Nematode neuropeptides as transgenic nematicides

Abstract

Plant parasitic nematodes (PPNs) seriously threaten global food security. Conventionally an integrated approach to PPN management has relied heavily on carbamate, organophosphate and fumigant nematicides which are now being withdrawn over environmental health and safety concerns. This progressive withdrawal has left a significant shortcoming in our ability to manage these economically important parasites, and highlights the need for novel and robust control methods. Nematodes can assimilate exogenous peptides through retrograde transport along the chemosensory amphid neurons. Peptides can accumulate within cells of the central nerve ring and can elicit physiological effects when released to interact with receptors on adjoining cells. We have profiled bioactive neuropeptides from the neuropeptide-like protein (NLP) family of PPNs as novel nematicides, and have identified numerous discrete NLPs that negatively impact chemosensation, host invasion and stylet thrusting of the root knot nematode Meloidogyne incognita and the potato cyst nematode Globodera pallida. Transgenic secretion of these peptides from the rhizobacterium, Bacillus subtilis, and the terrestrial microalgae Chlamydomonas reinhardtii reduce tomato infection levels by up to 90% when compared with controls. These data pave the way for the exploitation of nematode neuropeptides as a novel class of plant protective nematicide, using novel non-food transgenic delivery systems which could be deployed on farmer-preferred cultivars.

Fig 1. Exogenous neuropeptides disrupt normal Meloidogyne incognita chemotaxis, plant invasion and stylet thrusting.

(A) 100 M. incognita infective stage juveniles (J2s) were incubated in selected uNLPs, and subsequently challenged with an agar plate chemosensory assay (plant root exudate attractant / water control). Each assay of 100 nematode juveniles was repeated ten times. (B) Ten tomato seedlings were individually challenged with 500 M. incognita J2s incubated in selected uNLPs. Numbers of invading M. incognita J2s were normalised against the negative control group, and expressed as a relative percentage. (C) 100 M. incognita J2s were incubated in selected uNLPs and the frequency of stylet thrusting in response to 5 mM serotonin was counted. Data were normalised to control treated groups. Data shown represent the mean±SEM. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001 (One-Way ANOVA & Fisher’s LSD; Graphpad Prism 6).

Fig 2. Exogenous neuropeptides disrupt normal Globodera pallida chemotaxis, plant invasion and stylet thrusting.

(A) 100 G. pallida infective stage juveniles (J2s) were incubated in selected uNLPs, and subsequently challenged with an agar plate chemosensory assay (plant root exudate attractant / water control). Each assay of 100 nematode juveniles was repeated ten times. (B) Ten tomato seedlings were individually challenged with 500 G. pallida J2s incubated in selected uNLPs. Number of invading G. pallida J2s were normalised against the negative control group, and expressed as a relative percentage. (C) 100 G. pallida J2s were incubated in selected uNLPs and the frequency of stylet thrusting in response to 2 mM serotonin was counted. Data were normalised to control treated groups. Data shown represent the mean±SEM. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001 (One-Way ANOVA & Fisher’s LSD; Graphpad Prism 6).

Fig 3. Mi- NLP-15b potently inhibits the chemotaxis and infectivity of Meloidogyne incognita.

(A) Serial dilutions of Mi-NLP-15b indicate that J2 chemotaxis is inhibited by low picomolar concentrations. (B) Mi-NLP-15b significantly reduced J2 invasion levels at nanomolar concentrations. Data shown represent the mean±SEM. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001 (One-Way ANOVA & Fisher’s LSD; Graphpad Prism 6).

Fig 4. Transgenic microbes secreting uNLPs protect tomato against Meloidogyne incognita and Globodera pallida.

(A) Nine independent Chlamydomonas reinhardtii transformants secreting two distinct nematode neuropeptides (Mi-NLP-9f and Mi-NLP-15b) significantly inhibited the ability of M. incognita J2s to infect tomato plants, with up to 90% protection. (B) Bacillus subtilis cultures secreting either Mi-NLP-15b or Mi-NLP-40 also conferred significant protection against M. incognita J2 invasion. (C) C. reinhardtii transformants secreting Gp-NLP-15b (identical to Mi-NLP-15b) significantly inhibited the ability of G. pallida J2s to invade tomato plants. (D) B. subtilis cultures secreting Gp-NLP-15b also protected tomato plants from G. pallida J2 invasion. Data shown represents mean±SEM. *, P<0.05; **, P<0.01; ***, P<0.001 (One-way ANOVA & Fisher’s LSD; Graphpad Prism 6).

Fig 5. Plant parasitic nematode (PPN) unamidated neuropeptide-like proteins (uNLPs) do not alter Caenorhabditis elegans chemotaxis or Steinernema carpocapsae host-finding

Chemotaxis of mixed stage C. elegans towards the attractants sodium acetate (A), pyrazine (B), benzaldehyde (C), and diacetyl (D) are unaffected by exposure to selected PPN uNLPs. (E) Chemotaxis of S. carpocapsae towards the insect host Galleria mellonella is also unaffected by exposure to selected PPN uNLPs. Data shown represent mean ±SEM (One-way ANOVA & Fisher’s LSD; Graphpad Prism 6).