Siderophores ppt

Welcome

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Advances in siderophores: Plant pathogen interaction

SEMINAR-I

SundareshUHS13PGM396Plant pathology

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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.

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Siderophore producing organism

• Azotobacter

• Pseudomonas

• Bacillus

• Streptomyces

Among these microbes Pseudomonas species is the active siderophore producer.

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Metals used by organisms

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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

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Siderophores

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

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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 hydroxamatedesferrioxamine and mutants defective indesferrioxamine biosynthesis show tissue-specificreduced virulence

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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.

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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+ uptake10

Mechanism of siderophore mediated iron uptake inbacteria

(Syed Sajeed Ali, 2013)11

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

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.

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Types of siderophores

• Hydroxamate,

• Catecholate and

• Carboxylate

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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.

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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.

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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.

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Siderophore structure in bacteria

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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

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Representative fungal siderophores, Peptide and ester bonds separatingN5 – acyl- N5- hydroxyornithine 21

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

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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.

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Bio control potential of Pseudomonas

fluorescens against coleus root rot disease

(Vanitha and Ramjegathesh, 2014)

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Case study- 1

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

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Table: Antibiotics, Siderophores and HCN

production of P. fluoresens strains

Sl. no PGPR strains

Fluorescein Pyocyanin Siderophoreproduction

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

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Outcome

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

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Isolation of Siderophore producing bacteria from rhizosphere soil and their antagonistic activity against selected fungal plant pathogens

(Jenifer et al., 2013)

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Case study- 2

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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)

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Case study- 3

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

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Values in a row followed by the same letter are not significantly different (P < < 0.05)according to Duncan’s multiple range tests.

Table: Suppression of Fusarium wilt of pepper (Capsicum

annuum L.) in potting soil by B. subtilis CAS15

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Treatments - Fe + Fe

Diseaseincidence (%)

Disease severity (%)

Diseaseincidence(%)

Diseaseseverity (%)

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)

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

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Mean follows by a common letter in the same column are not significantly different at P = 0.01.

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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

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

Cont.

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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 thatobserved in SA-treated leaves, used as positive controls.

The activation of PR1 expression is correlated with an accumulation of SA, wasmeasured by HPLC in Arabidopsis leaves 24 h after CB treatment.

The siderophore treatment resulted in a 2- to 3-fold increase in SA content 24hpi 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.

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

Cont.

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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.

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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)

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Case study- 5

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., 200642

• 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)

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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

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5 dpi

Reduction in virulence of Δnps6 strains of C. miyabeanus(CmΔnps6),

A.brassicicola (Abnps6) and C. heterostrophus (Chnps6)

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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

Exogenous Application of Iron Enhances the Virulence of Δnps6Strains Of C. heterostrophus to Each Host.

(Oide et al., 2006) 47

Effect on virulence of Δnps6 strain of A.brassicicola by exogenous application of siderophores.

4 dpiDFO= 0.25mM, Ferric citrate= 0.50mMN-dimethylcoprogen=0.20mM

(Oide et al., 2006)48

Application of Ferric Citrate Enhances the Virulence of the F. graminearumΔnps6 Strain to Wheat.

(Oide et al., 2006)49

Production of microbial iron chelator (siderophores) by

fluorescent pseudomonads

(Sayyad et al., 2005)

Treatments Root length Shoot length Germination

mm % increasein mm

mm % increasein mm

Percentage (%)

% increase

Test 6.95 16.25 9.5 34.5 90 10

Control 5.9 - 7.5 - 80 -

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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

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.

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THANK YOU

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