Hyaloperonospora arabidopsidis (Hpa) is a naturally occurring oomycete pathogen on Arabidopsis thaliana. It is related to downy mildews of economically important crops such as cabbage, kale and broccoli, belonging to the Brassicaceae family. Downy mildew pathogens are obligate biotrophs that extract nutrients exclusively from living plant cells. As a part of its obligate biotrophy lifestyle, Hpa has lost the ability to assimilate inorganic nitrogen and sulfur. It thus has to acquire these nutrients from the host in an organic form; possibly amino acids. Using a reverse genetic approach, I was able to identify two host amino acid transporters that are up-regulated during Hpa infection: AAP3 and AAP6. Both of these transporters are localized in the vasculature of the plant, AAP3 mostly in the root, and AAP6 in the roots and shoots. Using transgenic lines of Arabidopsis containing transcriptional and translational reporter fusion constructs for these genes, I found that AAP3 displays increased mRNA accumulation which is attributable to an increased promoter activity in regions of shoot tissue colonized by Hpa. On the other hand, AAP6 displays a mild increase in mRNA accumulation under Hpa infection, but the induction becomes more prominent at the protein level as seen by fluorescence from GFP fused to AAP6. Surprisingly, null mutants of AAP3 did not impact Hpa growth whereas null mutants of AAP6 made the plant more susceptible to Hpa. Furthermore, aap6 mutants accumulate fewer free amino acids in the phloem compared to wild-type plants when infected with Hpa. Together, these results suggest that AAP6 acts a nutritional starvation gene for the pathogen and hence aids the plant during infection. While we now know more about AAP3's regulation during infection, its function remains to be elucidated. To successfully colonize a plant, a pathogen must be able to achieve both suppression of plant immunity and acquisition of nutrients from the plant host. While the former has been well studied, research on the latter is sparse. This work was a step in the direction to increase our understanding of potential players in nutrient acquisition by pathogens. / Doctor of Philosophy / A key aspect of achieving and maintaining food security is sustainable agricultural production. This is endangered by plant diseases that lead to large losses in crop production. All plant pathogens have to acquire food and nutrients from the plants they infect. Understanding how they acquire nutrients from the plant at a molecular level can give us insight into potential methods to prevent this and hence reduce the impact of plant diseases. One such nutrient is nitrogen. Nitrogen is essential to all of an organism’s cellular and metabolic processes. Organisms utilize nitrogen by converting it from inorganic forms such as nitrates to organic forms such as amino acids. Some plant pathogens, such as Hyaloperonospora arabidopsidis (Hpa), which causes downy mildew disease on the model plant Arabidopsis, complete their entire life cycle on a living plant. They are also unable to convert the inorganic nitrogen to organic forms and hence depend on acquiring organic forms of nitrogen from the plant. Thus, it is important to understand how they acquire amino acids from the plant. Plants use amino acid transporters that serve as a siphon or a pump in moving amino acids from one region of the plant to another. It is possible that pathogens manipulate plant’s amino acid transporters to move amino acids towards the infection site while, at the same time, plants might use another set of transporters to move amino acids away from the pathogen. This work was an attempt at understanding this potential role of plant amino acid transporters in plant-pathogen interactions using the model system of Hpa and Arabidopsis.

