advances in siderophores: plant pathogen interaction

1. Welcome
1

2. Advances in siderophores: Plant
pathogen interaction
SEMINAR-I
Sundaresh
UHS13PGM396
Plant pathology
2

3. Introduction
• Biological control of plant pathogens has been the subject
of much research in recent years.
• It can potentially help us limit the use of chemical
pesticides that are harmful to the environment.
• The use of plant growth-promoting rhizobacteria (PGPR),
such as siderophore-producing bacteria, represents a
potentially attractive alternative disease management
approach, since they have the capacity to increase yield
and protect crops simultaneously.
• Few organisms like Pseudomonas fluorescens, P. putida are
a special group of organisms which are widely used as bio
control agents.
3

4. Siderophore producing organism
• Azotobacter
• Pseudomonas
• Bacillus
• Streptomyces
Among these microbes Pseudomonas species
is the active siderophore producer.
4

5. Metals used by organisms
5

6. Importance of iron in microbial
metabolism
• Iron is a cofactor for essential cellular
processes in nearly all microorganisms.
• growth-limiting nutrient because of the low
solubility of ferric iron under aerobic
conditions
6

7. Siderophores
• The special low molecular weight , iron
chelating structures produced by bacteria
under Iron restricted condition.
7

8. History of Sidirophores
• The role of microbial siderophores in virulence to
plant hosts was first demonstrated for the bacterial
pathogen Erwinia chysanthemi which produces
the catecholate chrysobactin and the carboxylate
achromobactin
• Erwinia amylovora synthesizes the hydroxamate
desferrioxamine and mutants defective in
desferrioxamine biosynthesis show tissue-specific
reduced virulence
8

9. Role of siderophores
 High affinity system of Fe3+ acquisition, utilization
and storage.
 Sometimes, required for virulence.
 Helps in growth, colonization and asexual
sporulation.
 Elicit the plant defense through an antagonism
mechanism between SA and JA signaling cascades.
9

10. MECHANISM OF IRON ACQUISTION
BACTERIA
FUNGI
Reduction of Fe(III) to Fe(II)
Direct acquisition
By iron binding proteins
ferric siderophores
Siderophore-mediated Fe3+ uptake
RIA (reductive iron assimilation)
heme uptake
direct Fe2+ uptake
10

11. Mechanism of siderophore mediated iron uptake
inbacteria
(Syed Sajeed Ali, 2013) 11

12. Postulated fungal siderophore biosynthetic pathway
NRPS (Nonribosomal Peptide Synthetase) : Large multifunctional
enzymes that synthesize peptides from proteinogenic and
nonproteinogenic precursors independently of the ribosome. 12

13. Detection of siderophore production
• widely used method for detection of
siderophore production by microorganisms in
solid medium is the universal chrome azurol S
(CAS)-agar plate assay.
13

14. 14

15. Types of siderophores
• Hydroxamate,
• Catecholate and
• Carboxylate
15

16. Hydroxamate
• Hydroxamate group-bearing siderophores are mainly
synthesized by fungi and Gram-positive filament-
forming bacteria (streptomycetes).
• In fungal systems the hydroxamic acid chelating
group is commonly derived from acylated Nδ-acyl-
Nδ-hydroxy-L-ornithine.
16

17. Catecholate
• Each catecholate group provides two oxygen atoms for
chelation with iron so that a hexadentate octahedral complex is
formed as in the case of the hydroxamate siderophores. Linear
catecholate siderophore are also produced in certain species.
• Agrobactin and parabactin are produced by Agrobacterium
tumefaciens and Paracoccus denitrificans respectively.
17

18. Carboxylate
• The best characterized carboxylate type siderophore with a
novel structure is rhizobactin.
• Rhizobactin is produced by Rhizobium meliloti strain DM4
and is an amino poly (carboxylic acid) with
ethylenediaminedicarboxyl and hydroxycarboxyl moieties as
ironchelating groups.
• Staphyloferrin A, produced by Staphylococcus hyicus
DSM20459, is another member of this class of complexon
siderophores.
18

