Structural and functional genomics of plant

Structural and functional genomics of plant-associated Bacillus strains used for biocontrol of plant diseases Rainer Borriss, ABiTEP GmbH and Humboldt University Berlin KSPP International Conference 2012

CDS: 3693, 8.5 % are devoted to non-ribosomal synthesis of secondary metabolites

gb: CP000560

1

Secondary metabolites with biocontrol and plant growth promoting activities OH

O N H

I-Y-Q-P O

HN

fengycin

C10-12CHCH2CO=E-L-L-V-D-L-L

O

C11-13CHCH2CO=N-Y-N-P-E-S-T

C13-14CHCH2CO=E-O-Y-T-E-A

surfactin

O

bacillomycinD

OH

OH

bacilysin O H2N

Srf-NRPS O

Fen-NRPS

OH

H2C-C-COO-

1

H2C-C-COO-

Bae-MS

OH

H3C-C-COO- 2

H3C-CH

H3C-C

H3C-C

O

O

2x Pyruvat

Sfp

O

2-Acetolactat

OH

Trp

Dif-M S

OH

Acetoin

?

CH2COOH

Dbe-NRPS

DHB

O

O HO

macrolactin

CH3 O

DHB-Gly-N H

HO

Chen et al. 2007: Nature Biotechnology

H3C

O CH3

R

CH2COOH

Gly-DHB HN

difficidin

2,3-butandiol

IAA

H

OH

H3C-CH OH

3

N

Mln-MS

O

?

OH H3C-CH

IAA P

COOH

H

O

NRS-NRPS

O

N

Bac-NRPS Bmy-NRPS

NH

HO

O

O

bacillaene

O O

O

O N-Gly-DHB

CH3

OH

HO

N H

O OH

H

bacillibactin

1. Antimicrobial secondary metabolites 2

Giant gene clusters are devoted to non-ribosomal peptide synthesis. More than 8.5% of the whole genome are occupied by gene clusters involved in non-ribosomal synthesis of secondary metabolites.

1. Antimicrobial secondary metabolites

3

Giant gene clusters devoted to non-ribosomal polyketide synthesis


4

Gene clusters devoted to synthesis of plantazolicin in FZB42 and closely related strains

Plantazolicin is a ribosomally synthesized thiazol/oxazol peptide suppressing closely related Bacillus sp. including Bacillus anthracis 1. Antimicrobial secondary metabolites 5

Gene clusters devoted to mersacidin synthesis in FZB42 and closely related strains. FZB42contains only a fragmentary part of the whole gene cluster and is unable to synthesize mersacidin.


6

B. amyloliquefaciens type strain DSM7

2. Comparative Genomics 7

Venn diagram: DSM7 (1), FZB42 (2), B. subtilis (3): 3049 genes are shared by all three strains.  core genome

2. Comparative Genomics 8

Chromosomal locations of genome plasticity regions identified by different methods.

A, B, C: putative horizontally transferred genomic islands are depicted as predicted by IslanViewer (red spots), IslandPath (blue spots), SIGI-HMM (yellow spots) and SeqWord Sniffer (green spots). D graph shows a global alignment of bacterial chromo-somes built by the M-GCAT program. Indels are depicted by red rectangles. 9

