EEffffeecctt ooff ddrroouugghhtt oonn tthhee mmeettaabboolliissmm ooff ttaabbaaccccoo ppllaannttss (Nicotiana tabacum L.) Diploma thesis - abstract Lucia Miedzińska, Bc. Work Supervisor: Doc. RNDr. Helena Ryšlavá, CSc. Consultant: RNDr. Veronika Doubnerová, PhD. Drought stress is one of the most frequent form of plant stress, which occurs not only under condition of limited water availability, but also under reduced ability to accept water by roots, for example in salted or cold soils. In this thesis the changes in enzyme activities of: NADP-malic enzyme (EC 1.1.1.40; NADP-ME), phosphoenolpyruvate carboxylase (EC 4.1.1.31; PEPC) and pyruvate, phosphate dikinase (EC 2.7.9.1; PPDK) in tobacco plants (Nicotiana tabacum L., cv. W38) after drought were investigated. Enzyme activities in tobacco leaves were significantly increased during 11 days of stress, PEPC 2-fold, PPDK 3,3- fold and NADP-ME 4-fold compared to control plants. The regulation of NADP-ME and PEPC activities were studied on transcriptional level - by the real-time PCR method and on translational level - immunochemically. The expression of NADP-ME protein and transcription of mRNA for chloroplast NADP-ME isoform were increased, but mRNA for cytosolic isoform was not affected. The protein expression of PEPC was slightly increased, transcription of...

Phosphoenolpyruvate carboxylase (EC 4.1.1.31; PEPC) plays many roles in plants; it connects the metabolism of saccharides and amino acids. PEPC is regulated at many levels including phosphorylation of serine residue near N-terminus. The aim was to found out, if the composition of cultivation medium affects the activity of PEPC and metabolically related enzymes such as NADP-malic enzyme (EC 1.1.1.40), pyruvate, phosphate dikinase (EC 2.7.9.1; PPDK) and enzymes participating in nitrogen assimilation, thus nitrate reductase (EC 1.7.1.1; NR), glutamine synthetase (EC 6.3.1.2; GS), glutamate synthase (EC 1.4.1.14; GOGAT), NAD-glutamate dehydrogenase (1.4.1.2; NAD-GDH) and NADP-glutamate dehydrogenase (1.4.1.4; NADP-GDH). Tobacco plants (Nicotiana tabacum L., cv. Petit Havana SR1) were grown in vitro in Murashige-Skoog agar. The effect of the presence of 2% succrose, reduced content of nitrogen compounds or phosphate and limited supply of CO2 was followed. Sucrose added to Murashige-Skoog medium caused the increase of enzyme activity of PEPC, NADP-ME and most of enzymes related to nitrogen uptake and metabolism - NR, GS, GOGAT, NAD-GDH a NADP-GDH. Reduced content of nitrogen compounds and phosphate in cultivation medium decreased activity of all monitored enzymes. Enzyme activities were also affected by the...

1. Abstract ARP2/3 complex is well studied in case of animals, it plays key roles in motility of cells and intracellular organels. It's malfunctions result in severe growth disorders and even lethality of affected cells. On the contrary, plant cells do not exhibit such dramatic phenotype of ARP2/3 complex mutations like it is by animals. It is possible that just the different life strategies of plants and animals contribute to differences in a way how animal and plant cells use their cytoskeleton, where ARP2/3 complex is it's part as well. It is highly conserved 7 protein complex from yeast to human. His main functions are creation of new "de novo" actin filaments, actin branched filaments network. Some of the parasite organisms are capable of missusing its nucleator activity to actively move inside of host cell. Because of the plant cells are sourounded by the cell wall, which give them support in creating various shapes and also hinders active movement of the whole cell body, it is likely that ARP 2/3 complex could be possibly involved in novel plant specific functions as well. If we think about the different life strategy of plants and animals we can not ignore all the things these two kingdoms have in common regarding to cytoskeleton processes. That is the need both for vesicular transport and...

Salicylic acid is widely studied for its role in biotic stress signaling in plants. Several SA-binding proteins, including SABP2 (salicylic acid-binding protein 2) has been identified and characterized for their role in plant disease resistance. SABP2 is a 29 kDA tobacco protein that binds to salicylic acid with high affinity. It is a methylesterase enzyme that catalyzes the conversion of methyl salicylate into salicylic acid required for inducing a robust systemic acquired resistance (SAR) in plants. Methyl salicylic acid is one of the several mobile SAR signals identified in plants. SABP2-interacting protein 428 (SIP428) was identified in a yeast two-hybrid screen using tobacco SABP2 as a bait. In silico analysis shows that SIP428 possesses the SIR2 (silent information regulatory 2)-like conserved motifs. SIR2 enzymes are orthologs of sirtuin proteins that catalyze the NAD+-dependent deacetylation of Nε lysine-acetylated proteins. The recombinant SIP428 expressed in E. coli exhibits SIR2-like deacetylase activity. SIP428 shows homology to Arabidopsis AtSRT2 (67% identity), which is implicated in SA-mediated basal defenses. Immunoblot analysis using anti-acetylated lysine antibodies showed that the recombinant SIP428 is lysine acetylated. The expression of SIP428 transcripts was moderately downregulated upon infection by TMV. In the presence of SIP428, the esterase activity of SABP2 increased modestly. The interaction of SIP428 with SABP2, it's regulation upon pathogen infection, and similarity with AtSRT2 suggests that SIP428 is likely to play a role in stress signaling in plants.