Plant pathogens are one of the biggest threats to plant health and food security worldwide. To effectively contain plant disease outbreaks, classification and precise identification of pathogens is crucial to determine treatment and preventive measurements. Conventional methods of detection such as PCR may not be sufficient when the pathogen in question is unknown. Advances in sequencing technology have made it possible to sequence entire genomes and metagenomes in real-time and at a relatively low cost, opening an opportunity for the development of alternative methods for detection of novel and unknown plant pathogens. Within this dissertation, an integrated approach is used to reclassify a high-impact group of plant pathogens. Additionally, the application of metagenomics and nanopore sequencing using the Oxford Nanopore Technologies (ONT) MinION for fungal and bacterial plant pathogen detection and precise identification are demonstrated. To improve the classification of the strains belonging to the Ralstonia solanacearum species complex (RSSC), we performed a meta-analysis using a comparative genomics and a reverse ecology approach to accurately portray and refine the understanding of the diversity and evolution of the RSSC. The groups identified by these approaches were circumscribed and made publicly available through the LINbase web server so future isolates can be properly classified. To develop a culture-free detection method of plant pathogens, we used metagenomes of various plants and long-read nanopore sequencing to precisely identify plant pathogens to the strain-level and performed phylogenetic analysis with SNP resolution. In the first paper, we used tomato plants to demonstrate the detection power of bacterial plant pathogens. We compared bioinformatics tools for detection at the strain-level using reads and assemblies. In the second paper, we used a read-based approach to test the feasibility of the methodology to precisely detect the fungal pathogen causing boxwood blight. Lastly, with the improvement in nanopore sequencing, we used grapevine petioles to investigate whether we can go beyond detection and identification and do a phylogenetic analysis. We assembled a metagenome-assembled genome (MAG) of almost the same quality as the genomes obtained from cultured isolates and did a phylogenetic analysis with SNP resolution. Finally, for the cases where there may be no related genome in the database like the pathogen in question, we used machine learning and metagenomics to develop a reference-free approach to detection of plant diseases. We trained eight different machine learning models with reads from healthy and infected plant metagenomes and compared the classification accuracy of reads as belonging to a healthy or infected plant. From the comparison, random forest was the best model in terms of computational resources needed while maintaining a high accuracy (> 0.90). / Doctor of Philosophy / Microbes are present in every environment on the planet and have been on Earth for billions of years. While some microbes are beneficial, others can cause diseases. To differentiate the ones causing diseases from those who do not, looking into the evolutionary forces making them different is crucial to classify and identify them correctly. Although microorganisms cause diseases in humans and animals, the ones causing diseases in plants are one of the biggest threats to plant health and food security worldwide. In a perfect world, plant diseases would be diagnosed by eye or simple procedures. However, when a plant disease is present, it is not always obvious which organism, if any, is causing the disease making it hard for outbreaks to be detected and contained promptly. With technological advances, it is now possible to obtain all the genetic information of not only one organism but all the organisms living in an environment at a time. This genetic information can then be used to precisely identify what organism is causing a disease in a plant for faster disease diagnosis and, consequently, more efficient disease prevention and control. In this dissertation, we used the bacterial group, called Ralstonia solanacearum species complex, which can cause different diseases in more than 200 crops, to investigate and understand the evolution and diversity of the members of this group. We also used newly developed technologies to obtain the genetic material of all the organisms living in multiple important plants including tomato, grapevine, and the ornamental bush, boxwood. Using this genetic material, we developed a methodology for the detection of bacteria and a fungus causing plant diseases. While this works well when the suspected organism or a similar one is available for comparison, the detection of plant diseases in cases where this information is not available is challenging. Machine learning models, where computers can learn complex patterns from data, have the potential to detect pathogens without the need to compare the sequences to sequences of other pathogens. Here we also used the genetic material to train and compare different machine learning models to classify plants as either being infected or healthy.

