Dissertations / Theses on the topic 'Symbiosis related plant genes'

The arbuscular mycorrhizal (AM) association results from a successful interaction between the genomes of the two symbiotic partners. In this context, the aim of my research was to better characterize the role of the late stage symbiosis-related pea genes PsSym36, PsSym33 and PsSym40 in the functional AM (i) by investigating the effect of mutations in the three genes on fungal and plant gene responses and (ii) by creating conditions for the localization of two of the genes, PsSym36 and PsSym40, on the pea genetic map for future map-based cloning. The expression of a subset of ten fungal and eight plant genes,previously reported to be activated during mycorrhiza development, was compared in Glomus intraradices-inoculated roots of wild type and Pssym36, Pssym33 and Pssym40 mutant pea plants. Most of the fungal genes were down-regulated in roots of the Pssym36 mutant where arbuscule formation is defective, and several were upregulated with more rapid fungal development in roots of the Pssym40 mutant. Microdissection of mycorrhizal PsSym40 roots corroborated preferential expression of the three G. intraradices genes SOD, DESAT and PEPISOM in arbuscule-containing cells. Inactivation of PsSym36 also resulted in down regulation of plant genes whilst mutation of the PsSym33 and PsSym40 genes affected plant gene responses in a more time-dependent way. Results thus indicate an implication of the investigated pea SYM genes in the modulation of plant and fungal molecular interactions linked to signaling, nutrient exchange or stress response regulation during AM symbiosis formation and functioning. Conditions for localization of the PsSym36 and PsSym40 genes on the pea genetic map were developed for their future map-based cloning. Based on the molecular markers obtained, it was possible to conclude that localization of the PsSym40 gene most likely resides outside the linkage groups I, II, III or V of the genetic map of pea.

L’association mycorhizienne à arbuscules (AM) est le résultat d’une interaction compatible entre les génomes des deux partenaires symbiotiques. Dans ce contexte, le but de mes recherches a été de mieux caractériser le rôle des gènes de pois liés aux stades tardifs de la symbiose, PsSym36, PsSym33 and PsSym40, dans le fonctionnement de la symbiose MA (i) en étudiant l’effet des mutations de ces trois gènes sur l’expression des gènes de la plante et du champignon, et (ii) en créant les conditions pour positionner deux de ces gènes, PsSym36 and PsSym40, sur la carte génétique afin d’envisager leur clonage futur. L’expression d’un groupe de dix gènes fongiques et de huit gènes de plante, déjà décrits pour être activés durant le développement de la mycorhize, a été comparée dans les racines de pois inoculées avec G. intraradices chez les plantes de génotypes sauvages, ou les mutants Pssym36, Pssym33 et Pssym40. L’expression de la plupart des gènes fongiques a été inhibée dans les racines du mutant Pssym36 où la formation des arbuscules est avortée, tandis que l’expression de plusieurs d’entre eux a été activée lorsqu’il existe un développement plus rapide du champignon dans les racines du mutant Pssym40. Des microdisséquats obtenus à partir de racines mycorhizées du mutant PsSym40 confirment l’expression préférentielle de trois gènes de G. intraradices (SOD, DESAT et PEPISOM) dans les cellules contenant les arbuscules. L’inactivation du gène PsSym36 provoque également une inhibition des gènes de plante alors que la mutation des gènes PsSym33 and PsSym40 affecte l’expression des gènes de plante plutôt de façon temporelle. Les résultats indiquent ainsi une implication des gènes SYM de pois dans la modulation des interactions moléculaires entre la plante et le champignon impliquées au niveau de la signalisation, des échanges nutritifs ou de la régulation des réponses au stress durant la formation et/ou le fonctionnement de la symbiose AM. Les conditions pour la localisation des gènes PsSym36 and PsSym40 sur la carte génétique du pois ont été développées pour leur clonage basé sur la cartographie. En utilisant les marqueurs moléculaires obtenus, il a été possible de conclure que la localisation du gène PsSym40 réside vraisemblablement à l’extérieur des groupes de liaison I, II, III ou V de la carte génétique du pois
The arbuscular mycorrhizal (AM) association results from a successful interaction between the genomes of the two symbiotic partners. In this context, the aim of my research was to better characterize the role of the late stage symbiosis-related pea genes PsSym36, PsSym33 and PsSym40 in the functional AM (i) by investigating the effect of mutations in the three genes on fungal and plant gene responses and (ii) by creating conditions for the localization of two of the genes, PsSym36 and PsSym40, on the pea genetic map for future map-based cloning. The expression of a subset of ten fungal and eight plant genes,previously reported to be activated during mycorrhiza development, was compared in Glomus intraradices-inoculated roots of wild type and Pssym36, Pssym33 and Pssym40 mutant pea plants. Most of the fungal genes were down-regulated in roots of the Pssym36 mutant where arbuscule formation is defective, and several were upregulated with more rapid fungal development in roots of the Pssym40 mutant. Microdissection of mycorrhizal PsSym40 roots corroborated preferential expression of the three G. intraradices genes SOD, DESAT and PEPISOM in arbuscule-containing cells. Inactivation of PsSym36 also resulted in down regulation of plant genes whilst mutation of the PsSym33 and PsSym40 genes affected plant gene responses in a more time-dependent way. Results thus indicate an implication of the investigated pea SYM genes in the modulation of plant and fungal molecular interactions linked to signaling, nutrient exchange or stress response regulation during AM symbiosis formation and functioning. Conditions for localization of the PsSym36 and PsSym40 genes on the pea genetic map were developed for their future map-based cloning. Based on the molecular markers obtained, it was possible to conclude that localization of the PsSym40 gene most likely resides outside the linkage groups I, II, III or V of the genetic map of pea
Формирование арбускулярной микоризы (АМ) является результатом успешного взаимодействия между геномами двух симбиотических партнёров. Целью моего исследования являлось изучение роли поздних симбиотических генов гороха PsSym36, PsSym33 и PsSym40 в формировании функционального АМ симбиоза. Для этого было проведено исследование эффекта мутаций в генах PsSym36, PsSym33 и PsSym40 на экспрессию грибных и растительных генов, предположительно (по литературным данным) вовлечённых в процессы формирования АМ, а так же проведена работа по локализации генов PsSym36 и PsSym40 на генетической карте гороха для последующего более точного картирования и позиционного клонирования данных генов. Экспрессия десяти грибных и восьми растительных генов была определена в корнях растений дикого типа и PsSym36, PsSym33 и PsSym40 мутантов, инокулированных G. intraradices. В корнях PsSym36 мутанта, имеющего дефект развития арбускул, большая часть грибных генов была супрессирована, в то время как в корнях PsSym40 мутанта, для которого характерна более быстрая по сравнению с диким типом микоризация, был отмечен более высокий уровень экспрессии грибных генов. Использование метода микродиссекций позволило выделить клетки, содержащие арбускулы, из микоризованных корней мутанта PsSym40 и подтвердить, что гены G. intraradices SOD, DESAT и PEPISOM преимущественно экспрессируются в клетках, содержащих арбускулы. Мутация в гене PsSym36 также привела к подавлению экспрессии большинства вовлечённых в анализ растительных генов, тогда как мутации в генах PsSym33 и PsSym40 оказали влияние на ксперессию растительных генов в меньшей степени. Полученные результаты свидетельствуют о роли исследуемых SYM генов гороха в контролировании растительно-грибных молекулярных взаимодействий, связанных с сигналингом, обменом питательными веществами и стрессовыми реакциями в процессе формирования и функционирования АМ симбиоза. Проведённое генетическое картирование не привело к локализации генов PsSym36 и PsSym40 на генетической карте гороха. Однако разработка и использование молекулярных маркеров для картирования позволили исключить локализацию гена PsSym40 в I, II, III и V группах сцепления с высокой долей вероятности

The plasmids pDN211 and pDNll, isolated from the gene bank of the Rhizobium japonicum strain I-110, have been reported to complement two different Nif+ Hup· (nitrogen fixation positive and hydrogen uptake negative) mutants. A 5.9-kb Hindiii DNA fragment of the cosmid pHU52, isolated from the gene bank of R. japonicum strain 122DES, has been reported to code for the two polypeptide subunits of uptake hydrogenase. To determine homology between the structural genes of uptake hydrogenase of the two strains, a Southern blot of the Hindiii restriction fragments of the plasmids pDN211 and pDN11 was hybridized to the 5.9-kb Hindiii fragment. A 6.0-kb HindIII DNA fragment of pDN11 was observed to be homologous to the hup DNA probe. Thus, the hup genes of the two Rhizobium strains are conserved. Colony hybridization with the 5.9-kb DNA as the probe was used to detect the homologous hup genes in alfalfa-, chickpea- and pigeonpea- Rhizobium species. These Rhizobium species were also successfully derepressed for uptake hydrogenase in free living conditions. It was found that 30% of the alfalfa-, 30% of the chickpea- and 21% of the pigeonpea- Rhizobium strains tested were Hup+ as determined by the methylene blue (MB) reduction assay. All but one strain of alfalfa- (Celpril Ind. 3623) and one strain of pigeonpea- Rhizobium (IC3282) that showed strong homology to the hup DNA probe also exhibited MB reduction activity. The Hup+ strains of alfalfa- and pigeonpea- Rhizobium produced significantly higher yields as compared to the Hup- strains, whereas those of the chickpea-Rhizobium strains produced significantly lower yields as compared to the Hup- strains. Two of the alfalfa-Rhizobium strains, USDA1024 and CmRm~, exhibited Hup activities greater than any reported previously for this bacterial species. The cosmid-borne hup genes of R. japonicum were successfully expressed in all strains tested but the enzyme activities were very low in alfalfa-Rhizobium compared to those in chickpea- and pigeonpea-Rhizobium species. The relative efficiency of N2-fixation was significantly increased by the transfer of hup genes into the chickpea- and pigeonpea- Rhizobium strains.

