Essa pesquisa objetivou avaliar métodos para estimar o vigor das sementes de fumo (Nicotiana tabacum L.) variedade Virgínia, cultivar CSC 439, nuas e peletizadas representadas por cinco lotes de sementes. Essas sementes foram submetidas aos seguintes testes de vigor: condutividade elétrica (0,5; 0,8; e 1,0 g e 2,5; 4,0 e 5,0 g de sementes nuas e peletizadas, respectivamente, hidratadas por 2, 4, 6, 8 e 24h em 25 mL de água destilada à 25 °C), envelhecimento acelerado (41 °C e 43 °C por 12 e 24h) com água (100% UR) e com solução salina de NaCl saturada (76% UR) e deterioração controlada (graus de umidade de 20% e 24% para sementes nuas e 8% e 12% para peletizadas, a 40 °C e 43 °C por 24 e 48h). As avaliações foram realizadas aos 7, aos 10 e aos 16 DAS (dias após a semeadura). Adicionalmente foi determinado o grau de umidade e realizados os testes de germinação, de primeira contagem de germinação e, a emergência da plântula e a velocidade de emergência da plântula. O delineamento experimental foi o inteiramente casualizado com quatro repetições. Os dados foram submetidos separadamente à análise de variância e a comparação das médias pelo teste de Tukey a 5% de probabilidade. Todas as análises foram repetidas uma vez. Conclui-se que o teste de condutividade elétrica não é eficiente para ordenar os lotes de semente de fumo, nuas e peletizadas, em diferentes níveis de vigor. Para o teste de envelhecimento acelerado as condições mais adequadas são 41 ºC por 12 horas de exposição com avaliação aos 7 dias após a semeadura, utilizando água (100% UR) para as sementes nuas e solução salina de NaCl (76% UR) para as sementes peletizadas. Para o teste de deterioração controlada, as combinações mais adequadas para as sementes nuas são 24% de água, exposição a 43 °C por 24h e avaliação aos 7 dias após a semeadura e para as sementes peletizadas 8 % de água a 43 °C por 48h e avaliação aos 16 dias. / The objective of this research was to evaluate methods for estimating the physiological quality of tobacco seeds (Nicotiana tabacum L.) \'Virginia variety, CSC 439\' cultivar. For this, five original seeds lots and five coated seed lots were used. The seed vigor were evaluated by electrical conductivity test (0.5, 0.8, and 1.0 g and 2.5, 4.0 and 5.0 g of original and coated seeds, respectively, hydrated for 2, 4, 6, 8 and 24 hours in 25 mL of distilled water at 25 ° C), accelerated aging test (41 ° C and 43 ° C for 12 and 24 hours) with water and saturated salt solution (NaCl) and controlled deterioration test (moisture content 20% and 24% for original seeds and 8% and 12% for coated seeds at 40 ° C and 43 ° C for 24 and 48). The evaluations were performed at 7, 10 and 16 DAS (days after sowing). Additionally, it was determined the seed the moisture content, germination test, first counting, seedling emergence and speed of seedling emergence. The experimental design was a completely randomized and the means were compared by Tukey test (5%). In conclusion, the electrical conductivity test is not efficient to sort lots of original and coated tobacco seeds in different levels of vigor. On the accelerated aging test the most adequate conditions are observed at 41 ºC for 12 hours of exposition and evaluations performed at 7 days after sowing, by using water (100% HR) for the original seeds and NaCl saturated salt solution (76% HR) for coated seeds. On the controlled deterioration test for the tobacco seeds the most adequated conditions are observed with the combinations of 24% of moisture content for the original seeds at 43 °C for 24 hours on evaluations performed at 7 days after sowing and 8% moisture content at 43 °C for 48 hours of exposition for coated seeds.

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...

Thorium is an element belonging to heavy metals, which is characterized by its radioactivity similarly to uranium and radon. Thorium is not commonly used in industry, but becouse of its radioactive properties it has a great potentional for future use in nuclear energetics. Thus, increasing release of Th into the soil, water and atmosphere can be expected in near future and through plant biomass it could become a part of food chains and webs and, thus, to represent a considerable health risks to humans. Studies devoted to research on thorium and its effects on plants has not been published much yet. The majority of these studies focus mainly only on plant abilities to accumulate thorium and/or monitoring distribution of thorium in plant body. But till now, a study is still missing, which would monitor the effect of thorium on the physiological characteristics of plants. Plants of Nicotiana glutinosa (L.) medium Thorium accumulatin were hydroponically cultivated Hoagland nutrient media differedin the presence of thorium, tartaric acid, putrescine and phosphates. In first part of the present study I monitored accumulation of thorium by tobacco plants under the influence of the above-mentioned modifications of media. In the second part, I studied the Th effect on the photosynthetic apparatus (contents...

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.