Whole genome sequences of Bacillus amyloliquefaciens

Strain

note

NCBI

gene bank

Size bp

GC protein

rrna

tRNA

gene

Source

Bacillus amyloliquefaciens subsp.amyloliquefaciens DSM7

type strain

NC_014551 FN597644

3,980,199

46.1 %

3893

30

94

4044 DSMZ

LL3

chromosome NC_017190 CP002634

3,995,227

45.7 %

4219

22

72

4346 Nankai Univ.

plasmid

NC_017189 CP002635

6758

42.0 %

9

TA208

guanosine

NC_017188 CP002627

3,937,511

45.8 %

4089

18

70

4177 CAS Beijing

XH7

purine

NC_017191 CP002927

3939203

45.8 %

4190

21

75

4286 Guangzhou

NC_009725 CP000560

3,918,589 46.5 %

3693

30

87

3813 ABiTEP

30

86

4014 Lallemand

9

Bacillus amyloliquefaciens subsp. plantarum FZB42

type strain

IT-45

chromosome

CM001433

3,925,087 46.6 %

3898

plasmid

CM001434

8,009 40.5 %

9

CAU B946 rice isolate

NC_016784 HE617159

4,019,861 46.5 %

3823

30

95

3948 CAU Beijing

YAU - Y2

CeBiTec

NC_017061 HE774679

4,242,774

45.9 %

3989

30

91

4110 YAU Kunming

BGI

NC_017912 CP003332

4,238,624

45.9 %

4238

29

85

4352

419617 bp

46.0 %

4193

30

92

4315 NAU Nanjing

NZ_AJST01000 3899657 bp 46.6 %

3898

30

86

KRIBB

NAU B3

draft genome unpublished

9

Bacillus 5_B6

cherry tree

10

Phylogenetic tree based on core genomes

2. Comparative Genomics

11

2. Comparative Genomics

Within the „plantarum“ group ,13 gene clusters are involved in synthesis of antibiotics (polyketides, lipopeptides, bacteriocins, and lantibiotics)

12

Conclusions part 1 1. Comparative genomics has been proven as a valuable mean for improving bacterial taxonomy. Discriminating between the plant-associated and the non-plant- associated representatives of B. amyloliquefaciens has revealed two subspecies: „plantarum“ and „amyloliquefaciens“ (Borriss et al. 2011.IJSEM 61, 1786-1801). Subspecies plantarum is distinguished by a higher genomic capacity to synthesize nonribosomally antimicrobial metabolites. 130 genes have been identified as being unique for the subspecies. Besides comparative genomics, other methods of functional genomics have to apply for further elucidating molecular factors important for rhizosphere competence of those bacteria. 2. Due to the importance of the „plantarum“ subspecies, we propose: a) to choose Bacillus amyloliquefaciens FZB42 as a model strain for PGP bacilli. The strain is freely available for basic research from BGSC (strain number 10A6) and DSMZ (strain number DSM 23117) b) to intensify research concerning factors affecting root colonization and to screen for bacilli with improved ability to colonize plant roots c) In order to reduce the costs for registration, it would be highly desirable to permit generally use of representatives of this species as bicontrol agents without performing the whole set of investigations necessary today. 13

Colonization of Arabidopsis roots by FB01mut

Tips of the primary root [C], and of root hairs (D) are colonized by the bacteria 3. Rhizosphere competence 14

gfp-insertion into FZB42

•

Stable integration of gfp into the chromosome of FZB42

•

All tested colonies are emitting green light about 30 generations without selective pressure

•

No differences in growth between gfp-labeled FZB42 and wild type was observed

Fan et al. 2011, JBT Vol 151, Issue 4

3. Rhizosphere competence 15

Colonization of Lemna minor One day: Colonization mainly at root tip.

Five days: Increasing number of microcolonies at roots.

Nine days: Sunken spaces are surrounded by Chloroplast bearing parenchyma cells. GFP labeled cells are colonizing sunken space aerea of the Lemna fronds. 16

Colonization of Arabidopsis by FZB42 and mutant strains

A) Root tip of the primary root and B) primary root surface with a emerging lateral root colonized by FB01mut

G) Border cells of primary root tip and H) emerging lateral root colonized by ΔdegU-mutant.

I) Border cells of primary root tip and J) emerging lateral root colonized by ΔpznA-mutant.

M) Border cells of primary root tip and N) emerging lateral root colonized by ΔRBAM017410-mutant


GFP-labeled FZB42 colonizing maize roots

A: Junction between primary root and lateral roots is heavily colonized.

B: Presence of FZB42 in a concavity of maize root surface.

C,D: Bacteria colonizing root hairs. Bacteria grow along a root hair.

