Coniothyrium zuluense is the causal agent of a serious Eucalyptus stem canker disease in South Africa (Wingfield et al., 1997). Eucalypts are the most important hardwood plantations in the world, and in South Africa these hardwoods occupy approximately 1.5 million hectares of plantation area, an area that is soon to be increased by an additional 600 000 hectares. As exotics, Eucalyptus plantations are constantly exposed to infection by fungal pathogens such as C. zuluense, which by secreting cell-¬wall degrading enzymes contribute to the degradation of plant cell walls and subsequent reduction and in the quality of timber produced. This ultimately affects the South African paper, pulp and timber industries. Selection of resistant clones through traditional breeding methods is the most common method currently employed in overcoming the problem of fungal infection. The genetic manipulation of Eucalyptus trees for enhanced resistance to fungal diseases is an alternative to the time-consuming and tedious approach of conventional breeding. The identification of several antifungal proteins, particularly polygalacturonase-inhibiting proteins (PGIPs) from various plant species including Eucalyptus, lead to the hypothesis that over-expression of these proteins could potentially reduce pathogen attack. However, prior to the expression of PGIPs in plants, isolation and molecular characterization of these genes are required. The aims of this study were therefore (l) to clone and characterize the complete Eucalyptus grandis pgip gene, (2) to transform Nicotiana tabacum (tobacco) plants with the E. grandis pgip gene and (3) to test for inhibition of C. zuluense PGs by PGIPs extracted from transgenic tobacco plants. This forms the first step towards the generation of E. grandis clones that are more disease tolerant. A review of the role of fungal endopolygalacturonases and polygalacturonase¬inhibitors in plant-pathogen interactions are presented in chapter I. Strategies employed to isolate and characterize pgip genes from a range of plant species are highlighted and the importance ofPGIPs in disease resistance is discussed. In chapter 2, the molecular cloning and characterization of the E. grandis pgip gene is discussed. The work presented in this chapter is a follow up on work previously conducted by Chimwamurombe (2001). Previously, a partial Eucalyptus pgip gene sequence was obtained with the use of degenerate oligonucleotide primers. In this study, the complete Eucalyptus pgip gene was obtained through the employment of genome walking strategies. Transformation of Nicotiana tabacum cv LA Burley plants with the Eucalyptus pgip gene and the molecular characterization of transgenic tobacco plants is discussed in chapter 3. The transformation and expression of foreign genes in tobacco plants is a well-established protocol, making tobacco the most appropriate candidate plant for assessing the functionality of the plant transformation construct. The production of endopolygalacturonases from virulent C. zuluense isolates and the subsequent PGIP assays conducted to determine levels of PG inhibition are included in this chapter. This thesis consists of three independent chapters representing studies on the molecular characterization of an E. grandis pgip gene and focusing on the potential for inhibition of PGs produced by C. zuluense by Eucalyptus PGIP extracted from transgenic tobacco plants. Repetition of certain aspects in the individual chapters has been unavoidable and the thesis is presented following a uniform style. / Dissertation (MSc)--University of Pretoria, 2003. / Genetics / Unrestricted

Sclerotinia sclerotiorum is a pathogenic fungus that infects around 400 species of host    plants. Stem rot disease caused by this fungus is economically disastrous for Brassica napus cultivators in Sweden. Due to the lack of disease resistant cultivars, disease management has been solely dependent on fungicide application. The current disease  prediction models are not scientifically accurate and take into account factors such as   weather, previous disease incidence, and conomic effects which often result in unnecessary and excessive use of fungicides by cultivators. Real-Time Polymerase Chain Reaction has proven to be the fastest, most accurate and reliable technique for detecting plant pathogens as it gives an idea about disease severity by measuring pathogen concentration in environmental samples. Reproducible and able qPCR assays have the potential of being the main principle on which more scientifically accurate plant disease prediction and management models an be developed. The aim of this study was to validate a previously established qPCR assay to detect S. sclerotiorum. An absolute quantification experiment     was performed by using plasmid DNA cloned with a target gene as template. Further,   three different qPCR machines  were compared  to make a plausible conclusion regarding    their sensitivity and efficiency in detecting minuscule amounts of DNA from the   environment. While a solid conclusion could not be reached regarding the sensitivity of    each of these machines, this study pointed out some basic trends about each machine    that may help researchers in selecting the most efficient qPCR system when working with detection of plant pathogens.

