Parasitic plant infection is partially controlled through symbiotic pathways

DOI: 10.1111/j.1365-3180.2009.00749.x

Parasitic plant infection is partially controlled through

symbiotic pathways1

M FERNANDEZ-APARICIO*, N RISPAIL�, E PRATS�, D MORANDI�,

J M GARCIA-GARRIDO*, E DUMAS-GAUDOT�, G DUC§ & D RUBIALES�*CSIC, Estacion Experimental del Zaidın, Granada, Spain, �CSIC, Institute for Sustainable Agriculture, Alameda Obispo s ⁄ n, Cordoba,

Spain, �UMR 1088 INRA ⁄CNRS 5184 ⁄UB, Plante-Microbe-Environnement, Dijon CEDEX, France, and §UMR 102, INRA, Genetique

et Ecophysiologie des Legumineuses, Dijon CEDEX, France

Received 15 December 2008

Revised version accepted 1 September 2009

Summary

Legumes are unique in interacting with Rhizobium,

arbuscular mycorrhizal (AM) fungi, and parasitic

plants. To dissect common parts of these three plant–

organism interactions, infection by Orobanche crenata

was studied in mutants with altered symbiotic pheno-

types of Medicago truncatula and Pisum sativum. Oro-

banche crenata inoculation of mutant lines carrying

defective mutation in the genes dmi2 ⁄ sym19 and dmi3

resulted in an increase in O. crenata establishment.

Similarly, inoculation of mutants carrying mutation in

the gene sunn ⁄ sym29 that controls the autoregulation

mechanism of the symbiosis, also lead to a significant

increase in haustoria formation. Altogether, our results

suggest that parasitic plant infection is partly controlled

by both the conserved symbiotic pathway that mediates

symbiont recognition and establishment and the auto-

regulation mechanism that regulates the extent of

colonisation by Rhizobium and AM fungi.

Keywords: Medicago truncatula, Pisum sativum, Oroban-

che crenata, bean broomrape, Rhizobium, mycorrhiza,

symbiosis, parasitism.

FERNANDEZ-APARICIO M, RISPAIL N, PRATS E, MORANDI D, GARCIA-GARRIDO JM, DUMAS-GAUDOT E, DUC G &

RUBIALES D (2009). Parasitic plant infection is partially controlled through symbiotic pathways. Weed Research.

Introduction

Plants are constantly interacting with organisms present

within their rhizosphere. The arbuscular mycorrhizal

(AM) fungi of the genus Glomeromycota establish

symbiotic relationships with most plant species from

mosses to dicotyledons concomitantly with the land

colonisation by terrestrial plants (Kistner & Parniske,

2002). More recently during evolution, bacteria from the

genus Rhizobium evolved the ability to induce the

formation of a specific root organ, the nodules, in a

more restricted number of plants, the Fabaceae. The

nodules fix atmospheric nitrogen in exchange for plant

carbohydrates (Kistner & Parniske, 2002). Host mutants

impaired in both nodulation and mycorrhization have

indicated that the signalling pathways controlling these

symbiotic processes are largely overlapping (Duc et al.,

1989; Sagan et al., 1995; Kistner & Parniske, 2002).

Apart for these beneficial interactions, plants are also

constantly challenged by pathogenic organisms, includ-

ing the parasitic plants of the Striga, Orobanche and

Phelipanche genera (Rubiales, 2003; Parker, 2009).

These parasites have lost their autotrophic way of life

during evolution and have evolved to parasitise most

monocotyledonous and dicotyledonous plants. As with

symbiotic interactions, a complex dialogue has to occur

for efficient parasitisation of the host (Bouwmeester

et al., 2003; Rispail et al., 2007; Fernandez-Aparicio

W R E 7 4 9 B Dispatch: 5.10.09 Journal: WRE CE: Aftab Begum

Journal Name Manuscript No. Author Received: No. of pages: 7 PE: Sathiyaseelan

Correspondence: M Fernandez-Aparicio, CSIC, Estacion Experimental del Zaidın, Profesor Albareda 1, 18008 Granada, Spain.