Yes / Dickeya chrysanthemi is well known as a plant pathogen that caused major blackleg in the European potato industry in the 1990s. D. chrysanthemi strain L11 was discovered in a recreational lake in Malaysia. Here, we present its draft genome sequence. / University of Malaya High Impact Research (HIR) Grants UM C/625/1/HIR/MOHE/CHAN/01 (grant no. A-000001-50001) and UM C/625/1/HIR/MOHE/CHAN/14/1 (grant no. H-50001-A000027)

Thesis (MScAgric (Plant Pathology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: A survey was undertaken on apple and pear trees in the Western Cape Province to determine the aetiology of trunk diseases with reference to trunk diseases occurring on grapevine. Grapevine trunk diseases cause the gradual decline and dieback of vines resulting in a decrease in the vine’s capability to carry and ripen fruit. In recent years, viticulture has been expanding into several of the well established pome fruit growing areas. The presence of trunk pathogens in pome fruit orchards may affect the health of the pome fruit trees as well as cause a threat to young vineyards planted in close proximity to these potential sources of viable inoculum. Several genera containing species known to be involved in trunk disease on pome fruit and grapevine were found, including Diplodia, Neofusicoccum, Eutypa, Phaeoacremonium and Phomopsis. Diplodia seriata and D. pyricolum, were isolated along with N. australe and N. vitifusiforme. Four Phaeoacremonium species, P. aleophilum, P. iranianum, P. mortoniae and P. viticola, two Phomopsis species linked to clades identified in former studies as Phomopsis sp. 1 and Phomopsis sp. 7, and Eutypa lata were found. In addition, Paraconiothyrium brasiliense and Pa. variabile, and an unidentified Pyrenochaetalike species were found. Of these the Phaeoacremonium species have not been found on pear wood and it is a first report of P. aleophilum occurring on apple. This is also a first report of the Phomopsis species and Eutypa lata found occurring on pome trees in South Africa Two new coelomycetous fungi were also found including a Diplodia species, Diplodia pyricolum sp. nov., and a new genus, Pyrenochaetoides gen. nov. with the type species, Pyrenochaetoides mali sp. nov., were described from necrotic pear and apple wood. The combined ITS and EF1-α phylogeny supported the new Diplodia species, which is closely related to D. mutila and D. africana. The new species is characterised by conidia that become pigmented and 1-septate within the pycnidium, and that are intermediate in size between the latter two Diplodia species. Phylogenetic inference of the SSU of the unknown coelomycete provided bootstrap support (100%) for a monophyletic clade unrelated to known genera, and basal to Phoma and its relatives. Morphologically the new genus is characterised by pycnidial with elongated necks that lack setae, cylindrical conidiophores that are seldomly branched at the base, and Phoma-like conidia. The phylogenetic results combined with its dissimilarity from genera allied to Phoma, lead to the conclusion that this species represents a new genus. A pathogenicity trial was undertaken to examine the role of these species on apple, pear and grapevine shoots. N. australe caused the longest lesions on grapevine shoots, while Pyrenochaetoides mali, Pa. variabile, D. seriata and P. mortoniae caused lesions that were significantly longer than the control inoculations. On pears, D. pyricolum and N. australe caused the longest lesions, followed by D. seriata and E. lata. On apples, the longest lesions were caused by N. australe and P. iranianum. D. seriata, D. pyricolum, E. lata, N. vitifusiforme, Pa. brasiliense, P. aleophilum and P. mortoniae also caused lesions on apple that were significantly longer than the control. The study demonstrated that close cultivation of grapevine to apple and pear orchards may have inherent risks in terms of the free availability of viable inoculum of trunk disease pathogens. / No Afrikaans abstract available.