19. Siderophore structure in bacteria
19

20.  All fungal siderophores identified so far are hydroxamates
 Fungal hydroxamates are derived from the
nonproteinogenic amino acid ornithine and different acyl
groups,
 grouped into four structural families
(I) Rhodotorulic acid
(ii) Fusarinines
(iii) Coprogens
(iv) Ferrichromes
SIDEROPHORES
IN PLANT PATHOGENIC FUNGI
20

21. Representative fungal siderophores, Peptide and ester bonds separating
N5 – acyl- N5- hydroxyornithine 21

22. Characterized Pathogenic Fungal Siderophore NRPS
NRPS name Fungal species Siderophores Reference
Ferrichrome NRPS
Sid2 U. maydis Ferrichrome Yuan et al., 2001
Nps2 C. heterostrophus Ferricrocin Oide et al., 2007
Nps2 F. graminearum Ferricrocin Oide et al., 2007
Ssm1 M. grisea Ferricrocin Hof et al., 2007
SidFA/Fer3 U. maydis Ferrichrome A Eichhorn et al., 2006
Coprogen NRPS
Nps6 A.brassicicola N-dimethylcoprogen Oide et al., 2006
Nps6 C.heterostrophus Coprogen Oide et al., 2006
Nps6 C.miyabeanus nd Oide et al., 2006
Fusarinine NRPS
Nps6 F.graminearum Fusarinine C Oide et al., 2006
22

23. Mechanism of siderophores in bio
control of plant pathogens
• Siderophores produced by a microorganism can bind iron with
high specificity and affinity, making the iron unavailable for
other microorganisms; thereby limiting their growth.
• Competition for iron by siderophore production is an
important antagonistic trait found in many of the bacterial bio
control agents against plant pathogens.
• Microbial siderophores may stimulate plant growth directly by
increasing the availability of iron in the soil surrounding the
roots or indirectly by competitively inhibiting the growth of
plant pathogens with less efficient iron-uptake systems.
23

24. Bio control potential of Pseudomonas
fluorescens against coleus root rot disease
(Vanitha and Ramjegathesh, 2014)
24
Case study- 1

25. Materials and Methods
• Isolation of pathogen and Pseudomonas
strains
• Siderophores production assay
• Screening of antagonistic bacteria under in
vitro condition
• Preparation of talc-based formulation of bio
control agents
• Greenhouse studies
25

26. Table: Antibiotics, Siderophores and HCN
production of P. fluoresens strains
Sl. no PGPR
strains
Fluorescein Pyocyanin Siderophore
production
HCN
1 Pf1 + + + +++
2 CPF1 + + + +++
3 CPF2 - - + +
4 CPF3 - - + -
5 CPF4 + + + +
6 CPF5 + + + +
7 CPF6 + + - ++
8 CPF7 - - + -
9 CPF8 + + + ++
10 CPF9 + + + +
11 CPF10 - - - +
+ = Produced; - =Not produced
+++ =Strong; ++ =Medium; + =Low production 26

27. 27

28. 28

29. Outcome
• Siderophore-mediated and antibiotic-
mediated suppression of soil borne plant
diseases are the two most employed
mechanisms involved in biocontrol
mechanism of Pf1.
29

30. Isolation of Siderophore producing bacteria
from rhizosphere soil and their antagonistic
activity against selected fungal plant pathogens
(Jenifer et al., 2013)
30
Case study- 2

31. 31

32. The siderophore-producing bacterium,
Bacillus subtilis CAS15, has a biocontrol effect
on Fusarium wilt and promotes the growth of
pepper (Capsicum annuum L.)
(Xianmei Yu et al., 2011)
32
Case study- 3

33. Table: Effect of CAS15 on spore germination of
Fusarium oxysporum f. spp. capsici
Concentration of cell suspension ( cfu /ml)
0 103 104 105 106 107 108
Germinated
spores
50 46 43.7 35 29.3 20 18
Germination
percentage (%)
100 a 92 ab 87.3 70 c 58.6 d 40 e 36 e
Reduction (%) - 8 12.7 30 41.4 60 64
33
Values in a row followed by the same letter are not significantly different (P < < 0.05)
according to Duncan’s multiple range tests.

