CAN WE USE SYNTHETIC BIOLOGY AS AN
ALTERNATIVE TO PESTICIDES?
(An Unexpected Journey)
Keith G Davies
14 March 2018
Café Scientifique Hatfield
School to Rothamsted (10 years)from applied field ecology to the glasshouse
Rothamsted years (25 years)from the glasshouse to the laboratory
from the laboratory to the Eppendorf tube
from the Eppendorf tube to the genome
Structure of my talk
Post Rothamstedfrom the genome to synthetic biology
OPEC 1973 Oil Crisis
World Population< 4 billion (1975)
School to Rothamsted
One of 2 institutions at the time offering a degree with specialism in ecology
The only institution offering a degree in ecology with an industrial placement
School to Rothamsted
School to Rothamsted
European legislation regarding the use of pesticides
New legislation to replace Directive 91/414
A) New Registration regulation Directive 1107/2009B) Sustainable use Directive 2009/128/EC
Came into effect 14 June 2011
Also water framework directive
Priority to be given to non-chemical methods
Need for alternative nematode control methods
CONTEXT
School to Rothamsted
Rothamsted years
Plant-parasiticnematodes causelosses of around
$100 billionannually
C elegans movie
Bacterial wart diseasePasteuria penetrans
Increase in tomato yield following application of Pasteuriapenetrans to root-knot infected soils in Equador
adapted from Trudgill et al., (2000) Nematology 2, 823-845
Two Major Problems
1) Mass production
2) Infection and host specificity
Fallow Tomato Tomato
J2 per g Soil 94 64 7
%J2 +spores 47 89 100
Yield Kg/plot Na 11 25
Rhizoids
Granular masses
Penetration peg
Adhesion to cuticle
Sporulation
Matureendospores
Maturation
Rod
The life-cycle of Pasteuria penetrans: on root-knot Nematodes (Davies et al., Nematology 2011)
Meloidogyneincognita
Meloidogynearenaria
Reaction of second-stage juvenile cuticle by a polyclonalantibody
Davies & Danks Parasitology 105, 475-480
ENDOSPORE ATTACHMENT
Percentage of nematodes without Pasteuria spores in standard attachment bioassays with Meloidogyne arenaria ( ) and M. incognita ( )
Davies et al., 2001 Parasitology 122, 111-120
No fluorescence Low fluorescence High fluorescence
Davies et al., Let. Appl. Microb. 19, 370-373
Immunofluorescence of Pasteuria endospores
Immunofluorescence of Pasteuria endosporesattached to nematodes
Polyclonal antibody Monoclonal antibody
(Adapted from Preston et al., J. Nematology)
Cuticle characterisation using Mabs to Pasteuria
M. arenaria Race 1: peanutM. incognita Race 1: tobacco
Davies et al., Let. Appl. Microb. 19, 370-373
M. javanica: tobacco M. incognita Race 3: cotton
M. arenaria Race 1: peanutM. incognita Race 1: tobacco
M. incognita Race 3: cotton
Cuticle characterisation using Mabs to Pasteuria
Genomics
Caenorhabditis elegans Sequencing consortium 1998Meloidogyne incognita Abad et al., 2008Meloidogyne hapla Opperman et al 2008Heterodera glycines MonsantoBursaphenenchus xylophilus Kikuchi et al 2011Globodera pallida Urwin et al 2014Pratylenchus coffeae Opperman et al., 2015
Nematodes whole genomes
Endospore B. cereus(A. Moir)
Genomics
Caenorhabditis elegans Sequencing consortium 1998Meloidogyne incognita Abad et al., 2008Meloidogyne hapla Opperman et al 2008Heterodera glycines MonsantoBursaphenenchus xylophilus Kikuchi et al 2011Globodera pallida Urwin et al 2014Pratylenchus coffeae Opperman et al., 2015
Nematodes whole genomes
Selected Firmicutes whole genomes
Staphylococcus spp >100Clostridium spp >100Bacillus cereus >100Paenibacillus spp 51Bacillus subtilis 37Bacillus anthracis 38Bacillus thuringiensis 37
Endospore B. cereus(A. Moir)
Pasteuria genome survey sequences 4600
2 um
Spore attachment: Velcro-like mechanism
Davies, 2009, Advances in Parasitology 68, 211-245
A schematic diagram illustrating a possible model for the exosporium of the B. cereus family.
