Oomycetes are fungi-like eukaryotic microorganisms, which are actually phylogenetic relatives of diatoms and brown algae, within the kingdom Stramenopila. Many oomycete species, mainly in the genera Phytophthora, Pythium and downy mildews, are devastating plant pathogens that cause multibillion-dollar losses to agriculture annually in the world. Some oomycetes are also animal pathogens, causing severe losses in aquaculture and fisheries, and occasionally causing dangerous infections of humans. Phytophthora species, represented by the Irish Potato Famine pathogen P. infestans and the soybean pathogen P. sojae, are arguably the most destructive pathogens of dicotyledonous plants among the oomycete species and thus have been extensively studied. This dissertation focuses on the model oomycete pathogen P. sojae to investigate specific aspects of its molecular biology and establish an efficient genetic manipulation tool. Specifically, in Chapter 1, I briefly introduce the basic concepts of oomycete biology and pathology, and summarize the experimental techniques used for studies of oomycete genetics over the past two decades. Because the approach to studying fungi and oomycetes are similar (indeed they were incorrectly placed in the same taxonomic group until recently), a special section reviews the emerging genome editing technology CRISPR/Cas system in these organisms together. Chapter 2 and Chapter 3 focus on one of the most important intracellular activities, nuclear localization of proteins, and describe the characterization of nuclear localization signals (NLSs) in P. sojae. This focus stemmed from my early work on genome editing in P. sojae, when I discovered that conventional NLS signals from SV40 used to target the TAL effector nuclease (TALEN) to the nucleus worked poorly in P. sojae. In the first part of this work (Chapter 2), I used confocal microscopy to identify features of nuclear localization in oomycetes that differ from animals, plants and fungi, based on characterization of two classes of nuclear localization signals, cNLS and PY-NLS, and on characterization of several conserved nuclear proteins. In the second part (Chapter 3), I determined that the nuclear localization of the P. sojae bZIP1 transcription factor is mediated by multiple weak nuclear targeting motifs acting together. In Chapter 4 and Chapter 5, I describe my implementation of nuclease-based technology for genetic modification and control of P. sojae. In Chapter 4, I describe the first use of the CRISPR system in an oomycete, including its use to validate the function of a host specificity gene. This is of particular importance because molecular techniques such as gene knockouts and gene replacements, widely used in other organisms, were not previously possible in oomycetes. The successful implementation of CRISPR provides a major new research capability to the oomycete community. Following up on the studies described in Chapter 4, in Chapter 5, I describe the generalization and simplification of the CRISPR/Cas9 expression strategy in P. sojae as well as methods for mutant screening. I also describe several optimized methodologies for P. sojae manipulation based on my 5 years of experience with P. sojae. / Ph. D.

L’une des principales maladies attaquant le soya est la pourriture phytophthoréenne, causée par l’agent pathogène Phytophthora sojae. La méthode de lutte la plus efficace à ce jour pour contrer cet agent pathogène est la lutte génétique. Des gènes de résistance (Rps) se trouvant naturellement dans certaines lignées de soya sont introgressés dans des cultivars ayant un attrait pour l’agriculture. Cependant, pour définir quel gène Rps utiliser, il est essentiel de connaître les pathotypes de P. sojae se trouvant dans le sol, puisque les gènes Rps reconnaissent les gènes Avr caractérisant les différents pathotypes. Actuellement, les méthodes d’identification des nombreux pathotypes de l’agent pathogène sont des techniques de phénotypage longues et parfois imprécises. Cette étude présente donc le premier outil moléculaire ayant pour but de diagnostiquer rapidementet précisément les pathotypes de P. sojaese trouvant dans un échantillon de sol ou de tissus végétaux infecté. Une étude exhaustive de 31 isolats de P. sojae préalablement réalisée a permis d’identifier des marqueurs discriminants entre les haplotypes de virulence et d’avirulence pour les sept principaux gènes Avr retrouvés en Amérique. Des amorces spécifiques aux différents marqueurs ont été créées. Elles ont par la suite été adaptées afin de pouvoir être utilis.es simultanément dans une PCR multiplexe. Un taux d’efficacité à identifier les gènes d’avirulence présents chez différents isolats de P. sojae de 96% a été atteint lors de l’étude, Avr3a étant le seul gène à présenter des résultats aléatoires. Cela a donc ouvert la porte à d’éventuelles études plus approfondies sur l’interaction entre les gènes Rps3a et Avr3a. Le test sera de plus un outil précieux dans la prise de décision du cultivar à semer pour les producteurs, qui auront désormais accès à plus d’informations quant aux souches de P. sojaese trouvant dans leurs champs.