The production of strawberries can be severely limited by soilborne plant pathogens, insects and weeds. Macrophomina phaseolina is a problematic soilborne fungal pathogen in California strawberry production inciting the disease Macrophomina crown rot. When established, the pathogen can cause extensive plant decline and mortality. Host resistance will be a critical tool for managing this disease and guiding breeding programs in the post methyl bromide era. Evaluation of host resistance in strawberry germplasm to M. phaseolina was evaluated through phenotypic assessments of disease incidence. A total of 90 strawberry cultivars and elite selections were included in a replicated field trial conducted in artificially inoculated soils to assess host resistance. Significant differences in levels of resistance and susceptibility were observed among genotypes tested in this trial. The five most resistant strawberry genotypes from highest to lowest in percent plant mortality were: UC-R, UC-G, UC-V, Manresa, and Osceola. The five most susceptible strawberry genotypes with the highest percent mortality in ranking order from highest to lowest were: UC-J, Ruby June, Festival, UC-Y, and UC-A. Of the genotypes tested in this trial UC-V, Manresa and Osceola could be characterized as highly resistant, but no complete resistance was observed. An additional study was conducted to correlate host symptom expression with the extent of pathogen colonization in different strawberry tissues, and to determine if resistant germplasm can contribute to secondary inoculum production in the field. An established qPCR method was utilized to quantify M. phaseolina colonization of strawberry tissues. There were significant effects for cultivar (P < 0.0001) as well as a significant two-way interaction of cultivar x sample time (P= 0.0083) on the concentration of M. phaseolina DNA detected in strawberry tissues. Expression of the resistant phenotype in strawberry cultivars was associated with limited plant colonization by M. phaseolina. The extent of colonization of a specific cultivar by M. phaseolina was dependent on the sample time after inoculation with the pathogen. In addition, the roots and crowns of a specific strawberry cultivar were equally colonized on a per plant tissue weight basis, but this provides only speculation into the mechanisms conferring host resistance. A third study was conducted to integrate host resistance of strawberry genotypes with the use of organic amendments in effort to mutually enhance the efficacy of each factor for the control of Macrophomina crown rot. Artificially inoculated potting substrate was amended with Brassica juncea mustard seed meal at a rate of 4.94 tons ha-1(MSM), and anaerobic soil disinfestation utilizing rice bran at a rate of 22.24 tons ha-1 (ASD) were compared to a non-amended (UTC) and steam controls. The soil assay indicated that the ASD and steam treatments were able to reduce the CFU g-1 potting substrate of M. phaseolina by 99.7-100%. In addition, there were significant effects of soil treatment on the fresh biomass of weed seedlings recovered from the potting substrate. However, disease severity and host colonization of multiple strawberry cultivars by M. phaseolina was not reduced when grown in the treated potting substrate. The effect of strawberry cultivar on the extent of pathogen colonization was highly significant (P < 0.0001), in which cultivars characterized as resistant from phenotypic screenings possessed lower concentrations of M. phaseolina DNA. The suppression of M. phaseolina in response to organic amendments was limited but this study supports findings from previous experiments that genotype specific host resistance minimizes host colonization and reduces the production of secondary inoculum.

