Supplementary Tables
Table S1 Primers applied in this study.
Primers
HvUBQ5-F
HvUBQ5-R
HvPR1b-F
HvPR1b-R
HvPR10-F
HvPR10-R
HvJIP23-F
HvJIP23-R
HvKS1-F
HvKS1-R
HvKSL4-F
HvKSL4-R
HvDXS-F
HvDXS-R
TaAlpha-tubulin-F
TaAlpha-tubulin-R
ITS _Rh-F
ITS_ Rh-R
AtPDF1.2-F
AtPDF1.2-R
AtPR1-F
AtPR1-R
AtERF1-F
AtERF1-R
AtUBQ5-4-F
AtUBQ5-4-R
AtVSP2-F
AtVSP2-R
Sequence 5`- 3`
ACCCTCGCCGACTACAACAT
CAGTAGTGGCGGTCGAAGTG
GGACTACGACTACGGCTCCA
GGCTCGTAGTTGCAGGTGAT
GGAGGGCGACAAGGTAAGTG
CGTCCAGCCTCTCGTACTCT
GGAGTGTTTGGTACCCCCAT
GGCACCAGTGGCATTGTAGA
GACAGCCAAGGCTTTGAGAG
TGCACATCTTCCAGAACAGC
CGTCACCTTCTCCGAGACAT
GACGAACCTTCCTTGGGAGT
ACTTGCAGTGTTGGCACAAG
CACCAAACCCTCCTGTGACT
ATCTCCAACTCCACCAGTGTCG
TCATCGCCCTCATCACCGTC
TCAGCACATAACCACACCAATCGCG
TGCTTTGTACGCTCGGTAAGAAGGG
GTTTGCTTCCATCATCACCC
GGGACGTAACAGATACACTTG
AAGAGGCAACTGCAGACTCA
TCTCGCTAACCCACATGTTC
CGAGAAGCTCGGGTGGTAGT
GCCGTGCATCCTTTTCC
CCAAGCCGAAGAAGATCAAG
ATGACTCGCCATGAAAGTCC
CAAACTAAACAATAAACCATACCATAA
GCCAAGAGCAAGAGAAGTGA
5
1
Reference
Imani et al. (2011)
Imani et al. (2011)
Schäfer et al. (2009)
Schäfer et al. (2009)
Schäfer et al. (2009)
Schäfer et al. (2009)
Schäfer et al. (2009)
Schäfer et al. (2009)
Li (2012)
Li (2012)
Li (2012)
Li (2012)
this study
this study
Wen et al. (2013)
Wen et al. (2013)
Sharma et al. (2008)
Sharma et al. (2008)
Schikora et al. (2012)
Schikora et al. (2012)
Trusov et al. (2009)
Trusov et al. (2009)
Trusov et al. (2009)
Trusov et al. (2009)
Jacobs et al. (2011)
Jacobs et al. (2011)
Jacobs et al. (2011)
Jacobs et al. (2011)
Table S2 Comparison of the gene content of the genomes of RrF4 and C58 performed in the
EDGAR software (Blom et al., 2009). Presence of genomovar G8 specific gene clusters
(SpG8 gene clusters) as described by Lassalle et al. (2011).
