biological control and pasteuria penetrans: genomics and

53
Biological Control and Pasteuria penetrans: genomics and molecular biology Keith Davies Post-graduate International Nematology Course Ghent, Belgium www.rothamsted.bbsrc.ac.uk/ppi/staff/kgd.html Plant Pathology and Microbiology, Rothamsted Research, Harpenden, Hertfordshire, UK

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Page 1: Biological Control and Pasteuria penetrans: genomics and

Biological Control and Pasteuria penetrans:

genomics and molecular biology

Keith Davies

Post-graduate International Nematology Course

Ghent, Belgium

www.rothamsted.bbsrc.ac.uk/ppi/staff/kgd.html

Plant Pathology and Microbiology, Rothamsted Research, Harpenden, Hertfordshire, UK

Page 2: Biological Control and Pasteuria penetrans: genomics and

Structure of talk:

•Introduction and context

•Immunological approach to host specificity

•Genomics and taxonomy

•In vitro culture

•Collagen and the mechanism of attachment

•Three types of cuticle variation

Page 3: Biological Control and Pasteuria penetrans: genomics and

Generalised life cycle of a root-knot nematode Meloidogyne spp.

Page 4: Biological Control and Pasteuria penetrans: genomics and

The life-cycle of Pasteuria penetrans on root-knot nematodes (Advances in Parasitol. In press)

Rhizoids

Granular masses

Penetration peg

Adhesion to cuticle

Sporulation

Mature endospores

Maturation

Rod

Page 5: Biological Control and Pasteuria penetrans: genomics and

Increase in tomato yield following application ofPasteuria penetrans to root-knot infected soils in Equador

Fallow Tomato Tomato J2 per g Soil 94 64 7 %J2 +spores 47 89 100 Yield Kg/plot Na 11 25

adapted from Trudgill et al., (2000) Nematology 2, 823-845

Two Major Problems

1) Mass production

2) Infection and host specificity

Page 6: Biological Control and Pasteuria penetrans: genomics and

Pasteuria penetrans Differential Host Range Test

M. javanica

M. incognita

M. arenaria

H. avenae

H. glycines

H. schachtii

H. cajani

G. pallida

G. rostochiensis

PNG PP1 PMJ PCl PP4 PPW PPN PHC

+++ ++ ++ ++ + + + - +++ ++ ++ ++ + - - - + + + + + - - - - - - - + + - NA

- - NA - - + ++ + - - - - - + + ++ - - - - - NA NA +++ - - - - - NA NA +++ - - - - - ++ + +++

-, no attachment; +, 1-10 spores; ++, 11-20 spores; +++ > 21 spores

Page 7: Biological Control and Pasteuria penetrans: genomics and

Meloidogyne incognita

Meloidogyne arenaria

Reaction of second- stage juvenile cuticle by a polyclonal antibody

Davies & Danks Parasitology 105, 475-480

ENDOSPORE ATTACHMENT

Page 8: Biological Control and Pasteuria penetrans: genomics and

Davies et al., 2001, Parasitology 122, 111-120

Percentage of juveniles of Meloidogyne arenaria and M. incognita to which no spores attached

Page 9: Biological Control and Pasteuria penetrans: genomics and

Immunofluorescence of Pasteuria endospores

No fluorescence Low fluorescence High fluorescence

Davies et al., Let. Appl. Microb. 19, 370-373

Page 10: Biological Control and Pasteuria penetrans: genomics and

Immunofluorescence of Pasteuria endospores attached to nematodes

Polyclonal antibody Monoclonal antibody

(Adapted from Preston et al., J. Nematology)