La distribution des sucres est un processus clé dans le développement de la plante et cours des interactions plantes/microorganismes.Une recherche des acteurs moléculaires impliqués dans la répartition des ressources carbonées au cours de l'interaction avec le champignon nécrotrophe B. cinerea a été réalisée. Plusieurs familles de transporteurs de sucres et d'invertases ont été ciblées, permettant d'établir une cartographie des gènes régulés transcriptionnellement lors de l'interaction. Le rôle de certains gènes candidats a été étudié par une approche de génomique fonctionnelle afin de mettre en évidence une fonction biologique de l'allocation du carbone dans la résistance de la plante aux champignons nécrotrophes. Un système d'interaction simplifié, basé sur un dialogue moléculaire sans contact physique entre une culture cellulaire d'A. thaliana et B. cinerea, a été développé. Il a permis de mesurer les flux de sucres ainsi que les activités enzymatiques et métaboliques pour chaque partenaire. Nos résultats montrent que B. cinerea entraine une forte augmentation de l'activité invertasique pariétale dans les tissus infectés, indiquant qu'une transition source/puits a lieu. Plusieurs transporteurs de sucres sont différentiellement exprimés, certains d'entre eux modulant le devenir de l'interaction. L'activité d'absorption d'hexoses et le métabolisme primaire des cellules hôtes sont fortement stimulés, démontrant l'importance de la compétition pour les sucres à l'interface plante/agent pathogène. En conclusion, l'absorption des sucres alimente le métabolisme énergétique des cellules hôtes et participe aux mécanismes de défense de la plante. / During plant development and upon pathogen infection, sugar allocation is a key process in plant physiology. Cell wall invertases and sugar transporters, involved in the sink strength, likely play a major role in the metabolic plant response. Molecular actors involved in carbohydrates allocation upon B. cinerea interaction have been identified using a transcriptional approach. Some gene families of sugar transporters and invertases have been targeted, allowing the establishment of a cartography of genes regulated during the interaction. To understand the biological role of carbon allocation during the interaction between plants and necrotrophic fungi, candidate genes have been studied using a functional genomics approach.A simplified interaction system has been developped, allowing a molecular dialogue between Arabidopsis and B. cinerea cells, without any physical contact. This system enables the monitoring of radiolabelled sugar uptake rates and some enzymatic and metabolomic activities for both the host cells and the pathogen, independently.Globally, our results demonstrate that B. cinerea infection leads to the transition from a source to a sink tissue, with a strong increase in cell wall invertase activity. The expression of some sugar transporter genes is also affected, while some of them (AtSTP1 and 13) are involved in the disease development. Besides the increase in hexose uptake activity, primary metabolism is deeply affected, highlighting the competition for apoplastic sugars that takes place at the plant/pathogen interface. Sugar retrieval appears to be a key process, fuelling host cells with energy and signal molecules, contributing to the plant defense mechanisms.

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Dissertation (PhD)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Plants have developed sophisticated means of combating plant diseases. The events that prepare the plant for, and follow plant-pathogenic interactions, are extremely complex and have been the topic of intensive investigation in recent years. These interactions involve a plethora of genes and proteins, and intricate regulation thereof; from the host and pathogen alike. Studying the contribution of single genes and their encoded proteins to the molecular dialogue between plant and pathogen has been a focus of plant molecular biologists. To this end, a gene encoding a polygalacturonase-inhibiting protein (PGIP) was recently cloned from Vitis vinifera. These proteins have the ability to inhibit fungal endopolygalacturonases (ePGs), enzymes which have been shown to be required for the full virulence of several fungi on their respective plant hosts. The activity of PGIP in inhibiting fungal macerating enzymes is particularly attractive for the improvement of disease tolerance of