Dissertation

Natural and engineered resistance triggered by TAL effectors of Xanthomonas oryzae

Hummel, Aaron W

2013

Overview

Xanthomonas plant pathogenic bacteria cause yield-limiting disease in several important crops. Some species promote infection by secreting transcription activator-like (TAL) effectors directly into host cells where they interact with eukaryotic cellular apparatus to transactivate plant genes. Specific recognition occurs through direct, predictable interactions between hypervariable amino acid residues in the central DNA binding domain and adjacent nucleotides in the sense strand of the gene promoter, thus defining the length and sequence of the effector binding element (EBE). Activation of host susceptibility genes promotes disease, whereas induction of executor resistance (R) genes leads to plant defense. The vascular pathogen Xanthomonas oryzae pv. oryzae (Xoo) and the mesophyll pathogen Xanthomonas oryzae pv. oryzicola (Xoc) are causal agents of the devastating rice (Oryza sativa) diseases bacterial blight and bacterial leaf streak, respectively. To investigate whether executor R genes can be engineered for broader resistance, we added six predicted EBEs corresponding to TAL effectors from Xoo and Xoc to the promoter of Xa27. This modification resulted in specific activation of Xa27 in transgenic rice by Xoo, Xoc and each of the corresponding TAL effectors individually, as measured by quantitative Real Time RT-PCR (qPCR). It expanded the resistance of Xa27 to include additional strains of Xoo and all tested strains of Xoc. A bioinformatics analysis of sequences amended to the Xa27 promoter suggests the likely introduction of unwanted regulatory elements, highlighting the importance of EBE design to guard against spurious gene activation. During a screen of Xoc TAL effectors, we observed a hypersensitive reaction (HR) triggered by Tal2a when it was expressed heterologously in rice leaves by another Xanthomonas strain. The response was Tal2a-specific and dependent on gene activation, suggesting an executor R gene mechanism. EBE prediction, qPCR and next generation RNA sequencing studies identified three rice genes activated specifically in response to Tal2a. One, a ubiquitin carboxy-terminal hydrolase (UCH), was activated with designer TAL effectors (dTALEs) but was not sufficient to cause the HR. Testing of the remaining three genes through dTALE activation is ongoing. Expression from high and low copy plasmids points to a dose-dependent avirulence effect of Tal2a in Xoo and Xoc.

• Publisher: ProQuest Dissertations & Theses
• Subject: Cellular biology / Molecular biology / Plant Pathology
• ISBN: 9781303705458, 1303705451