Orchids are characterized by producing minute endosperm-lacking seeds, which depend on mycorrhizal fungi for germination and embryo development. Some aclorophyllous orchids remain dependent on the mycorrhizal association for carbon acquisition during their whole life history, whereasother orchids develop photosynthesis. Despite the biological significance of orchid mycorrhiza, gene expression studies are lacking. We have used different highthroughput approaches in order to understanding the mechanisms regulating orchid mycorrhiza development and functioning. Firstly, we have used a 2D-LC-MS/MS approach coupled to isobaric tagging for relative and absolute quantification (iTRAQ) to identify proteins with differential accumulation in Oncidium sphacelatum at different stages of mycorrhizal protocorm development (achlorophyllous and green protocorms) after seed inoculation with a Ceratobasidium sp. isolate. Quantitative analysis showed that the expected changes in carbon metabolism in green protocorms were accompanied by enhanced accumulation of proteins involved in the modulation of reactive oxygen species homeostasis, defense related responses, phytoalexins and carotenoid biosynthesis, suggesting that orchid protocorms undergo profound metabolic changes during the switch from the fully mycoheterotrophic to the photosynthethic stage. Secondly, three different proteomic techniques were carried out in independent experiments aiming to identify changes in protein accumulation in mycorrhizal roots of the terrestrial orchid Oeceoclades maculata.Finally, O. maculatamycorrhizal roots were used for transcriptome analyses. The data revealed a strong increase in general stress responses, accompanied by changes in signaling pathways possibly related to fungal recognition and establishment of a compatible interaction. Some of the upregulated genes may be involved in the reorganization of cell structure, likely related to accommodation of the fungal symbiont in the plant roots. We have also observed in mycorrhizal roots up-regulation of genes involved in carbon metabolism, including glycolysis/gluconeogenesis and amino sugars metabolism, as well as genes involved innitrogen assimilation. The down-regulation of genes involved in the jasmonate and ABA transduction pathways, and key genes encoding anti-fungal proteins, such as chitinase and a mannose-specific binding lectin, strongly suggests an alleviation of plant defense responses in O. maculata mycorrhizal roots. In general, our data suggest that the physiology of an orchid mycorrhiza is more similar to a compatible interaction than to an arm-race between plant and fungi. Overall orchid mycorrhiza have proved to be a promising model for investigating plantfungal interactions and further studies should now address the specific roles of the genes showing differential regulation in this study.
As orquídeas são caracterizadas por produzirem sementes diminutas, que não possuem endosperma. Necessitam, portanto, da interação com fungos micorrízicos para germinação e desenvolvimento do embrião. Algumas orquídeas aclorofiladas se mantêm dependentes dos fungos micorrízicos para a aquisição de carbono, enquanto outras desenvolvem a maquinaria fotossintética. Apesar do significado biológico das micorrizas de orquídeas, alterações na expressão gênica e no acúmulo de proteínas foram altamente negligenciads nos últimos anos. Neste trabalho, foram utilizadas diferentes técnicas sequenciamento e identificação de genes e proteínas em larga-escala para acessar as alterações moleculares responsáveis pela regulação das micorrizas de orquídeas. Uma abordagem baseada em 2D-LC MS/MS acoplada a técnica de quantificação absoluta e relativa iTRAQ, foiutilizada para identificar proteínas com acúmulo diferencial em Oncidium sphacelatum em diferentes estágios do desenvolvimento do protocormo (protocormos aclorofilados versus protocormos fotossintetizantes), após inoculação com um fungo do gênero Ceratobasidium. As análises mostraram que, as alterações esperadas no metabolismo do carbono foram acompanhadas de um acúmulo aumentado de proteínas envolvidas na modulação de espécies reativas de oxigênio, respostas de defesa, biossíntese de fitoalexinas e carotenóides, sugerindo que os protocormos de orquídeas passam por profundas alterações metabolicas durante a transição do metabolismo micoheterotrófico para o fotossintético. Posteriormente foram utilizadas três diferentes técnicas de proteômica quantitativa para explorar alterações fisiológicas em raízes micorrizadas e não-micorrizadas de Oeceoclades maculata.Este estudo foi ampliado, pela utilização de uma abordagem transcritômica ao mesmo modelo biológico. Em conjunto, os dados revelaram um forte aumento em respostas relacionadas ao estresse, acompanhadas de alterações em vias de transdução de sinal possivelmente relacionadas ao reconhecimento do simbionte fúngico e estabelecimento de uma interação compatível. Alguns genes com expressão aumentada devem estar envolvidos na reorganização celular, provavelmente ligada a acomodação do simbionte fúngico nas raízes das plantas. Também foi observado o aumento de genes envolvidos no metabolismo do carbono e de açúcares aminados, juntamente a genes relacionados a assimilação de nitrogênio em raízes micorrizadas. A expressão diminuída de genes envolvidas nas vias do jasmonato e ácido abscícico, juntamente a genes-chave que codificam para proteínas anti-fúngicas sugerem fortemente uma atenuação das respostas de defesa da planta em raízes micorrizadas de Oeceoclades maculata. No geral, parece que as micorrizas de orquídeas são fisiológicamente mais próximas de uma simbiose compatível do que de uma interação unilateral em favor da planta. Sobretudo, este sistema biológico provou ser promissor para investigação de interações planta-fungo e, próximas pesquisas devem agora ser focadas em funções específicas dos genes que mostraram regulação diferencial neste estudo.

The plant hormone auxin plays a major role in shaping plant morphology and development, but the gene networks regulating its synthesis and transport are incompletely known. The maize BARREN INFLORESCENCE 1 (BIF1) gene has recently been cloned and shown to play an important role in the early stages of polar auxin transport. Auxin is synthesized in shoot tips and transported basipetally through the plant shoot and acts as a morphogen by facilitating the degradation of transcriptional repressors in a concentration dependent manner. The AUX/IAA gene family encodes transcriptional repressors that regulate a subset of plant developmental responses governed by the transcription of early auxin inducible genes in plants. Although the maize BIF1 gene is a member of the AUX/IAA gene family, the co-ortholog(s) of BIF1 in Arabidopsis thaliana was not known prior to this research.

Bayesian phylogenetic reconstruction placed maize BIF1 in a clade sister to Arabidopsis thaliana AtIAA15. The BIF1 lineage has undergone two gene duplications since the divergence of the early grasses. Molecular evolutionary analyses by maximum likelihood suggest that the BIF1 alignment is under strong purifying selection with positive selection acting on a glutamine residue located in a functional region associated with AUX/IAA protein dimerization in one clade of BIF1 paralogs, the BIF1-Like2 (BIF1L2) clade. A character reconstruction analysis using maximum parsimony estimated an adenine to cytosine transversion at the base of the BIF1L2 clade changed a glutamine into an alanine residue in this functional region. Expression of BIF1 orthologs is conserved in floral meristems in the eudicot AtIAA15 clade containing the taxa Erianthe Guttata, Arabidopsis thaliana, Medicago truncatula, however grass BIF1L2 expression has diverged within the PACMAD – BEP clade, specifically in rice, where BIF1L2 expression is reported to have moved into root tissue. These results suggest that BIF1 paralogs has changed following a second round of gene duplication in the grasses. Taken together, a change in localized expression in these sequences, and positive selection acting on a glutamine-rich region of the protein-protein binding motif could imply that BARREN INFLORESCENCE1-like2 proteins are probably interacting with a new set or subset of AUXIN RESPONSE FACTOR (ARF) binding partners, and that neofunctionalization has occurred in the BARREN INFLORESCENCE1-like2 clade.

Over one billion people, more than 1/9th of the global population, are undernourished. Feeding the ever increasing population has to be the most important goal of plant sciences. Since cultivated areas are not likely to increase, I will need to produce more with what is available. This can be summarized in one word: yield. Unfortunately, wheat?s yield is expected to increase only 1.13% by 2019, a prediction that if converted into reality will likely indicate that I failed to cope with the world demographic increase. A new strategy to revolutionize wheat production is required, and some believe that this change might be represented by wheat hybrids. Achieving adequate commercial production of wheat hybrids has the potential to nearly double the yield of one of the world?s most important staple food. The first fundamental step toward this goal is to develop feasible methodologies to sterilize the male part of the complete wheat flowers. Two fertility-related genes are the primary target of this study, namely the species cytoplasm specific on chromosome 1D, and the desynaptic locus on chromosome 3B. This dissertation summarizes the important achievements obtained toward the cloning of the two loci by means of radiation hybrid functional analysis. Radiation hybrid is a technique that employs radiation to create genetic diversity along the targeted chromosome. Chapter 1 explains in details how this methodology can be applied to plants. The use of radiation hybrid mapping permitted creating a comprehensive map of wheat chromosome 3B, as discussed in Chapter 2, and then expanded the mapping information to identify the 2 Mb location of the desynaptic locus desw2, as discussed in Chapter 3. A similar approach on chromosome 1D allowed first to pinpoint the location of the species cytoplasm specific gene to a region of 2 Mb, as discussed in Chapter 4, and then ultimately to find a strong candidate for this locus, as discussed in Chapter 5. Now that the molecular locations of these genes have been unraveled by this study, their sequence can be streamlined into transformation to ultimately produce female wheat plants, and consequently hybrids.

