Verticillium wilt, caused by the soil-borne Verticillium dahliae Klebahn is a serious problem in the production of sunflower worldwide. To date, information on sunflower resistance to Verticillium spp. is very scarce, although it is critical for an effective management of this pathogen. In this study, two highly aggressive (Vd1396-9 and Vd1398-21) and two weakly aggressive V. dahliae isolates (Vs06-07 and Vs06-14) were used to inoculate moderately resistant (IS6111) and susceptible (IS8048) sunflower hybrids. VdNEP (V. dahliae necrosis and ethylene-inducing protein), an elicitor from V. dahliae, was also used to infiltrate sunflower plants. Our results indicate that VdNEP has a dual role in the interaction between sunflower and V. dahliae. VdNEP acted not only as a pathogenicity factor on sunflower by inducing wilting symptoms such as chlorosis, necrosis and vascular discoloration, but also as an elicitor triggering defense responses of the host. VdNEP induced the hypersensitive cell death in Nicotiana benthamiana leaves and sunflower cotyledons. Moreover, VdNEP activated the production of reactive oxygen species and the accumulation of fluorescent compounds in sunflower leaves. Pathogenesis-related genes (Ha-PR-3, and Ha-PR-5), two defensin genes (Ha-PDF and Ha-CUA1) and genes encoding Ha-ACO, Ha-CHOX, Ha-GST and Ha-SCO were up-regulated by VdNEP, suggesting that multiple signaling pathways are involved in this interaction. Two SA-related genes (Ha-PAL and Ha-NML1) were slightly suppressed after infiltration with VdNEP, suggesting a possible involvement of VdNEP in affecting sunflower defenses.

Bacterial endosymbionts can have profound impacts on their host’s ecology. Notably, endosymbionts can protect their hosts against natural enemies and influence host plant interactions. The endosymbiont Candidatus Arsenophonus infects a wide taxonomic range of arthropod hosts, and is suspected of an uncharacterized mutualistic role in hemipterous insects. In the soybean aphid, Aphis glycines, an introduced pest of soybeans in the United States, Arsenophonus is the sole facultative endosymbiont. The focus of this dissertation is to characterize the role of Arsenophonus in the aphid, with an overall emphasis on its impact on aphid management strategies. I first used diagnostic PCR to determine Arsenophonus infection frequency and strain diversity for native and introduced soybean aphids. I found that Arsenophonus infection is a uniform strain that is highly prevalent in soybean aphid. I then determined if Arsenophonus was a defense symbiont by curing two genotypes of soybean aphid of their natural Arsenophonus infection, resulting in infected and uninfected isolines within the same genetic background. I subjected these isolines to assays with three parasitoid species and a common aphid fungal pathogen, Pandora neoaphidis. I did not find differences in parasitism or fungal infections within the treatments. These results indicate that, although Arsenophonus is widespread, the symbiont should not interfere with biological control efforts. I next examined the influence of Arsenophonus on the ability of soybean aphid “biotypes” to colonize resistant Rag plants. I cured three additional soybean aphid biotypes. All isolines were subjected to growth rate assays on resistant Rag versus susceptible soybean. My results indicate that Arsenophonus infected soybean aphids have an increased population growth compared to uninfected aphids regardless of soybean plant type Finally, I induced soybean plants with jasmonic acid (JA) or salicylic acid (SA) to determine the effective plant defense against soybean aphid feeding. I also used Arsenophonus infected and uninfected aphids to determine any interaction between Arsenophonus and plant defense. I found SA treatment decreased soybean aphid population growth for one experiment, but had no effect when replicated. JA treatment had no effect, and there were no interactions between Arsenophonus infection and plant treatments.

