematic lineage of P. syringae appear to trigger less PTI on their host plant, tomato, than the ancestral allele suggesting that avoidance of PTI through allelic diversity in MAMPs is an effective alternative strategy to suppression of PTI through delivery of effectors. Finally, we start to elucidate a role for chemotaxis (chemical-directed movement) in P. syringae pathogenicity. Not only is chemotaxis required for pathogenicity of P. syringae on plants, but it also appears to contribute to delimiting the host range of several P. syringae pathovars. These results highlight that additional aspects of P. syringae pathogenicity, such as chemotaxis, can directly contribute to defining the host range of individual P. syringae pathovars. The current paradigm of P. syringae pathogenicity posits that MAMPS and the repertoire of effector proteins are the primary determinant of the host range of any P. syringae pathovar; in contrast these results inspire a more nuanced view of pathogenicity that considers multiple aspects of the infection process. / Ph. D.

Throughout evolution, plants have developed a sophisticated network of signaling pathways allowing the activation and regulation of immune responses. The identification of metabolic pathways which are involved in modulating the intensity of that immune responses is an important challenge in the field of plant-pathogen interaction. With this aim, we performed two genetic approaches in Arabidopsis thaliana against the disease caused by the hemibiotroph bacterial pathogen Pseudomonas syringae DC3000. We demonstrate that the regulation of two pathways, related between them, is crucial to activate an effective immune response. By means of a genetic screening of regulators components of plant immunity, we identified the mutant scs9 (suppressor of csb3) which shows an affected resistance that triggers a enhanced susceptibility to P.s. DC3000 through an independent pathway of salicylic acid (SA)-mediated immune response. The cloning and characterization of SCS9 reveals that it codes for 2'-O-ribose tRNA methyltransferase. Our results indicate that the SCS9-mediated methylation of nucleosides N32 and N34, located in the tRNAs anticodon loop, is crucial for the plant immunity effectiveness. On the other hand, with a chemical genetic screening of agonist molecules of the immune response, we identified the sulfonamides as priming inducer molecules that exhibit a faster and/or stronger activation of SA-related defense responses and enhanced resistance to P.s. DC3000. Analysis of the mechanism of action of these molecules reveals that synthesis and accumulation of folates exert a SA-independent negative control on the immune response to P.s. DC3000. Through comparative proteomic analysis we identified the 5-methyltetrahydropteroyltriglutamate homocysteine methyltransferase 1 (methione synthase, here named as METS1), enzyme responsible of the methionine synthesis in the folate-dependent 1C metabolism and overaccumulated in scs9 mutants, as modulator component in the immune response to P.s. DC3000. We observed that the overexpression of METS1 in transgenic plants of Arabidopsis suppresses plant immune responses and promotes enhanced susceptibility to P.s. DC3000. This repressor effect is due to a genome-wide increase in DNA methylation level, which is mediated by the overaccumulation of METS1 and the consequent increase of folate-dependent methionine synthesis. Therefore, the findings of this work provide a deeper knowledge about the mechanisms by which the DNA methylation and epigenetic regulation exert an influence on plant immunity through folate metabolism, particularly by METS1, whose synthesis is regulated through specific tRNA modifications mediated by SCS9. / Las plantas, a lo largo de la evolución, han desarrollado un sofisticado entramado de rutas de señalización que permiten la activación y el control de la respuesta inmune. Identificar qué procesos metabólicos participan en modular la amplitud de dicha respuesta inmune es un reto en el campo de la interacción planta-patógeno. Con este propósito, se han utilizado dos aproximaciones genéticas llevadas a cabo en Arabidopsis thaliana contra la infección por la bacteria hemibiotrofa Pseudomonas syringae DC3000. Los resultados ponen de manifiesto la importancia de la regulación de dos mecanismos, a su vez relacionados, para la activación de una respuesta inmune efectiva. Mediante un rastreo genético en busca de componentes reguladores de la inmunidad, identificamos el mutante que denominamos scs9 (supresor de csb3). scs9 muestra una resistencia afectada que conlleva un incremento en la susceptibilidad a P.s. DC3000 a través de un mecanismo