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.

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.

Experiments were conducted to test for ricin content in tissue samples from four castor cultivars, developing castor seed, germinating castor seedlings, and chemically and heat treated seed meal. Ricin content of each sample was examined via Western blotting with ricin A-chain specific antibodies. Results indicate that ricin is present solely within castor endosperm and is not present any other tissues. Samples from developing seed and germinating seedlings indicate ricin production begins around day 28 post pollination, and ricin is absent from the seedling 6 days after the onset of radicle emergence. This would seem to indicate that the purpose of ricin is to protect the seed and not the entire plant. Ricin content of seed meal treated separately with heat and chemicals was tested. It was found that hot-pressing of the seed was sufficient to denature ricin in the seed meal.