Patent Application
20200123208
This invention relates to nematode neuropeptides as transgenic nematicides.
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to PPN management has relied heavily on carbamate, organophosphate and fumigant nematicides which are now being withdrawn over environmental health and safety concerns.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.
Plant parasitic nematodes (PPN) reduce crop plant yield globally, undermining food security. Many of the chemicals used to kill these parasites are non-specific and highly toxic, and are being phased out of general use through governmental and EU regulation. The withdrawal of these chemicals is beneficial to the environment, but limits our ability to protect crops from infection. Efforts must now focus on developing environmentally safe PPN controls. PPNs can absorb various molecules directly from the environment into their nervous system, including peptides and proteins. Here we profiled the feasibility of using PPN neuropeptides, small signalling molecules, to interfere with normal PPN behaviour. We exposed PPNs to a variety of neuropeptides, and found that they could interfere with behaviours that are important to host-finding and invasion. We then developed soil-dwelling microbes that could generate and secrete these neuropeptides into the soil where the PPN infective juveniles are found. These transgenic microbes can protect host plants from infection, and represent a completely new approach to controlling PPNs in crop plants. Importantly, these neuropeptides appear to have no impact on other beneficial nematodes found in the soil.
Plant parasitic nematodes (PPNs) are responsible for an estimated 12.3% reduction in crop yield each year, which equates to losses of around $US80 billion worldwide [1, 2]. Traditionally PPNs have been controlled through the use of fumigant, carbamate and organophosphate nematicides which are being withdrawn over environmental health and safety concerns, through global and EU level directives [3]. The fumigant methyl bromide was used extensively to control PPN infestations for more than 60 years, however the identification of ozone-depleting characteristics was recognised within the Montreal Protocol which aimed to eliminate methyl bromide use by 2010 [4]. Likewise, dibromochloropropane (DBCP), a highly lipophilic brominated organochlorine was first used as a nematicide in the mid 1950's before animal safety tests in the 1960's demonstrated endocrine disrupting, and carcinogenic properties, alongside an increased incidence of developmental defects following exposure. Later studies further demonstrated strong mutagenic properties, and workers at the Occidental Chemical plant in California, which produced DBCP, displayed significantly higher rates of spermatogenic abnormalities relative to the rest of the population [5]. The carbamate nematicide aldicarb also triggers toxicity in non-target organisms through disruption of cholinergic neurons. Initial withdrawal of use across the USA in 1990 was followed by re-introductions to counteract a serious shortfall in alternative control options in 1995; similar dispensations have been afforded to EC states. The extensive withdrawal of frontline nematicides has left a significant shortfall in our ability to control PPNs.
Transgenic approaches could provide a cost-effective means of PPN control. Much effort has focused on the development of in planta RNA interference (RNAi) to silence PPN genes necessary for successful parasitism [6-9]. Whilst many such studies have shown promise, concerns surround the persistence of RNAi trigger-expressing traits. It remains to be established if DNA methylation and transcriptional silencing of double stranded (ds)RNA-expressing transgenes is an issue in plants other than Arabidopsis thaliana [10]. Efforts to inhibit PPN nutrient acquisition through transgenic expression of cystatins that inhibit intestinal protease activity have also proven an effective strategy [6]. The utility of peptide resistance traits has also been demonstrated [7], resulting in field level resistance and high target specificity [8]. Indeed, stacking peptide and cystatin resistance traits has proven extremely effective in plantain, triggering a 99% reduction in PPN infection levels at harvest, with a corresponding 86% increase in plantain yield [9].
Peptides have traditionally been viewed as poor drug candidates due to issues surrounding cellular uptake and half-life. However it has long been known that nematodes display an unusual neuronal uptake mechanism which is exploited by amphid dye-filling methods [11]. The amphid neurons assimilate exogenous peptides which subsequently accumulate in cells of the central nerve ring [11], where they can interact with available receptors.
Neuropeptides are highly enriched and conserved amongst nematodes, coordinating crucial aspects of physiology and behaviour [12-21]. The model nematode Caenorhabditis elegans encodes at least 113 neuropeptide genes, producing over 250 mature neuropeptides [16]. It is thought that this neurochemical diversity underpins the wide array of complex behaviours which are found within such neuroanatomically simple animals [16, 22]. Many neuropeptides are known to be expressed within the anterior neurons of nematodes [16, 22-24], and it is likely that their cognate receptors are expressed in these or adjacent cells. The retrograde transport of exogenous peptides suggests that these receptors could be amenable to activation through signalling molecules following their uptake from the external environment. Conceptually, the mining of native neuropeptide complements for novel nematicides is an attractive prospect, based on the a priori assumption of bioactivity. An additional positive quality of neuropeptides is their characteristically high potency when acting on cognate receptors [13, 25-30]. Furthermore, the high degree of phylogenetic sequence conservation suggests that neuropeptides could represent broad-spectrum nematicides as they share significant sequence similarity within and between parasite species [17, 22, 31, 32]. Disrupting PPN behaviour through the dysregulation of native neuropeptide signalling could hinder the development of resistance traits anchored on target receptor mutation. Selective pressure drives the propagation of drug target variants which escape agonism/antagonism, or the development of enhanced efflux mechanisms [33, 34]. Conceptually, the development of resistance to neuropeptides which coordinate crucial aspects of PPN biology would seem less likely.