Plants have evolved an efficient mechanism to defend themselves against pathogens. Many biotic and abiotic agents have been shown to induce defense mechanism in plants. Acibenzolar-S-Methyl (ASM) is a commercially available chemical inducer of local and systemic resistance (SAR) response in plants. ASM functioning at molecular level is mostly unclear. This research was designed to investigate the mechanism of ASM action in plants. It was hypothesized that SABP2, a plant protein, plays an important role in ASM-mediated defense signaling. Biochemical studies were performed to test the interaction between SABP2 and ASM. Transgenic SABP2-silenced tobacco plants were used to determine the role of SABP2 in SAR induced by ASM. The expression of PR-1 proteins was used as a marker for SAR induction. Results showed that SABP2 converts ASM into acibenzolar that induces the expression of PR-1 proteins and develops the SAR response in ASM-treated plants.

Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis (M. tuberculosis), is a global threat and the leading cause of death among individuals infected with HIV. TB treatment requires multi-drug cocktails, due to the increasing rates of drug resistance of the bacterium. With multi-drug cocktails, strains have been documented to be resistant to all major drugs in the fight against TB. Since the strains are drug resistant, it calls for an increasing need for vaccine and treatment development for the purpose of preventing and managing the disease. The most widely distributed vaccine against TB is Bacillus Calmette-Gue´rin (BCG). Apart from being ineffective in certain individuals, BCG offers only a limited timeframe of protection, is unable to serve as a booster for extending this timeframe and due to the intradermal route of administration requires costly refrigeration and syringes. LipY protein, a M. tuberculosis cell wall lipase, may play a potential role as not only a drug target but a potential vaccine antigen. LipY is known to be up-regulated during both active infection and dormancy. In a previous study, sera from TB patients had shown an IgG and IgM response against it. In this study transplastomic Lactuca sativa and Nicotiana tabacum plants were generated by transforming the chloroplasts through the particle delivery system with pLsDv-LipY and pLD-LipY vectors respectively. The vectors were flanked by the native trnI and trnA gene sequence to facilitate homologous recombination into the chloroplast genome. The vector also contained the 16S rRNA promoter, the selectable marker gene, aadA for specitinomycin resistance, the rbcL untranslated region, the LsPpsbA (PpsbA in N. tabacum) promoter, and LsTpsbA (tpsbA in N. tabacum) untranslated region. iv Site specific integration of the LipY gene into the chloroplast genome was confirmed by PCR. Homoplasmy of transplastomic plants was confirmed by Southern blot analysis. These plants showed normal growth and were fertile, producing seeds. Once germinated, these seeds did not show Mendelian segregation of the transgene. Immunoblot analysis was performed to analyze the expression of the LipY protein. A 40kDa protein was produced in E.coli, and a 25kDa protein was produced in chloroplasts; a cleaved product in chloroplasts is still valuable as an antigen for vaccine production. Future studies will include testing this chloroplast derived antigen in animal models for vaccine development.