Thesis (MSc)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: In the field of agriculture, plant pathogens are a major concern because of the severe damage these organisms cause to crops yearly. Fundamental studies regarding plant pathogens and their modes of action made it possible for researchers in the field of molecular biology to investigate pathogens further on a molecular level. Botrytis cinerea, has been used to great effect as a model system to investigate various aspects regarding pathogenesis, also on a molecular level. Molecular research done on B. cinerea over the last few years has shown that the endopolygalacturonases (EPGs) of this fungus are key role players in pathogenesis. This hydrolytic enzyme family of six members, encoded by the Bcpg1-6 genes, are important in breaking down the complex cell wall polymers of host plants, enabling the fungus to penetrate its host sufficiently. It has been shown that both BcPG1 and 2 are crucial for virulence of B. cinerea. A leucine-rich repeat inhibitor protein situated in the cell wall of various plant species, the polygalacturonase-inhibiting protein (PGIP), has been proven to interact with and inhibit EPGs, and thus the necrotic actions of B. cinerea. From literature it was clear that specific data regarding individual interactions of fungal EPGs with PGIPs are lacking currently. Furthermore, most experiments regarding the effects of EPG as well as interaction and inhibition studies of EPGs and PGIPs, rely on in vitro methods, without the possibility to contextualize the results on an in vivo or in planta level. The scope of this study was to specifically address the issues of individual EPG:PGIP interactions and the use of possible in vivo methodology by using EPGs from a highly virulent South African strain of B. cinerea and the grapevine VvPGIP1 that has been previously isolated in our laboratory. This PGIP, originally isolated from Vitis vinifera cv Pinotage, has been shown to inhibit a crude EPG extract from this strain with great efficiency. The approach taken relied on heterologous over-expression of the individual Bcpg genes and the isolation of pure and active enzymes to evaluate the inhibition of the EPGs with VvPGIP1. The genes were all successfully over-expressed in Saccharomyces cerevisiae with a strong and inducible promoter, but active enzyme preparations have been obtained only for the encoding Bcpg2 gene, as measured with an agarose diffusion assay. The in vitro PGIP inhibition assay is also based on the agarose diffusion assay and relies on activity of the EPGs to visualize the inhibiting effect of the PGIP being tested. The active EPG2, however, was not inhibited by VvPGIP1 when tested with this assay. The EPG encoding genes from B. cinerea were transiently over-expressed also in Nicotiana benthamiana by using the Agrobacterium-infiltration technique. Transgene expression was confirmed by Northern blot analysis and EPG-related symptoms were observed five to eight days post-infiltration. Differential symptoms appeared with the various EPGs, providing some evidence that the symptoms were not random events due to the infiltration or a hypersensitive response. Moreover, the symptoms observed for EPG2 was similar to those that were reported recently by another group on the same host. In spite of the expression data and the clear symptoms that developed, active preparations, as measured with the agarose diffusion plate asay, could only be obtained for EPG2 again. In our search for a possible in vivo method to detect and quantify EPG activity and inhibition by PGIPs, we tested and evaluated a technique based on chlorophyll fluorescence to detect the effect of EPGs on the rate of photosynthesis. Our results showed that the over-expression of these genes reduced the rate of electrons flowing through photosystem II, indicating metabolic stress occurring in the plant. We used the same technique to evaluate possible interaction between VvPGIP1 respectively with BcPG1 and 2 and found that the co-expressing of the Vvpgip1 gene caused protection of the infiltrated tissue, indicating inhibition of EPG1 and 2 by VvPGIP1. For EPG2, the observed interaction and possible inhibition by VvPGIP1 is the first report to our knowledge of an interaction between this specific EPG2 and a PGIP. Moreover, to further elucidate the in planta interaction between VvPGIP1 and the EPGs from the South African B. cinerea strain, we tested for possible interactions by making use of a plant two-hybrid fusion assay, but the results are inconclusive at this stage. Previous studies in our laboratory have shown that several natural mutations exist between PGIP encoding genes from different V. vinifera cultivars. Based on this finding and the fact that these natural mutations could result in changes with regard to EPG inhibition and ultimately disease susceptibility, we isolated an additional 37 PGIP encoding genes from various grapevine genotypes, some of which are known for their resistance to pathogens. Combined, these results make a valuable contribution to understand plant pathogen interactions, specifically in this case by modeling the interactions of pathogen and plant derived proteins. The possibility to use in vivo methods such as chlorophyll fluorescence to follow these interactions on an in planta level, provides exciting possibilities to strenghten and contextualize in vitro results. / AFRIKAANSE OPSOMMING: Plantpatogene organismes veroorsaak jaarliks erge skade aan landbougewasse en word dus as ’n ernstige probleem in die landbousektor beskou. Diepgaande studies wat handel oor plantpatogene en hul metodes van infeksie het dit vir molekulêre bioloë moontlik gemaak om patogene nou ook