Tel: (+34) 958 181600 ext. 147; Fax: (+34) 957 499252; E-mail: [email protected]

� 2009 The Authors

Journal Compilation � 2009 European Weed Research Society Weed Research

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

Diego

Tachado

et al., 2008a). This includes: (i) the recognition of the

host by the symbiont ⁄parasite, leading to profound

metabolic and morphological changes that orient the

organism toward the host, (ii) attachment of the

symbiont ⁄parasite to the host root and simultaneous

perception of a putative symbiotic ⁄parasitic associated

signal by the host root and (iii) penetration and

establishment within the host root. In the case of

parasitic plants, perception by the parasite of exuded

host root stimulants triggers seed germination. Germi-

nation gives rise to the radicle that elongates toward the

host root scouting concentration gradient of the stim-

ulant and adheres to the host root through the forma-

tion of an appressorium. Then, the parasitic plant

penetrates through the cortex due to mechanical

pressure and enzyme activity, grows to the vascular

cylinder and develops the haustorium that acts as the

endophytic bridge through which all bidirectional trans-

fer between host and parasite is achieved. The outer part

of the seedling develops into a nodule that serves as

nutrient storage organ and gives rise to a flowering spike

that emerges from the soil (reviewed in Perez-De-Luque

et al., 2008).

Flavonoids and strigolactones exuded by the host are

known to mediate the early recognition step of nitrogen

fixing and mycorrhizal symbiosis, respectively (Becard

et al., 1992; Chabot et al., 1992; Akiyama et al., 2005;

Cooper, 2007). During coevolution of host and symbiont,

strigolactones were selected as a host location signal that

promotes branching of germinative hyphae of AM fungi.

Strigolactones are also a germination stimulant of Oro-

banche and Phelipanche seeds, suggesting that this more

recently evolved parasitic plant has hijacked this estab-

lished communication for its own purpose (Akiyama

et al., 2005; Akiyama & Hayashi, 2008). Since AM fungi

and Rhizobium share common genetic pathways, it is

tempting to speculate that these genetic pathways at least

partially overlap with that driving plant parasitism.

Legumes uniquely interact with Rhizobium, AM fungi

and parasitic plants. The model legume Medicago

truncatula Gaertn. (Barker et al., 1990) is a natural host

of the Rhizobium, Sinorhizobium meliloti, as well as

various AM fungal species including Glomus mosseae

and G. intraradices, and it can be infected by the

parasitic plants Orobanche crenata Forsk., Orobanche

foetida Poir., Phelipanche aegyptiaca (Pers.) Pomel (syn.

Orobanche aegyptiaca Pers.) and Phelipanche ramosa

(L.) Pomel (syn. Orobanche ramosa L.) (Fernandez-

Aparicio et al., 2008b). Pea (Pisum sativum L.) is an

important grain legume crop whose cultivation is

severely constrained by O. crenata infection (Perez-

De-Luque et al., 2005; Rubiales et al., 2009). Medicago

truncatula genes �doesn�t make infections� dmi1, dmi2

and dmi3 are required for both AM development and

nodulation with Sinorhizobium meliloti (Catoira et al.,

2000). Medicago truncatula gene dmi2 and its ortho-

logues in Lotus japonicus L. and pea, SYMRK and

sym19, respectively, encode a leucine-rich repeat-like

receptor kinase acting near a point of molecular

convergence of AM and legume-rhizobium signalling

(Endre et al., 2002; Stracke et al., 2002). This leucine-

rich repeat-like receptor kinase is also required for the

actinorhizal symbiosis involving Frankia bacteria

(Markmann et al., 2008) and facilitates root colonisa-

tion of the root-knot nematode Meloidogyne incognita

(Weerasinghe et al., 2005). dmi2 is located at the head of

a convergent pathway integrating all symbiotic signals

and is activated once the specific symbiotic signal is

perceived by its specific receptor. Upon activation, dmi2

induces a still poorly understood transduction pathway

that recruits the putative nuclear potassium channels

encoded by dmi1 (Ane et al., 2004) and its L. japonicus

and pea orthologues, CASTOR ⁄POLLUX (Imaizumi-

Anraku et al., 2005) and sym8, respectively (Imaizumi-

Anraku et al., 2005; Edwards et al., 2007). This

ultimately induces calcium spiking in the cell nucleus.