Nematode neuropeptide complements are organised into three broad groupings: i) the FMRF-amide Like Peptides (FLPs); the INSulin like peptides (INSs); and iii) the Neuropeptide-Like Proteins (NLPs). FLPs represent the most widely studied and best understood family, characterised by a C-terminal RFamide motif, and are known to coordinate motor and sensory function [14, 16, 22]. In particular, C-terminal amidation is necessary for biological function, and so precludes FLPs from most transgenic delivery methods. INSs coordinate and integrate sensory signals with developmental circuits [35] and they share characteristic domain organisation and tertiary structure with vertebrate insulin peptides [16, 36-40]. Specific proteolytic processing requirements suggest that INSs do not represent ideal candidates for transgenic delivery methods. The NLPs represent the least studied grouping of neuropeptides, comprising every neuropeptide that does not conform to the biosynthetic and structural characteristics of FLPs or INSs and encompassing multiple peptide families. Little is known about their function in nematodes, however many NLPs are expressed in anterior neurons and do not appear to require post-translational modifications [20, 24, 40-45], making them more amenable to generation and delivery by transgenic systems than FLPs or INSs. A key gap in assessing the potential of unamidated NLPs as nematicides is the lack of data on their bioactivity in PPNs.
Jarecki et al (Discovery of neuropeptides in the nematode Ascaris suum by database mining and tandem mass spectroscopy. Journal of Proteome Research. 2011. 10, pp 2098-3106): Tables 2 and 3 identify putative peptides, naming them as A suum nlp-1 to nlp-17, and as A suum nlp-18 to 23 and 34 to 46, respectively. Jarecki et al concludes that predicting and identifying the A suum nlps is an “important first step in understanding the vital role neuropeptides play in the nervous system of A suum”.
McVeigh et al (Neuropeptide-like protein diverstity in phylum Nematoda. International Journal for Parasitology. 2008, 38, pp 1493-1503) identifies nematode neuropeptide-like protein (nlp) sequelogs. Table 1 summarises EST-derived nlp sequelogs in phylum Nematoda and Table 2 indicates their distribution. McVeigh et al provides a first study of the nlp diversity of phylum Nematoda and brings the nematode nlp complement to 46 genes.
Nathoo et al (Identification of neuropeptide-like protein gene families in Caenorhabditis elegans and other species. PNAS. 2001, 98, pp 14000-14005) identifies 32 previously uncharacterised C elegans nlp genes. Nathoo et al concludes that further characterisation of the nlp genes is likely to provide a greater understanding of the mechanisms involved in neuropeptide function in development and behaviour.
Husson et al (Discovering neuropeptides in Caenorhabditis elegans by two dimensional liquid chromatography and mass spectrometry. Biochemical and Biophysical Research Communications. 2005. 335, pp 76-86) identified 21 peptides derived from formerly predicted neuropeptide-like protein precursors and 28 predicted FMRFamide-related peptides. Husson et al sequenced 11 novel peptides derived from 9 peptide precursors.
Warnock et al (Nematode neuropeptides as transgenic nematicides. PLoS Pathogens. 2017. 13 (2), pp 1-20 :e1006237. doi: 10.1371/journal.ppat.1006237. eCollection 2017 Feb.) was published on 27 Feb. 2017, being after the priority date of the present Application.
Here we aimed to characterise the NLP complements in silico for two economically important PPNs that display different modes of infection and parasitism, M. incognita and G. pallida. Subsequently we aimed to screen NLPs for their ability to dysregulate the normal behaviour of infective stage juveniles (J2s) when applied exogenously and, simultaneously, to develop and assess novel transgenic delivery methods as next generation plant protection platforms.
