3. ICGEB
The International Centre for Genetic Engineering and Biotechnology
provides a scientific and educational environment of the highest standard and
conducts innovative research in life sciences for the benefit of developing
countries. It strengthens the research capability of its Members through training
and funding programmes and advisory services and represents a
comprehensive approach to promoting biotechnology internationally.
Components:
Trieste, Italy: 15 labs, 400 people
New Delhi, India
Cape Town, South Africa
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60 Member States
4. Cell-Cell Communication in Bacteria
Solitary / Planktonic
Vulnerable
Community
Biofilm
Antibiotics
Virulence
Swarming
Symbiotic relationships
Communication = chemical signalling
Introduction
4
8. Non AHL-ProducersAHL-Producers
LuxR solos
LuxR-type proteins that are not paired with a cognate LuxI-type synthase
luxRS pipx b ca
luxRS b ca
luxR luxI b ca
LuxI
SdiA S. enterica, E. coli
PsoR P. fluorescens
OryR X. oryzae
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QscR P. aeruginosa
ExpR S. meliloti
BisR R. leguminosarum pv. viciae
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Introduction
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9. xccR pipx b ca
Inter-kingdom signalling:
Interactions between bacteria and their
eukaryotic hosts
Introduction
RSM
9
10. Inter-kingdom signaling:
Interactions between bacteria and their
eukaryotic hosts
Introduction
Best studied cases of inter-kingdom signalling in
plant-associated bacteria:
Rhizobia-legume plants signalling to initiate
nodulation
Agrobacterium pathogenesis and plant tumor
development
Emerging field of research
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11. LuxR solos
Subfamily of LuxR solos from plant-
associated bacteria (PAB)
Introduction
LuxR superfamily11
PAB LuxR solos
12. AHL-binding HTH
LuxR (V. fisheri) W57 Y61 D70 P71 W85 G113 E178 L182 G188
PsoR (P. flurorescens) W W D P W G E L G
OryR (X. oryzae) M W D P W G E L G
XccR (X. campestris) M W D P W G E L G
NesR (S. meliloti) M W D P W G E L G
Introduction
LuxR solos
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13. New subfamily of LuxR solos from plant-associated bacteria
LuxR solos
Introduction
13
15. Introduction
XccR of Xanthomonas campestris
Pip promoter activated by XccR in planta
Xcc contains a LuxR solo
named XccR
Xcc does not produce
AHLs
xccR is genetically linked
to the pip gene
XccR and PIP are
important for virulence
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15
XccR binds pip promoter
Gel mobility shift Supershift
16. LuxR solos in plant-associated bacteria
OryR
PsoR
Introduction
16
17. Introduction
Organisms under study
Xanthomonas oryzae pv. oryzae (Xoo)
Vascular pathogen of the rice plant (Oryzae
sativa) causal agent of bacterial blight of rice.
Economically important pathogen mostly in
Asia, but found world-wide
Enters plants by way of hydathodes and
wounds on the roots or leaves
Model organism for studying plant-
pathogenesis
Pseudomonas fluorescens
Found ubiquitously in soil, beneficial for plants
Produce strong siderophore, ie: fluorescent
pyoverdin
Produces biocontrol agents like: 2,4-DAPG,
pyrrolnitrin, hydrogen cyanide, and phenazine,
and some secretion enzymes such as
protease, phospholipase C, and chitinase
Plant-growth promoting bacteria (PGPR)
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18. OryR and PsoR
Biosensors: X.oryzae does not produce AHLs
Affinity chromatography: OryR is solubilized in the
presence macerated rice, not of AHLs
Promoter fusion: OryR activate target promoters in the
presence of rice macerate, not AHLs
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Ferluga et al, 2009. J Bact
OryR
Introduction
18Ferluga et al, 2007. Mol Plant Path
Cucumber Rice Wheat
PsoR
P. fluorescens do not produce AHLs
PsoR is solubilized in the presence of rice and wheat
macerate but not cucumber or AHLs
PsoR activates target XooPip promoter with rice and wheat
but not cucumber.
