The effector-triggered immunity (ETI) is activated at the site of pathogen infection and results in a state of enhanced immunity called systemic acquired resistance (SAR) in distal, uninfected plant organs. SAR relays on mobile signals transported from infected cells to distal organs, and on signal amplification which supports transcriptional re-programming associated with priming and execution of SAR. Previous research in our lab has identified the chloroplastic TOP1 and cytosolic TOP2 as salicylic acid (SA)–binding oligopeptidases, non-competitively inhibited by SA. We demonstrate that SAR triggered with P. syringae DC3000 AvrRpt2 is abolished in top2 whereas top1 top2 exhibits a SAR slightly but consistently stronger than wild type (WT) controls, indicating that top1 is epistatic to top2. In agreement with the observed SAR phenotypes, top2 is defective in the induction of SAR markers including SA and Pip synthesis and SA signaling genes, whereas top1 top2 shows significantly higher induction of these markers. SAR- phenotype of top2 is rescued by exogenous SA, H2O2 and Pip applications. Interestingly, neither top1 nor top 1top2 are unable to mount SAR in response to Pip and H2O2 treatments. Analysis of ROS-responsive transcription factors and antioxidant gene induction in infected and distal tissues reveal significantly dysregulated patterns in all mutants, with top2 and top1 top2 most affected, indicating that TOP1 and TOP2 function together to support a pattern of successive waves of oxidation and reduction during SAR. The local and systemic oscillations are anti-corelated in Wt. The local vs. systemic anti-correlation is lost in the mutant genotypes. The amplitude of the mRNA oscillations is significantly lower in top2 plants, and significantly increased in top1top2 plants. top1 and top1top2 lost the oscillation compared to WT but they are still able to keep the expression up in time. top2 is unable to support the expression of some of the genes and oscillations and continued the expression of these genes in time. Overall, our results argue for a defining role of TOP chloroplastic and cytosolic proteolytic pathways in maintaining redox signaling necessary for the induction of SAR transcriptional re-programming and execution.

RNA interference (RNAi) is widely used to specifically silence the expression of any gene to study its function and to identify and validate therapeutic targets. Despite the popularity of this technology, recent studies have shown that RNAi may also silence non-targeted genes. Here we demonstrate the utility of a quick, efficient and robust approach to directly validate the specificity of RNAi as an alternative to indirect validation of RNAi through gene expression profiling. Our approach involves reversing (complementing) the RNAi-induced phenotype by introducing a synthetic version of the target gene that is designed to escape silencing. This synthetic gene complementation approach can also be used for mutational analysis of the target gene, or to provide a functional version of a defective protein after silencing the defective gene by RNAi. Using this approach we demonstrate that the loss of systemic acquired resistance, a form of innate immunity in plants, is indeed due to the silencing of salicylic acid-binding protein 2 rather than to off-target effects.

Systemic acquired resistance (SAR) is a defense response induced by an initial localized infection that leads to the generation of long-distance immune signals that travel to distant leaves to provide enhanced resistance to subsequent infections. The lipid transfer protein (LTP) DEFECTIVE IN INDUCED RESISTANCE1 (DIR1) travels via the phloem from induced to distant leaves during SAR and may chaperone several long-distance signal candidates. In this thesis, the role of DIR1 during SAR is explored by examining the route of DIR1 movement, investigating the conservation of DIR1 structure and function, and by identifying DIR1-interacting proteins. I demonstrate that Arabidopsis plant lines with restricted cell-to-cell movement through plasmodesmata are negatively impacted in long-distance DIR1 movement, suggesting that cell-to-cell movement is important for DIR1 to access distant leaves. To elucidate the molecular function of DIR1, orthology analysis was performed with putative DIR1 orthologs. Structurally important amino acid residues that contribute to the hydrophobicity of the LTP cavity were identified, supporting the