According to the invention, there is provided a peptide comprising, or consisting of:
Optionally, the peptide comprises, or consists of:
Further optionally, the peptide comprises, or consists of:
Still further optionally, the peptide comprises, or consists of:
Optionally, the peptide comprises, or consists of:
According to the invention, there is provided a peptide comprising
Optionally, the peptide comprises, or consists of:
Further optionally, the peptide comprises, or consists of:
According to the invention, there is provided a peptide comprising A-AA2-D-AA4-AA5-AA6-AA7-AA8-AA9-AA10-AA11-AA12-AA13-AA14-AA15-AA16;
Optionally, the peptide comprises, or consists of:
Further optionally, the peptide comprises, or consists of:
According to the invention, there is provided a peptide that comprises, or consists of:
Optionally, the peptide comprises, or consists of:
According to the invention, there is provided a peptide that comprises, or consists of:
The invention also provides a nematicidal composition comprising the aforementioned peptide, or a mixture thereof, and a suitable carrier.
The invention also provides an expression vector comprising the aforementioned peptide.
Optionally, a promoter is operably linked to the aforementioned peptide.
The invention provides a transgenic microorganism for expression of the aforementioned peptide, the microorganism comprising the aforementioned vector. The aforementioned peptide can be provided in a plasmid or, alternatively, the transgene can be incorporated directly into the genome of the microorganism.
The invention provides a method of treating plant parasitic nematodes, the method comprising providing either the aforementioned peptide or the aforementioned nematicidal composition or the aforementioned transgenic microorganism on or adjacent the plant parasitic nematodes, optionally in the rhizosphere of the plant.
Optionally, in the aforementioned peptide, the aforementioned nematicidal composition, the aforementioned vector, the aforementioned transgenic microorganism or the aforementioned method, the peptide comprises, or consists of:
or a mixture thereof.
Further optionally, in the aforementioned peptide, the aforementioned nematicidal composition, the aforementioned vector, the aforementioned transgenic microorganism or the aforementioned method, the peptide comprises, or consists of:
In the drawings,
Microorganisms in soil affect soil structure and fertility. Soil microorganisms can be classified as bacteria, actinomycetes, fungi, algae and protozoa. Up to 10 billion bacterial cells inhabit each gram of soil in and around plant roots, a region known as the rhizosphere.
Bacteria
Bacteria and Archaea are the smallest organisms in soil apart from viruses. Bacteria and Archaea are prokaryotic. All of the other microorganisms are eukaryotic. A prokaryote has a very simple cell structure with no internal organelles. Bacteria and archaea are the most abundant microorganisms in the soil, and serve many important purposes, including nitrogen fixation. B. subtilis is commonly found in the upper layers of the soil—the density of spores found in soil is about 106 spores per gram.
Fungi
Fungi are abundant in soil, but bacteria are more abundant. Fungi are important in the soil as food sources for other, larger organisms, pathogens, beneficial symbiotic relationships with plants or other organisms and soil health. Fungi can be split into species based primarily on the size, shape and color of their reproductive spores, which are used to reproduce. Most of the environmental factors that influence the growth and distribution of bacteria and actinomycetes also influence fungi. The quality as well as quantity of organic matter in the soil has a direct correlation to the growth of fungi, because most fungi consume organic matter for nutrition. Fungi thrive in acidic environments, while bacteria and actinomycetes cannot survive in acid, which results in an abundance of fungi in acidic areas. Fungi also grows well in dry, arid soils because fungi are aerobic, or dependent on oxygen, and the higher the moisture content in the soil, the less oxygen is present for them.
Algae
Algae can make their own nutrients through photosynthesis. Photosynthesis converts light energy to chemical energy that can be stored as nutrients. For algae to grow, it must be exposed to light because photosynthesis requires light, so algae are typically distributed evenly wherever sunlight and moderate moisture is available. Algae, do not have to be directly exposed to the sun, but can live below the soil surface given uniform temperature and moisture conditions. Algae are also capable of performing nitrogen fixation. Algae can be split up into three main groups: the Cyanophyceae, the Chlorophyceae and the Bacillariaceae. The Cyanophyceae contain chlorophyll that absorbs sunlight and uses that energy to make carbohydrates from carbon dioxide and water and also pigments that make it blue-green to violet in colour. The Chlorophyceae usually only have chlorophyll in it which makes it green, and the Bacillariaceae contain chlorophyll as well as pigments that make the algae brown in colour. Blue-green algae, or Cyanophyceae, are responsible for nitrogen fixation. The amount of nitrogen they fix depends more on physiological and environmental factors rather than the organism's abilities. These factors include intensity of sunlight, concentration of inorganic and organic nitrogen sources and ambient temperature and stability. Chlamydomonas reinhardtii is a single-cell green alga. Chlamydomonas species are widely distributed worldwide in soil and fresh water.
