USING FUNCTIONAL AND APPLIED GENOMICS TO IMPROVE STRESS AND DISEASE RESISTANCE IN FRUIT TREES
Location: Appalachian Fruit Research Laboratory: Innovative Fruit Production, Improvement and Protection
Title: The chemically inducible expression of Erwinia amylovora bacterial effectors EopB1 and HopCEa in apple
Submitted to: Molecular Plant-Microbe Interactions
Publication Type: Abstract Only
Publication Acceptance Date: May 10, 2007
Publication Date: July 21, 2007
Citation: Lalli, D., Artlip, T.S., Farrell, Jr., R.E., Wisniewski, M.E., Norelli, J.L. 2007. The chemically inducible expression of Erwinia amylovora bacterial effectors EopB1 and HopCEa in apple. Molecular Plant-Microbe Interactions. Book of Abstracts, p.343.
Erwinia amylovora, the causal agent of fire blight disease, utilizes a type three secretion system to deliver effector proteins into plant host cells. To investigate the role of individual bacterial effector proteins, we have engineered an apple host that transgenically expresses the bacterial effector proteins EopB1 or HopCEa. EopB1 is a homolog of Yersinia pseudotuberculosis YopJ, and HopCEa is a homolog of Pseudomonas syringae pv tomato HopC1. Both bacterial effectors were directionally cloned from E. amylovora strain 273 into a Gateway compatible entry vector. Gene specific primers were used that were modified to incorporate a Kozak sequence in the 5' end of the effector gene to facilitate proper translation in a eukaryotic system and a His tag at the 3' end for protein detection. The cloned bacterial effector genes were subsequently cloned through Gateway technology into a binary vector, pBinPlusARS.XVE. This binary vector incorporates the regulatory elements of the estradiol-induced XVE gene expression system developed by Zuo et al. (2000) and was used in the Agrobacterium-mediated transformation of apple. Transgenic apple lines were confirmed through PCR analysis with effector specific primers and evaluated for Agrobacteria contamination with virG specific primers. Through RT-PCR, we show the inducible expression of eopB1 and hopCEa in the presence of 25 uM estradiol and the detection of EopB1 and HopCEa expressed protein by Western analysis. Currently, we are evaluating 15 transgenic apple lines. These lines will enable us to investigate the effect of EopB1 and HopCEa on host gene expression, bacterial growth, and susceptibility in an apple host, as well as, provide us with a system to test additional bacterial effectors in the future.