The mop2 gene is required for epigenetic silencing; it was originally defined as a mutation, Mop2-1, which when dominant prevented paramutation at b1. Paramutation is an allele communication that causes a mitotically and meiotically heritable change in gene expression. Mop2-1 was subsequently shown to be involved in maintaining the silenced paramutant state and to prevent dsRNA-mediated transcriptional gene silencing (activities revealed only when the mutation is homozygous). Understanding the product encoded by mop2 will help dissect the underlying mechanisms involved in paramutation and dsRNA-mediated transcriptional silencing. This dissertation describes map-based cloning and candidate gene approaches directed toward the eventual goal of identification of mop2.Initial mapping of mop2 placed it within a region delineated by the markers umc1823 and eks1. On the maize physical map this region contains 21 BAC (Bacteria Artificial Chromosome) clones, representing 2.9 Mb. Skim sequencing identified additional markers for mapping and revealed the gene content. Extensive candidate gene examinations, including gene sequencing, expression profiling with microarrays and RT-PCR, and complementation tests with mutant alleles did not identify any of the four chromatin and RNAi-related genes as mop2.The new markers developed from the skim sequence enabled further mapping and molecular genotyping, which revealed that the Mop2-1 mutation was unstable. Approxi¬mately 10% of phenotypic heterozygous plants were actually genotypic homozygous. Further mapping using only Mop2-1 homozygous plants reduced the mop2 interval to a region of nine BACs, containing 57 genes.The mop2 region is highly syntenic to a rice region of 1.25 Mb on chromosome 4. The gene alignment and repetitive sequence analyses between the syntenic regions in these two species revealed both syntenic and non-syntenic blocks of sequences. Analyses suggested several potential mechanisms for the collinearity breakage, including, but not limited to, tandem duplications of genes in one species but not the other and the presence of gene fragments in maize, but not in rice.The research described herein provides the basis for continued efforts to clone mop2. Fine-structure mapping with new markers and a larger population, as well as candidate gene sequencing in the Mop2-1 BAC library, should be pursued to clone mop2.

Upon sensing pathogens, plants initiating defense responses typically resulting in programmed cell death (PCD). PCD effectively subdues biotrophic pathogens but is hijacked by necrotrophs that colonize the resulting dead tissues. We showed that barley wall associated kinase (WAK) genes, underlying the rcs5 QTL, are manipulated by the necrotrophic fungal pathogen Bipolaris sorokiniana to cause spot blotch disease. The rcs5 genetic interval was delimited to ~0.23 cM, representing an ~234 kb genomic region containing four WAK genes, designated HvWak2, Sbs1, Sbs2, and HvWak5. Post-transcriptional gene silencing of Sbs1&2 in the susceptible barley cultivars Steptoe and Harrington resulted in resistance, suggesting a dominant susceptibility function. Sbs1&2 expression is undetectable in barley prior to pathogen challenge; however, specific upregulation of Sbs1&2 occurred in the susceptible lines post inoculation. Promotor sequence polymorphisms were identified in the allele analysis of Sbs1&2 from eight resistant and two susceptible barley lines, which supported the possible role of promotor regulation by virulent isolates contributing to susceptibility. Apoplastic wash fluids from virulent isolates induced Sbs1expression, suggesting regulation by an apoplastic-secreted effector. Thus, the Sbs1&2 genes are the first susceptibility/resistance genes that confer resistance against spot blotch, a disease that threatens barley and wheat production worldwide. The nec3 mutants of barley are hyper-susceptible to many necrotrophs and show distinctive cream to orange necrotic lesions that are induced by infection, representing aberrant PCD. The γ- irradiation induced necrotic mutant, nec3-γ1 (Bowman) was confirmed as a nec3 mutant by allelism tests. The F2 progeny of a cross of nec3 x Quest inoculated with B. sorokiniana segregated as a single recessive gene fitting a 3 WT: 1 mutant ratio. The homozygous F2 mutant progeny were genotyped with four SSR and 25 SNP markers at nec3 locus on chromosome 6H, a physical region spanning ~ 16.96 Mb containing 91 high and low confidence annotated genes. Exome capture sequencing of nec3 mutants failed to identify a candidate gene, however, RNAseq analysis identified two candidates in the nec3 region with >three-fold downregulation. We hypothesize that the underlying aberrant PCD mechanism in the nec3 barley mutant facilitates extreme susceptibility to multiple adapted fungal pathogens of barley.

