Identification of the Genes of the Plant Pathogen Pseudomonas syringae MB03 Required for the Nematicidal Activity Against Caenorhabditis elegans Through an Integrated Approach.

Muhammad Ali,Xiaowen Sun,Naeem Mahmood Ashraf,Anum Bashir,Xun Yu,Tong Gu,Zhiyong Wang,Lin Li

doi:10.3389/fmicb.2022.826962

Abstract

Nematicidal potential of the common plant pathogen Pseudomonas syringae has been recently identified against Caenorhabditis elegans. The current study was designed to investigate the detailed genetic mechanism of the bacterial pathogenicity by applying comparative genomics, transcriptomics, mutant library screening, and protein expression. Results showed that P. syringae strain MB03 could kill C. elegans in the liquid assay by gut colonization. The genome of P. syringae MB03 was sequenced and comparative analysis including multi locus sequence typing, and genome-to-genome distance placed MB03 in phylogroup II of P. syringae. Furthermore, comparative genomics of MB03 with nematicidal strains of Pseudomonas aeruginosa (PAO1 and PA14) predicted 115 potential virulence factors in MB03. However, genes for previously reported nematicidal metabolites, such as phenazine, pyochelin, and pyrrolnitrin, were found absent in the MB03 genome. Transcriptomics analysis showed that the growth phase of the pathogen considerably affected the expression of virulence factors, as genes for the flagellum, glutamate ABC transporter, phoP/phoQ, fleS/fleR, type VI secretion system, and serralysin were highly up-regulated when stationary phase MB03 cells interacted with C. elegans. Additionally, screening of a transposon insertion mutant library led to the identification of other nematicidal genes such as acnA, gltP, oprD, and zapE. Finally, the nematicidal activity of selected proteins was confirmed by heterologous expression in Escherichia coli.

Highlights

Caenorhabditis elegans has been widely utilized to study host–pathogen interaction
Pseudomonas syringae has been traditionally recognized as a plant pathogen; notable killing activity against the animal model C. elegans has been recently demonstrated for a P. syringae wild-type strain MB03 (Ali et al, 2016; Bashir et al, 2020, 2021)
Comparative genomics, transcriptomics, and transposon insertion mutant library analyses were applied for genomewide identification of common and unique virulence factors required for the nematicidal activity of P. syringae MB03 against C. elegans

Summary

Introduction

Caenorhabditis elegans has been widely utilized to study host–pathogen interaction. Pathogenicity mechanisms of numerous bacterial species of Bacillus (Geng et al, 2016), Burkholderia (Lee et al, 2011; Paiva et al, 2013), Pseudomonas (Dubern et al, 2015; Nandi et al, 2015; Ali et al, 2016), Salmonella (Alegado et al, 2011), Staphylococcus (Begun et al, 2005; Irazoqui et al, 2010) and Nematicidal Activity of Pseudomonas syringaeYersinia (Darby et al, 2002; Atkinson et al, 2011) have been reported against C. elegans. Bacterial species have acquired different mechanisms to kill C. elegans such as slow killing due to infection and colonization (Ali et al, 2016), biofilm formation (Darby et al, 2002; Atkinson et al, 2011), fast killing due to diffusible metabolites (Kirienko et al, 2015; Nandi et al, 2015), and secretion of proteins (Luo et al, 2012; Geng et al, 2016; Zhang et al, 2016). Several species of the genus Pseudomonas, including P. aeruginosa, P. chlororaphis, P. fluorescens, P. putida, and P. protegens have been reported for their pathogenicity against animals (Feinbaum et al, 2012; Burlinson et al, 2013; Fernandez et al, 2015; Wei et al, 2017). The killing of C. elegans by the strains of P. aeruginosa has been attributed due to various mechanisms such as lethal paralysis, agar-based fast killing, liquid killing, and red death, along with gut colonization (Darby et al, 1999; Mahajan-Miklos et al, 1999; Tan et al, 1999b; Gallagher and Manoil, 2001; Zaborin et al, 2009; Cezairliyan et al, 2013; Kirienko et al, 2013, 2015; Ray et al, 2015). It has been established that change in the physical form of the killing assay (from agar-based killing to liquid-based killing) altered the killing mechanism; for instance, agar-based killing was mediated by the phenazines (Cezairliyan et al, 2013) and gut colonization (Tan et al, 1999a), whereas liquid killing was facilitated by pyoverdine (Kirienko et al, 2013)

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