This spiking is sensed by the M. truncatula gene dmi3,

also known as CCaMK, which encodes a calcium ⁄ cal-

modulin dependent kinase and induces symbiotic gene

expression that finally leads to symbiont establishment

within the host root (Levy et al., 2004). On the other

hand, theM. truncatula sunn gene and its pea orthologue

sym29 (Levy et al., 2004) encode another leucine-rich

repeat receptor-like kinase that is required for the

symbiotic autoregulation system, which regulates the

extent of host colonisation by the symbiont (Schnabel

et al., 2005). In association with a supernodulation

phenotype, Morandi et al. (2000) observed more colo-

nisation by the mycorrhizal fungi Glomus mosseae of a

sym29 mutant of pea and the majority of sunn mutants

of M. truncatula, than their corresponding wild types,

suggesting common factors of regulation for both

symbioses. The objective of the present study was to

dissect common parts of these three plant–organism

interactions. We therefore monitored the susceptibility

to the plant parasite O. crenata of several M. truncatula

and pea mutants deficient for dmi2 ⁄ sym19, dmi3 and

sunn ⁄ sym29 genes.

Materials and methods

Plants and inoculation

The M. truncatula genotypes used were: the wild type

line J5, its non-nodulating and non-mycorrhizal mutants

TR89 (defective for dmi2 gene) (Sagan et al., 1995) and

TRV25-25 (defective for dmi3 gene) and its supernodu-

lated mutant TRV17 (defective for sunn4 gene). These

2 M Fernandez-Aparicio et al.



1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

Diego

Nota

orientate

mutants were selected after gamma ray irradiation and

described in Morandi et al. (2000, 2005) and Schnabel

et al. (2005). The pea genotypes used were: the wild type

Frisson, its non-nodulating and non-mycorrhizal

mutant P4 (defective for dmi2 orthologue), and its

supernodulated mutants P116 and P87 (defective for

sunn orthologue; Table 1). These mutants were selected

after EMS mutagenesis and described in Duc and

Messager (1989) and Krussel et al. (2002)2 .

Experiments were performed in mini-rhizotrons

(Fernandez-Aparicio et al., 2008a) in the absence of

rhizobia and mycorrhiza. Seeds ofM. truncatula and pea

were induced to germinate by means of scarification and

cold stratification for 48 h in Petri dishes. One 4-day-old

seedling was transplanted per square Petri dish filled

with sterile perlite and covered on the upper surface by a

sheet of glass-fibre filter paper (GFFP; Whatman

International Ltd, England3 ) (12 · 12 cm). Petri dishes

were previously punctured to allow development of host

seedling stems. The dishes were sealed with parafilm and

wrapped in aluminium foil and vertically stored for

15 days in a growth chamber (20 ± 2�C, 12 per 12 h

day ⁄night rhythm, 200 lmol m)2 s)1).

Orobanche crenata seeds were surface-sterilised by

immersion in 0.5% formalin with 1% Tween� for 2 h

and 20 min at 50�C, followed by three rinses in distilled

water. Seeds were air-dried before use. Fifty seeds per

square centimetre were sown on sheets of GFFP wetted

with sterile distilled water. Each sheet was placed on one

square Petri dish (12 · 12 cm). The Petri dishes were

sealed with parafilm and incubated in the dark for

11 days at 20 ± 2�C to promote necessary warm

stratification of seeds to germinate.

Then, the O. crenata seeds were evenly distributed at

a density of 50 seeds cm)2 on the GFFP on which the

plants were developing its roots. Medicago truncatula

accessions are known to induce very low percentages of

germination of O. crenata seeds (Fernandez-Aparicio

et al., 2008b). Thus, the synthetic germination stimulant

GR24 (provided by Dr Zwanenburg, University of

Nijmegen, the Netherlands) was added simultaneously

with O. crenata seed distribution at a concentration of

10 mg L)1 to increase the number of germinated para-

sites and the chance of starting the parasitic process. The

experiment consisted on six replications per accession.

Plants were irrigated using a Hoagland�s nutrient

solution (Hoagland & Arnon, 1950) twice per week.

Parasitic plant development monitoring

After 35 days of plant growth in Petri dishes, germi-

nated O. crenata seeds that were close (<3 mm) to the

host roots were observed under a stereoscopic micro-

scope at · 30 magnification. The percentage of radicle

contact was determined as the number of O. crenata

germlings that established contact with host root

surface. The percentage of established haustoria and

total number of O. crenata nodules per host plant were

then recorded five and ten days later, respectively.