Thesis (MScAgric)--Stellenbosch University, 2003.
ENGLISH ABSTRACT: The resistance of plants to infection by phytopathogenic microorganisms is the result of multiple defence reactions comprising both constitutive and inducible barriers. While disease is the exception, such exceptions can be costly and even devastating. In particular, fungal diseases remain one of the major factors limiting crop productivity worldwide, with huge losses that need to be weighed up against massive cash inputs for pesticide treatments. Part of the defence reactions of plants is the synthesis of pathogenesis-related proteins, such as the plant hydrolases, glucanases and chitinases. In recent years, attention has been paid to the implementation of these proteins in plant transformation schemes. The rationale for this approach was that these antimicrobial agents not only degrade the main cell wall components of fungi, but also produce glucosidic fragments that act as elicitors of the biosynthesis of defence metabolites by the host. Furthermore, since these active antimicrobial agents are individually encoded by single genes, these defence systems should and have been shown to be highly amenable to manipulation by gene transfer. In this study, yeast glucanases from Saccharomyces cerevisiae were evaluated for their potential as antifungal proteins. The glucanases tested for their antifungal activity against Botrytis cinerea were the yeast EXG1 and BGL2 genes, encoding an exoglucanase and an endoglucanase respectively. An in vitro assay performed on these glucanases indicated that exoglucanase had a more detrimental effect on B. cinerea hyphal development and growth than the endoglucanase; the former caused typical disruption of the cells and leakage of cell material. The yeast exoglucanase was subsequently subcloned into a plant expression cassette containing the strong constitutive 358 promoter, yielding plasm ids pEXG1 and pMJ-EXG1. The pMJ-EXG1 construct targeted the exoglucanase to the apoplastic region with a signal peptide from an antimicrobial peptide from Mirabilis jalapa, Mj-AMP2. The pEXG1 and pMJ-EXG1 constructs were mobilised into Agrobacterium tumefaciens to facilitate the subsequent tobacco transformation, which yielded transgenic tobacco lines designated E and MJE respectively. Transgene integration was confirmed with southern blot and PCR analyses for both the E and MJE lines. The expression and heterologous production of the EXG1-encoded exoglucanase in the E-transgenic lines was shown with northern blots and activity assays respectively. Moreover, the high level of expression of the yeast exoglucanase led to a decrease in susceptibility of the E lines to B. cinerea infection in comparison to the untransformed tobacco controls. An average decrease in disease susceptibility of 40% was observed in an in planta detached leaf assay. Crude protein extracts from the E lines were also analysed in an in vitro quantitive fungal growth assay, inhibiting in vitro fungal growth by average 20%, thus further confirming the antifungal nature of the yeast exoglucanase. Although integration of the MJ-EXG1 expression cassette was confirmed, no mRNA levels could be detected with northern blot or RT-PCR analysis of the MJE lines. These lines also did not show any in vitro antifungal activities or a decrease in susceptibility to B. cinerea infection in the detached leaf assay. It is suspected that this result is possibly linked to gene silencing, a phenomenon quite frequently associated with heterologous and/or overexpression of glucanases in plant hosts. It appears as if the targeted overexpression to the apoplastic space triggered the gene silencing response, since the intracellularly overexpressed product was produced and shown to display activity. The yeast exoglucanase thus joins the list of silenced glucanases in overexpression studies in plants. Overall, this study confirmed the antifungal characteristics of the Saccharomyces exoglucanase and provides valuable information of the possibility of utilising yeast glucanases in a transgenic environment. A decrease in the susceptibility of tobacco to B. cinerea infection, as shown by the overexpressed EXG1-encoded exoglucanases, merits further investigation into the use of this gene in the engineering of disease-resistant crops.
AFRIKAANSE OPSOMMING: Die weerstand van plante teen infeksie deur fitopatogeniese mikroórganismes is die resultaat van verskeie meervoudige verdedigingsreaksies wat beide konstitutiewe en induseerbare versperrings behels. Terwyl siekte die uitsondering eerder as die reël is, kan sulke uitsonderinge duur en selfs verwoestend wees. In die besonder is swamsiektes een van die vernaamste faktore wat gewasproduksie wêreldwyd beperk, met enorme verliese wat teen kontantinsette vir plaagdoders opgeweeg moet word. Deel van die verdedigingsreaksie van plante is die sintese van patogeen-verwante proteïene, soos die planthidrolases, -glukanases en -chitinases. In die onlangse tyd is aandag geskenk aan die implementering van hierdie proteïene in plant transformasieskemas. Die grondrede hiervoor was dat hierdie antimikrobiese agente nie net die hoof selwandkomponente van swamme kan afbreek nie, maar ook glukosidiese fragmente produseer wat as ontlokkers van metabolietbiosintese vir die verdediging van die gasheer kan optree. Aangesien hierdie aktiewe antimikrobiese agente individueel deur enkele gene enkodeer word, blyk hierdie verdedigingsisteme om hoogs ontvanklik vir manipulasie deur geenoordrag te wees. In hierdie studie is die gisglukanase van Saccharomyces cerevisiae vir hul potensiaal as antifungiese proteïene geëvalueer. Die glukanases wat vir hul antifungiese aktiwiteit teen Botrytis cinerea getoets is, was die gis EXG1- en -BGL2-gene, wat onderskeidelik vir "n eksoglukanase en 'n endoglukanase enkodeer. "n In vitro toets wat op hierdie glukanases uitgevoer is, het aangedui dat die eksoglukanase 'n meer skadelike effek op die hife-groei en -ontwikkeling van B. cinerea as die endoglukanase gehad het; eersgenoemde het die tipiese ontwrigting van die selle en die uitlek van selmateriaal tot gevolg gehad. Die gis-eksoglukanase is gevolglik in 'n plant uitdrukkingskasset wat die sterk konstitutiewe 35S promotor bevat, gesubkloneer, wat plamiede pEXG1 en pMJ-EXG1 opgelewer het. Die pMJ-EXG1-konstruk het die eksoglukanase na die apoplastiese gebied geteiken deur 'n seinpeptied vanaf "n antimikrobiese peptied van Mirabilisjalaba, Mj-AMP2. Die pEXG1- en pMJ-EXG1-konstrukte is in Agrobacterium tumefaciens gemobiliseer, wat die gevolglike tabaktransformasies gefasiliteer het wat die E en MJE transgeniese tabaklyne onderskeikelik gelewer het. Transgeen-integrasie is deur suidelike klad- en PKR-analises vir beide die E en MJE lyne bevestig. Die uitdrukking en heteroloë produksie van die EXG1-enkodeerde eksoglukanase is in die transgeniese E lyne deur noordelike klad en aktiwiteitstoetse onderskeidelik aangetoon. Verder het die hoë uitdrukkingsvlak van die gis-eksoglukanase tot 'n vermindering in die vatbaarheid van die E lyne vir B. cinerea-infeksie relatief tot die ongetransformeerde tabakkontroles gelei. 'n Gemiddelde vermindering in siektevatbaarheid van 40% is in 'n in planta verwyderde-blaartoets waargeneem. Ru proteïen-ekstrakte van die E lyne is ook in 'n in vitro kwantitatiewe swamgroeitoets geanaliseer en het in vitro swamgroei met tot gemiddeld 20% geïnhibeer, wat dus verder die antifungiese aard van die gis-eksoglukanase bevestig het. Alhoewel die integrasie van die pMJ-EXG1 uitdrukkingskasset bevestig is, kon geen mRNA-vlakke met die noordelike klad- of RT-peR-analises van die MJE-Iyne waargeneem word nie. Hierdie lyne het ook geen in vitro antifungiese aktiwiteite of 'n vermindering in die vatbaarheid vir B. cinerea-infeksie getoon nie, soos in die verwyderde-blaartoets uitgevoer is nie. Dit word vermoed dat hierdie resultaat moontlik aan geenstilmaking gekoppel is, 'n verskynsel wat gereeld met heteroloë- en/of ooruitdrukking van glukanases in plantgashere gekoppel word. Dit blyk dat die ooruitdrukking wat tot die apoplastiese ruimte geteiken is, tot die geenstilmaking-respons aanleiding gegee het, aangesien die intrasellulêre ooruitgedrukte produk gemaak is en aktiwiteit getoon het. Die gis-eksoglukanase word dus deel van die lys van stilgemaakte glukanases in die ooruitdrukkingstudies van plante. In die algemeen het hierdie studie dus die antifungiese kenmerke van die Saccharomyces eksoglukanase bevestig en waardevolle inligting oor die moontlike gebruik van gis-glukanases in 'n transgeniese omgewing verskaf. 'n Afname in die vatbaarheid van tabak vir infeksie deur B. cinerea, soos deur die ooruitdrukking van EXG1-enkodeerde eksoglukanase getoon is, verdien dus verdere ondersoek van die gebruik van hierdie geen in die skepping van siekteweerstandbiedende gewasse.

