“Examining the regulation of herbivore-induced defense genes in maize”

Project Summary:

Maize, one of the most common staple food crops, is susceptible to several major pests on the field. As a defense mechanism against insect herbivory, maize produces a mixture of volatile terpenes, including (E)-nerolidol, linalool and (E, E)-geranyllinalool. There is a significant difference in terpene production between the Ky21 and B73 maize inbred lines. Genetic mapping studies revealed a quantitative trait locus (QTL) for this trait in a bHLH (basic helix loop helix) transcription factor.  Sequence comparisons showed that this QTL coincides with a 41 amino acid deletion in Ky21 compared to B73. We hypothesized a role for the transcription factor in regulating the expression of both tps2 (terpene synthase2) and sm2 (salmon silk2), a gene that is involved in the production of maysin, a defensive compound in the maize silks that confers resistance to corn earworm (Helicoverpa zea). I used a yeast two-hybrid assay to determine whether the transcription factor alleles from the two inbred lines show a variation in the homodimerization ability. The bait and prey vectors containing either the B73 or the Ky21 allele were cotransformed into yeast cells and grown on double minimal medium, which is selective for only the cotransformed cells. Colonies from these plates were subcultured on triple minimal medium, which is selective for interactions between the genes of interest. The medium was supplemented with X-gal to quantify the level of protein interaction through the development of blue-colored colonies, caused by the transcription activation of the LacZ reporter gene in the bait vector. Preliminary results are showing better growth and stronger blue staining of co-transformed yeast cells carrying the bHLH B73 allele. To complement the yeast two-hybrid assay, a qRT PCR analysis was done on bHLH overexpression maize lines to compare the expression level of bHLH and measure the effect on tps2 expression genes.

Overall the comparison of yeast two-hybrid interactions and qRT-PCR data gave us interesting and conclusive results, which can be further used in studying gene regulation by the bHLH transcription factor. Also, in a bigger prospect, these results may be useful in developing maize lines by enhancing pest resistance.

My Experience:

I found BTI a place of unity in diversity, working here gave me an idea of how we should investigate science correlating them with problems in agriculture and try to have a big picture of whatever work we do in the lab. I am fortunately to have been accepted and appreciate working with my bright mentor, Annett Richter. She was a great guide to work with and she understood each and every difficulty I faced in the lab, explaining to me patiently. Working in the Jander lab is really amazing and includes many diverse and intellectual people. This internship really gave a chance to learn how to look for problems in agriculture, how to plan a research program and conduct it. Working here has given great confidence to move forward in my career. The kind of techniques and methodology I have learned here will always remain a foundation for my future steps in the field of plant biology. I am thankful to Georg Jander and Annett for there great support and guidance.