M.Sc. (Biochemistry) / Plant innate immunity occurs in two interconnected branches, the first being the recognition of pathogen conserved surface structures known as pathogen- or microbe-associated molecular patterns (P/MAMPs) by the plant plasma membrane pathogen recognition receptors (PRRs), leading to activation of P/MAMP-triggered immunity (P/MTI). The second branch involves the recognition of pathogen avirulence (Avr) genes by the corresponding plant disease resistance (R) genes, known as the ‘gene-for-gene‘ interaction, and results in a more efficient or stronger defence response, namely effector-triggered immunity (ETI). Lipopolysaccharide (LPS) acts as a P/MAMP that induces an innate immune response in both plants and animals. LPS, especially the lipid A component, has been shown to play a vital role in activating immune responses in animals. Other LPS components such as lipooligosaccharide (LOS) and the core-oligosaccharide have also been shown to trigger an immune response in plants such as Arabidopsis thaliana. In mammalian cells, LPS binds to the LPS-binding protein (LBP) forming a LPS-LBP complex, which binds to a Toll-like receptor 4/myeloid differentiation-2 (TLR4/MD-2) complex together with the co-receptor CD14, a glycosylphosphatidylinositol (GPI)-linked protein, and activates an immune response. To date, there is still no knowledge about the LPS receptor(s) in plants.....