Cytoplasrnic male sterility (CMS) is a maternally inherited defect in the production of pollen, the male gamete of the flower. This sterility can be suppressed by nuclear Restorer of Fertility (Rf) genes that normally downregulate the expression of the CMS-associated novel mitochondrial gene. In Brassica napus, nap CMS and pol CMS are associated with related chimeric mitochondrial genes orf222 and orf224, respectively. CMS in both nap and pol is associated with a polar loss of locule development, loss of synchronous locule development and clumping of sporogenous tissue away from the tapetal cell layer, as well as secondary effects on petal and bud formation. In nap CMS, early accumulation of orf222 transcripts in the locule regions of developing anthers is associated with sterility, while the absence of orf222 transcripts from the locules is associated with fertility restoration. Accumulation of novel antisense transcripts of atp6 in a cell specific manner which matches that of sense transcripts of orf222 and atp6 in nap CMS anthers may be indicative of a post-transcriptional regulatory mechanism associated with CMS in flower buds.
Restoration of fertility in Brassica napus nap and pol CMS is associated with nuclearly encoded genes Rfn and Rfp, respectively. These restorers are very closely linked to one another, and may be allelic. Further efforts to isolate Rfp have narrowed the genomic region to approximately 105 kb of a syntenic region in Arabidopsis thaliana. Cosmid clones isolated from a library of Brassica rapa genomic DNA introgressed with Rfp have been successfully sorted into contigs through the application of the amplified fragment length polymorphism technique. The region to which Rfp is mapped is syntenic to a region of Arabidopsis DNA that is a duplication of a second location at the 23 megabase region of chromosome 1 of that genome. This region contains pentatricopeptide (PPR) motif-encoding genes that are highly related to other restorers of fertility of other species. By inference, Rfp from Brassica napus may encode PPR motifs. The PPR genes related to these previously characterized restorers of fertility are often found alongside the restorer genes existing as mini-clusters of several PPR-encoding genes. This is likely caused by selective pressure acting on PPR-encoding genes that resulted in diversification and multiplication of these genes. In addition, the PPR genes of this duplicated region are not syntenically located, whereas the non-PPR-encoding genes maintain their syntenic locations. The same is true for orthologous comparisons between Arabidopsis and other plant species. PPR genes are therefore malleable and capable of alteration in response to changing environmental pressures, such as the evolution of sterility inducing genes.

Les gènes NODULE-ROOT de Medicago truncatula, BLADE-ON-PETIOLE d’Arabidopsis thaliana et COCHLEATA de Pisum sativum font partie d'un clade spécifique NOOT-BOP-COCH-LIKE1 (NBCL1) hautement conservé et qui appartient à la famille de gènes NON-EXPRESSOR OF PATHOGENESIS RELATED PROTEIN1 LIKE. Chez les légumineuses, les membres du clade NBCL1 sont connus comme les principaux régulateurs de l'identité des organes symbiotiques (nodules). Les membres du clade NBCL2 (MtNOOT2) jouent également un rôle clé dans l'établissement et le maintien de l'identité de l’organe symbiotique, en redondance avec les gènes NBCL1. Il a également été démontré que ces gènes végétaux NBCL sont impliqués dans l'abscission. Les gènes NBCL sont également conservés chez les plantes monocotylédones chez lesquelles ils contrôlent différents aspects du développement. Ce travail de thèse vise à mieux comprendre les rôles des gènes NBCL1 et NBCL2 dans le développement des plantes légumineuses et chez Brachypodium et à découvrir de nouveaux acteurs moléculaires impliqués dans la régulation de l'identité des nodules dépendante de NBCL1, en utilisant de nouveaux mutants d'insertion TILLING et Tnt1 chez deux espèces de légumineuses (Medicago et Pisum). En outre, nous avons utilisé les mutations KO CRISPR chez Brachypodium pour mieux comprendre leur rôle chez les plantes monocotylédones. Ce travail de thèse a permis d'élucider les nouvelles fonctions des gènes NBCL1 dans le développement des tiges et l'architecture des plantes. Nous avons également révélé que les membres du clade NBCL2, spécifique aux légumineuses, fonctionnent de manière redondante avec le clade NBCL1 et jouent des rôles importants dans le développement des feuilles, des stipules, des inflorescences et des fleurs. De plus, nous avons montré un rôle dans le développement, l'établissement et le maintien de l'identité des nodosités, et par conséquent dans le succès et l'efficacité de l'association symbiotique. Dans cette thèse, nous avons également exploré les rôles des gènes NBCL BdUNICULME4 et BdLAXATUM-A, dans le développement de B. distachyon à l'aide de doubles mutants. Nous avons confirmé les résultats précédents et révélé une nouvelle fonction pour ces deux gènes dans l'architecture des plantes, la formation des ligules et des inflorescences, ainsi que dans la teneur en lignine. Ce travail de thèse a finalement permis l'identification et la caractérisation de nouveaux mutants pour les gènes de M. truncatula ALOG (Arabidopsis LSH1 et Oryza G1). Les protéines ALOG sont des partenaires d'interaction potentiels pour les NBCLs. Nous avons montré que certains membres ALOG jouent un rôle important dans le développement des nodules et des organes aériens. Dans l'ensemble, ce travail de thèse suggère qu'au cours de l'évolution, le programme de développement des nodules a été recruté à partir de programmes de régulation préexistants pour le développement et l'identité des nodosités
The Medicago truncatula NODULE-ROOT, the Arabidopsis thaliana BLADE-ON-PETIOLE, and the Pisum sativum COCHLEATA genes are members of a highly conserved NOOT-BOP-COCH-LIKE1 (NBCL1) specific clade that belongs to the NON-EXPRESSOR OF PATHOGENESIS RELATED PROTEIN1 LIKE gene family. In legumes, the members of this NBCL1 clade are known as key regulators of the symbiotic organ identity. The members of the NBCL2 clade (MtNOOT2) also play a key role in the establishment and maintenance of the symbiotic nodule identity, redundantly with NBCL1 while without significant phenotype alone. These NBCL plant genes were also shown to be involved in abscission. In addition, NBCL genes are also conserved in monocotyledon plants in which they also control different aspects of development. The present thesis work aims to better understand the roles of the NBCL1 and NBCL2 genes in development in both legume and Brachypodium plants and to discover new molecular actors involved in the NBCL1-dependent regulation of the nodule identity using novel TILLING and Tnt1 insertional mutants in two legume species, Medicago, and Pisum. In addition we used CRISPR knock-out mutations in Brachypodium to better understand their roles in monocotyledon plants. This thesis work unraveled new functions of the NBCL1 genes in plant shoot development and plant architecture. We also revealed that the members of the legume-specific NBCL2 redundantly function with NBCL1 sub-clade and play important roles in leaf, stipule, inflorescence and flower development. In addition we showed a role in nodule development, identity establishment and maintenance, and consequently in the success and efficiency of the symbiotic association. In this thesis, we also explored the roles of the highly conserved NBCL genes, BdUNICULME4 and BdLAXATUM-A, in the development of B. distachyon using double mutants. We confirmed previous results and reveal a new function for these two genes in plant architecture, ligule and inflorescence formation, and also lignin content. This thesis work has finally allowed the identification and the characterization of new mutants for M. truncatula ALOG (Arabidopsis LSH1 and Oryza G1) genes. ALOG proteins are potential interacting partners for NBCL. We showed that some ALOG members play important roles in nodule and aerial organ development. Altogether, this thesis work suggests that during evolution, the nodule developmental program was recruited from pre-existing regulatory programs for nodule development and identity