Call number: LD2668 .T4 1984 C87 / Master of Science

The root epidermis, being the outermost cell layer of the organ, is in contact with the soil environment. The position of the epidermis determines its important roles, such as taking up water and ions from the surrounding soil, and defending against harmful microorganisms. What is the chemical composition of the walls in this layer? The chemical nature of the soybean epidermal wall modifying substance was investigated in this study with the use of histochemical tests coupled with electron microscopy, and chemical depolymerizations in combination with chromatography. Soybean (<em>Glycine max</em>) was used as a test species in the present studay. Results of histochemical and electron microscopical studies indicated that the epidermal walls are modified with suberin. The suberized epidermal walls were permeable to apoplastic tracers, differing from those of cells with suberized Casparian bands, possibly due to the spatial distribution or chemical components of the suberin. Suberin may occur in a diffuse form linked with other wall components in the epidermis. What is the chemical nature of this modification, and does it play a role in pathogen resistance? The root epidermal wall compositions of two soybean cultivars were compared; one (cv. Conrad) is resistant to <em>Phytophthora sojae</em> and the other (cv. OX 760-6) is susceptible to this root-rot oomycete. Their epidermal walls were isolated enzymatically and subjected to two different degradation methods, i. e. BF₃-MeOH transesterification and nitrobenzene oxidation. The compositions of depolymerisates of the cell walls determined by GC-MS indicated four dominant suberin monomers varying in chain length from C16 to C24. In all epidermal cell walls, &omega;-hydroxycarboxylic acids were more abundant than diacids, carboxylic acids and alcohols. Two of the monomers detected (hydroxycarboxylic acid and a,&omega;-dicarboxylic acid) are known to be characteristic suberin markers. The quantitative chemical compositions significantly differed in the epidermal cell walls of the two soybean varieties. Walls of the resistant cultivar (Conrad) had a greater quantity of both the aliphatic and aromatic components of the polymer than the susceptible cultivar (OX760-6), providing evidence to support the hypothesis that preformed suberin plays a role in plant defense.