idea that DIR1 binds a hydrophobic ligand during SAR. RNAi-mediated knockdown of the DIR1 paralog DIR1-like did not impact the SAR response, supporting the idea that DIR1- like plays a lesser role in SAR. In addition, targeted protein-protein interaction assays determined that LTP1 and LTP2 interact with DIR1, and SAR phenotypic analysis of an ltp2-1 mutant supported a role for LTP2 in SAR. Lastly, a comparative proteomics approach identified several proteins with differential abundance in phloem exudates collected during the induction of SAR. Of these proteins, m-type thioredoxins, a major latex protein-like protein, and the UV-B photoreceptor UVR8 were essential for the manifestation of SAR. Together, these data provide insight into DIR1 function by identifying the importance of cell-to-cell movement through plasmodesmata, the DIR1 hydrophobic cavity, and DIR1-interacting proteins for DIR1-mediated SAR. In addition, this work identifies new phloem-localized proteins that contribute to the SAR response, providing fundamental knowledge on protein composition within the phloem during biotic stress. / Dissertation / Doctor of Philosophy (PhD)

<I>Orobanche</I> (spp.) are parasitic plants that attack the roots of many important crops. <I>O. aegyptiaca</I> penetrates the host root (aided by digestive enzymes) and forms connections to the host vascular tissue, from which it will withdraw all of its water and nutrient requirements. In order to control this weed, it is important to understand the relationship between the host and the parasite. To investigate how parasitism effects host defense pathways, we are studying the patterns of expression of host genes known to be involved in various aspects of plant defense responses. With respect to local defense responses, two genes of the isoprenoid pathway were studied, one of which is expressed in wounded tissue (<I>hmg1</I>), and another that is induced in response to wounding yet repressed in response to pathogen elicitors (squalene synthase). Genes analyzed that are associated with systemic defense include <I>PR-1</I>, <I>PR-2</I>, and <I>PR-5</I>, all of which are induced in response to pathogen attack as part of the systemic acquired resistance (SAR) response. Plant gene expression was studied using transgenic tomato plants containing <I>hmg1</I>-GUS fusions, and northern hybridization analysis of tobacco and Arabidopsis roots using gene-specific probes. Results indicated that expression of <I>hmg1</I> is induced, <I>PR-2</I> and squalene synthase are repressed, and <I>PR-1a</I>, <I>PR-1</I>, and <I>PR-5</I> are not affected in tissue parasitized by <I>O. aegyptiaca</I>. Together, these results indicate a complex response to the parasite. Whereas <I>hmg1</I> induction is consistent with <I>O. aegyptiaca</I> inflicting a simple wound-like injury, the repression of squalene synthase is consistent with plant recognition of a pathogen attack. In contrast, the failure of <I>Orobanche</I> to induce SAR- related <I>PR-1</I> in tobacco and <I>PR-1</I>, <I>PR-2</I>, or <I>PR-5</I> in Arabidopsis indicates an ability to avoid or perhaps inhibit some defense-related pathways. By comparing the regulation of these defense genes in response to <I>O. aegyptiaca</I> attack, we are able to gain a greater understanding of the host plant response to parasitization and explore potential gene candidates for future engineering strategies to create <I>Orobanche</I> resistant crops. / Master of Science

Plants respond to pathogens both locally at the site of infection, as well as systemically in distant leaves. Systemic Acquired Resistance (SAR) is an immune response that involves the long-distance transport of SAR signal via the phloem from the site of infection to distant, uninfected leaves to establish long-lasting resistance. The Arabidopsis thaliana Defective in Induced Resistance 1 (DIR1) protein, which is required for SAR, accesses the phloem during SAR for long-distance travel to systemic leaves, and is thought to be part of a SAR signal complex. However, many questions remain about the long-distance movement of DIR1 during SAR – including the cellular route travelled to reach the phloem and whether other proteins are required for DIR1 movement. Fluorescent fusion lines of DIR1 and the related protein DIR1-like were previously created were investigated as potential tools to trace the movement of DIR1/DIR1-like during SAR. Immunoblot analysis of leaf extracts from these