Statistical analysis

Assays were developed using a completely randomised

design.Data recorded as percentages were transformed to

arcsine square roots (transformed value = 180 ⁄P ·

arcsine [�(% ⁄ 100)]) to normalise data and stabilise

variances throughout the data range, and subjected to

analysis of variance using GenStat 7.04 , after which

residual plots were inspected to confirm data conformed

to normality. For comparison between a specific mutant

and thewild type, a contrast analysis (Scheffe�s F) for each

parameter was performed.

Results and discussion

The DMI ( �doesn�t make infections�) pathway controls

O. crenata establishment

None of the dmi2 and dmi3 deficient mutants of

M. truncatula tested had a different percentage of

Table 1 Phenotypes and mutations of the different M. truncatula and pea genotypes used in this study

Species Genotype Mutated gene Gene function

Phenotype

Nod Myc Orobanche

M. truncatula J5 (wild type) ) ) + + +

M. truncatula TR89 Mtsym2 (dmi2) LRR receptor kinase ) ⁄+ ) ⁄+ ++

M. truncatula TRV25-25 Mtsyml3 (dmi3) CcaM kinase ) ) ++

M. truncatula TRV17 Mtsyml2 (sunn4) CLVl-like kinase +++ ++ +++

P. sativum Frisson (wild type) ) ) + + +

P. sativum P4 sym19 (orthologue for dmi2) LRR receptor kinase ) ⁄+ ) ⁄+ ++

P. sativum P87 sym29 (orthologue for sunn) CLVl-like kinase +++ +++ +++

P. sativum P116 sym29 (orthologue for sunn) CLVl-like kinase +++ +++ +++

+, normal phenotype; ), completely resistant phenotype; ) ⁄+, leaky resistant phenotype with rare infection events; +++,

hypersusceptible phenotype.

Symbiotic pathways control parasitic plants 3



1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

germinated O. crenata seeds that contacted with the host

roots compared with the wild type (Fig. 1A). Surpris-

ingly, mutations in the pea dmi2 orthologues, sym19,

increased the number of germinated O. crenata seeds

contacting the host roots having a synergistic effect on

GR24 activity (Fig. 2A). Further studies are needed to

clarify effects of sym19 mutation on root exudation. The

specialisation on host recognition is mediated in each

parasitic plant–host plant interaction by unique combi-

nations between the kind and amount of such stimulants

exuded by each host plant (Bouwmeester et al., 2003;

Fernandez-Aparicio et al., 2009). Gradients in concen-

tration might act as chemo-attractants towards host

roots (Whitney & Carsten, 1981). Specific mixtures of

different stimulants at specific combinations in each

plant species could have differential stimulatory activity.

On the other hand, infection of M. truncatula TR89

(defective for dmi2 gene) and TRV25-25 (for dmi3 gene)

Medicago truncatula genotypes

Rad

icu

le c

on

tact

(%)

Esta

blish

ed

hau

sto

ria (

%)

No

du

le n

um

ber

0

10

20

30

40

50

60

0

5

10

15

20

25

A

B

WT

TR89

TRV25-2

5

TRV17

0

10

20

30

40

50

*

C

***

***

A

B

*

C***

***

*

C

Fig. 1 Assessment of susceptibility ofMedicago truncatula

genotypes to Orobanche crenata. (A) Percentage of germinated

O. crenata seeds that contacted host root, (B) percentage of

contactingO. crenata that form an efficient haustorium and (C) total

number of mature parasite nodules. Vertical bars represent standard

error for n = 6. Stars indicate statistically significant differences

between the wild type and the mutant at the level of P £ 0.05,

P £ 0.01 and P £ 0.001, as determined by contrast analysis.

Pisum sativum genotypes

WT P

4

P116

P87

0

10

20

30

40

50

0

10

20

30

40

Rad

icu

le c

on

tact

(%)

Esta

blish

ed

hau

sto

ria (

%)

A

B

0

20

40

60

80

100

120

140

160

180

No

du

le n

um

ber

C

**

***

*

***

*

Fig. 2 Assessment of susceptibility of Pisum sativum genotypes

to Orobanche crenata. (A) Percentage of germinated O. crenata

seeds that contacted host root, (B) percentage of contacting

O. crenata that form an efficient haustorium and (C) total number

of mature parasite nodules. Vertical bars represent standard

error for n = 6. Stars indicate statistically significant differences

between the wild type and the mutant at the level of P £ 0.05,

P £ 0.01 and P £ 0.001, as determined by contrast analysis.




1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

Diego

Nota

not in italics

mutants led to a highly significant increase in the parasitic

nodule establishment, compared with wild type infection.