independiente a la respuesta inmune mediada por ácido salicílico (SA). La clonación y caracterización de SCS9 revela que codifica una 2'-O-ribosa metiltransferasa de tRNA. Nuestros resultados indican que la modificación por metilación mediada por SCS9 de los nucleósidos N32 y N34 de la región anticodón de los tRNAs, es clave para la inmunidad de la planta. Por otro lado, mediante un rastreo de genética química en busca de moléculas agonistas de la respuesta inmune, identificamos un grupo de sulfonamidas como moléculas activadoras de un mecanismo de priming. Este conlleva una más rápida y/o más intensa activación de la respuesta defensiva dependiente de SA y de un incremento de la resistencia frente a P.s. DC3000. El análisis del mecanismo de acción de dichas moléculas revela que la síntesis y acumulación de folatos ejerce un control negativo sobre la respuesta inmune frente a P.s. DC3000; y ese control es ejercido de manera independiente a la ruta de señalización mediada por SA. A través de un análisis proteómico comparativo identificamos la proteína 5-metiltetrahidropteroiltriglutamato homocisteína metiltransferasa 1 (metionina sintasa, denominada aquí METS1), responsable de la síntesis de metionina en el metabolismo C1 dependiente de folatos y sobreacumulada en los mutantes scs9. Esta proteína participa entonces como componente modulador de la respuesta inmune a P.s. DC3000. La sobreexpresión de METS1 en plantas transgénicas observamos que suprime la respuesta inmune y conlleva a un incremento en la susceptibilidad frente a P.s. DC3000. Dicho efecto represor de la resistencia acontece a raíz de un incremento del nivel de metilación de DNA en todo el genoma mediado por la sobreacumulación de METS1 y del consiguiente posible aumento en la síntesis de metionina dependiente de folatos. Por tanto, estos resultados ahondan en el conocimiento de cómo la metilación de DNA y el control epigenético ejercen una influencia sobre la respuesta inmune. Esta influencia puede ser controlada a través del metabolismo de folatos, y en particular a través de METS1, enzima cuya síntesis está a su vez controlada por determinadas modificaciones de tRNA mediadas por SCS9. / Les plantes, al llarg de l'evolució, han desenvolupat un sofisticat entramat de rutes de senyalització que permeten l'activació i el control de la resposta immune. Identificar quins procesos metabòlics participen en la modulació de l'amplitud d'aquesta resposta immune és un repte en el camp de la interacció planta-patogen. Amb aquest propòsit, s'han utilitzat dues aproximacions genètiques en Arabidopsis thaliana en resposta a la infecció pel bacteri hemibiotrofo Pseudomonas syringae DC3000. Els resultats posen de manifest la importància de la regulació de dos mecanismes, al seu torn relacionats, per a l'activació d'una resposta immune efectiva. Mitjançant un rastreig genètic per a la recerca de components reguladors de la immunitat, es va identificar el mutant que denominem scs9 (supresor de csb3). scs9 mostra una resistència afectada que comporta un increment en la susceptibilitat a P.s. DC3000 fent ús d'un mecanisme independent a la resposta immune mediada per l'àcid salicílic (SA). La clonació i caracterització de SCS9 revela que codifica una 2'-O-ribosa metiltransferasa de tRNA. Els nostres resultats indiquen que la modificació per metilació mediada per SCS9 dels nucleòsids N32 i N34 de la regió anticodó dels tRNAs, és clau per a la immunitat de la planta. D'altra banda, per mitjà d'un rastreig de genètica química per a la recerca de molècules agonistes de la resposta immune, es va identificar un grup de sulfonamidas com a molècules activadores d'un mecanisme de priming. Aquest, comporta una més rápida i/o més intensa activació de la resposta defensiva dependent de SA i d'un increment de la resistència enfront de P.s. DC3000. L'anàlisi del mecanisme d'acció d'aquestes molècules revela que la síntesis i acumulació de folats exerceix un control negatiu sobre la resposta immune davant el bacteri P.s. DC3000; i eixe control és exercit de manera independent a la ruta de senyalització mediada per SA. Amb un anàlisi proteòmic comparatiu es va identificar la proteïna 5-metiltetrahidropteroiltriglutamato homocisteína metiltransferasa 1 (metionina sintasa, denominada ací METS1), responsable de la síntesi de metionina al metabolisme C1 dependent de folats i sobreacumulada en els mutants scs9. Aquesta, així doncs, es troba participant com a component modulador de la resposta immune a P.s. DC3000. La sobreexpressió de METS1 en plantes transgèniques suprimeix la resposta immune i comporta a un increment en la susceptibilitat per P.s. DC3000. L'efecte repressor de la resistència succeïx arran