DIR1/DIR1-like fluorescent fusion lines revealed no signal, indicating that no fusion protein was present in these lines and therefore, they were likely not useful as a tool for assessing the movement of DIR1/DIR1-like during SAR. Lipid Transfer Protein 2 (LTP2) is required for SAR and interacted with DIR1 in a yeast-two-hybrid assay. To investigate if LTP2 is required for DIR1 movement into the phloem and long-distance, DIR1 signal was investigated by immunoblotting of phloem exudates from SAR induced ltp2-1 mutant plants. The presence of DIR1 signal in phloem exudates of local ltp2-1 leaves but not distant ltp2-1 leaves suggested that LTP2 may be required for the long-distance movement of DIR1 during SAR, but not for DIR1 to enter the phloem in induced leaves. Gene expression changes in the systemic, uninfected leaves are associated with the establishment of SAR, however, it remains less clear if there is a core set of genes important for SAR induction upregulated at the initial site of infection. To investigate this question, SAR was induced through differing treatments that first activated the PAMP-triggered immunity (PTI) pathway or Effector-triggered immunity (ETI) pathway. Common genes upregulated between all three SAR-inducing treatments were identified, revealing genes previously and currently under investigation by the Cameron lab, as well as genes that represent candidates for possible future studies. / Thesis / Master of Science (MSc)

The conserved metabolites, oleic acid (18:1), a major monounsaturated fatty acid (FA), and glycerol-3-phosphate (G3P) are obligatory precursors of glycerolipid biosynthesis in plants. In Arabidopsis, the SSI2-encoded SACPD is the major isoform that contributes to 18:1 biosynthesis. Signaling induced upon reduction in oleic acid (18:1) levels not only upregulates salicylic acid (SA)-mediated responses but also inhibits jasmonic acid (JA)- inducible defenses. I examined the transcription profile of ssi2 plants and identified two transcription factors, WRKY50 and WRKY51. Although the ssi2 wrky50 and ssi2 wrky51 plants were constitutively upregulated in SA-derived signaling, they were restored in JAdependent defense signaling. Not only did these plants show JA-inducible PDF1.2 expression, but they were also restored for basal resistance to the necrotrophic pathogen, Botrytis cinerea. Overall, my results show that the WRKY50 and WRKY51 proteins mediate both SA- and low 18:1-dependent repression of JA signaling in Arabidopsis plants. My studies also show that cellular G3P levels are important for plant defense to necrotrophic pathogens. I showed that G3P levels are induced in Arabidopsis in response to the necrotrophic fungal pathogen B. cinerea. G3P-dependant induction of basal defense is not via the activities of other defense-related hormones such as SA, JA or the phytoalexin camalexin. Arabidopsis mutants unable to accumulate G3P (gly1, gli1) showed enhanced susceptibility to B. cinerea. Previous studies in our lab identified acyl-carrier protein 4 (ACP4), a component of FA and lipid biosynthesis, as an important regulator of plant systemic immunity. ACP4 mutant plants were defective in systemic acquired resistance (SAR) because they contained a defective cuticle. I further investigated the role of the plant cuticle in SAR by studying the involvement of long-chain acyl-CoA synthetases (LACS), a gene family involved in long-chain FA and cuticle biosynthesis, in SAR. In all, eight lacs mutants (lacs1, lacs2, lacs3, lacs4, lacs6, lacs7, lacs8, lacs9) were isolated and characterized. Six mutants were compromised in SAR. Together, my studies show that the various LACS isoforms contribute differentially to both cuticle formation and systemic immunity in Arabidopsis.

A cultura da laranja doce é muito importante ao redor do mundo, em especial no Brasil, maior produtor mundial dessas frutas. A produção citrícola sempre esteve ameaçada por muitas doenças de grande importância, tais como, o cancro cítrico, a clorose variegada dos citros (CVC) e pinta preta. Entretanto, em 2004, surgiu o huanglongbing (HLB) ou greening, que tem devastado pomares, e para a qual ainda não foi encontrada uma solução definitiva. A transgenia pode ser uma técnica auxiliar no manejo desta doença com a busca de cultivares mais tolerantes, em especial ao HLB. Neste trabalho, as pesquisas de transgenia não envolveram genes exógenos à planta como, por exemplo, genes de outros organismos ou genes