Life in the xylem:

1
Life in the xylem secrets of a wilt pathogen
unveiled by in vivo gene expression
Darby Brown University of Wisconsin-Madison Depart
ment of Plant Pathology
2
Introducing Ralstonia solanacearum a bacterial
wilt pathogen
3
Geographic distribution of R. solanacearum
4
Infection and colonization of tomato by Ralstonia
solanacearum
5
R. solanacearum reaches high cell density within
the host plant xylem
6
Regulation of R. solanacearum virulence
Establishing a population early virulence
factors
motility
Polygalacturonases (PGs) (cell wall degrading
enzymes)
Type III secretion
7
Regulation of R. solanacearum virulence
as bacteria colonize the xylem a switch in
virulence factor expression occurs
3-OH-PAME
PhcA
PGs TTSS
Extracellularpolysaccharide (EPS)
8
What other virulence factors are required for R.
solanacearum colonization?

• Prediction
• R. solanacearum uses many more genes in 5.8 Mb
  genome to infect plants
• functions of these proteins are quantitative
  (additive) and/or redundant

9
Identifying new virulence factors requires a new
approach

• In vivo expression technology (IVET)

• Screen for genes that are up-regulated in the
  plant host (identifies redundant/additive genes)

• Identifies genes without loss of function (no
  need for mutant screen)

10
The R. solanacearum IVET Screen
Step 1 need strain that is no longer virulent
in the host
11
Disease assay of R. solanacearum tryptophan
auxotroph on tomato plants
D.L. Coplin PhD Thesis UW-Madison, 1972
12
The R. solanacearum IVET Screen
Step 1 need strain that is no longer virulent
in the host
Step 2 Integrative promoter trap that will
restore avirulent phenotype
13
Promoter-Probe IVET Vector
RBS
RBS
gusA
trpEG
XhoI
TGA
OriT
KmR
14
Heterologous promoters function in pIVETDGB to
restore virulence
15
The R. solanacearum IVET Screen
Step 1 need strain that is no longer virulent
in the host
Step 2 Integrative promoter trap that will
restore avirulent phenotype
Step 3 Output screen to identify promoters that
are specifically upregulated in the plant
16
IVET output screen
Rich medium
Diseased plant infected with pool of IVET strains
White plant induced
Blue constitutive
17
IVET output screen
18
The R. solanacearum IVET Screen
Avirulent strain
?trpEG
trpEG
gusA

Random chromosomal fragments (some will contain
plant induced promoters)
Promoter trapping vector
Introduce library to trpEG mutant
Pool strains containing IVET library and
introduce to plant
Host functions as selective medium
Output screen to identify plant-induced genes
19
Retrieving the integrated IVET vector
Retrotransfer (Rainey, et al. 1997. Mol Gen
Genet 25684-87)
DH5? recipient
R. solanacearum IPX strain
HB101 pRK600 helper
20
Mechanics of Retrotransfer
IVET
Integration of IVET vector created a
cis-merodiploid Low frequency of homologous
recombination within chromosome, especially
without antibiotic selection
21
Mechanics of Retrotransfer
IVET
Recombination causes the integrated IVET vector
to pop out of chromosome
22
Mechanics of Retrotransfer
recipient
IVET
helper
If an appropriate recipient and helper strain
present, the recovered IVET plasmid can be
transferred to E. coli
Plasmid can now be easily prepped for sequencing
IVET
23
Sequencing the IVET promoter
uidA
trpEG
promoter
Sequence fusion junction
OriT
KmR
Determine Identity of cloned promoter (ipx gene)
using the R. solanacearum GMI1000 genomic
sequence database http//sequence.toulouse.inra.fr
/
24
IVET screen results 153 in planta expressed
(ipx) genes of 133,000 screened

• Metabolic genes 20.9
• Known virulence genes 4.6
• Stress response 9.2
• Regulation 12.4
• Transmembrane 18.9
• Completely Unknown 34.0

25
Distribution of IVET genes
26
Which gene(s) to study next?
Stress?
Novel?
Regulation?

• Thematic approach to analyze ipx genes

27
Are ipx gene fusions actually plant induced?
28
IVET genes are plant induced
Brown and Allen Figure 5
Fold Induction GUS activity at T0/T5
Stress
112
34
136
81
56
54
53
48
46
41
35
33
32
29
28
126
124
114
108
105
102
89
84
73
71
69
66
65
64
63
60
58
22
17
15
13
9
6
3
2
1
CTL
IPX Fusion Strain
29
Are ipx genes regulated by known regulators of
virulence?
Approach Measure ipxgus fusion expression in
various regulatory mutant backgrounds
30
Assayed ipxGUS gene expression in 7 regulatory
mutants
polygalacturonases, type 4 pili, twitching
motility
3-OH PAME
A
PhcS
PhcR
PehS
PehR
RpoS
FlhDC
VsrA


XpsR
VsrC
VsrD
Swimming motility
EpsR
B
EPS
PLANT CELL
HrpB
Type III secretion system
31
Introduced 32 ipxgus fusions into regulatory
mutants
32
Example of qualitative GUS activity
33
(No Transcript)
34
ipx genes are specifically upregulated in planta
but not in minimal medium
35
Three IVET strains are affected in virulence
glycolate oxidase R. solanacearum cant grow on
glycolate
putative transmembrane proteins- function unknown
36
Narrowing the focus
14 likely stress response genes isolated
Are stress response genes expressed during xylem
colonization ?
37
What stressors might R. solanacearum encounter in
the host plant?
Involves the localized production of reactive
oxygen species (ROS) H2O2 and other
plant-derived toxins
38
Hypothesis Drug efflux pumps play a role in R.
solanacearum virulence
3 putative multi-drug efflux pumps were
identified in IVET screen 1. acrA (18-fold
induced) 2. dinF 3. small multidrug efflux
pump (2 fold induced)
Mutated the dinF and acrA genes
39
acrA and dinF mutants are reduced in virulence in
tomato
40
Hypothesis uvrA1, another ipx gene, plays a
role in R. solanacearum virulence

• transcription of uvrA1 was 8-fold induced in
  tomato
• mutated the uvrA1 gene
• mutant was sensitive to UV-light
• assessed virulence of uvrA1 mutant in susceptible
  tomatoes

41
The uvrA1 mutant is still virulent
But theres a second locus uvrA2!!!
42
Summary

• 153 plant induced genes identified in IVET screen

• most ipx genes are induced gt2fold in tomato xylem

• controlled by known virulence regulators

• two genes are regulated differently in planta
  than MM

• three ipx strains are reduced in virulence

• mutations in 2 putative multidrug efflux pumps
  reduced virulence

• uvrA1 mutant is virulent on tomato, but theres
  a second locus