by Shih Sheung Mei. / Thesis submitted in: July 2003. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 206-215). / Abstracts in English and Chinese. / Abstract --- p.ii / Achnoledgements --- p.vi / Abbreviations --- p.viii / List of contents --- p.viv / List of tables --- p.xiii / List of figures --- p.xv / Chapter Chapter One --- Literature Review / Chapter 1.1 --- Introducation of Lentinula edodes --- p.1 / Chapter 1.1.1 --- Life cycle of Basidiomycete --- p.1 / Chapter 1.1.2 --- Differentially Expressed Genes in stages of Lentinula edodes --- p.3 / Chapter 1.2 --- Relationship of Monokaryons and Dikaryons in Basidiomycetes --- p.4 / Chapter 1.2.1 --- Mating Type Gene in Filamentous Fungi --- p.4 / Chapter 1.2.3 --- Dikaryon Formation and Homeodomain Proteins --- p.6 / Chapter 1.2.4 --- Clamp Connection formation in Dikaryon --- p.9 / Chapter 1.3 --- Stuctural Protein of Mushroom --- p.11 / Chapter 1.3.1 --- Hydrophobin --- p.11 / Chapter 1.3.1.1 --- General Introduction --- p.11 / Chapter 1.3.1.2 --- Structure of hydrophobin --- p.11 / Chapter 1.3.1.3 --- Formation of Disulphide bonds and Glycosylation --- p.12 / Chapter 1.3.1.4 --- Functions of Hydrophobins --- p.13 / Chapter 1.4 --- Genomics of filamentous fungi --- p.15 / Chapter 1.5 --- Genetic analysis of filamentous fungi --- p.18 / Chapter 1.6 --- Objectives of the Project --- p.20 / Chapter Chapter Two --- Identification of Differentially Expressed Genes in Dikaryons of Lentinula edodes by Microarray of Primordium Expressed Sequence Tags / Chapter 2.1 --- Introduction --- p.23 / Chapter 2.2 --- Materials and Methods --- p.27 / Chapter 2.2.1 --- Construction of EST database --- p.27 / Chapter 2.2.2 --- Construction of EST Microarray cDNA gene-chip --- p.27 / Chapter 2.2.2.1 --- Amplification of the primordium EST clones --- p.27 / Chapter 2.2.2.2 --- Purification of the amplified EST clones --- p.28 / Chapter 2.2.2.3 --- Spotting of the amplified EST clones onto chips --- p.29 / Chapter 2.2.3 --- Screening of the Differentially Expressed Genes in Dikaryons by Primordium Microarray --- p.31 / Chapter 2.2.3.1 --- Mycelium Cultivation and Preparation of Total RNA --- p.31 / Chapter 2.2.3.2 --- cDNA synthesis and labeling --- p.32 / Chapter 2.2.3.3 --- cDNA purification --- p.33 / Chapter 2.2.3.4 --- Probe Storage Conditions --- p.34 / Chapter 2.2.3.5 --- cDNA analysis --- p.35 / Chapter 2.2.3.6 --- Microarray hybridization --- p.37 / Chapter 2.2.3.7 --- Stringency washes --- p.39 / Chapter 2.2.3.8 --- Detection with TSA --- p.39 / Chapter 2.2.3.9 --- Microarray scanning and data anlysis --- p.41 / Chapter 2.3 --- Results --- p.45 / Chapter 2.3.1 --- Amplification of primordium ESTs --- p.45 / Chapter 2.3.2 --- Purification of PCR products --- p.45 / Chapter 2.3.3 --- Data Analysis of Microarray Data --- p.47 / Chapter 2.3.3.1 --- Generation of Primordium EST Microarray Image for analysis --- p.47 / Chapter 2.3.3.2 --- Normalization of the Data --- p.49 / Chapter 2.3.3.3. --- Transciption Profile of Dikaryon compared with Monokaryon --- p.79 / Chapter 2.3.3.4. --- Differentially Expression of Dikaryon L54 --- p.80 / Chapter 2.4 --- Discussion --- p.85 / Chapter Chapter Three --- Enrichment of Genes with Differentially Expression in Dikaryons by Construction of Full-length Subtractive Library / Chapter 3.1 --- Introduction of Subtraction Cloning --- p.93 / Chapter 3.2 --- Materials and Methods --- p.97 / Chapter 3.2.1 --- Construction of Full-length Dikaryotic Subtractive library --- p.97 / Chapter 3.2.1.1 --- Isolation of PolyA+ mRNA of Dikaryon for Subtraction --- p.97 / Chapter 3.2.1.2 --- Enrichment of Differentially Expressed Genes in Dikaryon L54 by Subtraction with Monokaryons A and B --- p.99 / Chapter 3.2.1.3 --- First-Strand cDNA Synthesis --- p.102 / Chapter 3.2.1.4 --- cDNA Amplification by Long-Distance PCR --- p.102 / Chapter 3.2.1.5 --- Proteinase K Digestion --- p.103 / Chapter 3.2.1.6 --- Sfi Digestion --- p.104 / Chapter 3.2.1.7 --- cDNA size fractionation by CHROMA SPIN-400 --- p.104 / Chapter 3.2.1.8 --- Determination of the Ligation Efficiency --- p.106 / Chapter 3.2.1.9 --- Ligation of cDNA to lamda TriplEx2 Vector --- p.107 / Chapter 3.2.1.10 --- Lamda-phage Packaging Reaction --- p.107 / Chapter 3.2.1.11 --- Titering the Unamplifled Library and Determining the Percentage of Recombinant Clones --- p.108 / Chapter 3.2.1.12 --- Library Amplification --- p.109 / Chapter 3.2.1.13 --- Conversion of λTriplEx2 Recombinant Clones to pTriplEx2 Recombinant Plasmids --- p.111 / Chapter 3.2.2 --- Screening of the Subtractive library --- p.114 / Chapter 3.2.2.1 --- Verification of the enrichment by Plaque Lifting hybridization --- p.114 / Chapter 3.2.2.1.1 --- Lifting the Plaques --- p.114 / Chapter 3.2.2.1.2 --- Synthesis of the Probes for Plaque Lift Hybridization --- p.115 / Chapter 3.2.2.1.3 --- Hybridization to the Membranes --- p.116 / Chapter 3.2.2.2 --- Screening the Subtractive library by Macroarray Hybridization --- p.117 / Chapter 3.2.2.2.1 --- Colony Picking by QPik System --- p.117 / Chapter 3.2.2.2.2 --- Gridding of Macroarray --- p.118 / Chapter 3.2.2.2.3 --- Filter Processing of Gridded Membrane --- p.119 / Chapter 3.2.2.2.4 --- Hybridization to the Macroarray Membrane --- p.120 / Chapter 3.3 --- Results and Discussion --- p.121 / Chapter 3.3.1 --- Enrichment of Differentially Expressed Genes in Dikaryon L54 by Subtraction with Monokaryons A and B --- p.121 / Chapter 3.3.2 --- Construction of the full-length subtractive library --- p.123 / Chapter 3.3.3 --- Conversion of A TriplEx2 Recombinant Clones to pTriplEx2 Recombinant Plamid --- p.124 / Chapter 3.3.4 --- Verification the Enrichment of Subtractive library by Plaque lifting Hybridization --- p.125 / Chapter 3.3.5 --- Screening of the Subtractive library by Macroarray --- p.125 / Chapter 3.4 --- Discussion --- p.126 / Chapter Chapter Four --- Identification of Genes with Differentially Expression in Dikaryons by Subtactive cDNA Library Microarray / Chapter 4.1 --- Introduction --- p.135 / Chapter 4.2 --- Materials and Methods / Chapter 4.2.1 --- Selection and Amplification of clonesin SubtractionLlibrary for Microarray screening --- p.140 / Chapter 4.2.2 --- PCR product Purification --- p.141 / Chapter 4.2.3 --- Generation of Subtractive Dikaryotic Library Microarray Chip --- p.142 / Chapter 4.2.4 --- Screening the Differentially Expressed Genesin Dikaryon L54 by the Subtraction Dikaryotic Library cDNA Microarray Analysis --- p.143 / Chapter 4.2.4.1 --- Preparation of Total RNA --- p.143 / Chapter 4.2.4.2 --- Synthesis and fluorescent labeling of total cDNA --- p.145 / Chapter 4.2.4.3 --- Purification of labeled cDNA --- p.146 / Chapter 4.2.4.4 --- Storage Condition of Probe --- p.147 / Chapter 4.2.4.5 --- Analysis of labeled total cDNA --- p.148 / Chapter 4.2.4.6 --- Microarray hybridization --- p.150 / Chapter 4.2.4.7 --- Stringency washes --- p.152 / Chapter 4.2.4.8 --- Detection with TSA --- p.153 / Chapter 4.2.4.9 --- Image generation and data analysis --- p.155 / Chapter 4.2.5 --- Sequence analysis of clones showing differentially expressed in dikaryons in microarray screening --- p.157 / Chapter 4.2.5.1 --- Single-pass partial sequencing of 3´ة-end of subtractive cDNA clones --- p.157 / Chapter 4.2.5.2 --- Compiling dikaryotic EST database --- p.158 / Chapter 4.2.6 --- Comparison microarray analysis with SAGE analysis of the differentially expressed genes --- p.159 / Chapter 4.3 --- Results --- p.161 / Chapter 4.3.1 --- Preparation of clones for microarray hybridization --- p.161 / Chapter 4.3.2 --- Screening the differentially expressed genesin dikaryon L54 by the subtractive dikaryotic library cDNA microarray analysis --- p.162 / Chapter 4.3.2.1 --- Image capture and microarray data analysis --- p.162 / Chapter 4.3.2.2 --- Comparision of dikaryon L54 with monokaryons A and B --- p.163 / Chapter 4.3.2.3 --- Sequenced and comparison of the differentially expressed genes in dikaryon --- p.166 / Chapter 4.3.3 --- Comparison microarray analysis with SAGE analysis of the differentially expressed genes --- p.169 / Chapter Chapter Five --- Conclusion and Future Perpectives --- p.198 / References --- p.206

