Nematicidal effects of a peptidase (NlpC/P60) from the phytopathogen Pseudomonas syringae MB03 on the model nematode Caenorhabditis elegans and plant-parasitic Meloidogyne incognita.

BACKGROUND: Plant-parasitic nematodes (PPNs) are severe threats to agricultural yields and continue to be challenging to treat in several crops worldwide. Microbial-based control has been suggested as a better alternative to chemical control. In this study, we aimed to identify and characterize nematicidal virulence factors of a common phytopathogenic bacterium, Pseudomonas syringae, mainly focusing on the nematicidal and suppressive activities of an NlpC/P60 family peptidase, namely, Peptidase03, against the model nematode Caenorhabditis elegans and an agriculturally important PPN, Meloidogyne incognita. METHODS AND RESULTS: Genome-wide virulence factor prediction of the P. syringae wild-type strain MB03 revealed numerous nematode pathogenic determinants. We selected 11 predicted nematicidal genes for cloning and induced expression in an Escherichia coli expression system and then performed comparative nematicidal bioassays on the model nematode C. elegans. The recombinant strain expressing Peptidase03 showed the highest level of toxicity against C. elegans, with 75.9% mortality, compared to the other tested strains. Purified Peptidase03 showed significant toxicity against C. elegans and M. incognita, with half lethal concentration (LC50) values of 147.9 µg/mL and 211.50 µg/mL, respectively. We also demonstrated that Peptidase03 could damage the intestinal tissues of C. elegans and exhibit detrimental effects on its growth, brood size, and locomotion. CONCLUSIONS: The Peptidase03 protein from P. syringae MB03 had significant nematicidal and suppressive activities against C. elegans and M. incognita, thereby showing potential for the development of an effective PPN-controlling agent for use in agricultural practice.

The Nif3-Family Protein YqfO03 from Pseudomonas syringae MB03 Has Multiple Nematicidal Activities against Caenorhabditis elegans and Meloidogyne incognita.

The nematicidal activity of the common plant-pathogenic bacterium Pseudomonas syringae against certain nematodes has been recently identified, but little is known about its virulence factors. In the current study, predictive analysis of nematode-virulent factors in the genome of a P. syringae wild-type strain MB03 revealed a variety of factors with the potential to be pathogenic against nematodes. One of these virulence factors that was predicted with a high score, namely, YqfO03, was a protein with structural domains that are similar to the Nif3 superfamily. This protein was expressed and purified in Escherichia coli, and was investigated for nematicidal properties against the model nematode Caenorhabditis elegans and an agriculturally important pest Meloidogyne incognita. Our results showed that YqfO03 exhibits lethal activity toward C. elegans and M. incognita worms, and it also caused detrimental effects on the growth, brood size, and motility of C. elegans worms. However, C. elegans worms were able to defend themselves against YqfO03 via a physical defense response by avoiding contact with the protein. Discovery of the diverse nematicidal activities of YqfO03 provides new knowledge on the biological function of a bacterial Nif3-family protein and insight into the potential of this protein as a specific means of controlling agricultural nematode pests.

The Pathogenicity of Pseudomonas syringae MB03 against Caenorhabditis elegans and the Transcriptional Response of Nematicidal Genes upon Different Nutritional Conditions.

Different species of the Pseudomonas genus have been reported for their pathogenic potential against animal cells. However, the pathogenicity of Pseudomonas syringae against Caenorhabditis elegans has never been reported. In this study, the interaction of P. syringae MB03 with C. elegans was studied. Different bioassays such as killing assay, lawn leaving assay, food preference assay, L4 growth assay and newly developed "secretion assay" were performed to evaluate the pathogenic potential of P. syringae on different growth media. The results of the killing assay showed that P. syringae MB03 was able to kill C. elegans under specific conditions, as the interaction between the host and the pathogen varied from non-pathogenic (assay on NGM medium) to pathogenic (assay on PG medium). The lawn leaving assay and the food preference assay illustrated that C. elegans identified P. syringae MB03 as a pathogen when assays were performed on PG medium. Green fluorescent protein was used as the reporter protein to study gut colonization by P. syringae MB03. Our results suggested that MB03 has the ability to colonize the gut of C. elegans. Furthermore, to probe the role of selected virulence determinants, qRT-PCR was used. The genes for pyoverdine, phoQ/phoP, phoR/phoB, and flagella were up regulated during the interaction of P. syringae MB03 and C. elegans on PG medium. Other than these, the genes for some proteases, such as pepP, clpA, and clpS, were also up regulated. On the other hand, kdpD and kdpB were down regulated more than threefold in the NGM - C. elegans interaction model. The deletion of the kdpD and kdpE genes altered the pathogenicity of the bacterial strain against C. elegans. Overall, our results suggested that the killing of C. elegans by P. syringae requires a prolonged interaction between the host and pathogen in an agar-based assay. Moreover, it seemed that some toxic metabolites were secreted by the bacterial strain that were sensed by C. elegans. Previously, it was believed that P. syringae could not damage animal cells. However, this study provides evidence of the pathogenic behavior of P. syringae against C. elegans.