crop species. The VvPGIP-encoding gene was subsequently transferred to Nicotiana tabacum for high-level expression of VvPGIP. These transgenic plants were found to be less susceptible to infection by Botrytis cinerea in an initial detached leaf assay. Also, it was shown that ePG inhibition by protein extracts from these lines correlated to the observed decrease in susceptibility to B. cinerea. This study expands on previous findings by corroborating the antifungal nature of the introduced PGIP by whole-plant, timecourse infection assays. Six transgenic tobacco lines and an untransformed wildtype (WT) were infected and the lesions measured daily from day three to seven, and again at day 15. The transgenic lines exhibited smaller lesions sizes from three to seven days post-inoculation, although these differences only became statistically significant following seven days of incubation. At this point, four of the six lines exhibited significantly smaller lesions than the WT, with reductions in disease susceptibility ranging between 46 and 69% as compared to the WT. Two of the lines exhibited disease susceptibility comparable to the WT. In these resistant plant lines, a correlation could be drawn between Vvpgip1 expression, PGIP activity and ePG inhibition. These lines were therefore considered to be PGIP-specific resistant lines, and provided ideal resources to further study the possible in planta roles of PGIP in plant defense. The current hypothesis regarding the role(s) of PGIP in plant defense is twofold. Firstly, PGIPs have the ability to specifically and effectively inhibit fungal ePGs. This direct inhibition results in reduced fungal pathogenicity. Alternatively, unhindered action of these enzymes results in maceration of plant tissue and ultimately, tissue necrosis. Subsequently, it could be shown that, in vitro, the inhibition of ePGs prolongs the existence of oligogalacturonides, molecules with the ability to activate plant defense responses. Thus, PGIPs limit tissue damage by inhibition of ePG; this inhibition results in activation of plant defense responses aimed at limiting pathogen ingress. Several publications reported reduced susceptibility to Botrytis in transgenic plant lines overexpressing PGIP-encoding genes. However, none of these publications could expand on the current hypotheses regarding the possible in planta roles of PGIP in plant defense. In this study we used transgenic tobacco lines overexpressing Vvpgip1 as resources to study the in planta roles for PGIP. Transcriptomic and hormonal analyses were performed on these lines and a WT line, both before and following inoculation with Botrytis cinerea. Transcriptomic analysis was performed on uninfected as well as infected tobacco leaf material utilizing a Solanum tuberosum microarray. From the analysis with healthy, uninfected plant material, it became clear that genes involved in cell wall metabolism were differentially expressed between the transgenic lines and the WT. Under these conditions, it could be shown and confirmed that the gene encoding tobacco xyloglucan endotransglycosylase (XET/XTH) was downregulated in the transgenic lines. Additionally, genes involved in the lignin biosynthetic pathway were affected in the individual transgenic lines. Biochemical evidence corroborated the indication of increased lignin deposition in their cell walls. Additionally, phytohormone profiling revealed an increased indole-acetic acid content in the transgenic lines. These results show that constitutive levels of PGIP may affect cell wall metabolism in the Vvpgip1-transgenic lines which may have a positive impact on the observed reduced susceptibilities of these plants. An additional role for PGIP in the contribution to plant defenses is therefore proposed. PGIP may directly influence defense responses in the plant leading to the strengthening of cell walls. This might occur by virtue of its structural features or its integration in the cell wall. These reinforced cell walls are thus “primed” before pathogen ingress and contribute to the decrease in disease susceptibility observed in lines accumulating high levels of PGIP. Transcriptional and hormonal analyses, at the localized response, were performed on Botrytis-infected leaf tissue of the transgenic lines and a WT line. Several Botrytis responsive genes were found to be upregulated in both the WT and the transgenic lines. Although limited differential expression was observed between the two