Page 11: Biological Control and Pasteuria penetrans: genomics and

Cuticle characterisation using Mabs to Pasteuria

M. arenaria Race 1: peanut M. incognita Race 1: tobacco

M. javanica: tobacco M. incognita Race 3: cotton

Davies et al., Let. Appl. Microb. 19, 370-373

Page 12: Biological Control and Pasteuria penetrans: genomics and

M. arenaria Race 1: peanut M. incognita Race 1: tobacco

M. javanica: tobacco M. incognita Race 3: cotton

Cuticle characterisation using Mabs to Pasteuria

Davies et al., Let. Appl. Microb. 19, 370-373

Page 13: Biological Control and Pasteuria penetrans: genomics and

M. incognita Race 2

0

20

40

60

80

100

120

Antibodies

% f

luo

rescen

t sp

ore

s

Pp/12

Pp/53

Pp/84

Pp/117

Pp/134

Con+ve

M. incognita Race 3

0

20

40

60

80

100

120

Antibodies

% f

luo

rescen

t sp

ore

s

Pp/12

Pp/53

Pp/84

Pp/117

Pp/134

Con+ve

Davies et al., Let. Appl. Microb. 19, 370-373

Characterisation of endospores attached to different nematode populations by 5 Mabs

Cuticle characterisation using Mabs to Pasteuria

M. arenaria race 1

0

20

40

60

80

100

120

Antibodies

% f

luo

rescen

t sp

ore

s

Pp/12

Pp/53

Pp/84

Pp/117

Pp/134

Con+ve

Page 14: Biological Control and Pasteuria penetrans: genomics and

Mixed population of nematodes: PEANUT susceptible to Race 1 of M. arenaria Increase in Race 1 M. arenaria pathogenic Pasteuria

Tritrophic interaction

M. arenaria Race 1: peanut

(After Davies, K.G., 2005, Advances in Applied Microbiology 57, 53-78)

Page 15: Biological Control and Pasteuria penetrans: genomics and

Mixed population of nematodes: TOMATO universally susceptible maintaining a diverse Pasteuria population

Tritrophic interaction

(After Davies, K.G., 2005, Advances in Applied Microbiology 57, 53-78)

Page 16: Biological Control and Pasteuria penetrans: genomics and

DNA extraction by bead beating

Unbeaten

Beaten

Genomics

DNA Extraction based on robust endospores 1) Infected females dissected from roots 2) Homogenised to extract spores 3) Enzyme digestion Proteinase K, lyzozyme, DNAase, RNAase 4) Heat treatment, protinase K, heat treatment 5) Bead beating 6) DNA amplification using kit 7) Sequencing: Sanger (2003) and 454 (2008)

Page 17: Biological Control and Pasteuria penetrans: genomics and

Sanger-2003 454-2008

Sequence 2.5 Mbp 8.6 Mbp

Contigs 1500 5964 (782 > 2 kb)

Largest contig 2.5 kb 54.7 kb

GC content 62 % 45 %

THE BIG PICTURE

Genomics

Bacterial genomes range from: < 1 Mbp Mycoplasma genitalium to > 9 Mbp Nostoc punctiforma

Page 18: Biological Control and Pasteuria penetrans: genomics and

Bacillus anthracis

Bacillus cerus

Bacillus subtilis

Bacillus halodurans

Pasteuria penetrans Staphylococcus aureus

Lactococcus lactis

Streptococcus pyogenes

Mycoplasma pneumoniae

Mycoplasma genitalium

Mycobacterium leprae

Mycobacterium tuberculosis

Aquifex aeolicus

Campylobacter jejuni

Heliobacter pylori

Rickettsia prowazekii

Neisseria meningitidis

Xylella fastidiosa

Pseudomonas aeruginosa

Escherichia coli

Buchnera aphidicola

Pasteurella multocida

Haemophilus influenzae

Chlamydophila pneumoniae

Chlamydia trachomatis

Chlamydia muridarum

Borrelia burgdorferi

Treponema pallidum

Phylogenetic analysis undertaken using

27 full-length genes

Genomics

Charles et al., 2005, J. Bact.

Gram positive

Gram negative

Page 19: Biological Control and Pasteuria penetrans: genomics and

Two major problems prohibiting Pasteuria penetrans from being developed into a

commercial product related to endospore production

1) In vitro mass production

2) Spore ‘type’ and attachment specificity

“Forget about the genome. And let’s say, ‘It’s great. We’ve got

all these sequences. Thank you very much for all the people

that helped to get them - please get us some more.’ And let’s

get on with the biology.”