Verticillium wilt, caused by the soil-borne Verticillium dahliae Klebahn is a serious problem in the production of sunflower worldwide. To date, information on sunflower resistance to Verticillium spp. is very scarce, although it is critical for an effective management of this pathogen. In this study, two highly aggressive (Vd1396-9 and Vd1398-21) and two weakly aggressive V. dahliae isolates (Vs06-07 and Vs06-14) were used to inoculate moderately resistant (IS6111) and susceptible (IS8048) sunflower hybrids. VdNEP (V. dahliae necrosis and ethylene-inducing protein), an elicitor from V. dahliae, was also used to infiltrate sunflower plants. Our results indicate that VdNEP has a dual role in the interaction between sunflower and V. dahliae. VdNEP acted not only as a pathogenicity factor on sunflower by inducing wilting symptoms such as chlorosis, necrosis and vascular discoloration, but also as an elicitor triggering defense responses of the host. VdNEP induced the hypersensitive cell death in Nicotiana benthamiana leaves and sunflower cotyledons. Moreover, VdNEP activated the production of reactive oxygen species and the accumulation of fluorescent compounds in sunflower leaves. Pathogenesis-related genes (Ha-PR-3, and Ha-PR-5), two defensin genes (Ha-PDF and Ha-CUA1) and genes encoding Ha-ACO, Ha-CHOX, Ha-GST and Ha-SCO were up-regulated by VdNEP, suggesting that multiple signaling pathways are involved in this interaction. Two SA-related genes (Ha-PAL and Ha-NML1) were slightly suppressed after infiltration with VdNEP, suggesting a possible involvement of VdNEP in affecting sunflower defenses.

As sessile organisms, plants are unable to escape from attack by herbivorous insects. To cope with this pressure, plants have evolved several defense strategies, including the production of secondary metabolites, specialized chemicals with ecological functions. Most studies have focused on the role of secondary metabolites in plant defense against chewing insects. Little is known about what compounds are present in phloem sap and how they affect phloem feeding insects. Therefore, I investigated the effects of phenolic compounds on phloem feeders, using Chaitophorus aphids in bioassays with wildtype and transgenic poplar overexpressing the transcription factor MYB 134, which results in elevated levels of tannins and reduced levels of phenolic glycosides. Aphids produced significantly more offspring on MYB 134 plants but showed a significant preference for lower tannin leaf tissue. Analysis of poplar phloem exudates and aphid extracts provides direct evidence that the phenolic glycosides salicin, salicortin and tremulacin are present in poplar phloem and are ingested by aphids. These results are discussed in relation to what is driving the differences in aphid fecundity and choice between plant types. / Graduate / 0309 / 0307 / 0329 / alphareghanwong@gmail.com

Myzus persicae (Sülzer), commonly known as green peach aphid (GPA), utilizes its slender stylet to penetrate the plant tissues intercellularly and consume copious amounts of photoassimilates present in the phloem sap causing extensive damage to host plants. The compatible interaction between GPA and Arabidopsis thaliana enabled us to characterize plant response to aphid infestation. Upon GPA infestation, Arabidopsis PAD4 (PHYTOALEXIN DEFICIENT4) gene modulates premature leaf senescence, which is involved in the programmed degradation of cellular components and the export of nutrients out of the senescing leaf. Senescence mechanism is utilized by plants to limit aphid growth. In addition, PAD4 provides antixenosis (deters insect settling and feeding) and antibiosis (impair aphid fecundity) against GPA and adversely impact sieve element availability to GPA. Basal expression of PAD4 contributes to antibiosis, and the GPA-induced expression of PAD4 contributes to antixenosis. Mutation in the Arabidopsis stearoyl-ACP desaturase encoding SSI2 (suppressor of SALICYLIC ACID [SA] insensitivity2) gene that results in an accelerated cell death phenotype and dwarfing, also conferred heightened antibiosis to GPA. Results of this study indicate that PAD4 is required for the ssi2-mediated enhanced antibiosis to GPA. The PAD4 protein contains conserved Ser, Asp and His residues that form the catalytic triad of many α/β fold acyl hydrolases. Arabidopsis plants expressing mutant versions of PAD4 [PAD4(S118A) and PAD4(D178A)] supported higher numbers of GPA as compared to wild type (WT) plants in no-choice tests. Furthermore, Electrical Penetration Graph (EPG) studies revealed that S118 residue in PAD4 is essential to limit GPA feeding from the sieve elements. However, the ability to deter insect settling in choice tests was not impacted by the PAD4(S118A) and PAD4(D178A) mutations, thus suggesting that PAD4s involvement in deterring insect settling and in antibiosis are determined by separate regions of PAD4. The MPL1 (MYZUS PERSICAE INDUCED LIPASE1) gene is another critical component of Arabidopsis defense against GPA. Like PAD4, MPL1 expression is induced in response to GPA infestation. However, MPL1 is required only for antibiosis and is not essential for antixenosis against GPA. EPG analysis suggests that the mpl1 mutant allele does not impact aphid feeding behavior. Since, MPL1 exhibits lipase activity, and ssi2 petiole exudates contain elevated levels of antibiosis, we propose that antibiosis to GPA requires a lipid(s), or a product thereof.