genotypes, the analyses identified a gene which was upregulated two-fold in the transgenic lines, as compared to WT. This was confirmed by quantitative Real-Time PCR. This gene is involved in the lipoxygenase pathway, specifically the 9-LOX branch, leading to the synthesis of the divinyl ether oxylipins colneleic and colnelenic acid, which show inhibitory effects on Botrytis spore germination. Phytohormone profiling revealed that the transgenic lines accumulated more of the defense-related hormone pool of jasmonates. These are formed via the 13-LOX pathway and have been shown to be important for the restriction of Botrytis growth at the site of infection. Collectively, the results from the infection analyses indicate that in these transgenic lines, both branches of the lipoxygenase pathway are differentially induced at the level of the localized response to Botrytis infection. Similarly, an increased induction of the synthesis of the defense-related hormone salicylic acid could be observed, although this hormone did not accumulate to significantly higher levels. These results are the first report of differential induction of a defense-related pathway in pgip-overexpressing lines and substantiate the proposal that following ePG inhibition by PGIP, signaling which activates plant defense responses, takes place. Taken together, these results significantly contribute to our understanding of the in planta role of PGIP in plant defense responses. / AFRIKAANSE OPSOMMING: Plante het deur evolusie gesofistikeerde meganismes teen die aanslag van plantsiektes ontwikkel. Die gebeure wat die plant voorberei, asook dié wat op plant-patogeen interaksies volg, is uiters kompleks en vorm die kern van verskeie navorsingstemas die afgelope paar jaar. Etlike plant- én patogeengene en proteïene is by hierdie interaksies betrokke en aan komplekse reguleringsprosesse onderworpe. Die bestudering van die bydrae van enkelgene en hul gekodeerde proteïene tot die molekulêre interaksie tussen ‘n plant en patogeen is ‘n sterk fokus van plant-molekulêre bioloë. Met hierdie doel as fokus, is ‘n geen wat vir ‘n poligalakturonaseinhiberende proteïen (PGIP) kodeer, van Vitis vinifera gekloneer. Hierdie proteïene beskik oor die vermoë om fungiese endopoligalakturonases (ePG's), ensieme wat benodig word vir die virulensie van verskeie fungi op hul gasheerplante, te inhibeer. Die inhibisie van ePG's deur PGIP en die gepaardgaande verminderde weefseldegradasie is ‘n baie belowende strategie vir die verbetering van verboude gewasse se patogeentoleransie. Die VvPGIPenkoderende geen is gevolglik na Nicotiana tabacum oorgedra vir hoëvlakuitdrukking van VvPGIP. Daar is gevind dat hierdie transgeniese plante minder vatbaar vir Botrytis cinerea-infeksies was in ‘n inisiële antifungiese toets wat gebruik gemaak het van blaarweefsel wat van die moederplant verwyder is. Daar is ook ‘n korrelasie gevind tussen B. cinerea-siekteweerstand en ePG-inhibisie deur proteïenekstrakte van die transgeniese populasie. Die huidige studie bou voort op en bevestig vorige bevindinge betreffende die antfungiese aard van die heteroloë PGIP in die heelplant en oor tyd. Ses transgeniese tabaklyne en 'n ongetransformeerde wilde-tipe (WT) is geïnfekteer en die lesies is vanaf dag drie tot sewe, en weer op dag 15, gemeet. Die transgeniese lyne het in die tydperk van drie tot sewe dae ná-inokulasie kleiner lesies as die WT getoon, alhoewel hierdie verskille slegs statisties beduidend geword het na sewe dae van inkubasie. Op daardie tydstip het vier van die ses lyne aansienlik kleiner lesies as die WT getoon, en verlagings in siektevatbaarheid het, in vergelyking met die WT, van 46% tot 69% gewissel. Twee van die lyne het siektevatbaarheid getoon wat vergelykbaar was met dié van die WT. In die siekteweerstandbiedende plantlyne was daar 'n verband tussen Vvpgip1-ekspressie, PGIP-aktiwiteit en ePG-inhibisie. Hierdie plantlyne is dus as PGIP-spesifieke siekteweerstandslyne beskou en dien dus as ideale eksperimentele bronne vir die ontleding van die moontlike in plantafunksies van PGIP in plantsiekteweerstandbiedendheid. Die huidige hipotese betreffende die funksie(s) van PGIP in plantsiekteweerstand is tweeledig. Eerstens het PGIP die vermoë om fungusePG's spesifiek en doeltreffend te inhibeer. Hierdie direkte inhibisie veroorsaak ‘n vermindering in patogenisiteit van die fungus op die gasheer. Indien ePG's egter hulle ensimatiese aksie onverstoord voortsit, sal weefseldegradasie