Sydney Brenner, January 2003, San Diego, California

Plant and Animal Genome Conference

Page 20: Biological Control and Pasteuria penetrans: genomics and

Comparison of endospore formation in Bacillus thuringiensis and Pasteuria penetrans

From Chen et al., (1997) Phytopathology 87, 273-283

Page 21: Biological Control and Pasteuria penetrans: genomics and

Sm all-molecule metabolism

Broad regulatory functions Macromolecular metabolism

Cell processes Sporulation

Transposons

Other

Conserved hypothetical Unknown function

6 -7 %

Genomics GO analysis

Driks, 2002 Trends Microbiol. 10: 251

Sporulation governed by Spo0F

Page 22: Biological Control and Pasteuria penetrans: genomics and

KinA

Spo0B

Spo0F

Spo0A Spo0A

RapB

DNA binding domain

DNA binding domain

KinB

RapA

Phosphatases

Inactive Active

Sporulation

abrB

Diagram of proteins involved in the phosphorelay required for initiating the sporulation signal transduction pathway in B. subtilis (from Feher et al., 1997). The red arrows represent environmental signals which initiate the transfer of the phosphoryl group via the Phosphorelay pathway.

Phosphorelay pathway

Kinases

Nutrient Starvation Temperature or pH extremes Cell crowding Antibiotic exposure

Page 23: Biological Control and Pasteuria penetrans: genomics and

Bsu MMNEK--ILIVDDQYGIRILLNEVFNKEGYQTFQAANGLQALDIVTKERPDLVLLDMKIPGMDG

Ban VMEGK--ILIVDDQYGIRVLLHEVFQKEGYQTFQAANGFQALDIVKKDNPDLVVLDMKIPGMDG

Bha -MN-K--ILVVDDQYGIRVLLNEILQKDGYQMFQAANGIQALAIVEEETPDLVLLDMKIPGMDG

Ppe MMNEKKLILIVDDQYAIRLLLKEIFSQDGIIVLQAAGGXEAIELVAQQQPDLMLLDMKMPGMDG

___________ ______________ ____ ____________ _____ __

b1 a1 b2 a2 b3

Bsu IEILKRMKVIDENIRVIIMTAYGELDMIQESKELGALTHFAKPFDIDEIRDAVKKYLPLKSN

Ban IEILKHVKEIDESIKVILMTAYGELDMIQEAKDLGALMHFAKPFDIDEIRQAVRNELAVEA-

Bha LEILRRIKDMNPNIEVIMMTAYGELNMINEAMQLGAVTHFAKPFDIDDVRAVIAENMKSS—-

Ppe LEILRRVRQVIPALKVIVMTAYGELEMMEKIRSLGVVMHFPKPFDVRAVRCGVLRYLXXXXX

_________ ______ ________ ____ ______________

a3 b4 a4 b5 a5

Residues in red are identical between Ppe and the Bacillus species. Residues in green are conservative exterior substitutions. The positions of b-strands and a-helices are indicated. Conserved active-site residues are highlighted in bold (arrows). Kojetin, et al., 2005, Biometals 18, 449-466

spo0F, Pasteuria and Bacillus species

Page 24: Biological Control and Pasteuria penetrans: genomics and

STRUCTURE OF Spo0F charged aspartic acids line the pocket of the

active site

• A divalent metal is required

• Thought to be Mg2+ or Mn2+

• Without the metal ion, phosphorylation will not occur

WHAT ABOUT OTHER IONS?

