Identification And Characterization Of Nematicidal Proteins From Pseudomonas Syringae MB03 Mutant Library

Plant-parasitic nematodes which are widely distributed,and are generally important pathogens that have been the major prohibitive factors restricting the further development of agricultural production globally.The harmful interaction between plant and parasitic nematode results in huge loss in crop yield,deterioration of product quality,ultimately,leading to massive economic loss in agricultural and forestry production.The traditional agricultural pest control measures are not ideal to control parasitic nematode as these practices require heavy workload and complex operation.Later,simple and feasible chemical control methods were developed however,these methods posse great threats to the environment and ecological balance.Microorganisms that can infect and cause the death of nematodes have gained much interest of scientific community because of the great developmental prospect of nematicidal products.To date,a large number of microorganisms with higher nematicidal activities have been reported,such as Pseu,domonas aeruginosa and Bacillus thuringiensis.However,no report on the toxicity of Pseudomonas syringae to nematodes has been documented.A strain of Pseudomonas syringae MB03 was isolated and identified by our laboratory,and the whole genome sequencing of this strain was completed.We found this strain has effective toxic activity against Caenorhabditis elegans and Meloidogyne incongnita by bioassay.Pathogenicity of Pseudomonas syringae is well reported against plants while for the first time,its toxicity against animals has been documented by our laboratory.To identify genes and related molecular mechanism involved in killing of worms,transposon mutant libraryof P.syringae strain MB03 was constructed using pUT-mini Tn5-Km2 transposon kit.Liquid killing assay reveled 12 mutants with reduced toxicity to C.elegans from the mutant library of 1256 mutant strains.The sequence of flanking region of Tn5 insertion site was determined.With two commonly used nucleic acid sequence analysis software vectorNTI and Bioedit,the nucleotide sequences were alignment with MB03 whole genome sequence.Finally,accurate insertion position of Tn5 transposons in seven mutants and their downstream genetic information were figured out.Based on the above experiments and bioinformatics analysis,we hypothesized that these genes might directly or indirectly involved in the pathogenicity of MB03 against C.elegans.In order to verify the assumption,complementary strains and over expressing strains of P.syringae were constructed.Moreover,proteins were also expressed in Escherichia coli.Different bioassays such as brood size assay,growth assay,food choice assay,locomotion assay,gut colonization assay,were performed using recombinants strains and purified proteins against C.elegans as model host.Finally the experimental results show that these mutants have less toxicity than MB03 on worms,a hypothetical protein as well as a lipoprotein can kill C.elegans,while ATPase and outer membrane prion play a key role in killing C.elegans.This study can lead to the development of new mechanisms biological control of plant nematodes,and also can provide genetic resources for the future development of nematode-resistant plants.Current study provides an insight into the molecular mechanism of pathogenicity of P.syringae MB03 against C.elegans.