Protozoa
Protozoa are eukaryotic organisms that were some of the first microorganisms to reproduce sexually, a significant evolutionary step from duplication of spores, like those that many other soil microorganisms depend on. Protozoa can be split up into three categories: flagellates, amoebae and ciliates. Flagellates are the smallest members of the protozoa group, and can be divided further based on whether they can participate in photosynthesis. Nonchlorophyll-containing flagellates are not capable of photosynthesis because chlorophyll is the green pigment that absorbs sunlight. These flagellates are found mostly in soil. Flagellates that contain chlorophyll typically occur in aquatic conditions. Flagellates can be distinguished by their flagella, which is their means of movement. Some have several flagella, while other species only have one that resembles a long branch or appendage. Amoebae are larger than flagellates and move in a different way. Amoebae can be distinguished from other protozoa by their slug-like properties and pseudopodia. A pseudopodia or “false foot” is a temporary obtrusion from the body of the amoeba that helps pull it along surfaces for movement or helps to pull in food. The amoeba does not have permanent appendages and the pseudopodium is more of a slime-like consistency than a flagellum. Ciliates are the largest of the protozoa group, and move by means of short, numerous cilia that produce beating movements. Cilia resemble small, short hairs. They can move in different directions to move the organism, giving it more mobility than flagellates or amoebae.
BLAST Identification of PPN uNLPs
The predicted NLP complement of C. elegans [16] was used in a simple BLASTp and tBLASTn analysis of available genomic/transcriptomic sequence data of G. pallida and M. incognita [46, 47]. All returned hits were curated by eye, and NLPs identified as per McVeigh et al. [17].
PPN Maintenance
M. incognita were maintained in tomato plants (cv. Moneymaker) under greenhouse conditions. 8 weeks post infection M. incognita eggs were harvested from the roots by washing away excess soil and by briefly treating cleaned roots in 5% sodium hypochlorite to soften the root tissue and release the eggs. Eggs were cleaned from debris by passage through nested sieves (180 micron, 150 micron and 38 micron) and washed thoroughly with water. Eggs were separated from remaining soil/silt by centrifugation (2000 rcf for 2 minutes) in 100% sucrose solution and collected in a thin layer of spring water (autoclaved and adjusted to pH 7). Eggs were treated in antibiotic/antimycotic solution (Sigma) overnight, placed in a nylon net with a 38 micron pore size, immersed in spring water and maintained in darkness at 23° C., until infective juveniles emerged. Freshly hatched juveniles were used for each assay.
G. pallida were maintained in potato (cv. Cara) at the Agri-Food and Biosciences Institute (AFBI), Belfast. Soil was collected surrounding potato roots, dried for one week and washed through sieves to collect cysts. Cysts were incubated in potato root diffusate in the dark at 17° C. until infective juveniles emerged. Freshly hatched juveniles were used for each assay.
PPN uNLP Screen
Predicted uNLPs from both M. incognita and G. pallida were synthesised by EZBiolab and dissolved into pH adjusted ddH2O to make a 5 mM stock which was aliquoted and stored at −20° C. J2s of both M. incognita and G. pallida were incubated for 24 hours in 200 μl of each peptide in a 24 well plate (SPL Lifesciences, South Korea) at a defined concentration.
PPN uNLP Screen: Chemosensory Assays
A 60 mm Petri dish was divided into two segments, a positive and a negative side, with a 0.5 cm ‘dead zone’ either side of the centre point. The petri dish was filled with 15 ml of 0.25% w/v agar which was allowed to solidify. 3 ml of 0.25% w/v agar slurry in spring water (pH 7, agitated with a magnetic stirrer for several hours to give a smooth consistency) was added to the petri dish and spread evenly over the surface. Root diffusate (attractant) and water only (control) 0.25% agar plugs were embedded in the agar slurry, either side of the assay arena. Root diffusate was collected from 10 tomato plants, aged 3-6 weeks in 1 litre pots, by pouring 500 ml of ddH2O through the soil three times. Diffusate from each plant was combined, filter sterilised and stored at 4° C. for a maximum of 1 month. Root diffusate agar plugs were made by melting 1.25% agar in ddH2O, cooling to 50° C. before mixing with 4 parts of root diffusate. The agar was then allowed to solidify at room temperature. 100 uNLP pre-treated M. incognita or G. pallida J2s were added by pipette to the centre of the plate. J2s which moved out of the ‘dead zone’ after 3 hours were counted and their location (+/−) scored. The distribution of J2s were used to create a chemotaxis index [68] for each plate, which formed one replicate, a total of 10 replicates where completed for each uNLP treatment.