Kojetin, et al., 2005, Biometals 18, 449-466

Page 25: Biological Control and Pasteuria penetrans: genomics and

Divalent Metal Dependent Phosphorylation

0 200 400 600 800 1000

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Mg KinA~P

Mg OF~P

uM

0 200 400 600 800 1000

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Cu KinA~P

Cu OF~P

uM

0 200 400 600 800 1000

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Zn KinA~P

Zn 0F~P

uM

MgCl2 ZnCl2 CuCl2

Rel

ativ

e am

ount

of

Kin

A~P

or S

po0

F~P

Phospho-transfer from KinA~P to Spo0F

KinA~P Spo0F~P

Kojetin, et al., 2005, Biometals 18, 449-466

Page 26: Biological Control and Pasteuria penetrans: genomics and

Growth of Pasteuria in culture is affected by divalent cations

+ Cu+2 - Cu+2

Kojetin, et al., 2005, Biometals 18, 449-466

Page 27: Biological Control and Pasteuria penetrans: genomics and

Davies et al., 2001, Parasitology 122, 111-120

Percentage of juveniles of Meloidogyne arenaria and M. incognita to which no spores attached

Page 28: Biological Control and Pasteuria penetrans: genomics and

Pasteuria penetans endospore

Bacillus anthracis endospore

Attachment is a key determinant for infection

Davies, Advances in Parasitol. In prep.

Page 29: Biological Control and Pasteuria penetrans: genomics and

B. Anthracis Strain

770

818

Ames

53169

9602R, A2R, 6183R

6602

4229

5725R

0

0

41

41

G-x-y repeat region

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

Number Filament G-x-y length repeats (nm)

Structure of BclA collagen-like repeats and their relationship to filament length in Bacillus anthracis (adapted from Sylvestre et al., J Bact. 185; 2003)

GENOMICS & ENDOSPORE ATTACHMENT

Page 30: Biological Control and Pasteuria penetrans: genomics and

X X X X X X X

X

X

X

X

cotZ-1 fab1 rfbD rfbB rfbC BclA

X X X X

yjbX cotY

yjbV

X

X

cotZ-2

X

yjcA yjcB

X

X X X

X X X

yjcD yjcE yjcF yjcG yjcH yjcC yjcC cotV cotW cotX

rfbA cloP

Ba

Bt

Bs

Ba

Bt

Bs

Gene organisation for the encoding proteins of the exosporium for Bacillus anthracis, B. thuringiensis & B. subtilis

Solid red genes denote BLAST hits (e-14) to Pasteuria (dashed red e-4)

COMPARATIVE GENOMICS as of 2003

(Davies & Opperman, IOBC Bulletin, 2006, )

Page 31: Biological Control and Pasteuria penetrans: genomics and

X X X X X X X

X

X

X

X

cotZ-1 fab1 rfbD rfbB rfbC BclA

X X X X

yjbX cotY

yjbV

X

X

cotZ-2

X

yjcA yjcB

X

X X X

X X X

yjcD yjcE yjcF yjcG yjcH yjcC yjcC cotV cotW cotX

rfbA cloP

Ba

Bt

Bs

Ba

Bt

Bs

Gene organisation for the encoding proteins of the exosporium for Bacillus anthracis, B. thuringiensis & B. subtilis

Solid red genes denote BLAST hits (e-14) to Pasteuria (dashed red e-4)

COMPARATIVE GENOMICS as of 2008

(Davies & Opperman, IOBC Bulletin, 2006, )

Page 32: Biological Control and Pasteuria penetrans: genomics and

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

Page 33: Biological Control and Pasteuria penetrans: genomics and

Prediction of Pasteuria filament length from the equation y = 0.1829x + 3.0747

GENOMICS & ENDOSPORE ATTACHMENT

Pasteuria Amino Filament

Contig G-x-ys upstream downstream acids length (nm)

Con 1_2 79 Yes Yes 430 81.7

Con 374 35 Yes Yes 221 43.5

Con 3_4 62 Yes Yes 260 50.6

y = 0.6005x - 26.252

R2 = 0.9998

y = 0.1829x + 3.0747

R2 = 0.9539

0

20

40

60

80

100

120

140

160

180

0 50 100 150 200 250 300 350

Number of amino acids in BclA

Nu

mb

er

of

Gxy r

ep

eats

0

10

20

30

40

50

60

70

co

llag

en

filam

en

t le

ng

th (

nm

)

(Davies & Opperman, IOBC Bulletin, 2006, )

Page 34: Biological Control and Pasteuria penetrans: genomics and

2 um

Spore attachment: Velcro-like mechanism

Davies, Advances in Parasitology In press.