34. Table: Suppression of Fusarium wilt of pepper (Capsicum
annuum L.) in potting soil by B. subtilis CAS15
34
Treatments - Fe + Fe
Disease
incidence (%)
Disease
severity (%)
Disease
incidence
(%)
Disease
severity (%)
B. subtilis 40 1.03 56 1.54
Control 72 2.01 64 1.82
Disease severity was assessed based on a 0-5 scale. (0-no symptoms, 5-plant dead)

35. Table: Plant height of pepper in the pot culture test after
planting.
Treatments Plant height (cm)
7 days 14days 21 days 28 days 40 days
B. subtilis 6.62 a 14.20 a 31.17 a 43.72 a 59.20 a
Control 5.73 a 11.16 b 20.17 b 28.32 b 39.25 a
Increase
percent (%)
15.53 27.24 54.53 54.38 50.83
35
Mean follows by a common letter in the same column are not significantly different at
P = 0.01.

36. 36
Microbial siderophores exert a subtle role in Arabidopsis during infection
by manipulating the immune response and the iron status
(Dellagi et al., 2009)
 E. chrysanthemi is an enterobacterium causing soft rot disease
Under iron deficiency, E. chrysanthemi releases two siderophores:
1) hydroxycarboxylate achromobactin ( iron limiting condition)
2) catecholate chrysobactin (severe iron deficiency)
The role of CB is characterized by Arabidopsis- E. chrysanthemi pathosystem.
Roles:
Elicit SA-mediated signaling pathway.
Modulate plant defenses through an antagonistic mechanism between SA and
jasmonic acid signaling cascades.
Promote bacterial growth in plant.
Case study- 4

37. PR1 gene expression and SA production in Arabidopsis
leaves following CB treatment
Cont.
37

38. Conti…..
Findings:
 24 h post infiltration (hpi), CB strongly activates the expression of the SA
marker gene PR1.
 No significant modification in the expression of ET/JA marker gene PDF1.2.
 The intensity of GUS staining in leaves treated with CB was similar to that
observed in SA-treated leaves, used as positive controls.
 The activation of PR1 expression is correlated with an accumulation of SA, was
measured by HPLC in Arabidopsis leaves 24 h after CB treatment.
 The siderophore treatment resulted in a 2- to 3-fold increase in SA content 24
hpi compared with control leaves.
Conclusion:
 CB activates a signaling pathway leading to PR1 up-regulation that is
dependent on SA production via ICS1/SID2 pathway.
38

39. Effects of CB on the expression of PR1 and PDF1.2 genes during E.
chrysanthemi infection
Cont.
39

40. Findings:
• The PR1 gene was strongly up-regulated by the wildtype bacteria
compared with the control plants.
• Infection by the siderophore-deficient mutant resulted in reduced
expression of PR1 gene.
• Expression of PDF1.2 that is not activated by wild-type bacteria 24 h
after infiltration, was strongly up-regulated in response to the
siderophore-deficient mutant.
• Preinfiltration of CB stimulates bacterial growth.
• In the control leaves preinfiltrated with water, E. chrysanthemi grew
by less than 1 order of magnitude.
Conclusion:
o CB represses the expression of PDF1.2. [ JA/ET Pathway(ISR)]
o CB activates the expression of PR1 [SA Pathway]
o But due to higher accumulation and amplification of SA , the wild
type bacteria take the advantage of antagonism between SA and
JA/ET pathway promoting its own growth.
Cont.
40

41. NPS6, encoding a non ribosomal peptide
synthetase involved in siderophore mediated
iron metabolism, is a conserved virulence
determinant of plant pathogenic ascomycetes
(Oide et al.,2006)
41
Case study- 5