When imbibed at their optimum germination temperature of 25°C, achenes of Tagetes minuta L. germinate over a period of approximately 48 h. At temperatures of between 35°C and 39°C, the achenes do not germinate but enter into a state of thermoinhibition. These supra-optimal conditions do not harm the achenes, however, and when the temperature is reduced below 35°C radicle emergence may be observed within 4 h. Achenes which have been thermoinhibited for periods of 24 h or more show "accelerated germination" which takes only 24 h, although the actual germination curve is identical to that of normally germinated achenes. This suggests that the achenes are metabolically active at thermoinhibitory temperatures and undergo most of the processes of normal germination, but that at some point any further development is halted, preventing radicle emergence. When the temperature is reduced, this block on germination is removed and since the achenes are already primed for germination, this occurs within a short time. An analysis of the proteins produced by germinating and thermoinhibited achenes was conducted using SDS-polyacrylamide gel electrophoresis (SDS-PAGE). This procedure was able to resolve approximately 40 different protein bands, but no differences were observed between thermoinhibited and germinating achenes. Two dimensional polyacrylamide gel electrophoresis (20-PAGE) was able to resolve approximately 200 individual polypeptides. The vast majority of polypeptides in T. minuta achenes are acidic, although the number of neutral to basic polypeptides increases as germination progresses. Ten polypeptides were identified which were specific to thermoinhibited achenes. These formed two distinct groups on the twodimensional gels. The larger group contained seven proteins, ranging in size from 22 kDa to 26.7 kDa and with isoelectric points of between 3.0 and 4.0. The smaller group contained three polypeptides with molecular weights of about 14 kDA and a pi of approximately 3.0. These polypeptides were all extremely specific to thermoinhibited achenes and declined rapidly when the incubation temperature was reduced, in a manner which correlated with an increase in the germinability of the achenes. Several characteristics of the expression of these polypeptides were similar to characteristics of embryo-dormancy in seeds where dormancy is thought to be actively imposed by the expression of specific dormancy-associated genes. This, along with the very tightly-regulated nature of these 10 polypeptides, suggests that thermoinhibition in T. minuta may be regulated through gene expression and that these ten polypeptides may represent the products of genes responsible for preventing radicle emergence at unfavourable temperatures. Since these polypeptides were only resolved using silver-staining and could not therefore be used for amino acid sequence analysis, this hypothesis was further investigated using differential display of mRNA to isolate genes which are expressed specifically in thermoinhibited achenes. A large number of cDNA fragments which were specific to either germinating or thermoinhibited achenes were identified and extracted from the differential display gels. Those cDNAs specific to the thermoinhibited achenes were taken for further analysis. Of the 62 fragments excised from the gels, 25 could be reamplified to generate single bands of the correct size on agarose gels. A further 22 cDNAs produced multiple bands, where one band was much brighter than the others and correlated with the size of the original fragment. Thirteen of the cDNAs which' generated single bands were cloned into the plasmid vector pGEM®-T Easy and transformed into either Escherichia coli JM109 or E. coli XL1-Blue. Recombinant colonies were identified using blue-white colour selection and the presence of the insert confirmed by colony blotting and restriction analysis. Three clones were chosen for each of the cDNAs. Reverse northern analysis confirmed that all 39 clones were specific to the mRNA pool of thermoinhibited achenes. High quality sequence data were obtained for 27 of the cDNA samples, the remainder appeared to have been degraded in transit. Alignment of the various sequences revealed that a total of 14 different sequences had been cloned, indicating that several of the bands isolated from the differential display gels contained multiple sequences. Electronic homology searches tentatively identified three of the sequences, whilst the remainder did not show significant homology to any known sequences. Of the cDNAs identified in this way, one may encode a plant transcription factor-like or nuclear RNA-binding protein whilst the other two may encode an RNase-L Inhibitor-like protein and a miraculin homologue. The potential roles of such genes in the imposition or maintenance of the thermoinhibited state are discussed. Although further research needs to be conducted to isolate full length cDNA sequences and to determine their exact expression patterns in germinating and thermoinhibited achenes, these results are consistent with the hypothesis that thermoinhibition in T. minuta achenes is under positive genetic control in a manner analogous to embryo dormancy. This thesis represents the first molecular study of thermoinhibition as well as the first report of active control over this phenomenon in any species. Since thermoinhibition, unlike dormancy, can be rapidly imposed and released under strictly controlled conditions without the need for any dormancy breaking treatment, T. minuta achenes represent an excellent model system for studies on the molecular control of seed germination. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2003.

Thesis (Ph. D.)--University of Kentucky, 2008. / Title from document title page (viewed on May 12, 2008). Document formatted into pages; contains: xvi, 221 p. : ill. (some col.). Includes abstract and vita. Includes bibliographical references (p. 214-219).

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [116]-129).

Thesis (Ph. D.)--Ohio University, March, 2004. / Includes bibliographical references (leaves 202-227).

Thesis (Ph. D.)--University of Oregon, 2000. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 110-117). Also available for download via the World Wide Web; free to University of Oregon users. Address: http://wwwlib.umi.com/cr/uoregon/fullcit?p9963446.

Thesis (Ph.D.)--Ohio University, November, 2008. / Title from PDF t.p. Release of full electronic text on OhioLINK has been delayed until December 1, 2012. Includes bibliographical references (leaves 145-153)

Thesis (Ph.D.)--Ohio University, November, 2008. / Release of full electronic text on OhioLINK has been delayed until December 1, 2012. Includes bibliographical references (leaves 145-153)

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 78-94).