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Neuropeptide like protein of Globodera pallida

All data that is presented here is based on purely literature analysis. Due to the COVID19 restrictions, our iGEM team was unable to perform experiments in the lab that would contribute to the characterization of this part. Neuropeptide-like proteins (NLPs) are small peptides (aprox. 20 amino acids) known to play a role in synaptic modulation for nematodes (Lee, 2002). One of these nematodes is the parasite Globodera pallida that causes significant damage to potato crops every year. A recent study by Dalzell and colleagues suggest using NLP to dysregulate G. pallida’s chemoattractant behavior towards the potato root exudate.

Besides NLPs there are two other neuropeptide classes that the nematode utilizes for neuronal signaling (insulin-like peptides & FMRFamide-related peptides). Of the three, NLPs show the best potency to be used in agriculture because they don’t require post-translational modifications, are easily taken up by the nematodes, and, because of their short sequence, are easy to express by a host organism, making them suitable for large-scale production (Geary and Maude, 2011). However, NLPs are as well the least studied nematode neuropeptide class and much still unknown about their mechanism of action in the nematode and behavior in the soil.

To identify the neuropeptide-like proteins, Dalzell and colleagues used the sequence of neuropeptides predicted in the model organism nematode C. elegans to perform a BLAST search (BLASTp) through the genome of G. pallida (Li and Kim, 2008). From this, they were able to predict 25 NLPs that are present in this nematode. The NLPs discovered are listed in Table 1. Subsequently, Dalzell and colleagues tested the effect of same NLPs on the chemoattractant behavior of G. pallida towards the root exudate. To do this, they inoculated the nematodes with a NLP for up to three hours. Nematodes are known to be very good at taking up chemicals from the environment with the amphids located in their anterior nervous system (Wang et al, 2011). Inoculating the nematodes with the neuropeptides will therefore be enough for them to reach the nervous system.

Table 1. The predicted NLP complements of G. pallida (Adapted from Warnock et al, 2016)

After inoculation, the nematodes were dropped on an agar plate that was divided into three segments. The agar slurry at one end of the plate was mixed with root exudates prior to the plating, the agar slurry at the other end was mixed with the water that is normally used to collect the root exudate in and a small stroke in the middle consisted of normal agar without any modifications. The nematodes were dropped on the middle stroke with their movement monitored after 3 hours. By counting the location of the nematodes, a chemotaxis score was determined that reflects the effect of the NLPs on the chemoattractant behavior of Globodera pallida towards root exudates. After analyzing their results (Fig. 1), we decided to use NLP14a as an initial choice for repelling the nematodes away from the root exudate, and thus the potato plant.

Figure 1. Chemotaxis behavior of G. pallida in response to NLP (Warnock et al, 2016)

It has happened before that agrochemicals targeted at a receptor specific to one organism, presented off target effects on other organism due to epitope similarities between proteins/peptides. To address the possibility of this happening with NLP14a,we performed a BLAST search through the protein databases of Uniprot & NCBI. The results can be observed in Table 2. Both showed similar results with no highly significant hit on a protein that has been characterized in another organism. Although this lower the potential of off-target effect, it certainly doesn’t exclude it.

Table 2. BLAST results using the NLP14a as query. The most simillar regions are bolted and the number of different amino acids is written is paranthesys

Sequence and Features

Bibliography

1.Wang D, Jones LM, Urwin PE and Atkinson HJ. A Synthetic Peptide Shows Retro- and Anterograde Neuronal Transport before Disrupting the Chemosensation of Plant-Pathogenic Nematodes. PLoS ONE. 2011; 6 (3): e17475 doi: 10.1371/journal.pone.0017475 PMID: 21408216

2.Li C, and Kim K. 2008. Neuropeptides. The C. elegans Research Community, WormBook.

3.Geary TG and Maule AG. Neuropeptide Systems as Targets for Parasite and Pest Control. Advances in Experimental Medicine and Biology. 2011; 692: v–vi.

4.Lee DJ. The Biology of Nematodes. 2002.

5.Warnock ND, Wilson L, Patten C, Fleming CC, Maule AG, Dalzell JJ. Nematode neuropeptides as transgenic nematicides. PLoS Pathog. 2017;13(2).