en uiteindelik weefselnekrose die gevolg wees. Daar kon ook bewys word dat die in vitroinhibisie van ePG's deur PGIP die leeftyd van oligogalakturoniede, molekules wat die vermoë het om die plantweerstandsrespons aan te skakel, kan verleng. PGIP het dus nie net die vermoë om ePG's, en dus weefseldegradasie, te inhibeer nie; maar hierdie inhibisie lei ook daartoe dat plantweerstandsresponse aangeskakel word met die oog op die vermindering van patogeenindringing. Verskeie publikasies het reeds gerapporteer oor verminderde Botrytisvatbaarheid in PGIP transgeniese plantlyne. Geeneen van hierdie publikasies kon egter uitbrei op die huidige hipotese aangaande die moontlike in planta-funksie van PGIP in plantsiekteweerstand nie. In hierdie studie is transgeniese tabaklyne wat PGIP ooruitgedruk gebruik om hierdie moontlike in planta-funksies vir PGIP uit te klaar. Transkriptoom- en hormonale analises is op hierdie plantlyne en ‘n WT voor en ná inokulasie met die nekrotroof Botrytis cinerea uitgevoer,. Transkriptoomanalises is uitgevoer op ongeïnfekteerde, sowel as geïnfekteerde tabakblaarmateriaal deur gebruik te maak van ‘n Solanum tuberosum-mikroraster. Die analises met gesonde, ongeïnfekteerde plantmateriaal het daarop gewys dat gene betrokke by selwandmetabolisme tussen die transgeniese lyne en die WT verskillend uitgedruk was. Dit kon bewys word dat, sonder infeksiedruk, die geen wat xiloglukaan-endotransglikosilase (XET) kodeer, in die transgeniese lyne afgereguleer was. Gene wat betrokke is in die lignien-biosintetiese pad was ook in die individuele transgeniese lyne beïnvloed. Biochemiese toetse het ook die aanduiding van verhoogde ligniendeposisie in die transgeniese lyne se selwande bevestig. Addisionele fitohormoonprofiele het getoon dat hierdie lyne ook beskik oor verhoogde vlakke van indoolasynsuur (IAA). Hierdie resultate wys daarop dat konstitutiewe vlakke van PGIP selwandmetabolisme in die Vvpgip1-transgeniese lyne moontlik kan beïnvloed, wat plantsiekteweerstand in dié lyne positief kan beïnvloed. Dit wil dus voorkom asof PGIP 'n bykomende funksie in plantsiekteweerstand het. Plantweerstandsreponse kan direk deur PGIP beïnvloed word, wat tot die versterking van plantselwande kan lei; dit kan geskied by wyse van die strukturele eienskappe van die proteïen of die integrasie daarvan in die selwand. Hierdie selwande is dus “voorberei” alvorens patogeenindringing plaasvind en kon bydra tot die verminderde siektevatbaarheid wat waargeneem is in lyne wat hoë vlakke van PGIP akkumuleer. Transkriptoom- en hormonale analises is ook uitgevoer op Botrytisgeïnfekteerde blaarmateriaal van beide die transgeniese lyne en ‘n WT. Verskeie Botrytis-responsgene is in beide die transgeniese lyne en die WT opgereguleer. Differensïele geenekspressie tussen die twee genotipes was taamlik beperk, maar in die analises kon ‘n geen geïdentifiseer word wat tweevoudig in die transgeniese lyne opgereguleer was in vergelyking met die WT. Hierdie resultaat is ook bevestig met behulp van die “Real-Time” Polimerasekettingreaksie (PKR). Hierdie geen is betrokke in die lipoksigenase (LOX) -pad (spesifiek die 9-LOXarm), wat tot die sintese van die diviniel-eter oksilipiene “colneleic-” en “colnelenic”-suur lei. Daar is al bewys dat hierdie twee verbindings Botrytisspoorontkieming kan inhibeer. Fitohormoonprofiele van die geïnfekteerde plante het gewys dat die transgeniese lyne verhoogde vlakke van die poel van jasmonate wat plantsiekteweerstands-hormone is, ná inokulasie akkumuleer. Hierdie hormone word in die 13-LOX-arm van die lipoksigenase pad gevorm en is belangrik vir die beperking van Botrytis by die infeksiesetel. Die resultate van die analises wat op Botrytis-infeksie volg, dui daarop dat beide arms van die lipoksigenasepad in die transgeniese lyne verskillend by die lokale respons geïnduseer word. ‘n Verhoogde induksie van ‘n ander plantsiekteweerstandshormoon, salisielsuur, kon ook opgemerk word, alhoewel die totaal geakkumuleerde vlakke nie beduidend hoër was as dié van die WT nie. Hierdie resultate is die eerste wat onderskeidende induksie van ‘n siekteweerstandspad in enige van die pgip-ooruitgedrukte plantlyne rapporteer. Daarmee ondersteun dit ook die hipotese dat, seintransduksie wat plantweerstandsresponse aanskakel, ná inhibisie van ePG deur PGIP plaasvind. Die resultate wat met hierdie studie verkry is, dra dus beduidend by tot die huidige kennis van die in planta-funksie van PGIP in plantsiekteweerstandsresponse.