Large amounts of industrial fertilizers are used to maximize crop yields. Unfortunately, they are not completely consumed by plants; consequently, this leads to soil pollution and negative effects on aquatic systems. An alternative to industrial fertilizers can be found in legume plants that provide a nitrogen source that is not harmful for the environment. Legume plants, through their symbiosis with soil bacteria called rhizobia, are able to reduce atmospheric nitrogen into ammonia, a biological nitrogen source. Establishment of the symbiosis requires communication on the molecular level between the two symbionts, which leads to changes on the cellular level and ultimately results in nitrogen-fixing nodule development. Inside the nodules hypoxic environment, the bacterial enzyme nitrogenase reduces atmospheric nitrogen to ammonia. Medicago truncatula is the model legume plant that is used to study symbiosis with mycorrhiza and with the bacteria Sinorhizobium meliloti. The focus of this work is the M. truncatula nodulation mutant nip (numerous infections and polyphenolics). The NIP gene plays a role in the formation and differentiation of nodules, and development of lateral roots. Studying this mutant will contribute knowledge to understanding the plant response to infection and how the invasion by rhizobia is regulated. Previous genetic mapping placed NIP at the top of linkage group 1 of the M. truncatula genome. A NIP mapping population was established with the purpose of performing fine mapping in the region containing NIP. DNA from two M. truncatula ecotypes A17 and A20 can be distinguished through polymorphisms. Positional mapping of the NIP gene is based on the A17/A20 genetic map of M. truncatula. The NIP mapping population of 2277 plants was scored for their nodulation phenotype and genotyped with flanking molecular genetic markers 146o17 and 23c16d, which are located ~1.5 cM apart and on either side of NIP. This resulted in the identification of 170 recombinant plants, These plants' DNAs were tested further with different available genetic markers located in the region of interest, to narrow the genetic interval that contains the NIP gene. Segregation data from genotyping analysis of recombinant plants placed NIP in the region between 4L4 and 807 genetic markers.

No description available.

Self-incompatibility (SI) is an important genetic mechanism to prevent the inbreeding of flowering plants and also an excellent system for studying cell-cell recognition and signal transduction. During evolution, several SI systems have been evolved. A unique SI system widely spreads in the grasses. In the grasses, two unlinked, multi-allelic loci (S and Z) determine SI specificity. A putative self-incompatibility gene (Bm2) was previously cloned. In this study, the role of Bm2 in self-incompatibility was investigated first. The cDNA homologues of Bm2 were sequenced from two pollen-only mutants. The results indicated that Bm2 is not the one of SI genes in Phalaris, but represents a subclass of thioredoxin h. Thus a map-based cloning strategy was then adopted to clone the SI genes from Phalaris. Fine linkage maps of the S and Z regions were constructed. RFLP probes from wheat, barley, oat and rye were screened and the S locus was delimited to 0.26 cM and the Z locus to 1.0 cM from one side using specially designed segregating populations. The S locus was located to the sub-centromere region of triticeae chromosome group 1 and the Z locus to the middle of the long arm of group 2. Finally, barley and rice bacterial artificial chromosome (BAC) clones corresponding to the S and Z region were identified to analyse the chromosome structures and to seek candidate SI genes. The abundant repetitive sequences in the identified barley BAC clones limit their usefulness. Identification of Rice BAC clones orthologous to the S and Z regions open the gate to use rice genome information to clone SI genes from the grasses. A positive rice clone (139.9 kb) orthologous to the S region contained 19 predicted genes. Several of these genes might be involved in pollen tube germination and pollen-stigma interaction, which are the major parts of SI reaction. A positive clone (118.9 kb) orthologous to the Z region gave 16 predicted genes. The predicted genes on the outmost ends of these clones could be used to construct contigs to cover the S and Z regions and delimit the S and Z loci in the grasses. / Thesis (Ph.D.)--Department of Plant Science, 2001.