This was both in terms of haustoria formation, estimated

as the number of established parasites from 100 parasitic

individuals, which were in contact with host root

(P < 0.05 and P < 0.01, respectively; Fig. 1B), and

total number of O. crenata nodules per plant (P < 0.05

and P < 0.001, respectively; Fig. 1C). Similarly, pea P4

(defective for sym19) showed a significant increase in the

level of parasitic nodule establishment in terms of

haustoria formation and nodule number per plants

(P < 0.05 and P < 0.001; Fig. 2B,C). These data sug-

gest that mutants in these two genes are more susceptible

to the parasite infection than the wild type and that these

two genes may play an opposite role in response to

symbionts or to parasitic plants. The DMI pathway is

thus suggested to play a role as a negative regulator of

plant infection in legumes. This hypothesis is confirmed

by a recent study showing that L. japonicusNod) mutant

castor-5 (homologous to M. truncatula dmi1), but not

Ccamk-3 (homologous toM. truncatula dmi3), was more

susceptible to P. aegyptiaca than the wild type (Kubo

et al., 2009). This pathway has so far been shown to be

required for the establishment of several beneficial

symbionts within the host roots, including the nitrogen-

fixing bacteria Rhizobium, the cyanobacteria Frankia and

the AM fungi (Endre et al., 2002; Stracke et al., 2002;

Markmann et al., 2008). In addition, the L. japonicus

SYMRK have been shown to facilitate root colonisation

of the root-knot nematode Meloidogyne incognita in a

Nod factor signalling-like manner (Weerasinghe et al.,

2005). The dmi2 ⁄ symRK mutants have been shown to

have a higher sensitivity to touch (Esseling et al., 2004)

and an aberrant defence reaction during symbiont

establishment (Limpens et al., 2005). Thus, this receptor

in legumes has been recently described as a �guard� that

converts basal defence reactions to benefit microbial

accommodation (Holster, 2008). The negative role of

dmi2 and dmi3 on O. crenata infection detected here and

the high conservation of dmi2 in plants found even in

species that do not form symbiotic interactions, lead us to

speculate a much more general function of the DMI

pathways and in particular of DMI2 ⁄SYMRK during

plant root–organism interaction. Indeed, it may be

possible that DMI2 and its subsequent signalling path-

ways is the integrator of the secondary signals generated

by the recognition of interacting organisms, allowing

switching basal defence according to the nature of the

interacting organism. The extreme conservation of

DMI2 ⁄SYMRK in all plant species, including species

that do not form symbioses such as Arabidopsis thaliana

(L.) Heynh. (Markmann et al., 2008), favours this

more general function of this receptor as a basal defence

switch.

The autoregulation mechanism control the extent

of O. crenata infection

The extent of host root colonisation by its symbiont is

permanently and strictly controlled by the host to

balance the resulting carbohydrate cost with the bene-

ficial effect of the symbiont (Fig. 3). This regulatory

mechanism of symbiotic invasion, the autoregulatory

mechanism, is controlled by a still unknown shoot-

derived signal and is mediated by a leucine-rich repeat

receptor-like kinase encoded by the orthologue of the

A. thaliana clv1 gene, called sunn in M. truncatula and

har1 in L. japonicus. Sunn. har1 deficient mutants show a

hypernodulant and hypermycorrhization phenotype

(Table 1) (Nishimura et al., 2002; Morandi et al., 2005;

Schnabel et al., 2005). The TRV17 mutant harbouring

the sunn4 mutation that leads to a dead allele of the

SUNN protein was used to determine whether this

symbiotic autoregulation system also regulates the

response to parasitic plants (Morandi et al., 2000). This

mutant showed more haustoria (P < 0.001) and nodule

(P < 0.001) formation, with around four fold more

healthy O. crenata nodules observed on the mutant

roots, indicating that this mutant has also a hyperpar-

asitisation phenotype (Fig. 1B,C, Table 1). Interest-

ingly, a separate study of the TRV17–O. foetida

interaction also showed a higher level of haustoria and

nodule formation (data not shown). In addition, the

analysis of two sym29 pea mutant lines, P116 and P87,

carrying a mutation in the sunn orthologue had signif-

icantly more haustoria formation (P < 0.01 and

P < 0.001, respectively), although this did not lead to

a higher number of mature nodules, probably due to

early abortion of the infection events (Fig. 2B,C).