Host resistance has become a cornerstone of sustainable production of flue-cured tobacco in regions where root-knot nematodes present a threat to growers. Resistance to races 1 and 3 of M. incognita, historically the most significant root-knot nematode threat to tobacco production, is now widely available in commercially available flue-cured tobacco varieties, and is imparted by the gene Rk1. The same gene also provides resistance to race 1 of M. arenaria. The widespread deployment of this resistance has fostered a shift in root-knot nematode population dynamics, as a result of which M. arenaria race 2 has become the predominant root-knot nematode threat in Virginia. A second resistance gene known to impart resistance to M. javanica, Rk2, has also been incorporated into numerous released cultivars in combination with Rk1. This combination has been demonstrated to impart increased resistance to M. incognita and M. javanica relative to either gene alone. In the present work, eleven greenhouse trials conducted from 2017-2019, as well as two trials conducted in 2018 and 2019 on a cooperating farm, investigated the efficacy of this stacked resistance against M. arenaria race 2 and compared the effect of stacking both resistance genes to the effect of either gene alone relative to a susceptible cultivar. We also evaluated how these forms of resistance compare with resistance possessed by a breeding line with resistance reportedly derived from N. repanda to determine if additional, novel sources of resistance to root-knot nematodes previously identified from other species in the genus Nicotiana could play a role in expanding the genetic diversity of germplasm available for the refinement of host resistance in flue-cured tobacco. Additionally, in light of potential new rule making from the FDA mandating reduced nicotine content of cured tobacco leaf, we investigated the relationship between alkaloid (nicotine) content of flue-cured tobacco and root-knot nematode parasitism, while also evaluating nematode parasitism effects on carbohydrate content. Despite considerable variability in our results, particularly under field conditions, our results demonstrate that stacking Rk1 and Rk2 imparts greater resistance to M. arenaria race 2 than either gene alone, but that an entry possessing resistance reportedly derived from N. repanda exhibited significantly greater resistance to root-knot nematodes than the combination of Rk1 and Rk2 based on root galling, and egg mass and egg production. The alkaloid content of flue-cured tobacco did not appear to have an effect on root-knot nematode parasitism under greenhouse or field conditions, but the presence of the nematode did lead to increased accumulation of nicotine in the roots of plants, while translocation of nicotine to leaves was reduced. Conversely, root-knot nematode parasitism was reduced accumulation of carbohydrates in roots, while having no significant effects on leaf carbohydrate content. / Ph.D. / Root-knot nematodes (Meloidogyne spp.) are microscopic round worms that can cause considerable damage to flue-cured tobacco (Nicotiana tabacum L.), and while not typically responsible for killing plants outright, can reduce the quality of cured tobacco leaf and may predispose plants to a host of other issues, resulting in challenges and economic burdens on growers. Chemicals that effectively control nematodes, which are animals, pose inherent threats to human applicators and may harm the environment in a number of ways, so the use of tobacco varieties that are resistant to root-knot nematodes is increasingly common and essential to sustainable tobacco production. One form of root-knot nematode resistance, called Rk1, has become common and is found in all commercially grown flue-cured tobacco. This form of resistance is effective against 2 ‘races’ of the root-knot nematode M. incognita, which has historically caused tobacco growers the most issues. However, because this resistance is so widely employed, growers have controlled these nematodes, while another species, M. arenaria, has become more prevalent, particularly ‘race’ 2, which is not controlled by Rk1. We know from previous research that another gene, Rk2, provides resistance to some root-knot nematode that Rk1 does not effect, and that combining both genes seems to provide even greater root-knot nematode control than either gene alone. We investigated whether Rk2 is effective at controlling M. arenaria race 2 when it is combined with Rk1 in greenhouse and field experiments. We also investigated how a different, novel type of resistance, which comes from a species of tobacco related to cultivated tobacco, compares with the Rk1/Rk2 resistance in greenhouse trials. Additionally, the FDA has recently suggested that nicotine levels in tobacco leaf should be dramatically reduced to help mitigate adverse human health consequences associated with tobacco consumption. Nicotine may play some role in resistance to root-knot nematode in tobacco, and conversely, root-knot nematodes may impact levels of nicotine, as well as other important chemical constituents of tobacco. We also investigated these questions in greenhouse and field experiments. Our results ultimately demonstrate that combining both Rk1 and Rk2 gives flue-cured tobacco a higher level of resistance to root-knot nematodes than either gene alone, but also suggests that the form of resistance we evaluated from a related Nicotiana species could be even more effective in controlling these nematodes. We observed that the amount of nicotine present in tobacco did not impact nematode parasitism, but that nematode parasitism could lead to lower levels of nicotine in the leaves of plants because the nematodes, which feed on plants roots, cause damage to the plant that interferes in the movement of nicotine from roots to leaves.

Utilizing resistant cultivars is a main control strategy for root-knot nematodes in flue-cured tobacco (Nicotiana tabacum L.). Most commercial cultivars possess the Rk1 gene, providing resistance to races 1 and 3 of Meloidogyne incognita and race 1 of M. arenaria. This initiated a shift in root-knot populations to other species and races, creating a need for resistance to those populations. Numerous cultivars possess a second resistance gene, Rk2. Greenhouse experiments investigated whether possessing both Rk1 and Rk2 increases resistance to a variant of M. incognita race 3 compared to either gene alone, and if high soil temperatures impact their efficacy. Root galling, numbers of egg masses and eggs, and the reproductive index were compared from roots of Coker 371-Gold (susceptible), NC 95 and SC 72 (Rk1Rk1), T-15-1-1 (Rk2Rk2), and STNCB-2-28 and NOD 8 (Rk1Rk1 and Rk2Rk2). The same data were analyzed from plants in open-top root zone cabinet growth chambers set to 25ºC, 30ºC, and 35ºC to examine if resistance is temperature sensitive. Despite variability, Rk1Rk2 entries conferred greater resistance than entries with Rk1 or Rk2 alone. Entries with Rk1 alone reduced galling and reproduction compared to the susceptible control, whereas T-15-1-1 (Rk2) did not, but often suppressed reproduction. An apparent reduction in nematode reproduction was observed at 25ºC and 30ºC on entries possessing Rk1 and Rk1Rk2 compared to the control and Rk2. However, no apparent differences in reproduction occurred on Rk1 and/or Rk2 entries at 35ºC compared to the control, indicating parasitism increased on resistant entries at higher temperatures. / Master of Science in Life Sciences