Right: Preferred sites of growth in maize (A) and (B), Arabidopsis [C], and Lemna minor (D)


FZB42 (FB01mut) coloizing lettuce roots

1.5 cm from main root tip

2 cm from main root tip

4 cm from main root tip

Root hairs in vicinity of main root tip

3. Rhizosphere competence

Suppression of Rhizoctonia solani by FZB42 in a gnotobiotic system

R.solani

R.solani FZB42

Left: Cocultivation of FZB42 and R. solani in Petri dishes. Right: Gnotobiotic system in quartz sand (lettuce, R. solani and FZB42) established and used by IGZ, Helmholtz, and ABiTEP 4. Biocontrol 20

Detection of secondary metabolites by FZB42 in presence of Rhizoctonia

1054.0

441.2 459.4

1084.0

Bacillomycin D

1119.3

1086.0

1.5

1098.0

1.0

461.2

656.3

441.2

x10 4

2.0

1045.2

Bacillomycin D, Surfactin

2.0

Intens. [a.u.]

1084.2 1114.3

x10 4 2.5

1108.0

997.9 1032.0

861.5

905.6

690.3 709.2

603.2

550.5

423.2

459.4

0.0 400

600

800

1000

1200

1400

1600

m/z

Fengycin

1.0

FZB42 (LA + P) in presence of R. solani Extract of agar in vicinity of the mycelium

1544.8

1502.7 1464.6

1355.3 1375.2

1135.3

1010.0

893.5

843.7

721.7

666.3 621.5

525.2

704.3

477.2

1032.1

Plantazolicin

0.5

495.4

0.5

1.5

411.2

Intens. [a.u.]

(LA + P) by mass spectroscopy after 4 days growth

0.0

4. Biocontrol 400

600

800

1000

1200

1400

m/z

21

Inhibiting effect of FZB42 on growth of phytopathogenic fungus Rhizoctonia solani in a gnotobiotic quartz sand system

GFP-tagged FZB42 (FB01, green fluorescence) growing at lettuce roots suppress completely R. solani (left). Development of Rhizoctonia solani on lettuce roots only in absence of FZB42 (right).

4. Biocontrol 22

The impact of B. amyloliquefaciens FZB42 on lettuce growth in the field natural infested with the bottom rot pathogen R. solani 8

Shoot dry mass [g/plant] a

a

6 4 b 2

0 Control

R. solani

FZB42 +Rs

Under biotic stress (presence of the pathogen R. solani, +Rs) and in respect of applied spore numbers (106, 107, 108 spores/ml) of FZB42-Rif. Plants were cultivated at 22/15°C for 4 weeks. Dry mass followed by the same letter are not significantly different according to Dunnett’s test (P=0.1).

Golzow, Brandenburg Size 100 x 25 square meters, Alluvial loam

Chowdhury et al. submitted

4. Biocontrol 23

Conclusions: part 2  A chemically defined axenic system was established with Lactuca sativa grown on quartz sand  Inoculation studies with GFP-tagged B. amyloliquefaciens FZB42 showed long chain-like colonies and after 2-4 weeks biofilm like appearance in the oldest part of the roots  Co-inoculation with R. solani in model quartz sand showed that FZB 42 was able to inhibit fungal infection for up-to 2 weeks, with no visible signs of infection

 First mutant studies revealed that Surfactin is necessary for root colonization  Field trials revealed that FZB42 enhances yield of lettuce in presence of Rhizoctonia solani without affecting rhizosphere community.

24

Thanks to.... Humboldt University Berlin, Germany Dr. Oliwia Makarewicz Dr. Xiaohua Chen Dr. Alexandra Koumoutsi Dr. Lilia Carvailhis Dr. Kristin Dietel Dr.Romy Scholz Dr. Ben Fan and many others

Prof. Qi Wang, CAU Beijing China Prof. Yuewen Gao, NAU Nanjing China Prof. Xueqiu He, YAU, Kunming China Dr. ChongMing Ryu, KRIBB, South Korea This researcht was mainly supported by the BMBF Germany

Technical University Berlin, Germany Dr. Joachim Vater Prof. Dr. Roderich Süßmuth

Rheinische Friedrich-Wilhelms-University Bonn, Germany Prof. Dr. Jörn Piel Jana Moldenhauer Goettingen Genomics Laboratory, Germany Prof. Dr. G. Gottschalk Dr. H. Liesegang CeBiTec University Bielefeld Prof. Alfred Pühler Dr. Christian Rueckert Dr. Jochen Blom

25 25