Evolution has provided vast genetic diversity, enabling plants to surmount many biotic pressures. Plants have evolved various morphological and biochemical adaptations to cope with herbivores attacks. Despite that, yearly, around 40 % of worldwide crop production is lost due to pests and pathogens, with 13 % due to insects. Tuta absoluta has become a major pest threatening tomato crops worldwide and without the appropriated management it can cause production losses between 80 to 100 %. To cope with this threat, we need to strengthen plant defense arsenals. The incorporation to plants of defensive genes like proteinase inhibitors by means of genetic engineering is a promising alternative. In the first chapter of this work we investigated the inhibitory activity of two trypsin inhibitors from barley; BTI-CMe and BTI-CMc. Besides, we succeeded to increase the BTI-CMc in vitro inhibitory activity by introducing a single mutation in its putative reactive site. In the second chapter, we investigated the in vivo effect of (a serine proteinase inhibitor) BTI-CMe and a (cysteine proteinase inhibitor) Hv-CPI2 isolated from barley on Tuta absoluta and we examined the effect of their expression on the tomato defensive response. We found that larvae fed on the double transgenic plants showed a notable reduction in weight. Moreover, only 56% of the larvae reached the adult stage. The emerged adults showed wings deformities and reduced fertility. We also investigated the effect of proteinase inhibitors ingestion on the insect digestive enzymes. Our results showed a decrease in larval trypsin activity. Proteinase inhibitors had no harmful effect on Nesidiocoris tenuis; a predator of Tuta absoluta, despite transgenic tomato plants attracted the mirid. We investigated whether or not plant defensive mechanisms were activated in the transgenic tomato plants and found that, interestingly, the expression of the barley cysteine proteinase inhibitor promoted plant defense, inducing the tomato endogenous wound inducible proteinase inhibitor 2 (Pin2) gene. Moreover, glandular trichomes production was increased and the emission of volatile organic compounds was altered. Our results demonstrate the usefulness of the co-expression of different proteinase inhibitors for the enhancement of plant resistance to pests. / La evolución ha proporcionado una gran diversidad genética, permitiendo a las plantas superar muchas presiones bióticas. Las plantas han desarrollado diversas adaptaciones morfológicas y bioquímicas para hacer frente a los ataques de los herbívoros. A pesar de ello, anualmente, alrededor del 40 % de la producción mundial de cultivos se pierde debido a plagas y patógenos, siendo un 13 % debido a insectos. Tuta absoluta se ha convertido en una de las principales plagas que amenazan los cultivos de tomate en todo el mundo y sin la gestión adecuada puede causar pérdidas de producción entre el 80 y el 100 %. Para hacer frente a esta amenaza, necesitamos fortalecer los arsenales de defensa de las plantas. La incorporación a las plantas, mediante ingeniería genética, de genes defensivos como los inhibidores de proteinasas es una alternativa prometedora. En el primer capítulo de este trabajo se investigó la actividad inhibitoria de dos inhibidores de tripsina procedentes de cebada; BTI-CMe y BTI-CMc. Además, se logró aumentar la actividad inhibitoria in vitro de BTI-CMc mediante la introducción de una única mutación en su putativo centro reactivo. En el segundo capítulo, se investigó el efecto in vivo de un inhibidor de serin proteinasa (BTI-CMe) y un inhibidor de cisteín proteinasa (Hv-CPI2) aislado de cebada en Tuta absoluta y se examinó el efecto de su expresión en la respuesta defensiva del tomate. Se encontró que las larvas alimentadas con las plantas transgénicas dobles mostraron una notable reducción de peso. Además, sólo el 56 % de las