Similarly, har1 deficient mutant of L. japonicus was also

more infected by P. aegyptiaca (Kubo et al., 2009) and

by the root-knot nematode M. incognita (Lohar & Bird,

2003). Thus, the autoregulation mechanism seems to be

a general pathway to control the extent of �parasitisa-

tion� performed by the plant symbionts or real parasites.

In this more general context, this autoregulation mech-

anism would be a second level of defence acting once the

basal defence mechanism have been overwhelmed or

specifically switched off (Fig. 3).

Conclusion

In the present study, we showed that two different

pathways initially described for the symbiotic and the

closely related root-knot nematode interaction are also

involved in response to parasitic plant infection, as

already suggested by Kubo et al. (2009) in L. japonicus.

We determined that the DMI2 and DMI3, two main

components of the DMI pathway, play a negative




1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

function during O. crenata infection in the legume

species M. truncatula and pea by limiting the number of

infection events. This fact, together with the extreme

conservation and structural complexity of the DMI2

protein in plant species, favours a more complex role of

this pathway as a general switch of basal defence than

initially thought. In this sense, our findings open new

perspectives into the highly complex function of this

pathway in plants and bring preliminary evidence

explaining the diversity of the DMI2 protein. We also

extended the recent results of Kubo et al. (2009) by

showing that the autoregulation mechanism limiting

host root colonisation by the symbiont to below a

detrimental level is part of the defence mechanism

against parasitic plants, not only in L. japonicus but also

in M. truncatula and pea. These findings indicate that

this autoregulatory mechanism would be a second

general defence mechanism acting after the basal defence

mechanism.

Acknowledgement

This research was supported by projects AGL2008-

01239 ⁄AGR and P07-AGR-02883.

References

AKIYAMA K & HAYASHI H (2008) Plastid-derived strigolactones

show the way to roots for symbionts and parasites. New

Phytologist 178, 695–698.

AKIYAMA K, MATSUZAKI K & HAYASHI H (2005) Plant

sesquiterpenes induce hyphal branching in arbuscular

mycorrhizal fungi. Nature 435, 824–827.

ANE JM, KISS GB, RIELY BK et al. (2004) Medicago truncatula

DMI1 required for bacterial and fungal symbioses in

legumes. Science 303, 1364–1367.

BARKER DB, BIANCHI S, BLONDON F et al. (1990) Medicago

truncatula, a model plant for studying the molecular genetics

of the Rhizobium-legume symbiosis. Plant Molecular Biol-

ogy Reporter 8, 40–49.

BECARD G, DOUDS DD & PFEFFER PE (1992) Extensive in vitro

hyphal growth of vesicular-arbuscular mycorrhizal fungi

in the presence of CO2 and flavonoids. Applied and

Environmental Microbiology 68, 1260–1264.

BOUWMEESTER HJ, MATUSOVA R, ZHONGKUI S & BEALE ?????

(2003) Secondary metabolite signalling in host-parasitic

plant interactions. Current Opinion in Plant Biology 6,

358–364.5

CATOIRA R, GALERA C, DE BILLY F et al. (2000) Four genes

of Medicago truncatula controlling components of a

nod factor transduction pathway. The Plant Cell 12, 1647–

1665.

CHABOT S, BEL-RHLID R, CHENEVERT R & PICHE Y (1992)

Hyphal growth promotion in vitro of the VA mycorrhizal

fungus, Gigaspora margarita Becker & Hall, by the activity

of structurally specific flavonoids compounds under

CO2-enriched conditions. New Phytologist 122, 461–467.

COOPER JE (2007) Early interactions between legumes and

rhizobia: disclosing complexity in a molecular dialogue.

Journal of Applied Microbiology 103, 1355–1365.

DUC G & MESSAGER A (1989) Mutagenesis of pea (Pisum

sativum L.) and the isolation of nodulation and nitrogen

fixation mutants. Plant Science 60, 207–213.

Fig. 3 Schematic representation integrating the genetic control

of symbiotic and parasitic interactions.




1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

DUC G, TROUVELOT A, GIANINAZZI-PEARSON V & GIANINAZZI S

(1989) A first report of non mycorrhizal plant mutants

(Myc)) obtained in pea. Plant Science 60, 215–222.