L’association symbiotique entre les légumineuses et les rhizobia aboutit à la formation de la nodosité fixatrice d’azote. Cet organe symbiotique généré de-novo permet l’hébergement intracellulaire des rhizobia qui, grâce à leurs activités nitrogénase,réduisent l’azote atmosphérique en ammonium, une forme de l’azote directement assimilable par la plante hôte.Les mécanismes moléculaires sous-jacents à la reconnaissance entre les deux partenaires symbiotiques, au processus d’infection et à l’organogénèse de la nodosité sont bien décrits, cependantl’établissement et la maintenance de l’identité de cet unique organe souterrain restent incompris.Les gènes NODULE-ROOT de Medicago truncatula, BLADEON-PETIOLE d’Arabidopsis thaliana et COCHLEATA de Pisumsativum sont membres du clade spécifique très conservé NOOTBOP-COCH-LIKE1 (NBCL1) qui fait partie de la famille des gènesNON-EXPRESSOR OF PATHOGENESIS RELATED PROTEIN1-LIKE. Chez les légumineuses, les membres de ce clade NBCL1 sont connus comme étant des régulateurs clés de l’identité de l’organe symbiotique.Mon travail de thèse a eu pour but d’améliorer la compréhension des rôles des gènes NBCL1, à la fois chez des espèces formant des nodosités indéterminées et déterminées, ainsi que de découvrir de nouveaux acteurs moléculaires impliqués dans l’identité de la nodosité dont la régulation est dépendante des gènesNBCL1, essentiellement par l’utilisation de mutants TILLING, Tnt1et LORE1 originaux chez trois espèces de légumineuses: la luzerne tronquée, le petit pois et le lotier.Ce travail rapporte essentiellement l’identification et la caractérisation de nouveaux mutants affectés dans des gènes qui font partie d’un second sous-clade NBCL2 spécifique des légumineuses.Nous avons révélé que les membres de ce sous-clade spécifique des légumineuses NBCL2 jouent d’importants rôles dans le développement de la nodosité, dans l’établissement et la maintenance de l’identité de la nodosité et par conséquence dans le succès et l’efficacité de l’association symbiotique.Ce travail suggère aussi qu’au cours de l’évolution, le programme de développement de la nodosité a recruté des mécanismes de régulations préexistants afin de réguler le développement de la nodosité et son identité, tel que le module de régulation impliquant des interactions entre des protéines NBCL et des facteurs de transcriptions basic leucine zipper de type TGACG. Nous avons identifié le facteur de transcription MtPERIANTHIA-LIKE, comme un premier partenaire protéique interagissant avec des protéines NBLC dans un contexte de nodosité symbiotique. Les gènes NBCL sont aussi impliqués dans les réseaux de régulations qui contrôlent le développement et le déterminisme de nombreux organes végétatifs et reproductifs aériens et sont également impliqués dans la capacité d’abscission de ces organes.Finalement, ce travail thèse a eu pour objectif d’explorer les rôles de ces gènes NBCL très conservés, dans le développement de la graminée non-domestiquée, Brachypodium distachyon
The symbiotic interaction between legumes andrhizobia results in the formation of a symbiotic nitrogen fixingnodule.This de-novo generated symbiotic organ allows the intracellularaccommodation of the rhizobia which reduces through theirnitrogenase activity the atmospheric nitrogen in ammonium, anitrogen form usable by the host plant.The molecular mechanisms underlying the symbiotic partnersrecognition, the infection process and the nodule organogenesis arewell described, however the identity establishment and maintenanceof this unique underground organ remain mis-understood.The Medicago truncatula NODULE-ROOT, the Arabidopsisthaliana BLADE-ON-PETIOLE and the Pisum sativumCOCHLEATA genes are members of a highly conserved NOOTBOP-COCH-LIKE1 (NBCL1) specific clade that belongs to theNON-EXPRESSOR OF PATHOGENESIS RELATED PROTEIN1-LIKE gene family. In legumes, the members of this NBCL1 cladeare known as key regulators of the symbiotic nodule identity.The present thesis work aims to better understand the roles of theNBCL1 genes, in both indeterminate and determinate nodule formingspecies and to discover new molecular actors involved in theNBCL1-dependent regulation of the nodule identity essentially usingnovel TILLING, Tnt1 and LORE1 insertional mutants in three legumespecies, Medicago, Pisum and Lotus.This thesis work has allowed the identification and thecharacterization of new mutants for genes belonging to a secondarylegume-specific NBCL2 sub-clade. We revealed that the members ofthis legume-specific NBCL2 sub-clade play important roles in noduledevelopment, identity establishment and maintenance, andconsequently in the success and in the efficiency of the symbioticassociation.This thesis work also shows that during evolution, the noduledevelopmental program has recruited pre-existing regulatorymechanisms for the nodule development and identity, such as theregulatory module involving interactions between NBCL proteins andTGACG type basic leucine zipper transcription factors. We identifiedthe transcription factor, MtPERIANTHIA-LIKE, as a first interactingpartner of NBCL proteins in a context of root nodule symbiosis.NBCL genes are also involved in the regulatory networks thatcontrol the development and the determinacy of many abovegroundvegetative and reproductive organs and were also shown as involvedin their abscission ability.In this thesis we also explored the roles of these highly conservedNBCL genes in the development of the non-domesticated grass,Brachypodium distachyon

Analyse des polypeptides obtenus par traduction in vitro d'arn messagers extraits de racines non nodulees, de nodosites fixatrices d'azote ou de nodosites non fixatrices. Etude de l'expression des genes codant pour les leghemoglobines et analyse de l'organisation de ces genes chez medicago sativa, des hybrides somatiques, et chez des legumineuses apparentees. Utilisation de sondes d'adn isolees d'autres especes de plantes pour suivre l'expression des genes codant pour des proteines connues pour jouer un role dans la symbiose (glutamine synthetase) ou les interactions plantes-microorganismes (phenylalanine ammonia-lyase, chalcone synthase, extensine)

La mutagenese par le transposon tn5 d'un fragment de 30 kb du megaplasmide psym de r. Meliloti, a permis de mettre en evidence 3 regions portant des genes de nodulation dont on etudie les proteines correspondantes. Le transfert a r. Trifolii d'un plasmide portant les genes nodfeg et nodh de r. Meliloti rend la souche hybride capable de noduler la luzerne mais inapte a noduler le trefle

by Zeng Naiyan.
"December 2004."
Thesis (Ph.D.)--Chinese University of Hong Kong, 2004.
Includes bibliographical references (p. 185-213)
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Mode of access: World Wide Web.
Abstracts in English and Chinese.

