Development of a reporter system for the analysis of xylophilus ampelinus type III secreted effectors
Nyembe, Nompumelelo Philile Praiseworth
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Xylophilus ampelinus, the causal agent of bacterial blight and canker of grapevines, has long been a threat to the table grape industry in the Western Cape, leading to severe economic losses due to the reduced productivity and shortened lifespan of infected grapevines. Very little is known about the genetic makeup of the organism, especially with regard to the factors that contribute to its pathogenicity. Generally, bacterial pathogens directly inject the effector proteins into host cells via Type III secretion system (T3SS). In the attempts to identify and characterize the T3 secreted effectors, different reporter plasmid systems have been used to study the secretion and translocation mechanisms the effectors employ during pathogenicity. The aim of the study was to generate a T3 reporter plasmid system for X. ampelinus that will allow the identification and classification of potential pathogenicity factors as members of the Type III secretion class of effectors. First, the avrBs1 family genes avrBs1 and avrA were identified and characterized. The two avirulence genes induced HR on Nicotiana tabacum leaves. Due to the relatedness of the X. ampelinus avr sequences to those of xanthomonads, and the fact that Xanthomonas avrBs1 has been successfully used in a number T3 effector studies, it was decided to construct an X. ampelinus T3 effector reporter vector based on the avrBs1 gene. The minimal segment of the X. ampelinus AvrBs1 protein C-terminus, sufficient for recognition inside host cells and also responsible for HR-induction was identified and characterized using Agrobacterium-mediated transient expression. The AvrBs157-413 HR-inducing domain was cloned in-frame with the 3x FLAG epitope, into a broad-host range vector. To test the reporter vector, the full length avrBs1 sequences of X. ampelinus and Xanthomonas campestris pv. campestris were cloned ahead of the 3x FLAG epitope and the constructs were transferred into XaΔavrBs1 knockout mutant to test for protein secretion. Furthermore, the reporter construct was tested for Type III protein translocation on Bs1 resistant pepper cultivar STAR 6657. Optimization of protein secretion and translocation assays is however required for the improved results. This might include the application of an alternative protein tag to identify candidate X. ampelinus T3SS effectors.