Conserved defenses in Nicotiana tabacum in response to Helicoverpa zea and Manduca sexta feeding

Herbivory stimulates the plant's “immune-like” system involving the production of protease inhibitors and related proteins which affect the herbivores digestion and absorption of plants nutrients, and can ultimately lead to death of the insect. Studies have shown how plants respond to attackers at the molecular level through elicited transcript patterns in response to assailants. Others have also demonstrated that host plants transcript patterns vary according to the type of attacking organisms. Using microarray analysis, I investigated whether or not tobacco (Nicotiana tabacum) responded differently to feeding from two Lepidopteran species: the specialist caterpillar Manduca sexta (tobacco hornworm) or the generalist caterpillar Helicoverpa zea (tomato fruitworm). I also examined the similarities between the microarray results and phytohormones-induced defense responses in tobacco. In this study, I hypothesize that specialist herbivores would induce different defense responses in plants compared to their generalist counter parts. Three treatments (M. sexta, H. zea and the non-wounded control) with five replicate tobacco plants per treatment were used. After 24 hours of feeding, tobacco leaves were harvested and flash frozen in liquid nitrogen. Tobacco microarray analysis illustrated that ~5400 (8%) genes were altered (P<0.05) with more than half with unknown functions. Defense, hypersensitive, and stress genes such as arginase, serine decarboxylase, and pathogenesis-related proteins were generally up regulated in herbivory treated tobacco plants, while photosynthesis-related genes were generally down regulated irrespective of the type of herbivores. Overall, ~75% of herbivore altered gene expression was similar between the two caterpillar treatments, with the most of the differently altered gene expression being genes with unknown function. However, genes such as xyloglucan endotransglycosylase/hydrolase and 50S ribosomal protein L21 showed higher expression in the H. zea treatment. Q-RT-PCR (Quantitative Real-Time Polymerase Chain Reaction) verified that seven defense-related genes (acidic chitinase, basic chitinase, defensin, putrescine N-methyltransferase I, s-adenosyl-L-methionine synthase, s-adenosylmethionine decarboxylase, and s-adenosylmethionine synthtase) were up regulated (especially defensin and the chitinases) in tobacco plants fed on by caterpillars in comparison to their non-wounded controls. When comparing between caterpillar treatments, although most genes were up regulated similarly, H. zea feeding induced higher expression levels in both defensin and basic chitinase than M. sexta. It is known that plant defenses could be stimulated by phytohormone mediated signal transduction. The exogenous phytohormones application on tobacco leaves over 24hrs highly correlated to some caterpillar herbivory microarray results and Q-RT-PCR results. Expression levels of both acidic chitinase and defensin were significantly higher in Jasmonate/Salicylate-treated plants compared to any single treatment, mirroring the high expression of defensin and basic chitinase in H. zea treated plants and suggestive of the caterpillar triggering both the systemic acquired resistance (SAR) and the induced resistance (IR) pathways in N. tabacum. Compared to my predicted outcome for the study, these results showed a different picture that suggest the need for additional analysis into the small number of genes that showed differential expression when comparing N. tabacum responses to specialist versus generalist herbivores.