PsoR is important for biocontrol in wheat but not cucumber
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Subramoni et al; 2011 Appl Env Microbiol
19. LuxR
‘solos’
Organism
Role of
LuxR solo
Binding
molecule/
s
Functions
Regulated
Reference
XccR
Xanthomonas campestris
pv. campestris
Virulence
Plant Signal
molecule
Cabbage (+)
Proline imino peptidase
(pip) gene expression;
Virulence on Cabbage
Zhang et al., 2007
OryR
Xanthomonas oryzae pv.
oryzae
Virulence
Rice Signal
molecule
Rice (+)
Proline imino peptidase
(pip) gene expression;
Virulence on rice,
flagellation
Ferluga et al., 2007;Ferluga et al., 2009;González et al., 2012
NesR Sinorhizobium meliloti Symbiosis
Not
determined
Nutritional and
environmental stress
response, plant
nodulation
Patankar et al., 2009
PsoR
Pseudomonas fluorescens
Pf-5/ CHA0
Biocontrol
Plant signal
molecule
Rice (+)
Wheat (+)
Cucumber (-)
Anti-microbial agents
2,4-DAPG, chitinase
Subramoni et al., 2011
XagR Xanthomonas axonopodis Virulence
Plant signal
molecule
Soybean (+)
Rice (+)
Cabbage (+)
Proline imino peptidase
(pip) gene expression;
Virulence on soybean;
Adhesion
Chatnaparat et al., 2012
Introduction
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20. oryR pipx b ca
Xanthomonas oryzae
Pathogen
OryR
Plant signal molecule(s)
Aim 1.1: Identify targets of OryR
regulation
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21. Microarray
wt mut
Xoo XKK12 Wild type Xoo XKK12 OryR mutant
2 biological replicates of both the wild type and an
oryR mutant in rich media with rice macerate
Results
Established highest pip promoter
activity at an OD600 of 2.0
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22. Top negatively-regulated
Microarray
Results
330 genes regulated over 2 fold
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Gene locus
Fold
Change
Function
XOO1701 34.75 hypothetical protein
XOO1051 31.95 hypothetical protein
XOO3951 24.33 methyltransferase homolog M.XphI
XOO0608 23.24 endonuclease
XOO1455 20.15 hypothetical protein
XOO3472 13.30 hypothetical protein
XOO0224 11.21 hypothetical protein
XOO1452 9.93 hypothetical protein
XOO3489 9.27 hypothetical protein
XOO4759 8.22 hypothetical protein
XOO1454 7.94 hypothetical protein
XOO4758 7.85 hypothetical protein
XOO0697 7.10 hypothetical protein
XOO1774 6.54 hypothetical protein
XOO1273 6.23 quinol oxidase, subunit I
XOO0696 5.77 hypothetical protein
XOO0599 5.72 phage integrase family site specific recombinase
XOO3467 5.44 hypothetical protein
XOO1580 5.02 RhsD protein
XOO4885 5.02 hypothetical protein
XOO0606 4.67 XorII very-short-patch-repair endonuclease
XOO4439 4.58 hypothetical protein
XOO0050 4.32 hypothetical protein
110 negatively-regulated genes over 2 fold
23. Gene locus Fold Change Putative function
XOO3196 32.56 PilY1
XOO0329 25.83 polyvinylalcohol dehydrogenase
XOO0031 10.18 hypothetical protein
XOO1391 9.56 hypothetical protein
XOO0134 8.06 IS1479 transposase
XOO3050 7.96 hypothetical protein
XOO3195 7.14 type IV pilin
XOO3062 6.95 hypothetical protein
XOO3061 6.72 hypothetical protein
XOO2834 6.44 hypothetical protein