EDWARDS A, HECKMANN A, YOUSAFZAI F, DUC G & DOWNIE

A (2007) Structural implications of mutations in the pea

SYM8 symbiosis gene, the DMI1 orthologue, encoding a

predicted ion channel. Molecular Plant-Microbe interactions

20, 1183–1191.

ENDRE G, KERESZT A, KEVEI Z et al. (2002) A receptor

kinase gene regulating symbiotic nodule development.

Nature 417, 962–966.

ESSELING JJ, LHUISSIER FGP & EMONS AMC (2004) A

nonsymbiotic root hair tip growth phenotype in NORK

mutated legumes: implications for nodulation factor induced

signalling and formation of a multifaceted root hair pocket

for bacteria. The Plant Cell 16, 933–944.

FERNANDEZ-APARICIO M, ANDOLFI A, EVIDENTE A, PEREZ-

DE-LUQUE A & RUBIALES D (2008a) Fenugreek root

exudates show species-specific stimulation of Orobanche seed

germination. Weed Research 48, 163–168.

FERNANDEZ-APARICIO M, PEREZ-DE-LUQUE A, PRATS E &

RUBIALES D (2008b) Infection of barrel medic (Medicago

truncatula) by Orobanche species. Annals of Applied Biology

153, 117–126.

FERNANDEZ-APARICIO M, FLORES F & RUBIALES D (2009)

Recognition of root exudates by seeds of broomrape

(Orobanche and Phelipanche) species. Annals of Botany

103, 423–431.

HOAGLAND DR & ARNON DI (1950) The water culture method

for growing plant without soil. California Agricultural

Experimental Station Circular 1, 347.

HOLSTER M (2008) SYMRK, an enigmatic receptor guarding

and guiding microbial endosymbioses with plant roots.

Proceedings of the National Academy of Science, USA

105, 4537–4538.

IMAIZUMI-ANRAKU H, TAKEDA N, CHARPENTIER M et al.

(2005) Plastid proteins crucial for symbiotic fungal and

bacterial entry into plant roots. Nature 433, 527–531.

KISTNER C & PARNISKE M (2002) Evolution of signal

transduction in intracellular symbiosis. Trends in Plant

Science 7, 511–518.

KUBO M, UEDA H, PARK P, KAWAGUCHI M & SUGIMOTO Y

(2009) Reactions of Lotus japonicus ecotypes and mutants

to root parasitic plants. Journal of Plant Physiology 166,

353–362.

LEVY J, BRES C, GEURTS R et al. (2004) A putative Ca2+

and calmodulin-dependent protein kinase required for

bacterial and fungal symbioses. Science 303, 1361–1364.

LIMPENS E,MIRABELLA R, FEDOROVA E et al. (2005) Formation

of organelle-like N2-fixing symbiosomes in legume root

nodules is controlled by DMI2. Proceedings of the National

Academy of Science, USA 102, 10375–10380.

LOHAR DP & BIRD DM (2003) Lotus japonicus: a new model

to study root-parasitic nematodes. Plant and Cell Physiology

44, 1176–1184.

MARKMANN K, GICZEY G & PARNISKE M (2008) Functional

adaptation of a plant receptor-kinase paved the way for

the evolution of intracellular root symbiosis with bacteria.

Plos Biology 6, e68.

MORANDI D, SAGAN M, PRADOVIVANT E & DUC G (2000)

Influence of genes determining supernodulation on root

colonization by the mycorrhizal fungus Glomus mosseae

in Pisum sativum and Medicago truncatula mutants.

Mycorrhiza 10, 37–42.

MORANDI D, PRADO E, SAGAN M & DUC G (2005) Charac-

terisation of new symbiotic Medicago truncatula (Gaertn.)

mutants, and phenotypic or genotypic complementary

information on previously described mutants. Mycorrhiza

15, 283–289.

NISHIMURA R, HAYASHI M, WU GJ et al. (2002) HAR1

mediates systemic regulation of symbiotic organ develop-

ment. Nature 420, 426–429.

PARKER C (2009) Observations on the current status of

Orobanche and Striga problems worldwide. Pest Manage-

ment Science 65, 453–459.

PEREZ-DE-LUQUE A, JORRIN J, CUBERO JI & RUBIALES D

(2005) Resistance and avoidance against Orobanche crenata

in pea (Pisum spp.) operate at different developmental stages

of the parasite. Weed Research 45, 379–387.