Kailas L et al. PNAS 2011;108:16014-16019
©2011 by National Academy of Sciences
0
0
41
41
B. anthracisStrain
770
818
Ames
53169
9602R, A2R, 6183R
6602
4229
5725R
G-x-y repeat region
Number FilamentG-x-y lengthrepeats (nm)
314 445 162 60.8
260 391 130 47.3
251 382 124 NA
239 370 118 48.7
131 262 52 28.4
122 253 48 29.8
113 244 42 25.1
92 223 28 13.9
GENOMICS & ENDOSPORE ATTACHMENT
Total of 12 Pasteuria penetrans collagens with G-X-Y repeats
5 unique to RES147, 4 unique toFl-1, 3 in common
79 G-X-Y repeats
62 G-X-Y repeats
36 G-X-Y repeats
GENOMICS & ENDOSPORE ATTACHMENT
>Contig1_2 (Ppenetrans-147)IFLGTTIGRLEFSGREFALGREGQQEHRGGPTTGPPGPPGAHGIQGPPGAQGIQGPAGTP
GAQGIQGPPGPAGTPGAQGIQGPPGPAGPAGPAGAAGSPGTPGPAGPAGPAGAAGSPGTP
GSPGTPGPAGPAGPAGPAGTPGTPGSPGTPGPAGPAGPAGTPGTPGAAGSPGTPGPAGPA
GPAGPAGTPGTPGPAGPQGTPGAPGPAGPQGTPGAPGPAGPQGTTGAAGPTGPQGTTGPQ
GTQGTQGPQGIQGIQGPVGPQGATGATGPGLNTSMTIVAGGGADTQFITPTPEGATGTAV
TLETGNGQRYGSGDIQLIGTTDILLPSTGTYLMSFHIDANYTSAAGAPVAGAYGSYVAYF
RQFTTDFFFNQIVAFWVGPALVNDAFDSSISNTVLGCVSDIPPHGLNNHMVRHESDVANS
LNTRRSQPQX
>Contig2_1 (Ppenetrans-147)
LQDQTNSPFRLEPEMWSINNIHSFNHMNKRKKQNIFFHTFSLGGLEDNHFMKHWIGRNSG
CIHYKNNGKIRNTITHTPSPRRSEGNRFVKHWIGRKSVYINSDYRDQHNHHNSSRTTLYR
NCEKCDNNQYEEFDNDHCEEFDNNHCCDCCLCNRCKCRVTGPTGPTGPTGRTGSTGRTGP
TGPTGRTGSTGRTGPTGPTGPTGPTGPTGRTGFTGRTGSTGSTGRTGPTGSTGRTGSTGS
TGRTGPTGSTGRTGSTGSTGRTGSTGSTSRPLVGRRNSR
>Contig3_4 (Ppenetrans-147)VANSRLEGWTAGPAGAQGISGPPGEPGIQGPAGTPGAQGIQGPPGPAGTPGAQGIQGPPG
PAGPTGPAGAAGSPGTPGPAGPAGPAGAAGSPGTPGSPGTPGPAGPAGPAGPAGTPGAPG
PAGPQGTPGAPGPAGPQGTPGAPGPAGPQGTTGAAGPTGPQGTTGPQGTQGTQGPQGIQG
IQGPVGPQGATGATGPGLNTSMTIVAGGGADTQFITPTPEGPTGPGGTFEPGNGPKYREG
GELHLIGTHRFSSSRVPGPF
The Future: Synthetic Biotechnolgy??
