Transgenic expression of Erwinia amylovora effectors eop1 and hopC _Ea in apple

Erwinia amylovora (Ea), the causal agent of fire blight, uses a type three secretion system (TTSS) to deliver effector proteins into plant host cells. Once inside the host cell, these effector proteins are thought to be involved with suppressing host defense responses, redirecting normal host metabolism to facilitate pathogen multiplication and initiating cell necrosis. In order to investigate the role of individual bacterial effector proteins we have engineered an apple host that transgenically expresses the bacterial effector proteins Eopl or HopC_Ea, under the control of an inducible promoter. Each bacterial effector was directionally cloned from Ea 273 into a Gateway compatible entry vector using gene-specific primers that incorporate a Kozak sequence in the 5′ end of the effector gene for 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. The inducible expression of eop1 and hopC_Ea in the presence of 25 uM estradiol was evaluated by RT-PCR. A total of 14 transgenic apple lines have been screened to date. Select lines are currently being used to investigate the effect of Eop1 and HopC_Ea on host gene expression, bacterial growth and susceptibility in an apple host. Preliminary results indicate that Eop1 facilitates the in planta growth of an Ea TTSS minus mutant, suggesting it is involved with suppressing basal resistance. To date, no effect has been found for HopC _Ea on growth of Ea in planta.