op molekulêre vlak verder te bestudeer. Botrytis cinerea is baie effektief as modelsisteem gebruik om verskeie aspekte van patogenese verder te bestudeer, ook op ‘n molekulêre vlak. Molekulêre navorsing op B. cinerea, het getoon dat die endopoligalakturonases (EPGs) van dié swam kernrolbelangrik in patogenese is. Hierdie sesledige hidrolitiese ensiemfamilie word gekodeer deur die Bcpg1-6 gene en is belangrik vir die afbraak van die komplekse selwandpolimere van plantgashere, om suksesvolle gasheerpenetrasie te veroorsaak. Daar is aangetoon dat beide BcPG1 en 2 essensieël vir virulensie van die patogeen is. ’n Leusienryke-herhalings inhibitorproteïen wat in die selwand van verskeie plantspesies voorkom, die poligalakturonase-inhiberende proteïen (PGIP), het interaksie met en inhibeer EPGs en gevolglik ook die nekrotiserende aksies van B. cinerea. Uit die literatuur is dit duidelik dat spesifieke inligting aangaande individuele interaksies van fungiese EPGs met PGIPs tans nog ontbreek. Verder word daar op in vitro metodologie staatgemaak wannneer die effekte van EPGs asook die interaksie en inhibisie met PGIPs bestudeer word, sonder om die konteks van die in vivo- of in planta-omgewing in ag te neem. Die fokus van hierdie studie was om aspekte van individuele EPG:PGIP interaksies, asook die moontlike gebruik van in vivo metodologie te bestudeer deur EPGs, afkomstig van ’n hoogs virulente Suid-Afrikaanse ras van B. cinerea en die wingerd VvPGIP1, wat vroeër in ons laboratorium geïsoleer is, te gebrruik. Hierdie PGIP wat uit Vitis vinifera cv Pinotage geïsoleer is, inhibeer ’n kru EPG-ekstrak van bogenoemde ras baie effektief. Die benadering wat gevolg is het op die ooruitdrukking van die individuele Bcpg-gene in heteroloë sisteme staatgemaak en die gevolglike isolering van suiwer en aktiewe ensieme om EPG-inhibisie deur VvPGIP1 te beoordeel. Al die gene is suksesvol in Saccharomyces cerevisiae ooruitgedruk onder ’n sterk induseerbare promotor, maar volgens ’n agarose-diffundeerbare toets kon aktiewe ensiempreparate slegs vir die enkoderende Bcpg2 verkry word. Die in vitro PGIP-inhibisie toets is ook op die gemelde toets gebasseer en vereis EPG-aktiwiteit om die inhiberende effek van die PGIP, te visualiseer. Die aktiewe EPG2 is egter nie deur VvPGIP1 geïnhibeer met die aanleg van hierdie toets nie. Die EPG-enkoderende gene van B. cinerea is ook tydelik in Nicotiana benthamiana ooruitgedruk deur gebruik te maak van ’n Agrobacterium-infiltrasietegniek. Transgeenuitdrukking kon met die Noordelike kladtegniek bevestig word en EPG-verwante simptome is vyf tot agt dae na infiltrasie waargeneem. Verskillende simptome vir die verskillende EPGs is waargeneem, wat aanduidend is dat die simptome nie lukrake gevolge van die infiltrasies, of ’n hipersensitiewe respons is nie. Verder kon die simptome wat EPG2 vertoon het, gekorreleer word met dié wat onlangs deur ’n ander groep op dieselfde gasheer waargeneem is. Ten spyte van die ekspressiedata en die waargenome simptome, kon aktiewe ensiempreparate op die agarose-diffundeerbare toets, weereens slegs vir EPG2 waargeneem word. ’n Metode wat gebasseer is op chlorofilfluoressensie is getoets en geëvalueer as ’n moontlike in vivo metode om EPG aktiwiteit en inhibisie deur PGIPs waar te neem en te kwantifiseer. Die resultate het bevestig dat die ooruitdrukking van hierdie gene die elektronvloeitempo deur fotosisteem II verminder het wat ’n aanduiding is dat metaboliese stres in die plant heers. Dieselfde tegniek is gebruik om die moontlike interaksies tussen BcPG1 en 2 en VvPGIP1 te bestudeer en het aangetoon dat die mede-uitdrukking van die Vvpgip1-geen aanleiding gee tot ’n beskermende effek van die geinfiltreerde weefsel, wat aanduidend is van inhibisie van EPG1 en 2 deur VvPGIP1. In die geval van EPG2 is hierdie interaksie en moontlike inhibisie met ’n PGIP die eerste waarneming in die verband. In ’n verdere poging om die in planta-interaksie tussen VvPGIP1 en die EPGs van die Suid-Afrikaanse B. cinerea ras uit te klaar, is ’n plantgebasseerde twee-hibriede toets aangelê, maar geen klinkklare resultate kon verkry word nie. Vorige werk het bevestig dat verskeie natuurlike mutasies in PGIP-enkoderende gene, afkomstig van verskillende V. vinifera kultivars, voorkom. Hierdie resultaat en die feit dat hierdie mutasies verskille in EPG inhibisie en uiteindelik vatbaarheid vir siektes kan beïnvloed, het aanleiding gegee tot die isolering van ’n verdere 37 PGIP-enkoderende gene uit ‘n verskeidenheid druifplantgenotipes, sommige waarvan juis bekend vir hul weerstand teen patogene is. Die gekombineerde resultate wat in dié studie verkry is, maak ’n waardevolle bydrae tot die verstaan van plant-patogeeninteraksies, spesifiek met die modelering van interaksies van patogeen- en plantgebasseerde proteïene. Die moontlikheid om in vivo-metodes soos chlorofilfluoressensie te gebruik in in planta-analises, is besonder bemoedigend om in vitro-resultate te versterk en ook in konteks te plaas.