Phang Tsui-Hung.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2002.
Includes bibliographical references (leaves 146-162).
Abstracts in English and Chinese.
Thesis committee --- p.i
Statement --- p.ii
Abstract --- p.iii
Acknowledgement --- p.vi
Abbreviations --- p.viii
Table of contents --- p.xii
List of figures --- p.xviii
List of tables --- p.xx
Chapter 1. --- Literature Review --- p.1
Chapter 1.1 --- Salinity as a global problem --- p.1
Chapter 1.2 --- Formation of saline soil --- p.1
Chapter 1.3 --- Urgent need to reclaim saline lands --- p.2
Chapter 1.4 --- Cellular routes for Na+ uptake --- p.2
Chapter 1.4.1 --- Carriers involved in K+ and Na+ uptake --- p.2
Chapter 1.4.2 --- Channels involved in K+ and Na+ uptake --- p.4
Chapter 1.5 --- Adverse effects of high salinity --- p.5
Chapter 1.5.1 --- Hyperosmotic stress --- p.5
Chapter 1.5.2 --- Ionic stress --- p.6
Chapter 1.5.2.1 --- Deficiency of K+ --- p.6
Chapter 1.5.2.2 --- Perturbation of calcium balance --- p.7
Chapter 1.5.3 --- Toxicity of specific ions --- p.7
Chapter 1.5.4 --- Oxidative stress --- p.10
Chapter 1.6 --- Mechanisms of salt stress adaptation in plants --- p.11
Chapter 1.6.1. --- Maintenance of ion homeostasis --- p.12
Chapter 1.6.1.1 --- Regulation of cytosolic Na+ concentration --- p.12
Chapter 1.6.1.2 --- SOS signal transduction pathway --- p.15
Chapter 1.6.2 --- Dehydration stress adaptation --- p.17
Chapter 1.6.2.1 --- Aquaporins ´ؤ water channel proteins --- p.17
Chapter 1.6.2.2 --- Osmotic adjustment --- p.20
Chapter 1.6.2.2.1 --- Genetic engineering of glycinebetaine biosynthesis --- p.23
Chapter 1.6.2.2.2 --- Genetic engineering of mannitol biosynthesis --- p.27
Chapter 1.6.3 --- Morphological and structural adaptation --- p.28
Chapter 1.6.4 --- Restoration of oxidative balance --- p.29
Chapter 1.6.5 --- Other metabolic adaptation --- p.31
Chapter 1.6.5.1 --- Induction of Crassulacean acid (CAM) metabolism --- p.34
Chapter 1.6.5.2 --- Coenzyme A biosynthesis --- p.34
Chapter 1.7 --- Soybean as a target for studying salt tolerance --- p.36
Chapter 1.7.1 --- Economical importance of soybean --- p.36
Chapter 1.7.2 --- Reasons for studying salt stress physiology in soybeans --- p.38
Chapter 1.7.3 --- Salt tolerant soybean in China --- p.39
Chapter 1.7.4 --- Exploring salt tolerant crops by genetic engineering --- p.41
Chapter 1.8 --- Significance of this project --- p.47
Chapter 2. --- Materials and methods --- p.48
Chapter 2.1 --- Materials --- p.48
Chapter 2.1.1 --- Plant materials used --- p.48
Chapter 2.1.2 --- Bacteria strains and plasmid vectors --- p.48
Chapter 2.1.3 --- Growth media for soybean --- p.48
Chapter 2.1.4 --- Equipment and facilities used --- p.48
Chapter 2.1.5 --- Primers used --- p.48
Chapter 2.1.6 --- Chemicals and reagents used --- p.49
Chapter 2.1.7 --- Solutions used --- p.49
Chapter 2.1.8 --- Commercial kits used --- p.49
Chapter 2.1.9 --- Growth and treatment condition --- p.49
Chapter 2.1.9.1 --- Characterization of salt tolerance of Wenfeng7 --- p.49
Chapter 2.1.9.2 --- Samples for subtractive library preparations --- p.50
Chapter 2.1.9.3 --- Samples for slot blot and northern blot analyses --- p.50
Chapter 2.1.9.4 --- Samples for gene expression pattern analysis --- p.50
Chapter 2.2. --- Methods --- p.52
Chapter 2.2.1 --- Total RNA extraction --- p.52
Chapter 2.2.2 --- Construction of subtractive libraries --- p.53
Chapter 2.2.3 --- Cloning of salt-stress inducible genes --- p.53
Chapter 2.2.3.1 --- Preparation of pBluescript II KS(+) T-vector for cloning --- p.53
Chapter 2.2.3.2 --- Ligation of candidate DNA fragments with T-vector --- p.53
Chapter 2.2.3.3 --- Transformation --- p.54
Chapter 2.2.3.4 --- PCR screening --- p.54
Chapter 2.2.4 --- Preparation of recombinant plasmid for sequencing --- p.55
Chapter 2.2.5 --- Sequencing of differentially expressed genes --- p.55
Chapter 2.2.6 --- Homology search of differentially expressed genes --- p.56
Chapter 2.2.7 --- Expression pattern analysis --- p.56
Chapter 2.2.7.1 --- Preparation of single-stranded DIG-labeled PCR probes --- p.56
Chapter 2.2.7.2 --- Preparation of cRNA DIG-labeled probes --- p.57
Chapter 2.2.7.3 --- Testing the concentration of DIG-labeled probes --- p.58
Chapter 2.2.7.4 --- Slot blot --- p.58
Chapter 2.2.7.5 --- Northern blot --- p.59
Chapter 2.2.7.6 --- Hybridization --- p.60
Chapter 2.2.7.7 --- Stringency washed --- p.60
Chapter 2.2.7.8 --- Chemiluminescent detection --- p.60
Chapter 3. --- Results --- p.61
Chapter 3.1 --- Characterization of salt tolerance of Wenfeng7 --- p.61
Chapter 3.2 --- Identification of salt-stress induced genes from Wenfeng7 --- p.70
Chapter 3.2.1 --- Screening subtractive libraries of Wenfeng 7 for salt inducible genes --- p.70
Chapter 3.2.1.1 --- Results of homology search for salt inducible genes --- p.71
Chapter 3.2.1.2 --- Northern blot showing the salt inducibility of selected clones --- p.72
Chapter 3.3 --- Genes expression pattern of selected salt inducible genes --- p.104
Chapter 3.3.1 --- Expression of genes related to dehydration adjustment --- p.104
Chapter 3.3.1.1 --- Dehydration responsive protein RD22 (Clone no.: HML806) --- p.104
Chapter 3.3.1.2 --- Beta-amylase (Clone no.: HML767) --- p.104
Chapter 3.3.2 --- Expression of genes related to structural modification --- p.105
Chapter 3.3.3 --- Expression of genes related to metabolic adaptation --- p.105
Chapter 3.3.3.1 --- Subgroup 1: Gene related to protein synthesis --- p.105
Chapter 3.3.3.1.1 --- Translational initiation factor nsp45 (Clone no.: HML1042) --- p.105
Chapter 3.3.3.1.2 --- Seed maturation protein PM37 (Clone no.: HML931) --- p.106
Chapter 3.3.3.2 --- Subgroup 2: Genes related to phosphate metabolism (Clone no.: HML1000) --- p.107
Chapter 3.3.3.3 --- Subgroup 3: Genes related to storage and mobilization of nutrient resources --- p.107
Chapter 3.3.3.3.1 --- Vegetative storage protein A (Clone no.: HML762) --- p.107
Chapter 3.3.3.3.2 --- Cysteine proteinase (Clone no.: HML928) --- p.107
Chapter 3.3.3.4 --- Subgroup 4: Genes related to senescence --- p.109
Chapter 3.3.4 --- Expression of genes encoding novel protein (Clone no.: HML782) --- p.109
Chapter 4. --- Discussion --- p.125
Chapter 4.1 --- Evaluation of salt tolerance of Wenfeng7 --- p.125
Chapter 4.2 --- Isolation of salt inducible genes in Wenfeng7 --- p.127
Chapter 4.2.1 --- Genes associated with dehydration adaptation --- p.129
Chapter 4.2.1.1 --- Dehydration responsive protein RD22 --- p.129
Chapter 4.2.1.2 --- Beta-amylase --- p.130
Chapter 4.2.2 --- Genes associated with structural adaptation --- p.132
Chapter 4.2.3 --- Genes associated with metabolic adaptation --- p.133
Chapter 4.2.3.1 --- Subgroup 1: Genes related to protein synthesis --- p.133
Chapter 4.2.3.2 --- Subgroup 2: Genes related to phosphate metabolism --- p.137
Chapter 4.2.3.3 --- Subgroup 3: Genes related to storage and mobilization of nutrient resources --- p.138
Chapter 4.2.3.4 --- Subgroup 4: Genes related to senescence --- p.140
Chapter 4.2.4 --- Novel genes --- p.142
Chapter 4.3 --- Brief summary --- p.142
Chapter 5. --- Conclusion and perspectives --- p.144
References --- p.146
Appendix I: Screening for salt tolerant soybeans --- p.163
Appendix II: Major equipment and facilities used --- p.165
Appendix III: Major chemicals and reagents used in this research --- p.166
Appendix IV: Major common solutions used in this research --- p.168
Appendix V: Commercial kits used in this research --- p.170

Human society has benefited greatly from plant secondary metabolites, often utilizing a variety of compounds as dyes, food additives, and drugs. In particular, pharmaceutical development has benefited greatly from plant secondary metabolites. One example of this utility is paclitaxel, a highly substituted diterpene approved in the treatment of breast cancer, ovarian cancer, non-small cell lung cancer, and the AIDS-related Kaposi’s sarcoma. Demand of paclitaxel is likely to increase, due to the current examination of paclitaxel in numerous clinical trials against a variety of other cancers. Taxus cell culture represents a production source of paclitaxel to meet future demand. However, paclitaxel production through Taxus cell culture is often variable and low. Targeted metabolic engineering of Taxus to produce superior paclitaxel-accumulating lines is a viable strategy to address variable and low yields. To facilitate the production of genetically engineered Taxus cell lines, stable transformation is required to examine the long-term effect of gene expression in vitro. Additionally, suitable transient transformation systems are necessary to characterize novel Taxus genes related to paclitaxel accumulation. A transient particle bombardment-mediated transformation protocol was developed to introduce transgenes into Taxus cells in vitro. Additionally, agroinfiltration in Nicotiana benthamiana was examined as a system to express genes related to paclitaxel biosynthesis and lead to the accumulation of the first dedicated taxane, taxa-4(5), 11(12)-diene. In regard to stable transformation, an Agrobacterium -mediated transformation protocol was developed, though this method requires further optimization for reliability and increased transformation efficiency. These transformation technologies will aid in the creation of elite paclitaxel-accumulating Taxus cell lines.

Biotic stress is one of the most serious constraints on rice productivity. Strategy adopting regulators in signal transduction of systemic acquired resistance for conferring long-lasting disease resistance against broad spectrum of pathogens become highly favorable. To achieve this, signal transduction of disease resistance should be well characterized.
OsGPBP1 is a putative G-protein binding protein and interacts with a member of the YchF G-protein subfamily that has not been thoroughly studied in plants, while OsRHC1 is a novel RING zinc finger protein harboring multiple transmembrane domains at the N-half and a unique RING-HC domain at the C terminus. Both of them were induced in the bacterial blight resistant near isogenic rice line upon wounding. Gain-of-function tests in transgenic Arabidopsis thaliana showed that their ectopic expressions are able to trigger the expression of both defense marker genes mediated either by SA- or JA/ET-pathways and led to increased resistance toward the pathogen Pseudomonas syringae pv. tomato DC3000 and both of the two clones seemed to rely on NPR1 (disease resistance key regulator) for function. Furthermore, over-expressions of the two clones in its native system are also able to activate rice defense marker genes.
Suppression subtractive hybridization experiment, using RNA samples from a pair of near-isogenic rice lines either containing the R gene Xa14 (CBB14) or its susceptible recurrent parent (SN1033), were previously performed in our laboratory. Two gene candidates ( OsGPBP1 and OsRHC1) probably encoding two novel types of signal transduction components related to disease resistance are chosen for further analysis.
Cheung, Ming Yan.
"September 2007."
Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4555.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2007.
Includes bibliographical references (p. 148-168).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstracts in English and Chinese.
School code: 1307.