PPN uNLP Screen: Tomato Invasion Assays
Tomato seeds were sterilised with 2.5% NaOCl for 15 minutes, washed 5 times in ddH2O and germinated on 0.5% Murashige and Skoog plates at 23° C. An agar slurry was prepared by autoclaving 0.55% (w/v) agar (using autoclaved spring water adjusted to pH 7) which was mechanically agitated overnight until it had a smooth consistency. Invasion assays were performed by mixing 500 pre-treated M. incognita or G. pallida J2s with agar slurry and a single tomato seedling (2 days post germination) in a 6 well plate. Assays were left at 23° C. for 24 hours in the case of M. incognita and at 18° C. for 24 hours in the case of G. pallida under a 16 hour light and 8 hour darkness cycle. Seedlings were stained using acid fuschin [69] and the number of nematodes within the roots counted.
PPN uNLP Screen: Stylet Thrusting Assays
Stylet thrusting assays where performed by incubating 100 M. incognita or G. pallida J2s for 15 minute in 5 mM or 2 mM serotonin (Sigma Aldrich, USA), respectively. J2s were placed on a glass slide and stylet thrusts were counted for randomly selected J2s, for 1 minute each. Counting took place for a maximum of 15 minutes. Longer incubations yielded inconsistent results. At least 30 J2s were counted for each neuropeptide treatment.
B. subtilis and C. reinhardtii Plant Protection Assays
B. subtilis were grown overnight in LB media containing ampicillin (100 μg/ml) at 37° C. with shaking, and harvested in the log phase of growth determined by measuring OD600nm. Five ml of culture at 0.5 OD was spun down and the pellet mixed with 3 ml of agar slurry and 500 J2s from either G. pallida or M. incognita. C. reinhardtii clones were grown at 23° C. with shaking, cultures in the log phase were measured at OD750 and 5 ml of culture at 0.5 OD was pelleted by centrifugation. C. reinhardtii pellets were mixed with 3 ml of agar slurry and 500 J2s from either G. pallida or M. incognita. Plant invasion assays were performed as described above.
C. elegans Culture and Assays
C. elegans wild-type N2 Bristol strain were obtained from the C. elegans Genomics Center and maintained on a Escherichia coli (strain OP50) lawn on nematode growth medium (NGM) agar plates (3 g/l NaCl, 17 g/l agar, 2.5 g/l peptone, 5 mg/l cholesterol, 25 mM KH2PO4 (pH 6.0), 1 mM CaCl2, 1 mM MgSO4) at 20° C. [70]. Chemotaxis assays were performed in a 9 cm diameter Petri dish on NGM agar which was split into a positive and negative side with a central ‘dead zone’ of 1.5 cm diameter. 100 mixed-staged C. elegans were washed three times in M9 buffer and soaked in 100 μM PPN uNLP, or M9 vehicle control for 24 hours. 2 μl of 50 mM sodium acetate, 0.5% pyrazine, 0.5% benzaldehyde or 0.5% diacetyl was spotted onto the positive side, 2 μl of ddH2O was spotted onto the negative side. Pyrazine, benzaldehyde and diacetyl volatile attractants were assayed immediately whereas the water soluble sodium acetate was assayed 18 hours following addition to the plate. Assays were maintained in the dark at 20° C., and counted after 1 hour.
S. carpocapsae Culture and Host-Finding Assay
S. carpocapsae were cultured in Galleria mellonella at 23° C. Infective juveniles (IJs) were collected using a White trap [71] in PBS. Freshly emerged IJs were used for each assay. 100 IJs were incubated for 24 hours in 100 μM of selected uNLPs, and host-finding assays performed as in Morris et al. [45].
Construction of uNLP Expression/Secretion Plasmids
Codon optimised DNA sequences coding for the desired neuropeptide flanked by restriction sites necessary to clone into the C. reinhardtii expression vector pChlamy_3 (Life Technologies, USA) or the B. subtilits expression vector pBE-S (Clontech, USA) were synthesised by GeneArt® Gene Synthesis (Life Technologies, USA).
Transformation of C. reinhardtii
uNLP secretion inserts, and vector pChlamy_3 were digested using Kpnl/Xbal (New England Biolabs, USA), ligated using T4 ligase (New England Biolabs, USA), and cloned into Escherichia coli One Shot® TOP10 chemically competent cells (Life Technologies, USA) following manufacturer's instructions. Ampicillin (Sigma Aldrich, USA) was used to select E. coli containing the pChlamy_3 plasmid, which was subsequently extracted using the High Pure Plasmid Isolation Kit (Roche) and sequenced (Eurofins Genomics, UK) to identify correct clones. C. reinhardtii was transformed by electroporation following manufacturer's instructions (GeneArt® Chlamydomonas Engineering Kit, Life Technologies) and individual colonies grown on TAP-Agar-Hygromycin plates (10 μg/mL) (Sigma Aldrich, USA) at 23° C. Colonies were picked and grown at 23° C. in 100 ml TAP growth media (Invitrogen, USA) with constant orbital agitation. qRT-PCR was performed to identify clones with the highest level of uNLP expression, which were then selected for downstream assays (pChlamy universal FWD: CACTTTCAGCGACAAACGAG, nlp-15b REV: CTACTAGTCGAGGCCGGTA; Mi-nlp-9f REV: GAACGGGCGGATGAAGTAG).