Self-incompatibility (SI) is an important genetic mechanism to prevent the inbreeding of flowering plants and also an excellent system for studying cell-cell recognition and signal transduction. During evolution, several SI systems have been evolved. A unique SI system widely spreads in the grasses. In the grasses, two unlinked, multi-allelic loci (S and Z) determine SI specificity. A putative self-incompatibility gene (Bm2) was previously cloned. In this study, the role of Bm2 in self-incompatibility was investigated first. The cDNA homologues of Bm2 were sequenced from two pollen-only mutants. The results indicated that Bm2 is not the one of SI genes in Phalaris, but represents a subclass of thioredoxin h. Thus a map-based cloning strategy was then adopted to clone the SI genes from Phalaris. Fine linkage maps of the S and Z regions were constructed. RFLP probes from wheat, barley, oat and rye were screened and the S locus was delimited to 0.26 cM and the Z locus to 1.0 cM from one side using specially designed segregating populations. The S locus was located to the sub-centromere region of triticeae chromosome group 1 and the Z locus to the middle of the long arm of group 2. Finally, barley and rice bacterial artificial chromosome (BAC) clones corresponding to the S and Z region were identified to analyse the chromosome structures and to seek candidate SI genes. The abundant repetitive sequences in the identified barley BAC clones limit their usefulness. Identification of Rice BAC clones orthologous to the S and Z regions open the gate to use rice genome information to clone SI genes from the grasses. A positive rice clone (139.9 kb) orthologous to the S region contained 19 predicted genes. Several of these genes might be involved in pollen tube germination and pollen-stigma interaction, which are the major parts of SI reaction. A positive clone (118.9 kb) orthologous to the Z region gave 16 predicted genes. The predicted genes on the outmost ends of these clones could be used to construct contigs to cover the S and Z regions and delimit the S and Z loci in the grasses. / Thesis (Ph.D.)--Department of Plant Science, 2001.

Les cultivars modernes de canne à sont de hauts polyploïdes, aneuploïdes issus de croisements interspécifiques entre deux espèces polyploïdes, une espèce sucrée domestiquée Saccharum officinarum et une espèce sauvage Saccharum spontaneum. Le gène majeur de résistance durable à la rouille brune, Bru1 a été identifié chez le cultivar de canne à sucre R570. Une approche de clonage positionnel de ce gène a été entreprise et a permis de construire une première carte physique. Elle comprend sept haplotype hom(é)ologues dont un correspond à l'haplotype cible porteur du gène Bru1 qui comprend sept clones BAC qui ne se chevauchent que partiellement laissant deux espaces non couverts. Il a été montré que cette situation résulte de la présence d'une insertion dans l'haplotype porteur de Bru1. Pour combler les deux espaces présents sur l'haplotype cible, deux stratégies utilisant l'annotation des BAC ont été employées : (i) une se basant sur la conservation des gènes existante entre les différents haplotypes hom(é)ologues de la région de Bru1 et (ii) une en utilisant des marqueurs flanquants les deux espaces. Ces stratégies no us ont permis de combler un des deux espaces, de couvrir partiellement le deuxième espace et de montrer que le gène de résistance se situerait dans l'insertion. Nous avons identifié un gène candidat correspondant à une Sérine/Thréonine kinase située dans l'insertion. Des tests d'expression ont été effectués en condition normale afin de vérifier si ce gène est exprimé mais aucune amplification n'a été obtenue. Parallèlement, la recherche de l'origine de l'insertion présente sur l'haplotype cible a été entreprise en retraçant son origine dans la généalogie de notre cultivar d'étude R570 et en criblant une banque de clones de Saccharum contenant différentes espèces de canne à sucre. Les résultats sur la généalogie tendent à nous dire que cette insertion est ancienne et aurait été transmise à R570 via S. barberi. / Modern sugarcane cultivars are high polyploids, aneuploids derived interspecific crosses between two polyploid species, domesticated sugar species Saccharum officinarum and a wild species Saccharum spontaneum. The major gene for sustainable resistance to brown rust, Bru1 was identified in the modern cultivar R570. An map-based approach has been undertaken and has built a first physical map. It includes seven haplotype hom(e)ologous which one corresponds to the haplotype carrying Bru1 which includes seven BACs that overlap only partially, including two gaps. It was shown that this situation results from the presence of an insertion in the target haplotype. To fill the two gaps, two strategies using the annotation of BACs were used: (i) Based on the good genes conservation between haplotypes hom(e)ologous and (ii) by using markers flanking the two gaps. These strategies have enabled us to fill one of two gaps, partially cover the second and show that the resistance gene would be in the insertion. We identified a candidate gene corresponding to a serine/threonine kinase located in the insertion. Expression tests were performed in normal condition to see if this gene is expressed but no amplification was obtained. Meanwhile, the search for the origin of the insertion present on the target haplotype was undertaken by tracing its origin in the genealogy of R570 and analysing a library of clones of Saccharum spp containing different types of cane sugar. Results on the genealogy we tend to say that this insertion is old and were sent to R570 via S. barberi.