Understanding the complex metabolic networks present in organisms, through the use of high throughput liquid chromatography coupled mass spectrometry, will give insight into the physiological changes responding to stress. However the lack of a proper work flow and robust methodology hinders verifiable biological interpretation of mass profiling data. In this study a novel workflow has been developed. A novel Kernel based feature alignment algorithm, which outperformed Agilent’s Mass profiler and showed roughly a 20% increase in alignment accuracy, is presented for the alignment of mass profiling data. Prior to statistical analysis post processing of data is carried out in two stages, noise filtering is applied to consensus features which were aligned at a 50% or higher rate. Followed by missing value imputation a method was developed that outperforms both at model recovery and false positive detection. The use of parametric methods for statistical analysis is inefficient and produces a large number of false positives. In order to tackle this three non-parametric methods were considered. The histogram method for statistical analysis was found to yield the lowest false positive rate. Data is presented which was analysed using these methods to reveal metabolomic changes during plant pathogenesis. A high resolution time series dataset was produced to explore the infection of Arabidopsis thaliana by the (hemi) biotroph Pseudomonas syringe pv tomato DC3000 and its disarmed mutant DC3000hrpA, which is incapable of causing infection. Approximately 2000 features were found to be significant through the time series. It was also found that by 4h the plants basal defence mechanism caused the significant ‘up-regulation’ of roughly 400 features, of which 240 were found to be at a 4-fold change. The identification of these features role in pathogenesis is supported by the fact that of those features found to discriminate between treatments a number of pathways were identified which have previously been documented to be active due to pathogenesis

As plantas se defendem do ataque de patógenos através de um sistema imune composto por duas fases. A primeira delas é mediada por receptores localizados na membrana celular ou intracelularmente, os quais são conhecidos como receptores de reconhecimento padrão (do inglês, pattern recognition receptors - PRR). Esses receptores reconhecem moléculas derivadas de microrganismos, as quais são conservadas evolutivamente e são chamadas de padrões moleculares associados a patógenos (do inglês, pathogen-associated molecular patterns - PAMPs). Esse reconhecimento dispara uma resposta de defesa conhecida como PTI (do inglês, PAMP-triggerd immunity - PTI). Alguns patógenos foram aptos a sintetizar moléculas capazes de suprimir a PTI e essas moléculas são denominadas de efetores. A resposta que ocorre devido à ação dos efetores é chamada de susceptibilidade disparada por efetores (do inglês, effector-triggered susceptibility - ETS). Entretanto, plantas resistentes podem reconhecer os efetores através de proteínas de resistência localizadas intracelularmente, ativando a imunidade disparada por efetores (do inglês, effector-triggeredimmunity - ETI). De modo geral, as respostas advindas da PTI e da ETI são similares, mas a segunda é ativada mais rapidamente e é mediada por um único gene de resistência R. Por essa razão, a ETI é conhecida como uma resposta à doença qualitativa e as plantas não desenvolvem sintomas, caracterizando a interação incompatível. Por outro lado, a PTI é mediada por diversos genes e as respostas de defesa são tardias, possibilitando a disseminação do patógeno pelas células da planta e a ocorrência da doença, o que caracteriza a interação compatível. Nas respostas de defesa, moléculas como o óxido nítrico, as poliaminas e o ácido salicílico participam do processo de sinalização. O sistema antioxidante da planta é ativado de modo a mitigar os efeitos das espécies reativas de oxigênio e o metabolismo da planta é alterado. Dessa maneira, o estudo das respostas de defesa contra patógenos, pode ser uma ferramenta útil para estabelecer controles efetivos para as doenças de plantas / Plants defend themselves from pathogen attack through an active immunity system composed by two phases. The first is mediated by cell surface and intracellular pattern recognition receptors (PRR), which recognizes conserved molecules derived from microbes known as pathogen-associated molecular patterns (PAMPs). This recognition triggers a defense response called PAMP-triggered immunity (PTI). Throughout evolution, pathogens were able to synthesize molecules capable of suppressing PTI. These molecules are named effectors and they are responsible for effector-triggered susceptibility (ETS). However, resistant plants can recognize effectors by intracellular resistance (R) proteins, initiating effector-triggered immunity (ETI). In general, responses derived from