Sugarcane smut is a worldwide distributed disease important to agribusiness, since it can affect sugarcane yield drastically. The disease is caused by the Basidiomycete Sporisorium scitamineum, a biotrophic fungus that colonizes mainly sugarcane. Sugarcane-smut interaction has been extensively studied by this research group for the past few years in their various aspects, considering both the pathogen attack and plant defenses. This work aimed to functionally address fungal candidate effector proteins associated with this pathosystem. Effectors are essential to modulate host metabolism to allow pathogen colonization. The identification of such proteins may assist in recognition of resistance genes relevant to genetic breeding programs. Based on the complete genome sequence of S. scitamineum and the dual transcriptomic data candidate genes were selected in silico. Selection strategies were based on the predicted secretome and differential expression levels of the genes in planta. Candidate effectors were analyzed regarding their expression pattern, subcellular location and influence over basal plant defenses and plant immunity. The results showed that the S. scitamineum candidate effector genes are expressed under the influence of the host genotype. It was observed various expression patterns in the set of selected genes and differential subcellular localization patterns. These results will enable future researches considering virulence level of different isolates and also help decision making in plant breeding programs. / O carvão da cana-de-açúcar é uma doença cosmopolita de grande importância para o agronegócio, uma vez que pode afetar a produtividade da cultura. A doença é causada pelo basidiomiceto Sporisorium scitamineum, fungo biotrófico que coloniza exclusivamente a cana-de-açúcar. A interação cana-carvão vem sendo extensivamente estudada por este grupo de pesquisa nos últimos anos em seus vários aspectos, considerando as atividades de ataque e defesa do patógeno e da planta, respectivamente. Este trabalho teve como finalidade o estudo funcional de proteínas candidatas a efetores neste patossistema. Efetores são moléculas essenciais na manipulação do metabolismo e fisiologia do hospedeiro de forma a permitir sua colonização. A identificação de tais proteínas auxilia no reconhecimento de genes de resistência podendo gerar informações relevantes a programas de melhoramento genético na produção de variedades resistentes. A estratégia de seleção utilizada se baseia em características do secretoma predito e da expressão diferencial de genes do patógeno in planta. Os candidatos foram analisados quanto ao padrão de expressão gênica, à localização sub celular e sua influência sobre a defesa basal e imunidade em plantas. Os resultados demonstraram que a expressão dos genes que codificam para as proteínas efetoras de S. scitamineum e é influenciada pelo genótipo das plantas infectadas. Foram observadas variações no padrão de expressão entre o conjunto de efetores selecionados, bem como padrões diferenciais de localização sub celular e influência sobre a imunidade em plantas. Os resultados gerados por este trabalho servirão de subsídio para estudos futuros sobre os níveis de virulência dos diferentes isolados do patógeno bem como para auxiliar a tomada de decisão em programas de melhoramento genético de variedades resistentes ao carvão da cana.