PEREZ-DE-LUQUE A, MORENO MT & RUBIALES D (2008)

Host plant resistance against broomrapes (Orobanche spp.):

defence reactions and mechanisms of resistance. Annals

of Applied Biology 152, 131–141.

RISPAIL N, DITA M-A, GONZALEZ-VERDEJO C et al. (2007)

Plant resistance to parasitic plants: molecular approaches

to an old foe. New Phytologist 173, 703–712.

RUBIALES D (2003) Parasitic plants, wild relatives and the

nature of resistance. New Phytologist 160, 459–461.

RUBIALES D, FERNANDEZ-APARICIO M, PEREZ-DE-LUQUE A

et al. (2009) Breeding approaches for crenate broomrape

(Orobanche crenata Forsk.) management in pea (Pisum

sativum L.). Pest Management Science 65, 553–559.

SAGAN M, MORANDI D, TARENGHI E & DUC G (1995) Selection

of nodulation and mycorrhizal mutants in the model plant

Medicago truncatula (Gaertn.) after gamma-ray mutagene-

sis. Plant Science 111, 63–71.

SCHNABEL E, JOURNET EP, DE CARVALHO-NIEBEL F, DUC G

& FRUGOLI J (2005) The Medicago truncatula SUNN gene

encodes a CLV1-like leucine-rich repeat receptor kinase

that regulates nodule number and root length. Plant

Molecular Biology 58, 809–822.

STRACKE S, KISTNER C, YOSHIDA S et al. (2002) A plant

receptor-like kinase required for both bacterial and fungal

symbiosis. Nature 417, 959–962.

WEERASINGHE RR, BIRD DM & ALLEN NS (2005) Root-knot

nematodes and bacterial Nod factors elicit common signal

transduction events in Lotus japonicus. Proceedings of the

National Academy of Science, USA 102, 3147–3152.

WHITNEY PJ & CARSTEN C (1981) Chemotropic response

of broomrape radicles to host root exudates. Annals of

Botany 48, 919–921.




1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

Author Query Form

Journal: WRE

Article: 749

Dear Author,

During the copy-editing of your paper, the following queries arose. Please respond to these by marking up your

proofs with the necessary changes/additions. Please write your answers on the query sheet if there is insufficient

space on the page proofs. Please write clearly and follow the conventions shown on the attached corrections

sheet. If returning the proof by fax do not write too close to the paper’s edge. Please remember that illegible

mark-ups may delay publication.

Many thanks for your assistance.

Query

reference

Query Remarks

Q1 AUTHOR: Please check that all the information displayed in your figures

and table are displayed correctly and that they appear in the correct order.

Q2 AUTHOR: Krussel et al. (2002) has not been included in the Reference

List, please supply full publication details.

Q3 AUTHOR: Please give address information for Whatman International:

town, state (if applicable), and country.

Q4 AUTHOR: Please give manufacturer information for GenStat 7.0:

company name, town, state (if USA), and country.

Q5 AUTHOR: Please provide the forenames ⁄ initials for the author Beale for

reference Bouwmeester et al. (2003).

MARKED PROOF

Please correct and return this set

Instruction to printer

Leave unchanged under matter to remain

through single character, rule or underline

New matter followed by

or

or

or

or

or

or

or

or

or

and/or

and/or

e.g.

e.g.

under character

over character

new character

new characters

through all characters to be deleted

through letter or

through characters

under matter to be changed





Encircle matter to be changed

(As above)

(As above)

(As above)

(As above)

(As above)

(As above)

(As above)

(As above)

linking characters

through character or

where required

between characters or

words affected

through character or

where required

or

indicated in the margin

Delete

Substitute character or

substitute part of one or

more word(s)Change to italics

Change to capitals

Change to small capitals

Change to bold type

Change to bold italic

Change to lower case

Change italic to upright type

Change bold to non-bold type

Insert ‘superior’ character

Insert ‘inferior’ character

Insert full stop

Insert comma

Insert single quotation marks

Insert double quotation marks

Insert hyphen

Start new paragraph

No new paragraph

Transpose

Close up

Insert or substitute space

between characters or words

Reduce space betweencharacters or words

Insert in text the matter

Textual mark Marginal mark

Please use the proof correction marks shown below for all alterations and corrections. If you

in dark ink and are made well within the page margins.

wish to return your proof by fax you should ensure that all amendments are written clearly

Documents

Parasitic plant infection is partially controlled through symbiotic pathways