larvas alcanzó la etapa adulta. Los adultos emergentes mostraron deformidades de las alas y reducción de la fertilidad. También se investigó el efecto de la ingesta de inhibidores de proteinasa en las enzimas digestivas de los insectos. Nuestros resultados mostraron una disminución en la actividad tripsina larvaria. Los inhibidores de proteinasas no tuvieron efectos nocivos sobre Nesidiocoris tenuis(depredador de Tuta absoluta) a pesar de que las plantas transgénicas de tomate atrajeron al mirido. Se investigó si los mecanismos defensivos de las plantas se activaban en las plantas de tomate transgénico y se encontró que, curiosamente, la expresión de la cistatina de cebada promovía la defensa de la planta, induciendo el gen del inhibidor de proteasa 2 endógeno del tomate, inducible por herida (Pin2). Además, aumentó la producción de tricomas glandulares y se alteró la emisión de compuestos orgánicos volátiles. Nuestros resultados demuestran la utilidad de la co-expresión de diferentes inhibidores de proteinasas para el aumento de la resistencia de las plantas a plagas. / L'evolució ha proporcionat una gran diversitat genètica, permetent a les plantes superar moltes pressions biòtiques. Les plantes han desenvolupat diverses adaptacions morfològiques i bioquímiques per fer front als atacs dels herbívors. Tot i això, anualment, al voltant del 40 % de la producció mundial de cultius es perd a causa de plagues i patògens, amb un 13 % a causa de insectes. Tuta absoluta s'ha convertit en una de les principals plagues que amenacen els cultius de tomaca a tot el món i sense la gestió adequada pot causar pèrdues de producció entre el 80 i el 100 %. Per fer front a aquesta amenaça, necessitem enfortir els arsenals de defensa de les plantes. La incorporació a les plantes de gens defensius com els inhibidors de proteïnases per mitjà de l'enginyeria genètica és una alternativa prometedora. En el primer capítol d'aquest treball es va investigar l'activitat inhibitòria de dos inhibidors de tripsina aïllats a partir d'ordi; BTI-CMe i BTI-CMC. A més, es va aconseguir augmentar l'activitat inhibitòria in vitro de BTI-CMC mitjançant la introducció d'una única mutació en el seu lloc reactiu putatiu. En el segon capítol, es va investigar l'efecte in vivo d'un inhibidor de serin proteinasa (BTI-CMe) i un inhibidor de cisteïn proteinasa (Hv-CPI2) aïllats d'ordi en Tuta absoluta i es va examinar l'efecte de la seva expressió en la resposta defensiva del tomaca. Es va trobar que les larves alimentades amb les plantes transgèniques dobles van mostrar una notable reducció de pes. A més, només el 56 % de les larves va aconseguir l'etapa adulta. Els adults emergents van mostrar deformitats de les ales i reducció de la fertilitat. També es va investigar l'efecte de la ingesta d'inhibidors de proteinasa en els enzims digestius dels insectes. Els nostres resultats van mostrar una disminució en l'activitat tripsina larvària. Els inhibidors de proteïnases no van tenir efectes nocius sobre Nesidiocoris tenuis, un depredador de Tuta absoluta, tot i les plantes transgèniques de tomaca van atreure al mirid. Es va investigar si els mecanismes defensius de les plantes s'activaven a les plantes de tomaca transgènic i es va trobar que, curiosament, l'expressió de cistatina d'ordi promovia la defensa de la planta, induint el gen de l'inhibidor de proteasa 2 endogen de la tomaca, induïble per ferida (Pin2). A més, va augmentar la producció de tricomes glandulars i es va alterar l'emissió de compostos orgànics volàtils. Els nostres resultats demostren la utilitat de la co-expressió de diferents inhibidors de proteïnases per a l'augment de la resistència de les plantes a plagues. / Hamza, R. (2017). Enhancement of tomato resistance to Tuta absoluta by the expression of two barley proteinase inhibitors [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/92723 / TESIS