XOO1099 6.11 TonB-dependent receptor
XOO0020 6.10 IS1479 transposase
XOO1390 6.00 RtxA
XOO2384 5.99 IS1595 transposase
XOO0095 5.89 Hpa1
XOO3052 5.78 hypothetical protein
XOO1098 5.77 amylosucrase or alpha amylase
XOO3063 5.71 IS1404 transposase
XOO2580 5.70 flagellar hook-associated protein FlgL
XOO3051 5.29 hypothetical protein
Top positively-regulated
220 positively-regulated genes over 2 fold•
Microarray
Results
P value: 2.5 x 10-7
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24. GENE Fold Change FUNCTION
XOO2580 5.7 flagellar hook-associated protein FlgL
XOO2583 4.93 flagellar protein
XOO2622 4.57 chemotaxis protein
XOO2611 4.27 flagellar biosynthesis protein FliP
XOO2572 4.12 flagellar hook protein FlgE
XOO2623 3.8 chemotaxis related protein
XOO2558 3.78 chemotaxis protein
XOO2857 3.71 chemotaxis protein methyltransferase
XOO2579 3.48 flagellar hook-associated protein FlgK
XOO2566 3.38 flagellar protein
XOO2601 3.24 flagellar MS-ring protein
XOO2833 3.19 chemotaxis protein
XOO2574 3.15 flagellar basal body rod protein FlgF
XOO2620 3.14 flagellar biosynthesis switch protein
XOO2571 2.94 flagellar basal body rod modification protein
XOO2570 2.87 flagellar basal body rod protein FlgC
XOO2569 2.84 flagellar basal body rod protein FlgB
XOO2577 2.83 flagellar basal body P-ring protein
XOO2610 2.79 flagellar protein
XOO2624 2.72 chemotaxis related protein
XOO2604 2.72 flagellar protein
XOO2619 2.67 flagellar biosynthesis regulator FlhF
XOO2605 2.59 flagellar FliJ protein
XOO2830 2.59 flagellar motor protein
XOO2848 2.49 chemotaxis protein
XOO2582 2.43 flagellar protein
XOO2831 2.29 flagellar motor protein MotD
XOO2607 2.27 flagellar protein
XOO2606 2.26 flagellar protein
XOO2608 2.24 flagellar motor switch protein FliM
XOO2578 2.19 flagellar rod assembly protein/muramidase FlgJ
XOO1468 2.16 chemotaxis protein
XOO2575 2.16 flagellar basal body rod protein FlgG
XOO2836 2.15 chemotaxis protein
oryR
18% of positively regulated genes are
associated with movement
Flagellum (30 genes)
Chemotaxis (9 genes)
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Microarray
Results
P value: 2.5 x 10-7
P value: 0.055
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25. Class I Class II Class III
fleQ
rpon2
flgM
fliEFGHIJK
fliLMNOP
fliQR
flhBAF
fliC
fliD
flgM
Chemotactic
Positively regulate genes Non regulated genes Non-flagellar genes
Results
Xoo flagellar genes
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33. FlhF Flagellar regulator
Important but not crucial for motility in Xoo (Shen et al. 2001.MPMI)
Directs correct placement of flagella in P. aeruginosa (Dasgupta et al. 2003. Mol
Microbiol) and is essential for swimming and swarming (Murray & Kazmierczak.
2006. J Bact)
Mutations in flhF in P. putida change flagellation from polar to peritrichous
flagellation, and over-expression results in a multiflagellated phenotype (Pandza
et al. 2000. Mol Microbiol)
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37. Results
Iron acquisition
Is PsoR important for iron acquisition in Pseudomonas fluorescens?