Transformation of B. subtilis
uNLP secretion inserts, and vector pBE-S were digested using Xbal/Mlul (New England Biolabs, USA), ligated using T4 ligase (New England Biolabs, USA), and cloned into E. coli One Shot® TOP10 chemically competent cells (Life Technologies, USA) following manufacturer's instructions. Ampicillin (Sigma Aldrich, USA) was used to select E. coli containing the pBE-S plasmids, which were subsequently extracted using the High Pure Plasmid Isolation Kit (Roche) and sequenced (Eurofins Genomics, UK) to identify correct clones. B. subtilis RIK1285 competent cells (Takara, USA) were transformed according to manufacturer's instructions and grown overnight at 37° C. on kanamycin selective plates (10 μg/mL) (Sigma Aldrich, USA). Individual colonies were picked and grown in LB broth overnight at 37° C. qRT-PCR (pBE-S universal FWD: GGATCAGCTTGTTGTTTGCGT, nlp-15b REV: CCTGGCCCAGTGAAAGAGTC, Mi-nlp-40 REV: TACCGGCTGCCAAGATACCA) was performed to confirm the expression of uNLP secretion cassettes.
Statistical Analysis
Data pertaining to behavioural and invasion assays were assessed by Brown-Forsythe and Bartlett's tests to examine homogeneity of variance between groups. One-way ANOVA was followed by Fisher's Least Significant Difference (LSD) test. All statistical tests were performed using GraphPad Prism 6.
BLASTp Identification of Predicted NLPs
Pro-peptide sequences of C. elegans NLPs predicted to be unamidated (no C-terminal glycine; uNLPs) were used as queries to conduct a BLASTp analysis of the predicted protein complements of both M. incognita and G. pallida [46, 47]. A total of four nlp genes encoding 25 predicted uNLPs were found within the G. pallida genome, and seven nlp genes encoding 28 predicted uNLPs within the M. incognita genome (Table 1).
Meloidogyne incognita.
Globodera pallida NLPs*
Meloidogyne incognita NLPs*
uNLPs Dysregulate Key Behaviours of M. incognita J2s
Predicted uNLPs were synthesised and screened against M. incognita and G. pallida J2s for plant protective qualities. Chemotaxis, host-invasion, and stylet thrusting behaviours were assayed following J2 exposure to 100 μM of each uNLP for 24 h. Eleven of 27 tested uNLPs were found to disrupt normal chemotaxis towards root exudate: Mi-NLP-8a (Cl: 0.018+/−0.3438, p=0.0416), Mi-NLP-15a (Cl: −0.01067+/−0.06497, p=0.0299), Mi-NLP-15e (Cl: −0.01767+/−0.09428, p=0.0275), Mi-NLP-40 (Cl: −0.04+/−0.04726, p=0.021), Mi-NLP-15f (Cl: −0.05+/−0.3547, p=0.0185), Mi-NLP-9b (Cl: −0.07417+/−0.154, p=0.001), Mi-NLP-14b (Cl: −0.1155+/−0.1472, p=0.0004), Mi-NLP-18a (Cl: −0.1353+/−0.1129, p=0.006), Mi-NLP-9f (Cl: −0.26+/−0.224, p<0.0001), Mi-NLP-15b (Cl: −0.3408+/−0.2207, p<0.0001), Mi-NLP-15c (Cl: −0.359+/−0.277, p=0.0002) (
Likewise, 13 uNLPs were also found to disrupt M. incognita host invasion compared to controls: Mi-NLP-8b (61.38%+/−11.56, p=0.0113), Mi-NLP-9e (48.68%+/−10.87, p=0.0022), Mi-NLP-18b (47.81%+/−6.008, p=0.0019), Mi-NLP-18e (46.49%+/−7.391, p=0.0014), Mi-NLP-9a (45.59%+/−22.97, p=0.0039), Mi-NLP-14a (44.61%+/−12.25, p=0.0033), Mi-NLP-15e (38.71%+/−5.963, p=0.0003), Mi-NLP-18d (34.21%+/−12, p=0.0001), Mi-NLP-15b (32.22%+/−8.122, p<0.0001), Mi-NLP-8d (29.31%+/−12.57, p<0.0001), Mi-NLP-9f (28.22%+/−5.253, p<0.0001), Mi-NLP-18a (25.88%+/−8.695, p<0.0001), Mi-NLP-14c (20.96%+/−11.12, p<0.0001) (