Page 35: Biological Control and Pasteuria penetrans: genomics and

But what about

Specificity?

Spore attachment: Velcro-like mechanism

Davies, 2008, Advances in Parasitology in prep.

Page 36: Biological Control and Pasteuria penetrans: genomics and

Reproductive mechanisms in major groups of Root-knot nematodes Meloidogyne spp.

Nematode spp.

M. arenaria

M. incognita

M. javanica

M. hapla Race A

M. hapla Race B

Chromosome No.

30-46 polyploid

41-46; polyploid

42-48; polyploid

13-17; n

43-48; polyploid

Mode of reproduction

Mitotic parthenogenesis

Mitotic parthenogenesis

Mitotic parthenogenesis

Amphimixis & meiotic parth.

Mitotic parthenogenesis

After Evans, AAF (1998) In: The Physiology and Biochemistry of Free-

living and Plant-parasitic nematodes CABI (Eds RN Perry & DJ Wright)

Page 37: Biological Control and Pasteuria penetrans: genomics and

3 spore

8 single egg mass

populations

Tested against

PP3 & RES147

M. incognita

Single egg mass 02-08-04

Routinely subcultured

every 4-6 months

26 spore

8 single egg mass

populations

Tested against

PP3 & RES147

M. hapla VW8 & VW9

Single egg mass

18 generations of

single egg mass

VW8 single juveniles

2 single egg masses

from 2 single J2

populations

Tested against

PP3 & RES147

VW9 single juveniles

2 single egg masses

from 3 single J2

populations

Tested against

PP3 & RES147

Experimental design

Davies, Rowe & Williamson 2008 Int. J. Parasit.,38, 851 - 859

Page 38: Biological Control and Pasteuria penetrans: genomics and

Endospore attachment of Pasteuria populations RES147 & PP3 to Meloidogyne hapla strains VW8 & VW9 and M. incognita

VW8 VW9 Mi VW8 VW9 Mi

ANOVA P < 0.001

Davies, Rowe & Williamson 2008 Int. J. Parasit.,38, 851 - 859

Page 39: Biological Control and Pasteuria penetrans: genomics and

Spores, RES147 & PP3, attaching to single juvenile descent lines arising of M. hapla VW9K1 (light grey), VW9K2 (dark grey), VW9K3 (black)

ANOVA P < 0.001

Davies, Rowe & Williamson 2008 Int. J. Parasit.,38, 851 - 859

Segregation in spore attachment

Page 40: Biological Control and Pasteuria penetrans: genomics and

Endospore attachment of Pasteuria population PP3 to single juvenile descent lines of Meloidogyne incognita

ANOVA P < 0.001

Davies, Rowe & Williamson 2008 Int. J. Parasit.,38, 851 - 859

Somaclonal variation in spore attachment

Page 41: Biological Control and Pasteuria penetrans: genomics and

Endospore attachment of Pasteuria population RES147 to Meloidogyne incognita

ANOVA P < 0.001

Davies, Rowe & Williamson 2008 Int. J. Parasit.,38, 851 - 859

Somaclonal variation in spore attachment

Page 42: Biological Control and Pasteuria penetrans: genomics and

Relationship between juvenile age and spore attachment

Davies et al., 1991 Revue de Nematol 14, 616 – 618.

Page 43: Biological Control and Pasteuria penetrans: genomics and

Anne Millet and Jonathan Ewbank 2004 Curr. Opin. Immunol. 16, 4-9

DAF-2, IGF & innate immunity in worms

Page 44: Biological Control and Pasteuria penetrans: genomics and

An evolutionary conserved phosphorylation cascade involving the insulin/insulin-like growth factor (IGF) receptor is well characterised in C. elegans