The root epidermis, being the outermost cell layer of the organ, is in contact with the soil environment. The position of the epidermis determines its important roles, such as taking up water and ions from the surrounding soil, and defending against harmful microorganisms. What is the chemical composition of the walls in this layer? The chemical nature of the soybean epidermal wall modifying substance was investigated in this study with the use of histochemical tests coupled with electron microscopy, and chemical depolymerizations in combination with chromatography. Soybean (<em>Glycine max</em>) was used as a test species in the present studay. Results of histochemical and electron microscopical studies indicated that the epidermal walls are modified with suberin. The suberized epidermal walls were permeable to apoplastic tracers, differing from those of cells with suberized Casparian bands, possibly due to the spatial distribution or chemical components of the suberin. Suberin may occur in a diffuse form linked with other wall components in the epidermis. What is the chemical nature of this modification, and does it play a role in pathogen resistance? The root epidermal wall compositions of two soybean cultivars were compared; one (cv. Conrad) is resistant to <em>Phytophthora sojae</em> and the other (cv. OX 760-6) is susceptible to this root-rot oomycete. Their epidermal walls were isolated enzymatically and subjected to two different degradation methods, i. e. BF₃-MeOH transesterification and nitrobenzene oxidation. The compositions of depolymerisates of the cell walls determined by GC-MS indicated four dominant suberin monomers varying in chain length from C16 to C24. In all epidermal cell walls, &omega;-hydroxycarboxylic acids were more abundant than diacids, carboxylic acids and alcohols. Two of the monomers detected (hydroxycarboxylic acid and a,&omega;-dicarboxylic acid) are known to be characteristic suberin markers. The quantitative chemical compositions significantly differed in the epidermal cell walls of the two soybean varieties. Walls of the resistant cultivar (Conrad) had a greater quantity of both the aliphatic and aromatic components of the polymer than the susceptible cultivar (OX760-6), providing evidence to support the hypothesis that preformed suberin plays a role in plant defense.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2016-04-20T08:03:41Z No. of bitstreams: 1 texto completo.pdf: 351964 bytes, checksum: f91834fae1f8b64d3fed68307f2774b1 (MD5) / Made available in DSpace on 2016-04-20T08:03:42Z (GMT). No. of bitstreams: 1 texto completo.pdf: 351964 bytes, checksum: f91834fae1f8b64d3fed68307f2774b1 (MD5) Previous issue date: 2015-08-10 / A podridão radicular de fitóftora (PRF) é uma das doenças mais agressivas para a cultura da soja, causando danos que podem levar a morte das plantas. O uso de cultivares resistentes é a principal estratégia para redução das perdas causadas por Phytophthora sojae. Por este motivo, estudos de identificação de genes e QTLs (Quantitative Trait Loci) associados à resistência parcial à PRF e associação de marcadores moleculares ligados à estes são de extrema importância. Neste trabalho foi realizada a genotipagem ampla de 68 cultivares de soja utilizando 5353 marcadores SNPs (Single Nucleotide Polymorphism), objetivando encontrar marcadores associados a resistência parcial de soja à Phytophthora sojae através de metodologias de genética de associação. Como resultado, neste trabalho foram localizados via metodologia de modelos lineares mistos (MLM) 23 marcadores candidatos associados à resistência parcial à PRF, dispostos em 9 cromossomos e, dentre estes marcadores, destacou-se o Gm16_29528259_T_C, por ser o com maior valor de associação (-log10(p)=2,9175) e os marcadores Gm05_8656389_T_C e Gm05_8695812_A_G, que apresentaram os maiores valores de r3 (0,18769), explicando, individualmente, 18,769% da variação fenotípica. Via metodologia de stepwise foram localizados 5 marcadores candidatos associados à resistência parcial à PRF (Gm10_37469103_C_A, Gm07_5417760_T_C, Gm11_37106045_C_T, Gm09_41620640_G_T e Gm09_45586982_T_C), dispostos em 4 cromossomos. Estes marcadores juntos obtiveram alto valor de r2 (0,6884), explicando 68,84% da variação fenotípica. Os marcadores SNPs associados à resistência parcial à podridão radicular de fitóftora estão distribuídos em dez grupos de ligação, sendo que, os grupos O, M, B1, A1 e A2, ainda não foram descritos como portadores de regiões controladoras da resistência. Os marcadores candidatos associados à resistência parcial à PRF localizados neste trabalho, após devidamente validados, podem ser utilizados para estabelecer um eficiente programa de seleção assistida por marcadores moleculares para resistência parcial de soja à PRF. / Phytopthora root and stem rot (PRSR) is one of the most aggressive diseases for the soybean crop, causing damage that can cause plant death. The uses of resistant cultivars is the main strategy to reduce losses cause by Phytophthora sojae. For this reason, studies for identifying genes or QTLs (Quantitative Trait Loci) associated with the partial resistance between this disease and the association of molecular markers linked to these genes or QTLs are paramount. In this research was carried out wide genotyping of 68 soybean cultivars using 5353 SNPs (Single Nucleotide Polymorphism) markers. The objective of this research was to identify markers associated with partial resistance to Phytophthora sojae, in soybeans through genetic screening methodology. Were located in the mixed linear models (MLM) methodology 23 candidate markers associated with partial resistance to PRSR, arranged on 9 chromosomes. The SNP Gm16_29528259_T_C stands out for being the higher value of association (-log10(p)=2,9175), the SNPs Gm05_8656389_T_C and Gm05_8695812_A_G showed the highest r2 values (0,18769), explained individually 18,769% of the phenotypic variation. Using stepwise methodology 5 markers associated (Gm10_37469103_C_A, with partial resistance to Gm07_5417760_T_C, PRST were located Gm11_37106045_C_T, Gm09_41620640_G_T and Gm09_45586982_T_C), arranged on four chromosomes. These markers together achieve high r2 value (0,6884), explaining 68,84% of the phenotypic variation. SNPs markers associated with partial resistance to PRSR are spread in ten linkage groups, wherein then groups O, F, B1, A1 and A2 have not been described as having the resistance controlling regions. The candidates markers associated with partial resistance to PRSR located in this research, after being properly validated, can be used to establish and effective marker-assisted selection (MAS) program for partial resistance to PRSR in soybean. / Sem lattes e agência de fomento

No description available.