sintéticos, ou seja, foi baseado em tecnologias mais recentes já aplicadas em outras espécies vegetais, nas quais a transgenia é utilizada para super-expressar genes dos sistemas de defesa da própria planta. Estudos indicam que a super-expressão de genes do sistema de Resistência Sistêmica Adquirida (SAR - do inglês, \"Systemic Acquired Resistance\") promove a resistência de plantas a doenças. Um gene importante para esse sistema é o gene npr1 que controla a expressão das proteínas relacionadas à patogênese (PR), em especial a PR1. Junto do gene npr1, os genes npr3 e npr4 também são reguladores desse sistema, atuando sobre o gene npr1 de acordo com os níveis de ácido salicílico presentes na célula, nível este que varia de acordo com o nível de infecção de cada célula. Porém, a avaliação de um evento transgênico de citros pode levar muitos anos. Desta forma, para diminuir esse tempo de avaliação, pensou-se em usar plantas modelos. O sistema escolhido foi o tomateiro Micro-Tom (Solanun lycopersicum L. cv. Micro-Tom). Para a obtenção das construções gênicas, foram identificados os genes Csnpr1, Csnpr3 e Csnpr4 de Citrus sinensis L. Osbeck a partir dos genes Atnpr1, Atnpr3 e Atnpr4 de Arabidopsis thaliana L.. Os genes de citros foram obtidos a partir de uma planta de laranja doce por RT-PCR e clonados no vetor pCambia 2201, que foi então inserido em Agrobacterium tumefaciens para a transformação genética. Foi feita a transformação genética de plantas de laranja doce (Citrus sinensis L. Osbeck) e do tomateiro Micro-Tom. Após o crescimento dos brotos regenerados, foi feita a avaliação das plantas obtidas por meio de PCR. As plantas geneticamente modificadas foram aclimatizadas. As plantas de citros foram enxertadas e mantidas em casa de vegetação. As plantas de tomateiro Micro-Tom foram propagadas por sementes. A progênie foi avaliada aplicando o antibiótico de seleção canamicina, obtendo-se assim uma linhagem transgênica homozigota. / The sweet orange industry is very important worldwide, specially in Brazil, considered the world´s largest producer. The citrus production has always been threatened by several diseases of great importance, such as canker, CVC, and black spot. However, in 2004, the huanglongbing (HLB) or greening has been detected and devastated many citrus groves, and no definitive solution has been found yet. Transgenes may be a helpful tool for the management of this diseases, leading to the production of tolerant cultivars, especially to HLB. In this work, research on transgenic did not include the use of exogenous genes to the plant, such as genes from other organism or synthetic genes, i.e, it was based on new emerging technologies, already used on other crops, in which transgeny is used to super express genes from the plants own defense system. Studies indicate that a super expression of genes from the system called Systemic Acquired Resistance (SAR) promotes disease resistance. One important gene to this system is the npr1 gene, which controls the expression of the pathogen related proteins (PR), in special the PR1. Together with the npr1 gene, the genes npr3 and npr4 are also regulators of this system, regulating the action of the npr1 gene according to the levels of salicylic acid present in the cell, this level varies with the level of infection in each cell. Nevertheless, evaluating a citrus transgenic event may take several years. In order to shorten this time, model plants were used. The model chosen was the Micro-Tom tomato (Solanun lycopersicum L. cv. Micro-Tom). In order to obtain the genetic constructions, the genes Csnpr1, Csnpr3 e Csnpr4 were identified in Citrus sinensis L. Osbeck from the genes, Atnpr1, Atnpr3 and Atnpr4 present in the Arabidopsis thaliana L. genome. The citrus genes were obtained from the citrus genome using RT-PCR procedure and cloned separately into the pCambia 2201 vector, which was inserted into Agrobacterium tumefaciens in order to perform the genetic transformation. Sweet orange (Citrus sinensis L. Osbeck) and Micro-Tom plants were genetically modified. After the growth of the regenerated shoots, the evaluation of the obtained plants was done through PCR analysis. The genetically modified plants were acclimatized, the citrus plants were grafted and kept in the greenhouse, the Micro-Tom plants were propagated trough seeds and its progeny was evaluated by applying the selection antibiotic kanamycin, thus obtaining a homozygous transgenic line.