42. Characterized Pathogenic Fungal Siderophore NRPS
NRPS name Fungal species Siderophores Reference
Ferrichrome
NRPS
Sid2 U. maydis Ferrichrome Yuan et al., 2OO1
Nps2 C. heterostrophus Ferricrocin Oide et al., 2007
Nps2 F. graminearum Ferricrocin Oide et al., 2007
Ssm1 M. grisea Ferricrocin Hof et al., 2007
SidFA/Fer3 U. maydis Ferrichrome A Eichhorn et al.,
2006
Coprogen NRPS
Nps6 A.brassicicola N-dimethylcoprogen Oide et al., 2006
Nps6 C.heterostrophus Coprogen Oide et al., 2006
Nps6 C.miyabeanus nd Oide et al., 2006
Fusarinine NRPS
Nps6 F.graminearum Fusarinine C Oide et al., 2006
42

43. • NPS6, is a virulence determinant
• Deletion of NPS6 orthologs (Δnps6 )in the
Rice pathogen- Cochliobolus miyabeanus,
Wheat pathogen- Fusarium graminearum, and
Arabidopsis pathogen- Alternaria brassicicola,
resulted in reduced virulence
• Exogenous application of iron enhanced the virulence of
Δnps6 strains of C. heterostrophus, C. miyabeanus, F.
graminearum, and A. brassicicola
(Δ= partial or complete deletion of NPS6)
43

44. Hypersensitivity of Cochliobolus heterostrophus Δnps6 strain to
KO2, Fe chelators 2DP (2, 2΄-dipyridyl) and BPS
(Bathophenanthroline disulfonic acid)
Growth of the Δnps6 strain is completely inhibited on MM+ 12 mM KO2
Growth of the Δnps6 strain is completely inhibited on MM +150 µM 2DP.
Average colony diameters of wild-type and Δnps6 strains is lesser on MM
+100 µM BPS
44

45. 5 dpi
Reduction in virulence of Δnps6 strains of C. miyabeanus(CmΔnps6),
A.brassicicola (Abnps6) and C. heterostrophus (Chnps6)
45

46. 46
Introduction of the NPS6 ortholog from the saprobe Neurospora
crassa to the Δnps6 strain of C. heterostrophus restored wild-type
virulence to maize
5 dpi

47. Exogenous Application of Iron Enhances the Virulence of Δnps6
Strains Of C. heterostrophus to Each Host.
(Oide et al., 2006) 47

48. Effect on virulence of Δnps6 strain of A.brassicicola by exogenous
application of siderophores.
4 dpi
DFO= 0.25mM,
Ferric citrate= 0.50mM
N-dimethylcoprogen=0.20mM
(Oide et al., 2006)
48

49. Application of Ferric Citrate Enhances the Virulence of the F. graminearum
Δnps6 Strain to Wheat.
(Oide et al., 2006)
49

50. Production of microbial iron chelator (siderophores) by
fluorescent pseudomonads
(Sayyad et al., 2005)
Treatments Root length Shoot length Germination
mm % increase
in mm
mm % increase
in mm
Percentag
e (%)
% increase
Test 6.95 16.25 9.5 34.5 90 10
Control 5.9 - 7.5 - 80 -
50
Table: Influence of P. fluorescens NCIM 5096 inoculation on wheat germination and
growth
Conclusion: 10 % increase in germination,
16.25 % increase in root length,
34.5 % increase in shoot length
Case study- 6

51. Conclusion
• Siderophore system constitutes a key position in Iron- homeostasis in
many plant pathogens.
• The role of siderophores in Iron homeostasis depend largely on the
pathogen-host system.
• Siderophore system affects growth, oxidative stress resistance as
well as asexual and sexual development.
• Common virulence determinant, at least in some plant pathogenic
fungi and bacteria.
• Modulates plant defense through an antagonistic mechanism
between SA & JA signaling cascade.
51

52. THANK YOU
52