Abiotic stress leads to a change in the water content of plants. Salinity and osmotic stress affect both the morphology and physiology of plants. Plants have therefore responded to these environmental changes by adapting and tolerating them. The SABP2-interacting proteins (SIP) 428-silenced RNAi transgenic tobacco lines were subjected to various abiotic stresses (salinity, osmotic, and drought). The effect of SIP428-silencing on the tobacco plants subjected to these abiotic stresses was monitored. The results from the root growth data show that the sip428-silenced lines exhibit enhanced tolerance to the stressors compared to the wild-type plants. Interestingly, results of the relative chlorophyll content show no significant difference between the wild-type plants and sip428-silenced transgenic plants. In summary, based on the results presented in this study it could be concluded that SIP428 is a negative regulator of salinity, osmotic and drought stresses. Further studies are required to understand the mechanism.

Abiotic stresses are constantly rising and pose a very high risk to global agricultural productivity and food security. Some plants have evolved several innate pathways for defense against these stresses. Hence, understanding stress signaling pathways can help develop crop plants with higher stress tolerance. The salicylic acid-mediated signaling pathway is important in plants experiencing biotic and abiotic stresses. In previous studies, SABP2-Interacting Protein (SIP-428) has been shown to be a negative regular of plant growth under abiotic stress. This study aimed to investigate the roles of SIP-428 in the ROS signaling of tobacco plants. We investigated transgenic RNAi-silenced lines of SIP-428 and wild-type tobacco plants for the activities of guaiacol peroxidase and catalase enzymes in Mannitol and NaCl-stressed plants for 7 and 14 days. Our results showed that SIP-428 plays a significant role in ROS signaling in Mannitol and NaCl-stressed plants via the activities of guaiacol peroxidase.

No description available.

Salicylic Acid (SA) is an important plant hormone which acts as a therapeutic agent in the plant in response to biotic and abiotic stress. It plays a significant role in growth and development. SABP2, a methyl salicylate esterase is a key player in SA mediated defense signaling. It catalyzes the conversion of mobile methyl salicylate to salicylic acid. During infection, accumulation of salicylic acid in the distal organ in response to the primary infection elsewhere primes the plant to defend against subsequent infection by the mechanism known as Systemic Acquired Resistance (SAR). SIP68, one of the interacting proteins of SABP2 is a glucosyltransferase protein. Glucosyltransferase protein catalyzes the formation of the glycosidic bond by transferring glucose molecule from donor to acceptor molecules. Plant glucosyltransferase is widely distributed in nature playing the dual role of activating and inactivating enzymes. They are also associated with changing the protein stability and solubility of compounds. Since SABP2 has a role in SA mediated defense signaling and glucosyltransferase proteins are associated with physiological function thus, there is a possibility of SIP68 associated with the major or supportive role in either or both functions. The purified recombinant SIP68 protein was tested for glucosyltransferase activity using radioactive method. The purified SIP68 glucosylates various artificially available flavonoid compounds with highest activity detected with Kaempferol (flavonol) followed by quercetin but negligible activity with SA. HPLC based glucosyltransferase assay further verified SIP68 as a flavonoid UDP-glucosyltransferase, not SA glucosyltransferase. Our interest is to further characterize SIP68 and assess its role in plant defense mechanism. Knowing its expression pattern inside plant cell will help us to assess its activity pattern inside the cell. For this enhanced Green Fluorescent Protein (eGFP) tagged SIP68 was transiently expressed inside the plant cell. Confocal microscopy imaging suggests SIP68 likely to be localized in the cytoplasm which will be further confirmed by subcellular fractionation. To assess the role of SIP68 in plant defense mechanism transgenic line expressing altered SIP68 gene was generated using CRISPR Cas9 technique. Verified transgenic line challenged under different biotic and abiotic stress will help us to understand the role of SIP68 in plant defense mechanism. Our research will help us to understand defense mechanism in tobacco model system enabling us to use the knowledge to develop the resistant varieties of crops that are capable of withstanding the adverse condition of pathogenic as well environmental challenges.