Nos anos recentes, a atividade agrícola da citricultura vem enfrentando grandes problemas fitossanitários, principalmente, com relação à viabilidade econômica decorrente do controle das doenças. A bactéria Candidatus Liberibacter spp. está associada ao HLB, a principal doença que limita a produção das plantas cítricas. Assim, muitos pesquisadores têm voltado suas atenções para estudarem e encontrarem genes-alvo na resposta do hospedeiro a este patógeno para utilização no melhoramento genético. Nesse sentido, métodos de transformação genética das plantas cítricas são essenciais, porém características inerentes à espécie limitam seu cultivo in vitro e requerem um maior tempo para crescimento e propagação. Com isso, torna-se importante o estudo em plantas modelo, principalmente, para seguir protocolos de validação de genes. De acordo com o exposto, o gene EDS5 isolado de Citrus sinensis, associado ao mecanismo de Resistência Sistêmica Adquirida (SAR) foi superexpresso por meio da transformação genética em tomateiro (Solanum lycopersicum L. Micro-Tom). Após o crescimento dos brotos regenerados, foram identificadas as plantas positivas por meio de análise de GUS e PCR. Linhagens transgênicas homozigotas foram obtidas com avaliação da resistência ao antibiótico canamicina. / In the recent years, the agricultural activity of the citrus industry has been facing big phytosanitary problems, mainly with regard to economic viability arising from disease control. The bacterium Candidatus Liberibacter spp. is associated with HLB, the main disease that limits the production of citrus trees. Thus, many researchers have been returning their attentions to study and find target genes in the host response to this pathogen for use in the genetic improvement. In this way, methods of genetic transformation of citrus plants are essential, but the inherent characteristics of the species border your in vitro cultivation and require a longer time for growth and propagation. Therefore, it is important to study of model plants, mainly for genetic validation protocols. Thus, the EDS5 gene isolated from Citrus sinensis, associated with Systemic Acquired Resistance mechanism (SAR) was overexpressed by genetic transformation in tomato (Solanum lycopersicum L. Micro-Tom). After the growth of regenerated shoots, positive plants were identified by PCR and GUS analysis. Homozygous transgenic lines were obtained with evaluation of resistance to kanamycin.

Systemic acquired resistance (SAR) is a type of plant defense mechanism that is induced after a localized infection and confers broad-spectrum immunity against related or unrelated pathogens. During SAR, a number of chemical signals and proteins generated at the site of primary infection travel to the uninfected tissues and are thought to alert the distal sites against secondary infections. Glycerol-3-phosphate (G3P) is one of the chemical signals that play an important role in SAR. G3P is synthesized in the cytosol and chloroplasts via the enzymatic activities of G3P Dehydrogenase (G3Pdh) or Glycerol Kinase (GK). Interestingly, a mutation in three of the five G3Pdh isoforms or GK impairs SAR by lowering the pathogen induced G3P pool. This suggests that total cellular pool of G3P is critical for SAR. To determine factors contributing to G3P flux between various subcellular compartments I analyzed the role of putative G3P transporters in G3P flux and SAR. The Arabidopsis genome encodes five isoforms of G3P Permeases (G3Pp) and these transmembrane proteins are predicted to localize to plasma membrane, chloroplast or mitochondria. At least two G3Pp isoforms (G3Pp1 and G3Pp3) were able to complement the Escherichia coli mutant impaired in the uptake of G3P into the cytoplasm. Characterization of Arabidopsis G3Pp mutants showed that a mutation in G3Pp2, G3Pp3 and G3Pp4 compromised SAR but not local resistance. Furthermore, this SAR defect could only be complemented by exogenous application of G3P. The G3Pp mutants accumulated wild-type-like levels of G3P suggesting that the subcellular compartmentalization of G3P might contribute to the induction of SAR.