Gene
C58 CDSs1
cluster
G8-specific regions
SpG8-1a
Atu1398 - Atu1408
SpG8-1b
SpG8-2a
SpG8-2b
Atu1409 - Atu1423
Atu3054 - Atu3059
Atu3069 - Atu3073
SpG8-3
Atu3663 - Atu3691
SpG8-4
SpG8-5
Atu3808 - Atu3830
Atu3947 - Atu3952
SpG8-6a
SpG8-6b
Atu4196 - Atu4206
Atu4213 - Atu4221
SpG8-7a
Atu4285 - Atu4294
SpG8-7b
Atu4295 - Atu4307
Specific to G58 sporadic in G8
Cluster 1
Cluster 2
Atu1183 - Atu1194
Atu4606 – Atu4615
Cluster 3
Atu4864 – Atu4896
Prophages, sporadic in G8
ATPP-1
Atu0436-Atu0471
ATPP-2
Atu1183 - Atu1194
ATPP-3
Atu3831 – Atu3858
Predicted function1
RrF4 CDSs
SY94_1294 - SY94_1304
Sugar and amino acid transport;
sugar metabolism
SY94_1305- SY94_1319 Ferulic acid uptake and catabolism
SY94_3050 - SY94_3054 Curdlan EPS biosynthesis
SY94_3064 - SY94_3068 Secondary metabolite biosynthesis
Siderophore biosynthesis; ironSY94_3658 - SY94_3682 siderophore uptake
Ribose transport; monosaccharide
SY94_3809 - SY94_3828 catabolism and
SY94_3921 - SY94_3925 Opine-like compounds catabolism
Drug/toxic (tetracycline)
SY94_4165 - SY94_4174 resistance
SY94_4183 - SY94_4190 Drug/toxic resistance
SY94_4254
- Environmental signal
SY94_4263
sensing/transduction
Environmental signal
SY94_4264 - SY94_4276 sensing/transduction
SY94_1089
- Prophage
SY94_10932
SY94_4568 - SY94_4574 Lipopolysaccharide biosynthesis
SY94_4815
SY94_48353
Absent
Prophage
SY94_1089- SY94_10932 Prophage
SY94_3831
- Prophage
SY94_38372
10
1
Adapted from Lassalle et al. (2011)
2
C58 homologous genes are only partially present
3
C58 homologous genes partially present and rearranged
2
15
Table S3 Overview of chromosomally coded genes of RrF4 that encode proteins/enzymes
which are potentially involved in the interaction of RrF4 with host plants and belong to
functional groups of genes which are well known to be involved in the beneficial effect of
PGPRs (e.g. as reviews by Lugtenberg and Kamilova, 2009). The gene homologs of C58 are
20
also given. Predicted functions of genes detected in the circular and linear chromosomes were
based on literature data, based on genome analysis of C58 (Goodner et al., 2001; Wood et al.,
2001), or KEGG passed analysis performed in GenDB (Meyer et al., 2003). The functionality
of gene was not proofed experimentally. Location of RrF4´s genes: CDS <SY94_3000 are
located at the circular chromosome; CDSs between SY94_ 3000 and SY94_ 5000 are located
25
on the linear chromosome.
Gene
RrF4 CDS
C58 CDS
Predicted function
Reference of
experimental
evidence*
Matrix components for attachment and biofilm
ctpABCDEFGHI
SY94_0156SY94_0148
Atu0224Atu0216
Flp-type pili: form thin
Wang et al.
filaments, thinner than flagella, (2014)
arranged around the cell
surface and sheeted into the
external milieu
celABCG and
celDE
SY94_3304SY94_3302,
SY94_3301;
Atu3309Atu3307,
Atu8186;
Cellulose synthesis gene (two
opera);
Involved in surface
attachment; and binding of
plant cells
Matthysse
(1995);
Matthysse et
al. (2005)
SY94_3298SY94_3299
Atu3302Atu3303
uppABCDEF
SY94_1137SY94_1142
Atu1235Atu1240
Polysaccharide biosynthesis
cluster - UPP: Unipolar
polysaccharide (required for
surface binding and biofilm
formation)
Xu et al.
(2013)
exoC (pgm)
SY94_4051
Atu4074
Required for the synthesis of
extracellular ß-1, 2-glucan and
succinoglucan polysaccharides
(phosphoglucomutase)
Changelosi et
al. (1987);
Bahat-Samet
et al. (2004)
3
chvAB
SY94_2559,
SY94_2561
Atu2728;
Atu2730
Required for synthesis of
extracellular beta-1,2-glucan –
involved in binding of plant
cells;
Cangelosi et
al. (1987)
chvA: beta-1, 2-glucan export
ATP-binding protein; chvB:
beta-1, 2-glucan biosynthesis
protein
SY94_3050-
Atu3054–
Atu3059
(SpG8-2a1)
Curdlan (beta-1,3-glycan) EPS
biosynthesis
Lassalle et al.