<p>Phytophthora root and stem rot (PRSR), caused by oomycete <i>Phytophthora sojae</i>, is the most severe soil-borne disease of soybean (<i>Glycine max</i> (L.) Merr.) worldwide. The disease can be effectively managed by introducing resistance to <i>P. sojae</i> (<i>Rps</i>) genes into soybean cultivars by breeding, which requires continuous efforts on identification of resistance resources from soybean germplasm. Previously, two resistance genes, <i>Rps2-cas</i> (former name <i>Rps2-das</i>) and <i>Rps14 </i>(former name<i> Rps1-f</i>), were mapped by linkage analysis from soybean landraces, PI 594549 C and PI 340029, respectively. The resistance underlying PI 594592 also need further characterization given its broad resistance spectrum. In this study, <i>Rps-2cas</i> and <i>Rps14</i> were further mapped, and <i>Rps2-b</i>, was identified and initial mapped from PI 594592. Thus, this thesis research was divided into three parts for three <i>Rps</i> genes.</p><p>The first part mainly focuses advances on <i>Rps2-cas</i>. Marker-assisted spectrum analysis was performed for <i>Rps-2cas</i> to confirm its potential in disease management. A high-quality genome assembly of PI 594549 C was generated, and KASP markers were developed based on comparison between new reference and Williams 82 reference genome. The gene was further mapped to a 32.67-kb region on PI 594549 C reference genome harboring three expressed NLRs by 24 recombinants screened from a large F₄ population. Comparative genomics analysis suggests the only intact NBS-LRR gene in the fine mapping region is the best candidate gene for <i>Rps2cas</i>, and its function was validated by stable transformation. Evidences from other high-quality assembly genomes suggest <i>Rps2-cas</i> originated from an ancient unequal crossing over event.</p> <p>In the second part, <i>Rps14</i> was further mapped using 21 recombinants identified from a F₃population consisting of 473 plants. In commonly used Williams 82 reference genome, the assembly of fine mapping region was incomplete, and <i>Rps14</i> region showed drastic variation in size and copy number of NLRs in 23 high-quality genome assemblies, suggesting the complexity of <i>Rps14</i> region and high-quality reference sequence of donor line is required for isolation of <i>Rps14</i> candidate genes. Marker assisted resistance test showed <i>Rps14</i> had wider resistance spectrum to different <i>P. sojae </i>isolates comparing to other <i>Rps</i> genes on chromosome 3, and phylogenic analysis further supported the potential of <i>Rps14</i> to be a novel resistance gene. </p> <p>For the third part, an F₂population derived from a cross between PI 594592 and Williams was tested by <i>P. sojae</i> race 1. The 3:1 and 1:2:1 Mendelian segregation ratios were observed in F₂individuals and F_2:3families, respectively, suggesting a single dominant <i>Rps</i> gene in PI 594592. The gene was initially mapped to the distal end chromosome 16 overlapped with <i>Rps2</i>, and the gene was tentatively named as <i>Rps2-b</i>. Polymorphic SSR markers and InDel markers designed based on re-sequencing data of PI 594592 and Williams was used to genotyping all the F_2:3families, and a linkage map was constructed for <i>Rps2-b</i>. <i>Rps2-b</i> was mapped to a 461.8-kb region flanked by SSR marker Satt431 and InDel marker InDel3668 according to the reference genome (Wm82. a2). Marker-assisted resistance test showed <i>Rps2-b</i> hold a wide resistance spectrum. </p>

Phytophthora root and stem rot is the most destructive soybean soil-borne disease worldwide and can be managed using soybean cultivars with genes conferring resistance to <i>Phytophthora sojae</i>. Here we show that soybean <i>Rps11</i> is an ~27-kb nucleotide-binding site leucine-rich repeat (NLR) gene that confers broad-spectrum resistance to the pathogen. This giant gene is located in a genomic region containing 12 unusually large NLR genes of a single origin and was formed by rounds of intergenic/intragenic unequal recombination that involves the promoter regions and the LRR regions. Comparison of the genomic region in the Rps11 donor line with its corresponding regions in 29 diverse soybean genomes revealed drastic regional diversification including NLR copy number variation ranging from 5 to 23, and absence of allelic copy of <i>Rps11</i> in all 29 genomes. This study highlights innovative evolution and complexity of an NLR cluster and enables precise selection of <i>Rps11</i> for cultivar improvement.

L'objectif de ce projet visait à déterminer si le silicium (Si), reconnu pour son effet prophylactique contre les agents biotrophes, pouvait protéger le soja contre Phytophthora sojae, un oomycète hemi-biotrophe. Le défi initial consistait à développer une méthode d'inoculation qui reproduisait le processus d'infection naturelle tout en permettant aux plants d'absorber le Si. Dans un premier temps, l'inoculation de zoospores dans une solution hydroponique a permis de générer des infections reproductibles conséquentes avec les réponses phénotypiques attendues. Dans un deuxième temps, l'ajout de Si a permis de réduire significativement l'incidence de la maladie tout en augmentant le rendement des plants. Cet effet était surtout manifeste sur les cultivars possédant un niveau de résistance contre P. sojae ou sur ceux absorbant plus de Si. Nous proposons ainsi une nouvelle méthode d'inoculation de P. sojae représentative de l'infection naturelle et démontrons que le Si peut servir pour la lutte contre cet oomycète.