d'un increment del nivell de metilació de DNA en tot el genoma, mediat per la sobreacumulació de METS1 i del consegüent posible augment en la síntesi de metionina dependent de folats. Per tant, aquests resultats aprofundixen en el coneixement de com la metilació de DNA i el control epigenètic exerceixen una influència sobre la resposta immune. Aquesta influència pot ser controlada mitjançant el metabolisme de folats, i en particular a través de l'enzim METS1, la síntesi de la qual està al seu torn controlada per determinades modificacions de tRNA mediades per SCS9. / González García, B. (2017). Implicación de las modificaciones de tRNA y del metabolismo de los folatos en la respuesta inmune de Arabidopsis [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/86162

Most plant pathogens are specialists where only few plant species are susceptible, while all other plants are resistant. Unraveling the mechanisms behind this can thus provide valuable information for breeding or engineering crops with durable disease resistance. A group of Pseudomonas syringae strains with different host ranges while still closely related were thus chosen for comparative study. We confirmed their close phylogenetic relationship. We found evidence supporting that these strains recombined during evolution. The Arabidopsis thaliana and tomato pathogen P. syringae pv. tomato (Pto) DC3000 was found to be an atypical tomato strain, distinct from the typical Pto strains commonly isolated in the field that do not cause disease in A. thaliana, such as Pto T1. Comparing A. thaliana defense responses to DC3000 and T1, we found that T1 is eliciting stronger responses than DC3000. T1 is likely lacking Type III effector genes necessary to suppress plant defense. To test this, we sequenced the genomes of strains that cause and do not cause disease in A. thaliana. Comparative genomics revealed candidate effector genes responsible for this host range difference. Effector genes conserved in strains pathogenic in A. thaliana were expressed in T1 to test whether they would allow T1 to growth better in A. thaliana. Surprisingly, most of them reduced T1 growth. One of the effectors, HopM1, was of particular interest because it is disrupted in typical Pto strains. Although HopM1 has known virulence function in A. thaliana, HopM1 reduced T1 growth in both A. thaliana and tomato. HopM1 also increased the number of bacterial specks but reduced their average size in tomato. Our data suggest that HopM1 can trigger defenses in these plants. Additionally, transgenic detritivore Pseudomonas fluorescens that can secrete HopM1 shows dramatically increased growth in planta. The importance of genetic background of the pathogen for the functions of individual effectors is discussed. T1 cannot be manipulated to become an A. thaliana pathogen by deleting or adding individual genes. We now have a list of genes that can be studied in the future for the molecular basis of host range determination. / Ph. D.

Developing a flue-cured tobacco cultivar with high resistance to tobacco cyst nematode (TCN) is an important initiative in the Southern Piedmont of Virginia, where this pathogen causes severe yield losses. One hundred twenty eight lines representing a diverse geographic array of tobacco accessions, including cultivars from several types of tobacco, flue-cured-type tobacco introductions, and wild Nicotiana species were evaluated for TCN resistance under greenhouse conditions. Inheritance of TCN resistance has been reported to be closely linked or pleiotropic to inheritance of wildfire resistance. Consequently, accessions were also screened for wildfire resistance under greenhouse conditions to evaluate this relationship among a diverse group of tobacco accessions. Twenty-one accessions were identified with resistance to TCN. Response to the two pathogens was highly correlated. However, there was no relationship between resistance to the two pathogens for several accessions. 'KY 190', a fire-cured cultivar, possessed the N. longiflora source of wildfire resistance, but was found susceptible to TCN. This result seems to rule out pleiotropy and is consistent with the assertion that the two resistance genes are closely linked. six TCN resistant lines and two susceptible lines were selected and a diallel study was conducted to determine the inheritance of resistance to TCN. Additive gene action contributed significantly to inheritance of TCN resistance. Three accessions, 'Burley 64', 'Kutsaga 110', and 'Tl 1597', were determined to be the most promising parents for use in a breeding program designed to develop a flue-cured cultivar with a high level of TCN resistance. / Master of Science