“… the real challenge will start when we enter the synthetic biology phase of research in our field. We will then devise new control elements and add these new modules to the existing genomes or build up wholly new genomes.”
Szybalski, 1974
Gibson et al., 2010 Science 329: 52-56
Within the synthetic genome Mycoplasma mycoidesthere were ‘watermarks’
CRAIGVENTER coded as:TTAACTAGCTAATGTCGTGCAATTGGAGTAGAGAACACAGAACGATTAACTAGCTAA
VENTERINSTITVTE coded as:TTAACTAGCTAAGTAGAAAACACCGAACGAATTAATTCTACGATTACCGTGACTGAGTTAACTAGCTAA
“WHAT I CANNOT BUILD, I CANNOT UNDERSTAND.”
– attributed to Richard Feynman
“TO LIVE, TO ERR, TO FALL, TO TRIUMPH, TO RECREATE LIFE OUT OF LIFE.”
– from James Joyce’s
The Future: Synthetic Biotechnolgy??
“… the real challenge will start when we enter the synthetic biology phase of research in our field. We will then devise new control elements and add these new modules to the existing genomes or build up wholly new genomes.”
Szybalski, 1974
Scie
nce
Mar
ch 2
5 2
01
6
Life-cycle disruption
(a) Hatch and migration• nematicides
• root diffusates• rhizosphere microorganisms
(b) Feeding, maturation and fecundity• nematicides
• host plant susceptibility• rhizosphere and endophytic micro-
organisms
(c) Mate finding and reproduction• nematicides
• mating disruption
Biological control perspective
(Gair et al., 1969)
Suppressive soilsare usually the
product of more than one organism
Davies et al., 1990
May June
July August
Nematodes Pasteuria Fungi
Cereal Cyst Nematode decline phenomenon
Fred Gommers
Biological control organisms
1) Fungal parasitesPochonia chlamydosporiaPaecilomyces lilacinusArthrobotrys oligosporaTrichoderma spp.
2) Bacterial parasitesPasteuria penetransRhizobacteriaBacillus spp.
Example 2 Arthrobotrys
Tunlid and Ahrén2011: In Keith Davies and Yitzak Spiegel (Eds) Biological Control of
Plant –Parasitic Nematodes
Peptides “nemin”elicitors
adhesins
Lectins
Serineproteases
Subtilisins
Toxic metabolites(linoleic acid)
Example 1 Pochonia
Kerry & Hirsch 2011: In Keith Davies and Yitzak Spiegel (Eds) Biological Control of
Plant –Parasitic Nematodes
Example 5 Pasteuria
Davies K G 2009, Adv. In Parasitol. 68, 211 - 245
Velcro – like attachment
Collagen moleculeslectins
Carbohydrates
Signalling pathways
Glycosyl-transferases
Mucins
Innate immunity
Designer biological control agents?
Lipopolysaccarhide effectors
Mucin effectors
Protease enzymes
Adhesion factors
Bacteriocins
Siderophores
Antifungal agents
Hormonal peptideschitinases
Signalling molecules
What is now proved was once only imaginedWilliam Blake
1757 - 1827
From Frankenstein toVenterstein?
“WHAT I CANNOT BUILD, I CANNOT UNDERSTAND.”
– attributed to Richard Feynman
“TO LIVE, TO ERR, TO FALL, TO TRIUMPH, TO RECREATE LIFE OUT OF LIFE.”
– from James Joyce’s
AcknowledgementsUNIVERSITY OF OXFORD
Jonathan HodgkinMaria Gravato-Nobre
Delia O’RoukeFreddie Partridge
Dave Stroud
BBSRC: Institute Development
Fellowship
Indian Agric. Res. Inst.Junaid Khan
Uma Rao
UC Davis: Valerie Williamson
George Brunning
NCSU: Charlie Opperman
David BirdBetsy Scholl
Indian Council Agric. Res. Arohi Srivastava
www.youtube.com/watch?v=sls1NzoaXZg
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