The ecology and evolution of a species is often considered only within the context of pairwise interactions even though a species' distribution and abundance may be determined by interactions with many species within and between trophic levels. Multiple herbivores often share the same host and may interact indirectly by altering the relationships between herbivores, their host plants and their parasitoids. However, the relationships between parasitoids and herbivore hosts have typically been studied in isolation of other herbivore species. I examined how the outcomes of species interactions change when multiple relationships are considered. Chapter 1 examined the potentially conflicting selection pressures Manduca sexta exerts on Nicotiana tabacum (tobacco), since M. sexta has pollinating adults but herbivorous larvae. I demonstrated that high nectar amino acids do not affect floral visitation, but increased oviposition of herbivores on leaves. Thus, the relative costs and benefits of nectar rewards may depend on the community of pollinators and their life histories. In the remaining chapters I examined how feeding on tobacco by the aphid Myzus persicae altered the interactions between a parasitoid (Cotesia congregata) and its hornworm host (M. sexta). Chapter 2 demonstrated that aphids reduced hornworm abundance and parasitism. Changes in hornworm abundance were not due to density-dependent changes in moth oviposition, but the proportion of caterpillars attacked by parasitoids was inversely density dependent with hornworm density. Chapter 3 examined whether changes in hornworm abundance and parasitism reflected aphid-induced changes in host plant quality or volatile emissions. Aphids increased hornworm mortality, did not affect parasitoid performance, and increased parasitoid search time. In combination with Chapter 2, results suggest that aphids can mediate parasitoid-caterpillar interactions through changes in host plants that reduce hornworm survival and alter parasitoid behavior. Chapter 4 addressed how the outcome of interactions that are altered by aphids changed with spatial scale, and found no effect spatial scale on hornworm abundance and parasitism. In this system, aphids alter parasitoid-caterpillar interactions through changes in plant quality that reduce hornworm performance and abundance, and in turn, influence parasitoid attack. This work demonstrates that the outcome of multispecies interactions may not be predictable from pairwise interactions. / Ph. D.

Bacterial fruit blotch (BFB) of cucurbits is caused by Acidovorax citrulli, a Gram-negative seedborne bacterium that can cause up to 100% fruit yield losses in the field. Currently, BFB is a major problem for the cucurbits industry worldwide. Thus far, attempts to identify resistance in cucurbit germplasm for controlling BFB have been unsuccessful. Despite the importance of the disease, little is known about the molecular mechanisms of A. citrulli pathogenicity, due to a lack of molecular tools for studying the A. citrulli/cucurbit interaction. The genomic sequence of A. citrulli strain AAC00-1 has been determined, and the components of type III secretion system have been identified. The goal of this research was to develop molecular tools for studying the BFB disease. Nineteen putative type III effector genes were cloned from two representative A. citrulli strains (AAC00-1 and M6). The distribution of 19 type III effectors among A. citrulli strains, collected worldwide, was studied. A novel Gateway-compatible binary vector was developed for transient expression of A. citrulli type III effectors genes in planta. A set of modified vectors for marker-exchange mutagenesis in A. citrulli were constructed. The model plant species Nicotiana benthamiana was found to be susceptible to A. citrulli, while Nicotiana tabacum was resistance to A. citrulli, so therefore could carry nonhost resistance genes. Two T3S effectors, Aave1548 and Aave2166, triggered water soaking-like cell death in N. benthamiana, but HR-like cell death in N. tabacum. Bacterial mutagenesis and in planta disease assay confirmed that both Aave1548 and Aave2166 have significant virulence contributions to A. citrulli in N. benthamiana plant and melon seeds. Aave2166 encodes a putative acetyltransferase that belongs to the YopJ super family, which is conserved in both animal and plant pathogenic bacteria. Wild type but not the putative catalytic mutant (C232A) of Aave2166 can trigger cell death phenotype in N. benthamiana and N. tabacum. N. benthamiana yeast two-hybrid cDNA library screening using Aave2166 identified six N. benthamiana proteins/peptides which specifically interacted with Aave2166. Further characterization of these Aave2166 interactors may allow us to understand the virulence mechanism provided by Aave2166. The identification of nonhost resistance genes that can recognize Aave2166 and other type III effectors may help to develop novel strategies to control BFB disease of cucurbit. / Ph. D.