PTI and ETI are the same, but the latter is activated faster and is mediated by a single R gene. For this reason, ETI-response is also known as qualitative disease response (QDR) and plants do not develop disease symptoms, characterizing the incompatible interaction. On the other hand, PTI is mediated by several genes and the defense response is delayed, enabling the pathogen to spread out and to cause disease. This interaction is known as compatible. In defense responses, molecules like nitric oxide, polyamines and salicylic acid can participate in signaling process. The antioxidant system can be activated to quench the ROS effects and the plant metabolism is altered. In this sense, studying defense responses against pathogens can help to develop tools to establish effective control methods for plant disease

Xylella fastidiosa é o agente causal de uma série de doenças que ocorrem em plantas economicamente importantes como laranjeiras, videiras e cafeeiros, causando no Estado de São Paulo prejuízos relevantes à indústria citrícola. Esta bactéria Gram-negativa é restrita ao xilema das plantas e à porção anterior do trato digestório dos insetos vetores das famílias Cicadellidae e Cercopidae, conhecidos como cigarrinhas. Tentativas de elucidar os mecanismos de virulência e patogenicidade adotados por esta bactéria apontam a formação do biofilme como etapa fundamental para o estabelecimento da infecção e o consequente desenvolvimento da doença na planta, mas fatores adicionais parecem contribuir, tais como a produção de toxinas. As bacteriocinas são proteínas com atividade antibiótica contra cepas próximas à espécie produtora e que já foram associadas à virulência e patogenicidade de outras bactérias. Uma varredura in silico no genoma de X. fastidiosa 9a5c revelou 13 sequências codificadoras de microcinas putativas no cromossomo. Transcritos de todos esses genes foram detectados por RT-qPCR em culturas de X. fastidiosa 9a5c, e análises comparativas com genomas públicos (cepas Temecula1, Dixon, Ann-1, M23, M12, EB92-1 e GB514) e recém sequenciados por nosso grupo de pesquisa (cepas U24d, J1a12, 3124, Hib4, Pr8x e Fb7) revelaram que cada cepa possui seu próprio arsenal de bacteriocinas. Diferenças encontradas in silico entre os loci de bacteriocinas nas cepas foram demonstradas experimentalmente. Nossos resultados comprovam a variabilidade predita nos quatro clusters de bacteriocinas que identificamos, o que é esperado para genes relacionados à adaptação e patogenicidade. Destes loci, três foram detectados por RT-PCR como transcritos policistrônicos. Nossa tentativa de detectar essas proteínas em culturas de X. fastidiosa (através de sequenciamento de polipeptídeos por HPLC-MS/MS) foi capaz de identificar uma das bacteriocinas putativas e, portanto, o conjunto de nossas observações apóia a continuidade dos estudos para elucidar o papel das bacteriocinas na fisiopatologia de X. fastidiosa. / Xylella fastidiosa is the causal agent of diseases that affect several economically important crops such as sweet orange trees, grapevines and coffee trees, causing in the State of São Paulo considerable losses mainly to the citrus industry. This Gram-negative bacterium is restricted to the plant xylem and to the upper gastrointestinal tract of its insect vectors, the sharpshooters from the Cicadellidae and Cercopidae families. Attempts to elucidate the virulence and pathogenicity pathways employed by this bacterium point the biofilm formation as a fundamental step for the establishment of the infection and the consequent development of the plant disease, but additional factors seem to contribute to these processes, such as the production of toxins. Bacteriocins are proteinaceous antibiotics that act against closely-related species and have been previously associated with virulence and pathogenicity in other bacteria. An in silico screening of the X. fastidiosa 9a5c genome revealed 13 coding sequences as putative microcins in the chromosome. Transcripts from all those genes were detected through RT-qPCR in X. fastidiosa 9a5c cultures, and comparative analyses on the public genomes (Temecula1, Dixon, Ann-1, M23, M12, EB92-1 and GB514) plus the ones recently sequenced by our group (U24d, J1a12, 3124, Hib4, Pr8x and Fb7) revealed that each strain possesses its own arsenal of bacteriocins. Differences found in silico among the loci in all strains were experimentally confirmed. Our results demonstrated the predicted variability in the four bacteriocins clusters as expected for adaptation and pathogenicity-related genes. Three out of the four bacteriocins loci were detected by RT-PCR as polycistronic transcripts. Our attempt to detect these proteins in X. fastidiosa cultures (using HPLC-MS/MS polypeptide sequencing) identified one of the putative bacteriocins, and therefore our observations warrant further efforts to elucidate the role of bacteriocins in the X. fastidiosa physiopathology.