O Brasil é o maior produtor mundial de maracujá azedo (Passiflora edulis f. flavicarpa) sendo esta a espécie de maior expressão comercial dentre as passifloras cultivadas. A bacteriose do maracujazeiro, causada por Xanthomonas axonopodis pv. passiflorae (Xap), é uma das doenças mais severas da cultura, acarretando grandes prejuízos aos produtores. Atualmente, é incipiente o conhecimento sobre a interação maracujá azedo-Xap. Diante disso, a identificação e a caracterização dos genes envolvidos no processo de defesa são passos importantes para dar suporte ao desenvolvimento de variedades resistentes. Assim, o objetivo deste trabalho foi identificar e caracterizar genes de maracujá azedo diferencialmente expressos durante a resposta de defesa à Xap, bem como mensurar a sua expressão. Para isso, foram construídas duas bibliotecas subtrativas de cDNA (forward e reverse) usando o método SSH a partir de transcritos de folhas, que foram inoculadas com o patógeno ou solução salina (controle). Após o sequenciamento dos clones, o processamento e a montagem das sequências, as unisequências foram anotadas através da Plataforma PLAZA e do programa computacional Blast2GO. Genes envolvidos em diversos processos biológicos foram selecionados para a validação das bibliotecas por PCR quantitativo. Usando a Plataforma PLAZA, 78 % (764) das unisequências mostraram similaridade com proteínas de Arabidopsis thaliana, enquanto 87 % (866) delas apresentaram similaridade com proteínas putativas de diversas espécies vegetais, quando se utilizou Blast2GO. Na biblioteca forward, foram identificadas 73 proteínas relacionadas à resposta de defesa, dentre as quais estão proteínas envolvidas na sinalização intracelular, na ativação da transcrição e regulação da expressão de genes de defesa, bem como proteínas de defesa, de resistência e relacionadas à patogênese (PRs). Dentre os 22 transcritos validados, 95 % foram diferencialmente expressos em pelo menos um dos três períodos avaliados; os genes mais expressos em resposta à infecção pelo patógeno são os que codificam as enzimas lipoxigenase, (+)-neomentol desidrogenase e quitinase, as quais participam diretamente nas respostas de defesa vegetal. Dos genes cuja expressão foi mais reprimida, dois codificam proteínas relacionadas à fotossíntese e dois codificam proteínas envolvidas na detoxificação da amônia e do H2O2. Nossos resultados sugerem que a planta utiliza um arsenal de transcritos para responder à infecção; entretanto, este arsenal não é eficiente para impedir a ação do patógeno e, consequentemente, o desenvolvimento da bacteriose nas condições estudadas. Nosso estudo é inédito e gerou informações sobre a reprogramação transcricional durante a interação maracujá azedo-Xap, o que constitui um importante passo para o melhor entendimento sobre este patossistema. / Brazil is the main producer of yellow passion fruit (Passiflora edulis f. flavicarpa) worldwide, which is the most widely commercialized crop among the cultivated passifloras. The bacterial leaf spot induced by Xanthomonas axonopodis pv. passiflorae (Xap) is one of the most severe diseases of the crop, causing great losses to producers. Currently, we understand very little about the yellow passion fruit-Xap interaction. Therefore, the identification and characterization of genes involved in the defense process are important steps to support the development of resistant varieties. Thus, the objective of this study was identify and characterize differentially expressed genes during the defense response to Xap, as well as to measure their expression. For that, we constructed two subtractive cDNA libraries (the forward and the reverse) by performing the SSH method from leaf transcripts, which were inoculated with the pathogen or saline solution (control). After sequencing the clones and sequence data processing, sequences were assembled into unique sequences, which were annotated using the PLAZA Platform and the computational program Blast2GO. Genes involved in several biological processes were selected to validate the libraries by quantitative PCR. When PLAZA was used for sequence similarity searches, 78 % (764) of the yellow passion fruit unique sequences showed similarity to proteins of Arabidopsis thaliana; when Blast2GO was used, 87 % (866) of the unique sequences showed similarities to putative proteins of several plant species. For the forward library, 73 proteins related to defense response were identified, such as those involved in intracellular signaling, transcription activation and regulation of defense gene expression, as well as defense and resistance proteins, and pathogenesis-related proteins (PRs). Of the 22 validated transcripts, 95 % were differentially expressed during at least one of the three periods evaluated; the genes up-regulated in response to the pathogen infection were those that code for the enzymes lipoxygenase, (+)-neomenthol dehydrogenase and chitinase, which participate directly in plant-defense responses. Out of down-regulated genes, two code for photosynthesis-related proteins, and two for ammonia and H2O2 detoxification. Our results suggest the plant uses an arsenal of transcripts to respond to infection; however, this arsenal is not effective to prevent pathogen action and consequently the occurrence of bacterial leaf spot under the evaluated conditions. The present study is the first to produce information on the transcriptional reprogramming during the passion fruit-Xap interaction, which represents an important step for a better understanding of this pathosystem.