| The relationship between plants and insects is incredibly intricate, involving complicated cascades of biochemical pathways produced in the plant tissue that resist herbivory and pathogen attack. Cross-talk between three hormone signaling pathways, jasmonic acid, salicylic acid, and ethylene, in plants has been shown to be important in signal transduction in response to herbivory. In this experiment microarrays were utilized to study the gene expression in Nicotiana tabacum (cultivated tobacco) to determine the mechanism in which caterpillar saliva suppresses foliar nicotine accumulation in the plant. The tobacco plants were grown from seeds in an environmentally controlled growth chamber. Helicoverpa zea (Corn Earworm) caterpillars were surgically wounded for two different treatments: an ablated treatment, where the salivary glands were removed, and a mock treatment, where the surgery was mimicked but the salivary glands remained intact. The caterpillars were caged onto individual tobacco leaves and allowed to feed for 24 hours, periodically repositioning the cage to ensure optimal herbivory. The leaves were harvested and the RNA was purified from each leaf sample. Copied RNA was formed from the template RNA strand and labeled with cyanine 3 or cyanine 5 dyes. The samples were hybridized onto the Agilent tobacco oligo microarrays and analyzed. The analysis of the arrays produced hundreds of significantly altered genes from the non-wounded control in both the mock and ablated treatments. Substantial research of the 2,095 genes significantly altered showed major trends in the data that corresponded to different plant physiological pathways including defense, photosynthesis, metabolism and stress. In particular, 19% of the significantly altered plant genes were involved in the plant defense response, 16% were involved in plant growth and development, 21% were involved in the plant stress response, and 8% were involved in plant metabolism, to name a few. My results indicate that many widely known defense and pathogenesis-related genes such as basic chitinase (mock ∼5 fold, ablated ∼1 fold) and beta-1,3 glucanase (mock ∼ -6 fold, ablated ∼ -3 fold) were significantly altered between caterpillar herbivory treatments. Genes involved in ethylene synthesis and regulation were stimulated as a result of the caterpillar herbivory, including Indole-3-acetic acid (mock ∼ 3 fold, ablated ∼4 fold) and 3-hydroxyisobutyryl-coenzyme A hydrolase (mock ∼ 2 fold, ablated ~ 1 fold). Quantitative real-time PCR analysis resulted in significant differences in gene expression due to the labial saliva between the two experimental treatments in Defensin (mock ∼ 29 fold, ablated ∼ 4 fold), Putrescine N-methyltransferase (mock ∼ 15 fold, ablated ∼ 3 fold), Polyphenol oxidase (mock ∼ 17 fold, ablated ∼ 22 fold), and S-adenosylmethionine decarboxylase (mock ∼ 4 fold, ablated ∼ 11 fold). In conclusion, this study determined that H. zea caterpillar herbivory with and without labial saliva significantly altered 2, 095 genes from the control non-wounded treatment. These results show that the caterpillar oral elicitors impact the tobacco plant defense response. The microarray analysis provided in this study supports the concept that ethylene biosynthesis actively antagonizes and down regulates nicotine biosynthesis. |