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Grow assay: wild type, PsoR mutant and PsoR
over-expressing strains in the presence of the
iron chelator 2,2’$dipyridil+in+order+to+evaluate
the+role+of+PsoR+under+iron+stress
Indirect evidence that PsoR
plays a role in iron
acquisition in P. fluorescens
100 uM FeCl3
38. Classical promoter library screening method
Powerful: Heterologous E. coli system identifies direct
targets of regulation
Promoter trapping
Results
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pBBR
PsoR
E. coli (pBBRPsoR)
pQF50
lacZ Blue colony White colony
Possible target
of PsoR
regulation
-
Blue colony White colony
Constitutive
promoter
Possible target
of PsoR
regulation
Plate on X-gal
Plate on X-gal
E. coli
pQF50
lacZ
pBBR
39. After sequencing: found two additional targets of PsoR:
PFL_0133 large adhesive protein (lapA)
PFL_4057 Sensory box histidine kinase/response
regulator
XagR of X. axonopodis found to be important for
adhesion (Chatnaparat et al., 2012 MPMI)
Promoter trapping
E. coli (pBBRPsoR) E. coli (pBBR)
40. OryR is involved in the regulation of motility-related
genes in response to a plant molecule
OryR mutants show reduced swimming and swarming
in media supplemented with rice macerate
Flagellin production in planta is reduced in the OryR
mutant.
OryR probably acts through the regulation of the flhF
flagellar regulator through a luxbox-like element
PsoR is important for iron transport/acquisition
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Summary OryR and PsoR regulons
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42. How different
are the new group
of plant associated
solo LuxRs to
classical QS
LuxRs?
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Aim 2: Evaluate the
degree of conservation and
exchangeability between
QS-LuxRs and PAB-LuxRs
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45. Promoter switching
Both OryR and PsoR can activate the lasI
promoter of P. aeruginosa
None of the QS LuxR proteins tested
activated the Xoo pip promoter
Results
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46. Point mutations
AHL-binding HTH
Consensus W57 Y61 D70 P71 W85 G113 E178 L182 G188
PsoR M Y D P W G E L G
∆PsoR W Y D P W G E L G
OryR M W D P W G E L G
∆OryR W Y D P W G E L G
∆OryR
X. oryzae oryR-
∆PsoR
P. fluorescens psoR-
Results
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∆Solo
Reporter
Xoo pip prom
AHLs
X. oryzae oryR-
/ P. fluorescens psoR-Recover ability to bind AHLs?
Point mutations did not recover AHL
binding capacities or affect rice-binding
47. HTH
Rice molecule
Helix-turn-helix domain
Expression
E. coli heterologuos system
Results
Reporter
Xoo pip prom
HTH domain of some QS LuxRs can
activate transcription
Strain Avg M.U. SD
E. coli M15 (pMPXoopip) 33.3 3.4
E. coli M15 (pMPXoopip) (pQEOryRHTH) 37.8 6.1
E. coli M15 (pMPXoopip) 26.6 5.5
E. coli M15 (pMPXoopip) (pQEPsoRHTH) 28.0 2.8
HTH domain of PAB LuxRs cannot activate
transcription of target Xoo pip gene
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48. Summary plant associated luxR solos:
QS LuxR proteins do not induce activation of
Solo LuxR target pip promoter
OryR and PsoR can activate QS-target lasI
promoter of P. aeruginosa
LuxR solos contain substitutions in 1 or 2 key
amino acids in the autoinducer binding domain,
reverting to consensus QS LuxR sequence does
not recover AHL binding.
The HTH domains of OryR and PsoR cannot
activate target promoters on their own
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49. Aim 3: Method Developed to analyze X.
oryzae in planta expressed proteins
Giuliano Degrassi
Mike Myers
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IF: 4.878
50. In planta proteomics
Xoo cells can easily be retrieved from
infected plants
More accurately detects real set of
proteins being expressed by Xoo in planta.
