Functional characterisation and evaluation of Erwinia mallotivora bifunctional chorismate mutase via TargeTron® gene knockout system

Abu Bakar, N., Juri, N. M., Sohaime, M. Z. and Samsuddin, A. F.

Abstract
Erwinia mallotivora is the causal agent of papaya dieback disease, a devastating diseases affecting papaya production in Malaysia. Preliminary omics studies had successfully uncovered the key effectors of Erwinia mallotivora’s (Em’s) pathogenicity, where chorismate mutase (CM) was among the most prominent effectors in E. mallotivora. An in-depth in silico characterisation and functional analysis of the CM was carried out. Sequence analysis revealed that E. mallotivora’s CM protein contained two catalytic motifs; chorismate mutase type II (CM 2) and prephenate dehydratase (PDT) and a regulatory domain (ACT), where no signal peptide was detected in its sequence. Based on this characterisation, it is confirmed that our targeted CM is a non-secreted bifunctional chorismate mutase/prephenate dehydratase (Cmp). Sequence homology search showed that EmCmp is highly conserved (>80% identity) with various bifunctional Cmp from several Erwinia and Pantoea species (n ≥60). To confirm its role in Em virulency, a Cmp knockout mutant of Em (EmΔCmp) was generated via TargeTron® Gene Knockout system. The EmΔCmp strain significantly loss its virulency to papaya seedlings as compared to the wild E. mallotivora strain in the inoculation assay, revealing the evidence of EmCmp function and importance as the target gene for future research towards disease management strategy in papaya.

Keywords: Erwinia mallotivora, papaya, chorismate mutase, knock out

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