Die Identifizierung des albostrians Gens erfolgte mittels Karten-basiertem Klonieren. Begonnen wurde mit der Kartierung in zwei kleinen F2-Kartierungspopulationen, MM4205 und BM4205, die zur Lokalisierung des Genes auf dem langen Arm von Gerstenchromosom 7H führte. Durch Kartierung mit hoher Auflösung in Verbindung mit extensiver Markersättigung konnte der betreffende DNA-Bereich schrittweise von anfangs 14,29 cM auf schließlich 0,06 cM eingeschränkt werden, wobei insgesamt 1344 F2-Pflanzen analysiert wurden. Zwischen den nächsten flankierenden genetischen Markern konnte in einem Bereich von 46 Kbp ein einzelnes Gen identifiziert werden. Durch Sequenzvergleich des abgeleiteten Genprodukts mit Einträgen in Datenbanken konnte das Protein der CMF-Genfamilie putativer Transkritionsregulatoren mit DNA-bindenden oder Protein-Protein-Wechselwirkungs-Eigenschaften zugeordnet werden. Eine erste Bestätigung der Identität des Kandidatengens mit dem albostrians-Gen konnte durch Analyse einer EMS-induzierten TILLING-Population (abgeleitet von der Gerstensorte ‚Barke’) erreicht werden. Unter den 42 gefundenen induzierten Mutationen gab es eine Mutation, die zu einem vorzeitigen Stopcodon und damit nach der Translation potenziell zu einem verkürztem Protein führt. Die Nachkommenschaft dieser heterozygoten Mutante spaltete in grüne und albino Pflanzen auf. Der albino-Phänotyp war perfekt mit dem homozygoten Status der nonsense-Mutation in den untersuchten 245 M4-Nachkommen von fünf heterozygoten M3-Pflanzen der Mutantenfamilie verbunden. Nach transienter Transformation von Gerstenblatt-Epidermiszellen mittels biolistischem Cobombardement von ALBOSTRIANS::GFP-Fusionsprotein mit dem mCherry-markierten Organellenmarker pt-rk-CD3-999 konnte die Lokalisation des ALBOSTRIANS-Proteins in den Plastiden und im Kern beobachtet werden. / Map-based cloning was employed for identification of the albostrians gene. Starting with mapping in two small F2 mapping populations, MM4205 and BM4205, the locus could be assigned to the long arm of barley chromosome 7H. High-resolution genetic mapping in conjunction with extensive marker saturation allowed to reduce the genetic target interval iteratively from initially 14.29 cM to finally 0.06 cM by analyzing a total of 1344 F2 plants. A single gene could be identified in a physical distance of 46 Kbp between the closest flanking genetic markers. Functional annotation of the deduced protein revealed it to represent a member of the CMF gene family of putative transcriptional regulators comprising DNA binding or protein-protein interaction properties. The identified candidate gene was first confirmed by screening an EMS-induced TILLING population derived from barley cv. ‘Barke’. Among the 42 identified induced mutations a single mutation introduced a premature stop codon potentially resulting in a shorter protein upon translation. Progeny of this heterozygous mutant segregated for green and albino plants. The albino phenotype was perfectly linked with the homozygous state of the stop codon mutation in 245 M4 offspring of five heterozygous M3 plants of the mutant family. Transient transformation by biolistic co-bombardment of barley epidermal cells with an ALBOSTRIANS::GFP fusion protein and an mCherry labelled organelle marker pt-rk-CD3-999 revealed the ALBOSTRIANS protein is targeting to plastids and nucleus.

