Yellow rust, caused by Puccinia striiformis Westend. f. sp. tritici Eriksson is one of the most severe leaf diseases of wheat. Aim of this study is to illuminate the downstream signaling pathways upon incompetible infection of rust pathogen in wheat, thus to understand the genes involved in resistance mechanism. The strategy used is the dual bait yeast two-hybrid analysis which is the most powerful method for in vivo detection of protein-protein interactions. The bait proteins used are / the domains of Yr10 yellow rust resistance gene, Rad6 gene which is considered to have a critical role in R gene mediated signaling pathway, and WR5 gene fragment which is an unknown protein having homology to the WD40 repeat containing protein with apoptosis related activity. Screening of a yeast prey library with these baits revealed proteins having mostly apoptosis related functions (SRP72, POR1, CSE1), translation initiation control in response to stress conditions (Gcn2p, Eap1p), phosphorylation (SKY1) and dephosphorylation activities (GAC1), cell cycle control (FAR1), oxidative stress control (OXR1), protein degradation control (TOM1), protein folding control (CPR7) and ion homeostasis in the cell (POR1, GAC1). The significance of the study can be summarized as i) being the first yeast two hybrid analysis of a wheat R gene, ii) being able to detect interacting partners with anticipated functions, iii) most importantly, initiating further detailed analysis of the key interactors.

Relations among transcription, pre-mRNA processing and chromatin modifications are only partially understood. The human protein SNW1/SKIP belongs to factors which couple these processes. The protein plays role in pre-mRNA splicing and transcription on the level of both initiation and elongation. According to the hypothesis of K. Jones laboratory, it physically and functionally interacts with positive transcription elongation factor b during transcription elongation and influences methylation of histone H3 on lysine 4, a modification characteristic for active transcription (Bres et al., Genes Dev. 19:1211-26, 2005, Bres et al., Mol Cell. 36:75-87, 2009). The yeast ortholog of SNW1/SKIP, Prp45, was until now reported only in connection with splicing regulation. However, unpublished results from our Laboratory and others showed that it is employed in transcription elongation as well. The aim of the diploma project was to search for the relations between Prp45 and the factors regulating transcription. It was confirmed that the mutation prp45(1 169) results in the delay of PHO5 and PHO84 expression during transcriptional induction. Next, we discovered new genetic interactions between PRP45 and several genes encoding the effectors of chromatin modifications. How Prp45 influences the expression of PHO5 and PHO84...

DNA is subject to attack by DNA damaging agents from both environmental and endogenous sources. In response to DNA damage, living organisms enhance expression of many related genes to facilitate DNA repair and survival. The SOS response is a well-understood prokaryotic regulatory cascade that controls the expression of more than 30 genes in response to DNA damage. However, in eukaryotic organisms from simple budding yeast to human, such a regulatory network has not been reported.<p>Previous research in our laboratory found that among DNA repair mutants of <i>Saccharomyces cerevisiae</i>, only rad6 and rad18 defective in the post-replication repair pathway significantly affected DNA damage induction of several genes examined. Rad6 and Rad18 form a ubiquitin conjugation-ligase complex and are required for the cellular tolerance to damaged DNA. Since the Rad6-Rad18 complex binds to single-stranded DNA, it may act as a DNA damage sensor required for the activation of DNA damage-induced transcription. We performed microarray analysis and found that the induction of up to 379 genes, including those involved in DNA repair, control of replication and transcription, regulation of the cell cycle and cell metabolism, are compromised in the rad6 and rad18 mutants. Although Rad6/Rad18 monoubiquitinates proliferating cell nuclear antigen (PCNA) following DNA damage to initiate a damage tolerance response, PCNA ubiquitination is not required for DNA damage induction. In budding yeast, cell-cycle checkpoints are involved in the control of DNA damage induction of gene expression through phosphorylation of a protein kinase Rad53 by two pathways represented by Rad24 and Sgs1. The Rad6-Rad18 complex appears to function in the Rad24 pathway and parallel to Sgs1. We further demonstrated that the Rad17 subunit of the 9-1-1 complex is subject to Rad6/Rad18- and DNA damage-dependent mono-ubiquitination and that the Rad17-Lys197 residue with flanking sequences homologous to Lys164 of PCNA is absolutely required for the DNA damage induction by Rad6-Rad18. Hence, by ubiquitinating two DNA clamps, PCNA and 9-1-1, the Rad6-Rad18 complex plays a central role in the cellular response to DNA damage by coordinating translesion synthesis, error-free bypass, homologous recombination, as well as transcriptional regulation, reminiscent of roles of RecA in <i>E. coli</i> cells.<p>Several individual genes have also been examined in this study to elucidate the regulatory mechanisms acting on specific DNA damage-inducible genes. In the microarray analysis, DDI2 and DDI3, two identical genes located in duplicated chromosomal regions, were identified due to the highest induction ratio (122-fold) after MMS treatment. Interestingly, DDI2/DDI3 can only be highly induced by SN2-type alkylating agents. Promoter deletion analysis mapped the putative upstream acting sequence (UASDDI2) responsible for 40% of basal expression and 90% of induced expression by MMS.<p>The CRT10 gene was identified through screening of the yeast deletion library for hydroxyurea (HU) resistance. CRT10 encodes a putative 957 amino acid, 110 kDa protein with a leucine repeat and a WD40 repeat near the N-terminus. Deletion of CRT10 resulted in an enhanced resistance to HU reminiscent of the inactivation of two other ribonucleotide reductase (Rnr) suppressors, CRT1 and SML1, which regulate Rnr activity at transcriptional and translational levels, respectively. Epistasis analysis indicates that CRT10 belongs to the CRT1 pathway but not the SML1 pathway. Indeed, deletion of CRT10 enhanced the survival of the mec1 null mutant and increased basal level and DNA damage-induced expression of RNR2 and RNR3, suggesting that Crt10 regulates RNR genes at the transcriptional level. Furthermore, the dun1 mutation is epistatic to crt10 with respect to both HU sensitivity and RNR gene expression. Interestingly, the expression of CRT10 itself is induced by DNA damaging agents and this induction requires DUN1, suggesting that CRT10 plays a role in cellular response to DNA damage and replication blocks. The CRT10 function appears to be achieved by positive regulation of the CRT1 transcript level, indicating that CRT10 is a component of the regulatory circuit.