Wheat is the most economically important crop forming one quarter of Australian farm production. The wheat industry is severely affected by diseases, with fungal pathogens causing the most important economic losses in Australia. The application of fungicides and chemicals can control crop diseases to a certain extent, however, it is expensive and public concern for the environment has led to alternative methods of disease control to be sought, including the use of microorganisms as biological control agents. Microorganisms are abundant in the soil adjacent to plant roots (rhizosphere) and within healthy plant tissue (endophytic) and a proportion possess plant growth promotion and disease resistance properties. Actinobacteria are gram-positive, filamentous bacteria capable of secondary metabolite production such as antibiotics and antifungal compounds. A number of the biologically active endophytes belonging to the Actinobacteria phylum were isolated in our laboratory. A number of these isolates were capable of suppressing the wheat fungal pathogens Rhizoctonia solani, Pythium sp. and Gaeumannomyces graminis var. tritici, both in vitro and in planta indicating the potential for the actinobacteria to be used as biocontrol agents. The aim of this research was to investigate the molecular mechanisms underlying this plant-microbe interaction. The indigenous microbial populations present in the rhizosphere and endophytic environment are critical to plant health and disruptions of these populations are detrimental. The culture-independent technique Terminal Restriction Fragment Length Polymorphism (T-RFLP) was used to characterise the endophytic actinobacteria population of wheat roots under different conditions. Soils which support a higher number of indigenous microorganisms result in wheat roots with higher endophytic actinobacterial diversity and level of colonisation. Sequencing of 16S rRNA gene clones, obtained using the same actinobacteria-biased PCR primers that were used in the T-RFLP analysis, confirmed the presence of the actinobacterial diversity, and identified a number of Mycobacterium and Streptomyces species. It was found that the endophytic actinobacterial population of the wheat plants contained a higher diversity of endophytic actinobacteria than reported previously, and that this diversity varied significantly among different field soils. The endophytic actinobacteria have previously been shown to protect wheat from disease and enhance growth when coated onto the seed before sowing. As the endophytes isolated were recognised as potential biocontrol agents, the impact on the indigenous endophytic microbial population was investigated. Utilising the T-RFLP technique it was established that the use of a commercial microbial inoculant, containing a large number of soil bacterial and fungal strains applied to the soil, disrupts the indigenous endophyte population present in the wheat roots. The hypothesis is that non-indigenous microbes proliferate and dominate in the soil preventing a number of endophytic-competent actinobacterial genera from access to the seed and ultimately endophytic colonisation of the wheat roots. This dramatically reduces diversity of endophytes and level of colonisation. In contrast the use of a single endophytic actinobacteria endophyte inoculant results in a 3-fold increase in colonisation by the added inoculant, but does not significantly affect this indigenous population. Colonisation of healthy plant tissues with fungal endophytes has been shown to improve the competitive fitness with enhanced tolerance to abiotic and biotic stress and improved resistance to pathogens and herbivores. In this study the fungal endophyte population of wheat plants grown in four different soils was analysed using partial sequencing of 18S rRNA gene sequences. Sequence anlaysis of clones revealed a diverse range of fungal endophytes. In this diverse range of fungal endophytes a number sequences were highly similar to those of previously known fungal phytopathogens. A number of sequences detected were similar to fungal species previously identified in soil or plant material but not as endophytes. The remaining sequences were similar to fungal species without a known relationship with plants. Plants have developed an inducible mechanism of defence against pathogens. In addition to local responses plants have developed a mechanism to protect uninfected tissue through a signal that spreads systemically inducing changes in gene expression. In the model plant Arabidopsis thaliana activation of the Systemic Acquired Resistance (SAR) pathway and the Jasmonate (JA)/Ethylene (ET) pathway is characterised by the production of pathogenesis-related (PR) and antimicrobial proteins resulting in systemic pathogen resistance. Endophytic actinobacteria, isolated from healthy wheat roots in our laboratory, have been shown to enhance disease resistance to multiple pathogens in wheat when coated onto the seed before sowing. Real Time RT-PCR was used to determine if key genes in the SAR and JA/ET pathways were induced in response to inoculation with endophytic actinobacteria. Inoculation of wild-type Arabidopsis thaliana with selected strains of endophytic actinobacteria was able to �prime� the defence pathways by inducing low level expression of SAR and JA/ET genes. Upon pathogen infection the defence-genes are strongly up-regulated and the endophyte coated plants had significantly higher expression of these genes compared to un-inoculated plants. Resistance to the bacterial pathogen Erwinia carotovora subsp. carotovora was mediated by the JA/ET pathway whereas the fungal pathogen Fusarium oxysporum triggered primarily the SAR pathway. Further analysis of the endophytic actinobacteria-mediated resistance was performed using the Streptomyces sp. EN27 and Arabidopsis defence-compromised mutants. It was found that resistance to E. carotovora subsp. carotovora mediated by Streptomyces sp. EN27 occurred via a NPR1-independent pathway and required salicylic acid whereas the jasmonic acid and ethylene signalling molecules were not essential. In contrast resistance to F. oxysporum mediated by Streptomyces sp. EN27 occurred via a NPR1-dependent pathway but also required salicylic acid and was JA- and ET-independent. This research demonstrated that inoculating wheat with endophytic actinobacteria does not disrupt the indigenous endophytic population and may be inducing systemic resistance by activating defence pathways which lead to the expression of antimicrobial genes and resistance to a broad range of pathogens.