Forward genetic screen was performed in Arabidopsis thaliana to isolate novel genes involved in leaf development. The tarani (tni) mutant was selected for further study based on its unique cup-shaped lamina with +ve Gaussian curvature. We show that the larger size of tni leaves is due to rapid growth rate due to excess and prolonged cell division. We monitored the front of the receding cell division zone as a function of time and showed that the shape of the front is more concave compared to wild type, leading to positive curvature. Application of gibberellic acids (GA) synthesis inhibitor rescued the positive curvature of tni suggesting a role for GA in maintaining leaf flatness. Overexpression of cell cycle inhibitor KRP2 also flattened the leaf, confirming a role of cell division. The floral organs and seed are also larger in the tni mutant. Besides growth, tni trichomes are hyper-branched which usually happens when there is more endoreduplication. We found that the nuclei of tni trichomes are larger than wild type nuclei, suggesting increased DNA content. Genetic interaction studies showed that TNI works independent of other trichome branching genes such as with TRYPTICHON and FURCA1. Map-based cloning showed that tni is positioned on left arm of the 3rd chromosome. Using molecular markers, we narrowed down to interval to a 65 kb region, which codes for 19 genes. Sequencing several of them revealed a G→A transition at the 3rd intron - 4th exon junction of At3g20630 gene. RT-PCR analysis showed the presence of an additional full-length transcript with extra un-spliced 3rd intron. Overexpression of this un-spliced variant in wild type plants produced phenotypes like hyperbranched trichomes and cup-shaped leaves; plus additional phenotypes like organ fusion and organ polarity defects. Complementation and allelic tests confirmed that TNI codes for AtUBP14, an ubiquitin protease. The tni plants have longer stem and roots which grow at faster rate compared to wild type. Confocal microscopic analysis of mature embryos showed that both shoot (SAM) and root apical meristems (RAM) of tni plants are larger in size. In RAM, the numbers of quiescent center (QC) cells and stem cells have increased in tni plants. The tni inflorescence and flowers are bigger than wild type in size. Also the degree of axillary shoots has increased in the tni plants. Overexpression of the splice variant of TNI produced undifferentiated callus-like structures in the shoot apex and in hypocotyl. All these phenotypes show that TNI is involved in meristem proliferation. The tni siliques produced many un-fertilized ovules and shrunken and malformed seeds suggesting gametic and/or embryo lethality. We observed that tni embryos were mis-patterned at various stages of development. Following the cell division pattern shows that cells arising from the ‘basal cell’ of the embryo take apical cell fate in tni embryos. The topmost cell of the suspensor, which is also the precursor cell of RAM, is not specified as hypophysial cell in several tni embryos. In the forward genetic screen, we isolated another mutant called tooth (tth), which has deeper serrations at the leaf margin and narrower leaves compared to wild type. It has been mapped to the longer arm of the 2nd chromosome. Genetic interaction studies show that tth is not allelic to other serration mutants such as serrate and mir164a.

Cyclic nucleotide-gated channels (CNGCs) are non-selective cation channels that were first identified in vertebrate photosensory and olfactory neurons. Although the physiological roles and biophysical properties of animal CNGCs have been well studied, much less is known about these channels in plants. The Arabidopsis genome encodes twenty putative CNGC subunits that are postulated to form channel complexes that mediate various physiological processes involving abiotic and biotic stress responses, ion homeostasis and development. The identification of Arabidopsis autoimmune CNGC mutants, such as defense no death class (dnd1 and dnd2), and the constitutive expressor of pathogenesis related genes 22 (cpr22) implicate AtCNGC2, 4, 11 and 12 in plant immunity. Here, I present a comprehensive study of the molecular mechanisms involved in CNGC-mediated signaling pathways with emphasis on pathogen defense. Previously, a forward genetics approach aimed to identify suppressor mutants of the rare gain-of-function autoimmune mutant, cpr22, identified key residues that are important for CNGC subunit interactions and channel function. First, I present a structure-function analysis of one of these suppressor mutants (S58) that revealed a key residue in the cyclic nucleotide binding domain involved in the stable regulation of CNGCs. Second, I present a new suppressor screen using AtCNGC2 T-DNA knockout mutants that specifically aimed to identify novel downstream components of CNGC-mediated pathogen defense signaling. In this screen, I successfully isolated and characterized the novel Arabidopsis mutant, repressor of defense no death 1 (rdd1), and expanded this study to demonstrate its involvement in AtCNGC2 and AtCNGC4-mediated signal transduction. Additionally, I demonstrated for the first time, the physical interaction of AtCNGC2 and AtCNGC4 subunits in planta. The findings presented in this thesis broaden our current knowledge of CNGCs in plants, and provide a new foundation for future elucidation of the structure-function relationships and signal transduction mediated by these channels.