DNA is subject to attack by DNA damaging agents from both environmental and endogenous sources. In response to DNA damage, living organisms enhance expression of many related genes to facilitate DNA repair and survival. The SOS response is a well-understood prokaryotic regulatory cascade that controls the expression of more than 30 genes in response to DNA damage. However, in eukaryotic organisms from simple budding yeast to human, such a regulatory network has not been reported.<p>Previous research in our laboratory found that among DNA repair mutants of <i>Saccharomyces cerevisiae</i>, only rad6 and rad18 defective in the post-replication repair pathway significantly affected DNA damage induction of several genes examined. Rad6 and Rad18 form a ubiquitin conjugation-ligase complex and are required for the cellular tolerance to damaged DNA. Since the Rad6-Rad18 complex binds to single-stranded DNA, it may act as a DNA damage sensor required for the activation of DNA damage-induced transcription. We performed microarray analysis and found that the induction of up to 379 genes, including those involved in DNA repair, control of replication and transcription, regulation of the cell cycle and cell metabolism, are compromised in the rad6 and rad18 mutants. Although Rad6/Rad18 monoubiquitinates proliferating cell nuclear antigen (PCNA) following DNA damage to initiate a damage tolerance response, PCNA ubiquitination is not required for DNA damage induction. In budding yeast, cell-cycle checkpoints are involved in the control of DNA damage induction of gene expression through phosphorylation of a protein kinase Rad53 by two pathways represented by Rad24 and Sgs1. The Rad6-Rad18 complex appears to function in the Rad24 pathway and parallel to Sgs1. We further demonstrated that the Rad17 subunit of the 9-1-1 complex is subject to Rad6/Rad18- and DNA damage-dependent mono-ubiquitination and that the Rad17-Lys197 residue with flanking sequences homologous to Lys164 of PCNA is absolutely required for the DNA damage induction by Rad6-Rad18. Hence, by ubiquitinating two DNA clamps, PCNA and 9-1-1, the Rad6-Rad18 complex plays a central role in the cellular response to DNA damage by coordinating translesion synthesis, error-free bypass, homologous recombination, as well as transcriptional regulation, reminiscent of roles of RecA in <i>E. coli</i> cells.<p>Several individual genes have also been examined in this study to elucidate the regulatory mechanisms acting on specific DNA damage-inducible genes. In the microarray analysis, DDI2 and DDI3, two identical genes located in duplicated chromosomal regions, were identified due to the highest induction ratio (122-fold) after MMS treatment. Interestingly, DDI2/DDI3 can only be highly induced by SN2-type alkylating agents. Promoter deletion analysis mapped the putative upstream acting sequence (UASDDI2) responsible for 40% of basal expression and 90% of induced expression by MMS.<p>The CRT10 gene was identified through screening of the yeast deletion library for hydroxyurea (HU) resistance. CRT10 encodes a putative 957 amino acid, 110 kDa protein with a leucine repeat and a WD40 repeat near the N-terminus. Deletion of CRT10 resulted in an enhanced resistance to HU reminiscent of the inactivation of two other ribonucleotide reductase (Rnr) suppressors, CRT1 and SML1, which regulate Rnr activity at transcriptional and translational levels, respectively. Epistasis analysis indicates that CRT10 belongs to the CRT1 pathway but not the SML1 pathway. Indeed, deletion of CRT10 enhanced the survival of the mec1 null mutant and increased basal level and DNA damage-induced expression of RNR2 and RNR3, suggesting that Crt10 regulates RNR genes at the transcriptional level. Furthermore, the dun1 mutation is epistatic to crt10 with respect to both HU sensitivity and RNR gene expression. Interestingly, the expression of CRT10 itself is induced by DNA damaging agents and this induction requires DUN1, suggesting that CRT10 plays a role in cellular response to DNA damage and replication blocks. The CRT10 function appears to be achieved by positive regulation of the CRT1 transcript level, indicating that CRT10 is a component of the regulatory circuit.

