Development of Reporter Genes for Monitoring Pattern-Triggered Immunity

Project Summary

Environments rich in pathogens challenge plant growth. They reduce crop yields worldwide by 10-16%, costing $220 billion annually. Pattern-triggered immunity (PTI) is the first line of defense against pathogens. PTI relies on recognition of microbe-associated molecular patterns, essential for microbe survival, by host pattern recognition receptors. PTI activates signaling cascades leading to induced gene expression and other responsesUnderstanding signaling and regulation of PTI remains a challenge in plant pathology research and is important for crop improvement.

This investigation aimed to develop PTI-specific reporter constructs from two previously identified PTI-specific genes encoding a NAC domain protein (Solyc02g0699960) and a lipid particle serine esterase (Solyc04g077180). Towards this goal, promoter regions ~1 kb upstream of the each gene’s start site were cloned into a gusA construct. Syringe infiltration with Agrobacterium tumefaciens was used to transform the resulting plasmids into Nicotiana benthamiana and Solanum lycopersicum leaves. Constructs were tested for PTI-specific activity by treatment of transformed leaves with flg22 or P. flourescens. Gus was used as a histochemical readout of PTI-specific expression patterns. Results indicated successful cloning and functional promoters. However, non-PTI-specific expression of the reporters was observed and further experiments are necessary to determine PTI-specificity.

Successful development of PTI reporters will be useful in experiments elucidating PTI signaling and regulation processes: time and tissue-specific analysis of PTI, examination of transcription factor binding motifs regulating of gene transcription through promoter analysis, and high-throughput screening for breakdown of PTI response.

My Experience

Being an intern at the Martin lab was a terrific opportunity. I was able to appreciate how research is done at a top-tier lab and how to be a successful researcher. I deeply enjoyed both casual and formal discussions about ongoing research because they gave me a chance to think critically and learn about the hows and whys of research. Working with my mentor, Tom Jacobs, was invaluable as he struck the perfect balance between guidance and independence. I would like to thank him for giving me confidence in my research and problem-solving skills and for never sugar-coating the scientific method.  I learned and applied various molecular biology techniques including promoter sequence analysis and cloning towards a project that I found myself truly invested in (see above). As for career goals, this internship allowed me to narrow my areas of interest, gave me a realistic view of graduate school in the plant sciences, and helped me develop a plan after graduation.