The dynamics of a plant-pathogen interaction vary both within and among species. Both spatial structuring and specific genetic and life-history characteristics will affect the interaction and the outcome of a potential co-evolution between the two organisms. In this thesis I have studied the interaction between the wild perennial herb Silene dioica and its automictic, obligate anther smut Microbotryum violaceum MvSd. From the plant perspective, I have examined different aspects of biochemical resistance in S. dioica to M. violaceum MvSd. From the pathogen perspective, I have focused on the breeding system of M. violaceum MvSd and its connection to fitness and distribution of genetic diversity. I have used varying methods; glasshouse trails involving inoculation of plants with the pathogen, classical Mendelian analysis involving controlled crosses between plants, microscopic studies of spores and molecular DNA-analysis. With the results I demonstrate that resistance to M. violaceum MvSd in S. dioica can be specific to the attacking pathogen strain and also spatially highly diverse both within and among populations within a metapopulation. Together, these factors are likely to delay the establishment of the disease within host populations and reduce the spread and amount of disease, once it has been established. The results also suggest that the specific resistance expressed against two different M. violaceum MvSd strains were determined by separate gene systems and that, in both cases, the resistance was simply inherited. This implies a potential for relatively rapid response to M. violaceum-induced selection in S. dioica populations variable for resistance. My results also show that automixis clearly is the predominating breeding system of M. violaceum MvSd, similarly to what earlier has been shown for M. violaceum MvSl. Furthermore, I found lower levels of neutral genetic diversity in M. violaceum MvSd in the northern parts of Sweden, compared to what has been found in populations in more southern Europe. This result is consistent with predictions that populations in the outer regions of a species distribution have lower levels of genetic variation. Moreover, populations were highly differentiated in northern Sweden, which could have been generated by high selfing rates, genetic drift and high population turnover rates, all factors that coincide with life-history and ecology of M. violaceum MvSd. However, despite the general low variability in neutral genetic markers, I did find variation in fitness related traits, both within and among populations, as well as differences in infection ability between strains, suggesting there is a potential for co-evolution between S. dioica and M. violaceum MvSd in the area. To summarize, this thesis reflect a plant-pathogen system that is highly influenced by constant colonisation-extinction dynamics, which is likely to have influenced both the genetics of resistance in the plant and the breeding system of the pathogen and thus also the interaction between the two organisms.

Plant programmed cell death (PCD) is an essential process in plant-pathogen interactions. Importantly, PCD can have contrasting effects on the outcome depending on context. For example, plant PCD in plant-biotroph interactions is clearly beneficial to plants, whereas it could be detrimental to plants in plant-necrotroph interactions. Sclerotinia sclerotiorum is an agriculturally and economically important necrotrophic pathogen. Previous studies have shown that S. sclerotiorum secretes oxalic acid (OA) to enhance Sclerotinia virulence by various mechanisms including induction of PCD in plants. A recent study has also shown that reactive oxygen species (ROS) generation correlates with induction of PCD during disease development. These studies focus on links between ROS, oxalate, and PCD, and how they impact S. sclerotiorum disease development. I examined the involvement of ROS in pathogenic development of S. sclerotiorum. I identified and functionally characterized two predicted S. sclerotiorum NADPH oxidases (Nox1 and Nox2) by RNAi. Both nox genes appear to have roles in sclerotial development, while only Nox1-silenced mutants showed reduced virulence. Interestingly, the reduced virulence of the Nox1-silenced mutant correlated with decreased production of OA in the mutant. This observation suggests that regulation of ROS by S. sclerotiorum Nox1 may be linked to OA. The next study details the phenotype of plants inoculated with an S. sclerotiorum oxalate deficient mutant (A2), which showed restricted growth at the infected site. This response resembles the hypersensitive response (HR), and is associated with plant resistance responses including cell wall strengthening, plant oxidative burst, and induction of defensin genes. Conversely, leaves infected with wild type showed unrestricted spreading of cell death and were not associated with these resistant responses. Furthermore, previous work had shown that a Caenorhabditis elegans anti-apoptotic gene (ced-9) conferred resistance to wild type S. sclerotiorum, while this gene had negligible effects on the phenotype of plant leaves inoculated with A2 mutants. These findings suggest that HR-like cell death by A2 and PCD by wild type S. sclerotiorum may be regulated by different pathways. As a whole, these results reveal the importance of ROS, oxalate, and PCD in Sclerotinia disease development as well as the significance of interplay between them. These studies contribute to the understanding of the underlying mechanisms of Sclerotinia disease.