[ES] La Biología Sintética de Plantas tiene como objetivo rediseñar las plantas para que adquieran características y funcionalidades novedosas a través de circuitos reguladores ortogonales. Para lograr este objetivo, se deben desarrollar nuevas herramientas moleculares con la capacidad de interactuar con factores endógenos de manera potente y específica. CRISPR/Cas9 surgió como una herramienta prometedora que combina la capacidad personalizable de unión al DNA, a través de la versión catalíticamente inactivada de la proteína Cas9 (dCas9), con la posibilidad de anclar dominios autónomos de activación transcripcional (TADs) a su estructura para lograr una regulación específica de la expresión génica. Los activadores transcripcionales programables (PTAs) pueden actuar como procesadores específicos, ortogonales y versátiles para el desarrollo de nuevos circuitos genéticos en las plantas. En busca de dCas9-PTA optimizados, se llevó a cabo una evaluación combinatoria de diferentes arquitecturas dCas9 con un catálogo de varios TAD. La mejor herramienta resultante de esta comparación, denominada dCasEV2.1, se basa en la estrategia scRNA y la combinación de los dominios de activación EDLL y VPR con un bucle multiplexable gRNA2.1, que es una versión mutada del gRNA2.0 descrito previamente. En este trabajo, el activador programable dCasEV2.1 demostró ser una herramienta potente y específica, logrando tasas de activación más altas que otras estrategias dCas9 disponibles en plantas. Se observaron tasas de activación sin precedentes dirigidas a genes endógenos en N. benthamiana, acompañadas de una estricta especificidad en todo el genoma, lo que hace que esta herramienta sea adecuada para la regulación estricta de redes reguladoras complejas. Como prueba de concepto, se diseñaron cuatro programas de activación para distintas ramas de la ruta de los flavonoides, buscando obtener enriquecimientos metabólicos específicos en hojas de N. benthamiana. El análisis metabólico de las hojas metabólicamente reprogramadas mediante dCasEV2.1 reveló un enriquecimiento selectivo de los metabolitos diana y sus derivados glicosilados, que se correlacionaron con el programa de activación empleado. Estos resultados demuestran que dCasEV2.1 es una herramienta eficaz para la ingeniería metabólica y un componente clave en los circuitos genéticos destinados a reprogramar los flujos metabólicos. Finalmente, basándonos en dCasEV2.1, desarrollamos un sistema optimizado de regulación de genes inducidos por virus (VIGR) que utiliza un vector Potato Virus X (PVX) para el suministro de los programas de activación CRISPR codificados con gRNA. Este enfoque permite controlar el transcriptoma de la planta a través de una aplicación sistémica basada en aerosol de componentes CRISPR a plantas adultas. El nuevo sistema PVX-VIGR produjo una fuerte activación transcripcional en varios genes diana endógenos, incluidos tres factores de transcripción MYB-like seleccionados. Las activaciones específicas de MYB condujeron a perfiles metabólicos distintivos, demostrando que las aplicaciones potenciales de la herramienta dCasEV2.1 en plantas incluyen la obtención de perfiles metabólicos personalizados utilizando un suministro basado en aerosol de instrucciones de reprogramación transcripcional codificadas por gRNA. En resumen, esta tesis proporciona herramientas novedosas para la activación transcripcional fuerte, ortogonal y programable en plantas, con una caja de herramientas ampliada para el suministro de los programas de activación. / [CA] La Biologia Sintètica de Plantes té com objectiu redissenyar les plantes per que obtinguen característiques i funcionalitats innovadores mitjançant circuits reguladors ortogonals. Per arribar a aquest objectiu, s'han de desenvolupar noves ferramentes moleculars amb la capacitat d'interactuar amb factor endògens d'una manera potent i específica. CRISPR/Cas9 va sorgir com una ferramenta prometedora que combina la capacitat personalitzable d'unió al DNA, mitjançant la versió catalíticament inactivada de la proteïna Cas9 (dCas9), amb la possibilitat de fixar dominis autònoms de activació transcripcional (TADs) a la seua estructura per aconseguir una regulació específica de la expressió gènica. Els activadors transcripcionals programables (PTAs) poden actuar com a processadors específics, ortogonals i versàtils per al desenvolupament de nous circuits genètics a les plantes. Buscant dCas9-PTA optimitzats, es va realitzar una avaluació combinatòria de distintes arquitectures dCas9 amb un catàleg de diversos TAD. La millor ferramenta segons aquesta comparació, anomenada dCasEV2.1, es basa en la estratègia scRNA i la combinació del dominis d'activació EDLL i VPR amb un bucle multiplexable gRNA2.1, que es una versió mutada del gRNA2.0 descrit prèviament. En aquest treball, el activador programable dCasEV2.1 es va mostrar com una ferramenta potent i específica, aconseguint nivells d'activació majors que altes estratègies dCas9 disponibles en plantes. Es van observar taxes d'activació sense precedents dirigides a gens endògens en N. benthamiana, junt a una estricta especificitat en tot el genoma, indicant que aquesta ferramenta és adequada