Activation of phosphorylation pathway begins with binding of insulin-like ligand to DAF-2 receptor (38 present in Ce only a few characterised) DAF-2 activates AGE-1 which converts phosphatidylinositol biphosphate to a triphosphate PiP3 binds to the AKT-1/AKT2 complex that phosphorylates the Forkhead transcription factor DAF-16 DAF-16+P cannot be translocated to nucleus to activate DAF-2 pathways DAF-16 can enter nucleus L1 and L2 activates dauer formation L3 – adults activates stress response and anti-microbial genes (Millet & Ewbank, 2004)

Page 45: Biological Control and Pasteuria penetrans: genomics and

Alter lipid metabolism

Dauer formation

Alter fertility

Alter lifespan

Activate stress response

DAF-2 mutants can alter a number of important metabolic activities

Inhibition of DAF-2

Page 46: Biological Control and Pasteuria penetrans: genomics and

Alter lipid metabolism

Dauer formation

Alter fertility

Alter lifespan

Activate stress response

DAF-2 mutants can alter a number of important metabolic activities

Inhibition of DAF-2

Page 47: Biological Control and Pasteuria penetrans: genomics and

MCF-7 Cells

-40

-20

0

20

40

60

80

100

120

1.00E-12 1.00E-10 1.00E-08 1.00E-06

EPL001

(20 mer version)In

cre

ase

in

ce

ll n

um

be

r (%

)

EGF stimulated IGF-I stimulated

EPL001 is peptide that inhibits IGF:

72 hours after application EPL001 inhibits Epidermal Growth Factor (EGF) & Insulin Growth Factor (IGF) stimulated MCF-7 cells (breast carcinoma cell line)

John Haylor et al., in prep.

Page 48: Biological Control and Pasteuria penetrans: genomics and

Total larvae produced per day

0

20

40

60

80

100

120

140

160

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

days

Nu

mb

er

of

larv

ae

Control

EPL01

EPL030

Mean larvae per adult Control EPL001 EPL030 17 24 6 +43% -64% ANOVA P < 0.05

0

50

100

150

200

250

300

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Time days

Accu

mu

late

d l

arv

ae

Control

EPL001

EPL030

Davies and Hart (2008) Nematology 10, 103-112

Manipulation of life-span and fecundity in C. elegans

Page 49: Biological Control and Pasteuria penetrans: genomics and

The effects of EPL001 and EPL030 on the attachment of Pasteuria to root-knot J2s

•Meloidogyne incognita allowed to hatch in water •Treat with 1µM of EPL001 and EPL030 (water control) •At 0, 18, 21 and 27 hrs wash (x3) water •Endospore (strain RES147) attachment test by centrifugation •Count endospores adhering to the cuticle

Page 50: Biological Control and Pasteuria penetrans: genomics and

The effects of EPL001 and EPL030 on the attachment of Pasteuria to root-knot J2s

0

0.5

1

1.5

2

2.5

3

T0 T18 T21 T27

Sp

ore

s p

er J

2

ANOVA P < 0.001 WATER

EPL001

EPL030

Davies unpublished

Page 51: Biological Control and Pasteuria penetrans: genomics and

Endocuticle

Struts

Exocuticle

Epicuticle

Microvilli

Excretory/secretory products

DAF-2 modulation of E/S products

Modulation of surface coat through EPL001 and EPL030

Time line

Young Old

Davies unpublished

Page 52: Biological Control and Pasteuria penetrans: genomics and

Three types of cuticle variation

• Constituative variation: under genetic control and can segregate • Clonal vartiation: probable under epigenetic control • Induced variation: modulated by environmental parameters

Page 53: Biological Control and Pasteuria penetrans: genomics and

Acknowledgements ROTHAMSTED Sofia Costa Brian Kerry Rosa Manzanilla-Lopez Sharad Mohan Chris Rawlings Janet Rowe Shashi Sharma Andrew Warry

PASTEURIA BIOSCIENCES Tom Hewlett Susan Griswold Kelly Smith

Plant Nematode Genetics Group

North Carolina State University

NORTH CAROLINA S. U. David Bird Lauren Charles Charlie Opperman Jenn Schaff Betsy Scholl Jenora Waterman

U. C. DAVIS Valerie Williamson