Shuk-man Chow.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2004.
Includes bibliographical references (leaves 171-186).
Abstracts in English and Chinese.
Thesis committee --- p.i
Statement --- p.ii
Abstract --- p.iii
Acknowledgement --- p.viii
General abbreviations --- p.x
Abbreviations of chemicals --- p.xi
List of figures --- p.xii
List of Tables --- p.xiii
Table of contents --- p.xv
Chapter 1. --- Literature review
Chapter 1.1. --- General introduction to rice disease --- p.1
Chapter 1.1.1. --- Pathogenesis of Bacterial Leaf Blight (BLB) --- p.1
Chapter 1.1.2. --- Pathogenesis of rice blast --- p.2
Chapter 1.1.3. --- Control of rice diseases --- p.3
Chapter 1.2. --- Plant defense mechanisms --- p.4
Chapter 1.2.1. --- Basal resistance in plants --- p.4
Chapter 1.2.2. --- Wound induced defense response --- p.5
Chapter 1.2.3. --- Pathogen induced host defense response --- p.6
Chapter 1.3. --- Structure of R gene products --- p.7
Chapter 1.4. --- Recognition between R and Avr proteins in rice --- p.8
Chapter 1.5 --- Current knowledge on Xa resistance and AvrXa avirulence protein --- p.9
Chapter 1.6 --- Current knowledge on Pi resistance and AvrPi avirulence protein --- p.10
Chapter 1.7 --- Pathogen induced signal transduction cascade --- p.12
Chapter 1.7.1. --- R gene mediated signal transduction cascade --- p.12
Chapter 1.7.2. --- Signal events of G-protein activation --- p.12
Chapter 1.7.3. --- Signaling events for the accumulation of Ca2+ in cytosol --- p.13
Chapter 1.7.4. --- Signaling events for oxidative burst --- p.14
Chapter 1.7.5. --- MAPK cascade in defense signaling --- p.15
Chapter 1.7.6. --- Transcriptional regulation of disease resistance related genes --- p.16
Chapter 1.7.7. --- Translational regulation of disease resistance related genes --- p.17
Chapter 1.8. --- Defense responses and defense related genes --- p.19
Chapter 1.8.1. --- Pathogenesis related (PR) proteins --- p.20
Chapter 1.8.2. --- Phytoalexins --- p.21
Chapter 1.9. --- Disease resistance related genes common between rice blast and BLB resistance --- p.22
Chapter 1.10. --- SA induced signal transduction pathway in rice --- p.23
Chapter 1.11. --- Important tools facilitating the identification of disease resistance related genes from BLB resistant rice lines --- p.24
Chapter 1.12. --- Hypothesis --- p.26
Chapter 1.13. --- Project objective --- p.26
Chapter 2. --- Materials and Methods --- p.27
Chapter 2.1. --- Plant Materials --- p.27
Chapter 2.2. --- Pathogen Inoculation --- p.27
Chapter 2.3. --- RNA extraction --- p.29
Chapter 2.4. --- Denaturing gel electrophoresis --- p.29
Chapter 2.5. --- Subtraction libraries construction --- p.30
Chapter 2.5.1. --- Cloning of disease resistance related genes --- p.32
Chapter 2.5.1.1. --- pBluescript II KS (+) T-vector preparation --- p.32
Chapter 2.5.1.2. --- Ligation --- p.32
Chapter 2.5.1.3. --- Transformation --- p.32
Chapter 2.5.1.4. --- Colony picking --- p.33
Chapter 2.5.1.5. --- PCR amplification of DNA inserts --- p.33
Chapter 2.5.1.6. --- Purification of PCR products --- p.34
Chapter 2.6. --- Gene chips printing --- p.34
Chapter 2.7. --- Probes synthesis and gene chips hybridization --- p.35
Chapter 2.8. --- Standard-RNAs synthesis --- p.35
Chapter 2.9. --- Data collection and analysis --- p.36
Chapter 2.10. --- Sequencing --- p.36
Chapter 2.11. --- cDNA synthesis --- p.37
Chapter 2.12. --- RT-PCR --- p.38
Chapter 2.13. --- DNA gel electrophoresis --- p.39
Chapter 3. --- Results --- p.58
Chapter 3.1. --- Construction of BLB gene chips --- p.58
Chapter 3.1.1. --- Preparation of cDNA clones for gene chips construction --- p.58
Chapter 3.1.2. --- Purification of PCR products on microtiter plate --- p.59
Chapter 3.1.3. --- Gene chips construction --- p.59
Chapter 3.1.4. --- DNA immobilization --- p.62
Chapter 3.1.5. --- Probe synthesis --- p.62
Chapter 3.1.6. --- Gene chip analysis --- p.65
Chapter 3.1.6.1. --- Scanning --- p.65
Chapter 3.1.6.2. --- Data analysis --- p.65
Chapter 3.2. --- "Identification of disease resistance related genes commonly regulated by Xa2, Xal2 and Xal4 BLB resistance loci" --- p.70
Chapter 3.2.1. --- "Signal perception, transduction and regulatory elements" --- p.71
Chapter 3.2.1.1. --- Proteins involved in reversible phosphorylation cascade --- p.71
Chapter 3.2.1.2. --- Proteins potentiate signal transduction through specific protein-protein interaction --- p.72
Chapter 3.2.1.3. --- Other signal transduction components --- p.73
Chapter 3.2.2. --- Transcriptional and translational regulatory elements --- p.74
Chapter 3.2.2.1. --- Proteins involved in transcriptional regulation --- p.74
Chapter 3.2.2.2. --- Proteins involved in post-transcriptional regulation --- p.75
Chapter 3.2.2.3. --- Proteins involved in translational regulation --- p.76
Chapter 3.2.3. --- "Oxidative burst, stress, apoptotic related genes" --- p.77
Chapter 3.2.3.1. --- Stress related proteins --- p.77
Chapter 3.2.3.2. --- Proteins involved in induction of oxidative burst --- p.78
Chapter 3.2.3.3. --- PR proteins --- p.79
Chapter 3.2.3.4. --- Proteolysis related proteins --- p.79
Chapter 3.2.4. --- Cell maintenance and metabolic genes --- p.80
Chapter 3.2.4.1. --- Antioxidant --- p.80
Chapter 3.2.4.2. --- Metabolic genes --- p.81
Chapter 3.2.4.3. --- Molecular chaperone --- p.82
Chapter 3.2.4.4. --- Cell cycle regulators --- p.82
Chapter 3.2.4.5. --- Cell wall maintenance --- p.83
Chapter 3.2.4.6. --- Proteins involved in protein transport --- p.83
Chapter 3.2.5. --- Unclassified/others --- p.84
Chapter 3.3. --- Expression analysis of disease resistance related genes --- p.88
Chapter 4. --- Discussion --- p.141
Chapter 4.1. --- Differential expression of disease resistance candidates --- p.141
Chapter 4.2. --- Disease resistance signal transduction components --- p.143
Chapter 4.2.1. --- Reversible phosphorylation cascade --- p.143
Chapter 4.2.2. --- Signal transduction potentiated by protein-protein interaction --- p.144
Chapter 4.3. --- Other signaling molecules --- p.145
Chapter 4.3.1. --- PRL1-interacting factor G --- p.145
Chapter 4.3.2. --- Vacuolar-type H+-ATPasen subunit G --- p.146
Chapter 4.4. --- Regulation of expression of disease resistance candidates --- p.146
Chapter 4.4.1. --- Transcriptional regulation of disease resistance related genes --- p.146
Chapter 4.4.1.1. --- G-box binding protein --- p.147
Chapter 4.4.1.2. --- MYB TF --- p.147
Chapter 4.4.2. --- Post-transcriptional modification of disease resistance candidates --- p.148
Chapter 4.4.2.1. --- RNA splicing factor --- p.148
Chapter 4.4.2.2. --- Glycine rich RNA binding proteins --- p.149
Chapter 4.4.3. --- Translational regulation of disease resistance related genes --- p.149
Chapter 4.5. --- Induction of oxidative burst --- p.150
Chapter 4.6. --- PR proteins --- p.151
Chapter 4.7. --- Cell maintenance --- p.152
Chapter 4.7.1. --- Protein folding --- p.152
Chapter 4.7.2. --- Protein degradation --- p.153
Chapter 4.7.3. --- ROS scavenging --- p.154
Chapter 4.7.4. --- Regulation of cell cycle --- p.154
Chapter 4.8. --- "Confirmation and profiling of disease resistance related candidates commonly regulated in Xa2, Xal2 and Xal4 BLB resistance NILs at different time points" --- p.155
Chapter 4.8.1. --- Basal resistance related genes --- p.156
Chapter 4.8.2. --- General disease resistance related genes --- p.161
Chapter 4.8.3. --- Pathogen responsive genes --- p.164
Chapter 4.8.4. --- Prediction of novel genes functions --- p.168
Chapter 4.9. --- Future prospect --- p.169
Chapter 4.10. --- Conclusion --- p.169
References --- p.171
Appendix --- p.187