Eleven uNLPs were also found to disrupt the rate of serotonergic-induced M. incognita stylet thrusting (positively or negatively) compared with controls: Mi-NLP-40 (210%+/−21.35, p<0.0001), Mi-NLP-18c (175.5%+/−20.72, p<0.0001), Mi-NLP-2 (164.4%+/−20.3, p<0.0001), Mi-NLP-18d (147.7%+/−16.05, p<0.0001), Mi-NLP-18b (146.6%+/−7.609, p=0.0002), Mi-NLP-9c (143.2%+/−9.878, p=0.0005), Mi-NLP-14c (134.7%+/−19.51, p=0.0053), Mi-NLP-15b (129.9%+/−10.59, p=0.0159), Mi-NLP-8a (126.1%+/−9.278, p=0.0359), Mi-NLP-15f (75.18%+/−9.199, p=0.0456), Mi-NLP-14a (61.6%+/−7.86, p=0.002) (
uNLPs Dysregulate Key Behaviours of G. pallida J2s
12 of 25 tested uNLPs were found to disrupt chemotaxis of G. pallida J2s towards root exudate: Gp-NLP-21f (Cl: 0.1456+/−0.1232, p=0.0317), Gp-NLP-21g (Cl: −0.01357+/−0.1854, p=0.0248), Gp-NLP-21h (Cl: −0.01564+/−0.08072, p=0.0488), Gp-NLP-15b (Cl: −0.04762+/−0.1983, p=0.0345), Gp-NLP-14e (Cl: −0.0641+/−0.2329, p=0.0026), Gp-NLP-15h (Cl: −0.06784+/−0.1415, p=0.0052), Gp-NLP-21b (Cl: −0.117 +/−0.1936, p=0.0008), Gp-NLP-15a (Cl: −0.1451 +/−0.221, p=0.0035), Gp-NLP-21i (Cl: −0.1733 +/−0.02667, p=0.0074), Gp-NLP-15g (Cl: −0.2208 +/−0.1568, p=0.001), Gp-NLP-15c (Cl: −0.227+/−0.0776, p=0.0002), Gp-NLP-14a (Cl: −0.3765+/−0.1039, p<0.0001), Gp-NLP-21e (Cl: −0.3804+/−0.2762, p<0.0001) (
Five uNLPs disrupt G. pallida host invasion relative to controls: Gp-NLP-21d (2.01%+/−1.545, p=0.004), Gp-NLP-21c (14.07%+/−4.655, p=0.0115), Gp-NLP-21a (178.9%+/−48.52, p=0.0201), Gp-NLP-21b (6.897%+/−3.855, p=0.0086), Gp-NLP-21g (214.8%+/−30.31, p=0.0012) (
Three uNLPs were also found to modulate serotonergic-induced stylet thrusting of G. pallida J2s relative to controls groups: Gp-NLP-21 i (117.7%+/−4.497, p=0.0302), Gp-NLP-21 h (116.8%+/−4.876, p=0.0046), Gp-NLP-15c (56.07%+/−9.441, p<0.0001) (
Mi-NLP-15b Inhibits M. incognita Chemotaxis and Host Invasion with High Potency
The potency of Mi-NLP-15b-induced disruption of chemotaxis and host invasion was assessed by exposing M. incognita J2s to various concentrations of synthetic Mi-NLP-15b for 24 h. Normal chemotaxis of M. incognita towards root exudate was inhibited across a range of dilutions, indicating high potency: 100 μM, (Cl: −0.3782+/−0.07224, p=0.0031), 10 μM, (Cl: −0.03579+/−0.1504, p=0.0025), 1 μM, (Cl: −0.1344+/−0.1733, p=0.001), 100 nM, (Cl: −0.1195+/−0.1968, p=0.0014), 10 nM, (Cl: −0.1741+/−0.1724, p=0.0055), 1 nM, (Cl: −0.07105+/−0.1534, p=0.0035), 100 pM, (Cl: 0.03117+/−0.1594, p=0.0202), 10 μM, (Cl: −0.1553+/−0.2642, p=0.0037) (
Transgenic Microbes Secreting uNLPs Protect Plants from PPN Invasion
Innoculation of C. reinhardtii cultures secreting selected uNLPs into the tomato invasion assay arena inhibited M. incognita invasion relative to untransformed C. rehinhardtii: Mi-NLP-9f (10.32%+/−10.32, p<0.0001), Mi-NLP-15b (10.82%+/−6.574, p<0.0001) (
PPN uNLPs do not Alter Behaviours of Non-Target Nematodes
BLAST was used to identify NLP-15b homologues across available expressed sequence tags (ESTs) or genomes of PPNs and non-target nematode species. PPNs with diverse life history traits share high levels of NLP-15b sequence similarity, however sequence similarity is reduced in non-target nematode species (Table 2).