The first part of the thesis describes a systematic function analysis that identified in vitro E2 partners for ten different HECT E3 ligase proteins. Using mass spectrometry, the linkage composition for the resulting autoubiquitylation products of a number of functional E2-HECT pairs was determined. HECT domains from different subfamilies catalyze the formation of very different types of Ub chains, largely independent of the E2 in the reaction. The second part of the thesis describes the characterization of the RAD6-interactome. Using affinity purification coupled with mass spectrometry, I identified a novel RAD6-interacting E3 ligase, KCMF1, which binds to a different surface on RAD6 than the other RAD6-associated E3 ligases. KCMF1 also recruits additional proteins to RAD6, and this new complex points to novel RAD6 functions. Interestingly, the RAD6A R11Q mutant polypeptide, found in X-linked mental retardation patients specifically loses the interaction with KCMF1, but not with other RAD6-associated E3 ligases.

The first part of the thesis describes a systematic function analysis that identified in vitro E2 partners for ten different HECT E3 ligase proteins. Using mass spectrometry, the linkage composition for the resulting autoubiquitylation products of a number of functional E2-HECT pairs was determined. HECT domains from different subfamilies catalyze the formation of very different types of Ub chains, largely independent of the E2 in the reaction. The second part of the thesis describes the characterization of the RAD6-interactome. Using affinity purification coupled with mass spectrometry, I identified a novel RAD6-interacting E3 ligase, KCMF1, which binds to a different surface on RAD6 than the other RAD6-associated E3 ligases. KCMF1 also recruits additional proteins to RAD6, and this new complex points to novel RAD6 functions. Interestingly, the RAD6A R11Q mutant polypeptide, found in X-linked mental retardation patients specifically loses the interaction with KCMF1, but not with other RAD6-associated E3 ligases.

The main objective of this thesis dissertation is functionally characterizing the genes involved in biotic and abiotic stresses of plants at molecular level. Previously, upon pathogen attack Rad6 gene expression was found to be changed in wheat and barley plants. To functionally characterize the Rad6 gene, VIGS (Virus induced gene silencing) system was used. HR (Hypersensitive response) like symptoms was detected in every silenced barley and wheat plants. To figure out, transcriptomes and proteomes of Rad6 silenced plants were analyzed. 2-D PAGE analysis was also performed on silenced and control wheat plants. No pathogen growth was observed in Rad6 silenced barley lines. Additionally, the susceptible wild type Arabidopsis plants showed resistant phenotype when any of the Rad6 gene copies is mutated. This suggests that Rad6 gene has a negative regulatory role in plant disease resistance which was proved for the first time. Yeast two hybrid protein interaction study suggests that RAD6 carrying out its function by interacting with SGT1 protein and regulating resistance related genes. It has been first time reported in this thesis that E2 (Ubiquitin conjugating enzyme) takes role in plant disease resistance. Boron which is the other consideration in the scope of thesis as an abiotic stress factor at a very limited amount is necessary for the normal development of plants. This study is conducted on highly boron tolerant Gypsophila perfoliata L. collected from a location in the boron mining area. The plant samples were tested in the presence of high boron (35 mg/kg) concentrations. The transcriptomes of the plant samples treated with the excess levels of boron to that of the samples grown under normal concentration were compared using differential display PCR method. Thirty bands showing differential expression levels at varying time points were analyzed. 18 of them were confirmed via qRT-PCR.