(2011)
Atu3056 in
SpG8-2a
Putative beta-1,3-glucan
synthase (curdlan synthase,
CrdS)
Lassalle et al.
(2011)
Siderophore biosynthesis;
Rondon et al.
(2004)
SY94_3682
Atu3663–
Atu3691
(SpG8-31)
SY94_3675 SY94_3682
Atu3684–
Atu3691
Homologous to fecABCDE
genes involved in TonB
dependent reuptake of the
siderophores if chelated to iron
Braun et al.
(2006)
SY94_3675
Atu3684,
Atu3692,
FecAIR seemed to form a cell
surface signaling system,
whose homolog in Escherichia
coli was proposed to regulate
the whole system of
biosynthesis, release, and
reuptake of siderophores
Braun et al.
(2006)
SY94_3054
SY94_3052
Siderophore biosynthesis
SY94_3680-
SY94_3683
Atu3693
SY94_3684
iron-siderophore uptake
Metabolism (nutrient uptake and metabolism/catabolism)
SY94_1294 SY94_1304
Atu1398–
Atu1408
(SpG8-1a1)
Sugar and amino acid
transport; sugar metabolism
Lassalle et al.
(2011)
SY94_3809 SY94_3828
Atu3808–
Atu3830
(SpG8-41)
Ribose transport;
monosaccharide catabolism
and carbohydrate metabolism
Lassalle et al.
(2011), but
without
experimental
proof
SY94_1305 SY94_1319
Atu1409–
Atu1423
Ferulic acid uptake and
catabolism (uptake and
catabolism of plant derived
Lassalle et al.
(2011)
4
β-ketoadipate
pathway
Linear
chromosome
(SpG8-1b1)
phenolic compounds)
Present at the
linear
chromosome
Pathway for aromatic
compound degradation
(degradation of plant derived
compounds)
Goodner et al.
(2001) and
references
therein
Cell wall degradation, degradation of cellulose, hemicellulose, pectin, and/or lignin
Pectinase (kdgF)
SY94_3140
Atu3145
Pectine degrading protein
Wood et al.
(2001)
Ligninase (ligE)
SY94_1026
Atu1121
Lignin-degrading protein
Wood et al.
(2001)
Beta-endoglucanase (celC)
SY94_3302
Atu3307
Encoded within the cellulose
synthesis gene cluster
Goodner et al.
(2001)
(linear chr.)
Xylanase (xynA)
SY94_2222
Atu2371
Endo-1,4-beta-xylanase
Regulators of
pectinase and
cellulase (pecS/M)
SY94_0202/
SY94_0203
Atu0272/
Atu0273
transcriptional regulator, MarR
family
1
G8-specific regions defined by Lassalle et al. (2011).
* not tested/proofed for RrF4
5
30
Table S4 Quantification of root colonization by RrF4 using qPCR targeting the ITS of the
ribosomal operon. RrF4 cells were calculated per g root FW (three ITS targets per cell). Data
represent mean values and standard deviations (in brackets) of three independent biological
replicates. The upper 4 cm of roots were investigated. FW: fresh weight; nd: under detection
35
limit.
Time
Barley
Wheat
-1
[dpi]
[cells g FW ]
0
2.1 (±0.4) × 10
5
1.4 (±0.3) × 10
7
2.1 (±0.7) × 10
14
8.1 (±1.4) × 10
Arabidopsis
-1
[cells g FW-1]
[cells g FW ]
8
9
9
9
3. 3 (±2.0) × 10
1.4 (±0.2) × 10
3.1 (±0.6) × 10
6.0 (±2.5) × 10
8
9
9
9
nd
5.0 (±0.3) × 10
1.2 (±0.6) × 10
2.9 (±0.5) × 10
8
9
9
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7