Meloidogyne incognita
Meloidogyne javanica
Meloidogyne hapla
Meloidogyne chit woodi
Globodera paffida
Globodera rostochiensis
Heterodera glycines
Pratylenchus penetrans
Radopholus similis
Steinemema carpocapsae
Pristionchus pacifius
Caenorhabditis elegans
Incubation of mixed-stage C. elegans in selected PPN uNLPs (100 μM, 24 h) had no statistically significant impact on chemotaxis towards: (i) sodium acetate. Mi-NLP-9f (Cl: 0.5261+/−0.064, p=0.4164), Mi/Gp-NLP-15b (Cl: 0.3142+/−0.039, p=0.3578) or Mi-NLP-40 (Cl: 0.4820+/−0.115, p=0.6485) relative to control groups (Cl: 0.4269+/−0.094), (
Discussion
We have identified seven nlp genes which putatively encode 27 mature unamidated peptides in the root knot nematode, M. incognita. Likewise, four nlp genes predicted to encode 24 mature unamidated peptides were identified in the potato cyst nematode, G. pallida (Table 1). Several predicted unamidated NLPs share high levels of amino acid sequence similarity between M. incognita and G. pallida, with one predicted peptide, designated NLP-15b, perfectly conserved between the two. Indeed, NLP-15b is highly conserved at the sequence level across PPN species with diverse life history traits; less sequence similarity is observed between NLP-15b from PPNs and non-target species such as S. carpocapsae, C. elegans or P. pacificus for example (see Table 2).
Selected M. incognita and G. pallida peptides had a negative impact on PPN chemosensation and host-finding behaviours, but not on chemosensory or host-finding behaviours of mixed stage C. elegans or S. carpocapsae infective juveniles (
Whilst it is tempting to extrapolate something on native NLP functionality from these data, we do not know if the aberrant phenotypes observed are due to interactions between tested NLPs and their cognate receptors. However, we do observe that exogenous NLPs can interact with endogenous neurophysiological circuits, interfering with host-finding, invasion and serotonergic stylet-thrusting behaviours of both M. incognita and G. pallida juveniles (
In order to further assess the efficacy of exogenous NLPs as nematicides, we developed two transgenic synthesis and delivery systems which could be deployed in field, potentially through seed treatments or soil amendments. Gram positive Bacillus spp. are a major component of rhizosphere microbial communities [52, 53], and are frequently categorised as Plant Growth Promoting Rhizobacteria (PRPR) [54, 55]; B. subtilis has also been shown effective in controlling Meloidogyne species [56]. More generally, B. subtilis represents an important organism for many biotechnology applications, and is classified as GRAS (generally regarded as safe) by the FDA [57, 58]. It is increasingly well served by the development of synthetic biology tools [59], and can persist in soil for long periods through the production of spores [60]. We modified B. subtilis to secrete a number of PPN NLPs, and found that transformed B. subtilis cultures confer significant levels of protection on tomato cv. Moneymaker against both M. incognita and G. pallida infective juveniles (
We also utilised the soil-dwelling microalgae, C. reinhardtii as a novel synthesis and delivery platform. Like B. subtilis, C. reinhardtii benefits from an improving suite of synthetic biology tools [67]. C. reinhardtii cultures secreting selected PPN NLPs also provided significant levels of protection to tomato cv. Moneymaker when challenged by either M. incognita or G. pallida infective juveniles (
The NLP screening approach employed here may underestimate the efficacy achievable through a continuous transgenic delivery (
Our data demonstrate that unamidated NLPs represent a new class of potent and specific plant protective nematicide which could be deployed as a transgenic trait in crop plants, or through soil microorganisms such as the B. subtilis and C. reinhardtii systems developed here. In particular, these non-crop delivery approaches could facilitate rapid deployment to many different crop plant species and cultivars. A key consideration in the development of PPN resistance traits must be the maintenance of genetic diversity across crop cultivars and isolates. This reduces the chance of widespread pathology from other pests as a result of genetic bottlenecks introduced by a single preferred transgenic cultivars.
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The invention is not limited to the embodiments described herein but can be amended or modified without departing from the scope of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1701750.0 | Feb 2017 | GB | national |
This application is a national stage application, filed under 35 U.S.C. § 371, of International Application No. PCT/EP2018/052709 filed Feb. 2, 2018, which claims priority to GB Patent Application No. 1701750.0 filed Feb. 2, 2017, which applications are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2018/052709 | 2/2/2018 | WO | 00 |