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Results
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52. In planta proteomics
Results
Found most reported Xoo virulence factors
Hydrolytic enzymes
Cellulases
1,4,-beta cellobiosidase
Cysteine protease
Lipase/esterase
Motility/chemotaxis
Adhesion
Iron acquisition
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Robust method for analyzing in planta
expressed proteins of vascular pathogens
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17 hypothetical proteins; 36/64 proteins with known (or putative)
function in virulence or plant-bacteria interactions
53. In planta proteomics
Results
Generated mutants of 10 unreported/hypothetical proteins
to determine their role in virulence
Accession ORF Putative function
gi122879019 XOO0439 peptidase
gi122879107 XOO1487 cysteine protease
gi122879038 XOO0680 protease
gi58581605 XOO1982 protein U
gi58581474 XOO1851 periplasmic protease MucD
gi58579630 XOO0007 hypothetical protein
gi58584205 XOO4582 outer membrane protein ompP1
gi58581372 XOO1749 twitching motility protein pilJ
gi58583149 XOO3526 hypothetical protein
gi58581428 XOO1805 hypothetical protein
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55. In planta proteomics
Results
Protein U mutant significantly less
virulent than wild type
Virulence partially restored by in trans
complementation
Shows some similarity to a spore
coat/pili formation protein from
Myxococcus xanthus but its role in the
G-
Xoo is unknown; putative signal
peptide
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56. In planta proteomics
Results
Role of Protein U in planta
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MW PY+
Rice
PY M9+
Rice
M9
Slight increase of PrU in supernatants of
cultures of Xoo with rice macerate
Protein U promoter activity significantly
increases in media supplemented with rice
Protein U Gus promoter fusion
Protein U has a role in planta
57. Summary in planta proteomics
This is an effective method for identifying Xoo
proteins expressed in planta
A total of 324 unique proteins were detected, of
these 64 shared among all biological replicates
Protein U is involved in rice virulence, these effects
are plant-dependent
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58. Conclusions:
OryR and PsoR belong to a sub-family of LuxR proteins found
only in plant associated bacteria and represent a novel group of
proteins involved in inter-kingdom signaling between plants and
bacteria.
Xanthomonas oryzae is a vascular pathogen, apparently OryR plays
a role in informing the bacteria of its proximity to the plant and
initiates fast movement to allow rapid colonization of the vascular
system.
PsoR of Pseudomonas fluorescens is a LuxR solo shown to be
important for antimicrobial properties including iron-acquisition
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59. The subfamily of plant-associated bacteria LuxR solos share
common characteristics with QS LuxRs but represent a separate
cluster binding to a different molecule. Although they use a similar
DNA promoter region, for the most part have diverged enough to
not be interchangeable
Proteomics analysis is a robust method for analyzing in planta
expressed proteins of vascular pathogens
Protein U is a novel virulence-associated factor in Xanthomonas
oryzae
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Conclusions:
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60. Future perspectives:
(i) Identify the structure of the plant signal molecule(s). Once identified it will allow
answering questions such as the nature of the interaction with the solo. It will also reveal
whether the molecule(s) or family of molecule(s) is widespread or specific to a certain
group of plants.
(ii) It would also be interesting to find additional targets of OryR and PsoR, possibly in
different conditions, more representative of the interactions between the plant and the
bacteria (ie: in vivo). In addition the role of the hypothetical proteins regulated by OryR and
PsoR could reveal new loci involved in plant-bacterial interactions.
(iii) A more complete picture of OryR and PsoR regulation could be obtained by further
studies in respect to the nature of this regulation, whether it is direct or indirect with the
use of protein/DNA approaches like gel-mobility shift experiments.
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61. (iv) It would be interesting to follow the action of OryR and its targets (such as pip) in
planta through the use of techniques like as fluorescence microscopy in order to elucidate
the time/place of maximum PAB-solo activity.
(v) Electron microscopy could be used to elucidate the function of protein U by observing
possible changes in surface structure.
(vi) Finally, broaden study to other bacterial species in order to establish how plant-
associated bacteria from different phylogenetic groups use this novel inter-kingdom
signaling system.
Future perspectives:
61
62. Thank you
Bacteriology group ICGEB
Vittorio Venturi
Giuliano Degrassi
Giulia Devescovi
Iris Bertani
Daniel Passos
Bruna Goncalves Coutinho
Hitendra Patel
Veronica Parisi
Previous members:
Sara Ferluga
Zulma Suarez
Sujatha Subramoni
Carlos Nieto
Maja Grabusic
Giacomo Roman
Mike Myers Protein Networks group
Sandor Pongor Protein Structure and
Bioinformatics
Ghent University, Belgium
Monica Höfte
David De Vleesschauwer
University of Nova Gorica, Slovenia
Elsa Fabbretti