per a la regulació estricta de xarxes reguladores complexes. Como proba de concepte, se van dissenyar quatre programes d'activació per a diferent branques de la ruta dels flavonoides, cercant obtenir enriquiments metabòlics específics en fulles de N. benthamiana. L'anàlisi metabòlic de les fulles metabòlicament reprogramades mitjançant dCasEV2.1 va revelar un enriquiment selectiu del metabòlits diana i els seus derivats glicosilats que es correlacionen amb el programa d'activació emprat. Aquests resultats demostren que dCasEV2.1 és una ferramenta eficaç per a l'enginyeria metabòlica i un component clau als circuits genètics destinats a reprogramar els fluxos metabòlics. Finalment, en base a dCasEV2.1, desenvoluparem un sistema optimitzat de regulació de gens induïts per virus (VIGR) que utilitza un vector Potato Virus X (PVX) per al subministrament dels programes d'activació CRISPR codificats amb gRNA. Aquesta aproximació permet controlar el transcriptoma de la planta mitjançant l'aplicació sistèmica basada en aerosol de components CRISPR a plantes adultes. El nou sistema PVX-VIGR va produir una gran activació transcripcional en diversos gens diana endògens, inclosos tres factors de transcripció MYB-like seleccionats prèviament. Les activacions específiques de MYB conduïren a perfils metabòlics distintius, demostrant que les aplicacions potencials de la ferramenta dCasEV2.1 en plantes inclouen la obtenció de perfils metabòlics personalitzats emprant un subministrament basat en aerosol de instruccions de reprogramació transcripcional codificades per gRNA. En resum, aquesta tesis proporciona noves ferramentes per a l'activació transcripcional forta, ortogonal i programable en plantes, amb una caixa de ferramentes eixamplada per al subministraments dels programes d'activació. / [EN] Plant Synthetic Biology aims to redesign plants to acquire novel traits and functionalities through orthogonal regulatory circuits. To achieve this goal, new molecular tools with the capacity of interacting with endogenous factors in a potent and specific manner must be developed. CRISPR/Cas9 emerged as promising tools which combine a customizable DNA-binding activity through the catalytically inactivated version of Cas9 protein (dCas9) with the possibility to anchor autonomous transcriptional activation domains (TADs) to its structure to achieve a specific regulation of the gene expression. The Programmable Transcriptional Activators (PTAs) could act as specific, orthogonal and versatile processor components in the development of new genetic circuits in plants. In search for optimized dCas9-PTAs, a combinatorial evaluation of different dCas9 architectures with a catalogue of various TADs was performed. The best resulting tool of this comparison, named dCasEV2.1, is based on the scRNA strategy and the combination of EDLL and VPR activation domains with a multiplexable gRNA2.1 loop, which is a mutated version of the previously described gRNA2.0. In this work, the dCasEV2.1 programable activator was proved to be a strong and specific tool, achieving higher activation rates than other available dCas9 strategies in plants. Unprecedented activation rates were observed targeting endogenous genes in N. benthamiana, accompanied by strict genome-wide specificity that makes this tool suitable to perform a tight regulation of complex regulatory networks. As a proof of concept, a design of four activation programs to activate different branches of the flavonoid pathway and obtain specific metabolic enrichments in N. benthamiana leaves was performed. The metabolic analysis on the dCasEV2.1 metabolically reprogrammed leaves revealed a selective enrichment of the targeted metabolites and their glycosylated derivatives that correlated with the activation program employed. These results demonstrate that dCasEV2.1 is a powerful tool for metabolic engineering and a key component in genetic circuits aimed at reprogramming metabolic fluxes. Finally, based on dCasEV2.1, we developed an optimized Viral Induced Gene Regulation (VIGR) system that makes use of a Potato Virus X (PVX) vector for the delivery of the gRNA-encoded CRISPR activation programs. This approach offers a way to control the plant transcriptome through a spray-based systemic delivery of CRISPR components to adult plants. The new PVX-VIGR system led to strong transcriptional activation in several endogenous target genes, including three selected MYB-like transcription factors. Specific MYB activations lead to distinctive metabolic profiles, showing that the potential applications of the dCasEV2.1 tool in plants include the obtention of custom metabolic profiles using a spray-based delivery of gRNA-encoded transcriptional reprogramming instructions. In sum, this thesis provides novel tools for strong, orthogonal and programmable transcriptional activation in plants, with an expanded toolbox for the delivery of the activation programs. / Selma García, S. (2022). Development of CRISPR-based programmable transcriptional regulators and their applications in plants [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/185046 / TESIS

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.