Les trichothécènes de Fusarium appartiennent au groupe des sesquiterpènes qui sont des inhibiteurs la synthèse des protéines des eucaryotes. Les trichothécènes causent d’une part de sérieux problèmes de santé aux humains et aux animaux qui ont consommé des aliments infectés par le champignon et de l’autre part, elles sont des facteurs importants de la virulence chez plantes. Dans cette étude, nous avons isolé et caractérisé seize isolats de Fusarium de la pomme de terre infectée naturellement dans un champs. Les tests de pathogénicité ont été réalisés pour évaluer la virulence des isolats sur la pomme de terre ainsi que leur capacité à produire des trichothécènes. Nous avons choisi F. sambucinum souche T5 comme un modèle pour cette étude parce qu’il était le plus agressif sur la pomme de terre en serre en induisant un flétrissement rapide, un jaunissement suivi de la mort des plantes. Cette souche produit le 4,15-diacétoxyscirpénol (4,15-DAS) lorsqu’elle est cultivée en milieu liquide. Nous avons amplifié et caractérisé cinq gènes de biosynthèse trichothécènes (TRI5, TRI4, TRI3, TRI11, et TRI101) impliqués dans la production du 4,15-DAS. La comparaison des séquences avec les bases de données a montré 98% et 97% d'identité de séquence avec les gènes de la biosynthèse des trichothécènes chez F. sporotrichioides et Gibberella zeae, respectivement. Nous avons confrenté F. sambucinum avec le champignon mycorhizien à arbuscule Glomus irregulare en culture in vitro. Les racines de carotte et F. sambucinum seul, ont été utilisés comme témoins. Nous avons observé que la croissance de F. sambucinum a été significativement réduite avec la présence de G. irregulare par rapport aux témoins. Nous avons remarqué que l'inhibition de la croissance F. sambucinum a été associée avec des changements morphologiques, qui ont été observés lorsque les hyphes de G. irregulare ont atteint le mycélium de F. sambucinum. Ceci suggère que G. irregulare pourrait produire des composés qui inhibent la croissance de F. sambucinum. Nous avons étudié les patrons d’expression des gènes de biosynthèse de trichothécènes de F. sambucinum en présence ou non de G. irregulare, en utilisant le PCR en temps-réel. Nous avons observé que TRI5 et TRI6 étaient sur-exprimés, tandis que TRI4, TRI13 et TRI101 étaient en sous-exprimés en présence de G. irregulare. Des analyses par chromatographie en phase-gazeuse (GC-MS) montrent clairement que la présence de G. irregulare réduit significativement la production des trichothécènes par F. sambucinum. Le dosage du 4,15-DAS a été réduit à 39 μg/ml milieu GYEP par G. irregulare, comparativement à 144 μg/ml milieu GYEP quand F. sambucinum est cultivé sans G. irregulare. Nous avons testé la capacité de G. irregulare à induire la défense des plants de pomme de terre contre l'infection de F. sambucinum. Des essais en chambre de croissance montrent que G. irregulare réduit significativement l’incidence de la maladie causée par F. sambucinum. Nous avons aussi observé que G. irregulare augmente la biomasse des racines, des feuilles et des tubercules. En utilisant le PCR en temps-réel, nous avons étudié les niveaux d’expression des gènes impliqué dans la défense des plants de pommes de terre tels que : chitinase class II (ChtA3), 1,3-β-glucanase (Glub), peroxidase (CEVI16), osmotin-like protéin (OSM-8e) et pathogenèses-related protein (PR-1). Nous avons observé que G. irregulare a induit une sur-expression de tous ces gènes dans les racines après 72 heures de l'infection avec F. sambucinum. Nous avons également trové que la baisse provoquée par F. sambucinum des gènes Glub et CEVI16 dans les feuilles pourrait etre bloquée par le traitement AMF. Ceci montre que l’inoculation avec G. irregulare constitut un bio-inducteur systémique même dans les parties non infectées par F. sambucinum. En conclusion, cette étude apporte de nouvelles connaissances importantes sur les interactions entre les plants et les microbes, d’une part sur les effets directs des champignons mycorhiziens sur l’inhibition de la croissance et la diminution de la production des mycotoxines chez Fusarium et d’autre part, l’atténuation de la sévérité de la maladie dans des plantes par stimulation leur défense. Les données présentées ouvrent de nouvelles perspectives de bio-contrôle contre les pathogènes mycotoxinogènes des plantes.
Fusarium trichothecenes are a large group of sesquiterpenes that are inhibitors of eukaryotic protein synthesis. They cause health problems for humans and animals that consume fungus-infected agricultural products. In addition some of Fusarium trichothecenes are virulence factors of plant pathogenesis. In this study, sixteen Fusarium strains were isolated and characterized from naturally infected potato plants. Pathogenicity tests were carried out to evaluate the virulence of these isolates on potato plants and their trichothecene production capacity. We chose F. sambucinum strain T5 as a model for this study because it was the most aggressive strain when tested on potato plants. It induces a rapid wilting and yellowing resulting in plant death. This strain produced 4,15-diacetoxyscirpenol (4,15-DAS) when grown in liquid culture. We amplified and characterized five trichothecene genes (TRI5, TRI4, TRI3, TRI11, and TRI101) involved in the production of 4,15-DAS. Nucleotide BLAST search showed 98% and 97% sequence identity with trichothecene biosynthetic genes of F. sporotrichioides and Gibberella zeae, respectively. We used F. sambucinum to determine if trichothecene gene expression was affected by the symbiotic arbuscular mycorrhizal fungus (AMF) Glomus irregulare. We found that the growth of F. sambucinum was significantly reduced in the presence of G. irregulare isolate DAOM-197198 compared with controls that consisted of carrot roots without G. irregulare or F. sambucinum alone. Furthermore, inhibition of the growth F. sambucinum was associated with morphological changes, which were observed when G. irregulare hyphae reached F. sambucinum mycelium, suggesting that G. irregulare may produce compounds that interfere with the growth of F. sambucinum. Using real-time qRT-PCR assays, we assessed the relative expression of trichothecene genes of F. sambucinum confronted or not with G. irregulare. When G. irregulare was confronted with F. sambucinum, TRI5 and TRI6 genes were up-regulated, while TRI4, TRI13 and TRI101 were down-regulated. We therefore used GC-MS analysis to determine whether G. irregulare affects trichothecene production by F. sambucinum. We found that the production of 4,15-DAS trichothecene was significantly reduced in the presence of G. irregulare compared with controls that consisted of carrot roots without G. irregulare or F. sambucinum alone. Interestingly, 4,15-DAS pattern was reduced to 39 μg/ml GYEP medium by G. irregulare compared to 144 μg/ml GYEP with F. sambucinum grown with carrot roots or F. sambucinum alone respectively. We tested the AMF capacity to induce defense responses of potato plants following infection with F. sambucinum. The response of AMF-colonized potatoes to F. sambucinum was investigated by tracking the expression of genes homologous with pathogenesis-related proteins chitinase class II (ChtA3), 1,3-β-glucanase (gluB), peroxidase (CEVI16), osmotin-like protein (OSM-8e) and pathogenesis-related protein (PR-1). We found that the AMF treatment up-regulated the expression of all defense genes in roots at 72 hours post-infection (hpi) with F. sambucinum. We also found that a decrease provoked by F. sambucinum in gluB and CEVI16 expression in shoots could be blocked by AMF treatment. Overall, a differential regulation of PR homologues genes in shoots indicates that AMF are a systemic bio-inducer and their effects could extend into non-infected parts. In conclusion, this study provides new insight into on the interactions between plants and microbes, in particular the effects of AMF on the growth and the reduction of mycotoxins in Fusarium. It also shows that AMF are able to reduce the disease severity in plants by stimulating their defense. The data presented provide new opportunities for bio-control against mycotoxin-producing pathogens in plants.

When tomato Craigella is infected with Verticillium dahliae Dvd-E6 (Dvd-E6), a tolerant state is induced with substantial pathogen load, but few symptoms. Unexpectedly, these plants are more robust and taller with Dvd-E6 behaving as an endophyte. Some endophytes can protect plants from virulent pathogens. This research was undertaken to improve understanding of the cellular and molecular nature of Verticillium tolerance in tomato, especially whether infection by Dvd-E6 can protect Craigella from virulent V. dahliae, race 1 (Vd1). To permit mixed infection experiments a restriction fragment length polymorphism (RFLP)-based assay was developed and used for differentiating Dvd-E6 from Vd1, when present in mixed infections. The results suggested that protection involves molecular interplay between Dvd-E6 and Vd1 in susceptible Craigella (CS) tomatoes, resulting in restricted Vd1 colonization. Further studies showed a dramatic reduction of Vd1 spores and mycelia. To examine genetic changes that account for these biological changes, a customized DNA chip (TVR) was used to analyze defense gene mRNA levels. The defense gene response was categorized into four groups. Group 1 was characterized by strong induction of defense genes followed by suppression. However, Vd1-induced gene suppression was blocked by Dvd-E6 in mixed infections. These genes included some transcription factors and PR proteins such as class IV chitinases and beta glucanases which are known to target fungal spores and mycelia. Experiments also were repeated with a Craigella resistant (CR) isoline containing a fully active Ve locus (Ve1+ and Ve2+). The biological results showed that the presence of the Ve1+ allele resulted in restricted Vd1 colonization and, in a mixed infection with Dvd-E6, Vd1 was completely eliminated from the plant stem. Surprisingly, there was no significant increase in defense gene mRNAs. Rather, elevated basal levels of defense gene products appeared sufficient to combat pathogen attack. To investigate functional effects of the genetic changes observed, an inducible RNAi knockdown vector for a defense gene (TUS15G8) with unknown function (pMW4-TUS15G8) as well as the Ve2 resistance gene (pMW-Ve2) was prepared as a initial step for future transformation analyses. Taken together the results reveal intriguing but complex biological and molecular changes in mixed infections, which remain a basis for future experiments and potential agricultural benefits.
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