1. Control of Gene Expression
in Xylella fastidiosa
Donald A. Cooksey
Department of Plant Pathology and Microbiology
University of California, Riverside
Control of Gene Expression
in Xylella fastidiosa
• Cell-cell signaling
• Global regulatory genes
• Specific two-component regulatory
systems
• Chemosensory systems
• Iron regulation
• Temperature regulation

2. Differentially-expressed genes in regulatory
gene mutants of Xylella fastidiosa
• Identification of genes regulated by “global”
regulatory genes known to control a variety of
pathogenicity genes in other bacteria (rsmA,
algU, gacA)
• mRNA extracted from regulatory gene knock-
out strains hybridized to complete genome
microarray and compared with wild-type
strain
An rsmA mutant had enhanced biofilm
formation and reduced virulence
Wild type rsmA-

3. Differentially-expressed genes in an rsmA
mutant of Xylella fastidiosa
• Genes repressed by rsmA:
– Heat shock proteins: hspA, grpE, dnaJ, dnaK,
dksA
– ABC transporters: yadG, malF
– Fimbrial protein: pilA
– Type II protein secretion: secB
– Protease: PD1971
– Transcriptional regulator: cysB
– Other genes of unknown function
Phenotypic characteristics of algU-
90 b
80 WT
algU::nptII b b a
%Aggregated Cells
70
b
60 a
b a
50 b
a
40 a a
a
30 a
a
20 a
10
0
algU::nptII wild-type 0 3 6 9 12 15 18 21 24
Days
5 b
4.5 algU::nptII b
WT
4
3.5 b a
a
OD650nm
3
a
2.5 a
2
a a
1.5
a
1
0.5
0
algU::nptII wild-type 0 3 6 9 12 15
Days
Shi, X.Y., et al., Appl Environ Microbiol. 2007, 73(21): 6748–6756.

4. wild-type algU::nptII Water
7
algU::nptII b
6 b b
WT
Disease development of algU-
Disease severity
5
a
i) late appearance of leaf scorch 4
a
ii) slower disease progression 3 a a
a
2 a
a
1
0
4 8 12 16 20
Weeks
wild-type algU::nptII
Bacterial population in grapevines
Wild type
12
b algU::nptII
10
C F U /g tissu e (x10 6 )
Water
8
6
b
4
a b
2
a
0 a
Inoculated point 25cm 50cm
Samples site

5. Genes differentially expressed in X. fastidiosa algU- in vitro
Expression level
ORF Gene Description
in wild type
PD1926 pilus assembly fimbrial protein down
PD0095 rsmA RsmA homologue-regulate virulence determinants up
PD0066 hfq host factor-I protein, ubiquitous RNA-bing protein up
PD0347 algS sugar ABC transporter ATP-binding protein up
PD0120 algC Phosphomannomutase up
PD1153 algR two-component system, regulatory protein up
PD1276 algH transcriptional regulator up
PD1286 mucD periplasmic protease up
PD1709 mopB outer membrane protein up
PD1807 ompW outer membrane protein up
PD1065 SecB Type II secretion system-Preprotein translocase up
PD0664 clpS ATP-dependent Clp protease adaptor protein up
PD0665 clpA ATP-dependent Clp protease subunit up
PD1685 clpB ATP-dependent Clp protease subunit up
PD1371 grpE heat shock protein GrpE up
PD1370 dnaK heat shock protein-Hsp70 up
PD0750 gltA citrate synthase-Energy production and conversion up
PD1672 bfr Bacterioferritin up
PD0216 cvaC d colicin V antibacterial polypeptides toxin up
Phenotypic characteristics of gacA-
90 b
A05
80
DAC1984 b
% Aggregated Cells
70 b
60 b
50 b
b a
40
b
30 a
a
20 b
a
a
10 a
a a
gacA∆Gm wild-type 0
0 3 6 9 12 15 18 21 24
Days
4.5 b
b
4 DAC1984
A05
3.5 b
3
OD650nm
2.5
2
a
a a
a
1.5 a
a
1 a
0.5
0
wild-type gacA∆Gm 0 3 6 9 12 15
Days
Shi, X.Y., et al., Appl Environ Microbiol. 2008. Submitted.

6. Disease development of gacA-
i) late appearance of leaf scorch
ii) slower disease progression
wild-type gacA∆Gm Water
7
b
DAC1984 b b
6 A05
Disease Severity
5
4
a a
3 a
a
a
2
a a
1
0
4 8 12 16 20
Weeks
wild-type gacA∆Gm
Bacterial population in grapevines
Wild type
12
b gacA∆Gm
10 Water
CFU/g tissue (10 )
6
8
6
b
4
b
2 a
a a
0
Inoculated point 25cm 50cm
Samples sites

7. Genes differentially expressed in X. fastidiosa gacA- in vitro
Expression level
ORF Gene Description
in wild type
PD1926 pilus assembly fimbrial protein up
PD0731 XadA outer membrane protein-Autotransporter adhesin up
PD0744 hsf surface protein-Autotransporter adhesin up
PD1295 putative membrane protein-unknown function up
PD0264 oprO porin O precursor. up
PD1294 actP acetate permea-cotranscribed with the acs gene up
PD0216 cvaC d colicin V precursor-antibacterial polypeptides toxin up
PD0215 cvaC colicin V precursor-antibacterial polypeptides toxin down
PD1395 gumC protein involved in exopolysaccharide biosynthesis down
PD0243 Conserved Domains-Membrane-fusion protein down
PD1688 bioI cytochrome P450-like enzyme down
PD2039 Oxidoreductase-Putative multicopper oxidases down
PD1703 Conserved Domains: Lysophospholipase down
PD1702 Conserved Domains: LIP, Secretory lipase down
PD0244 Conserved Domains-acriflavin resistance protein down
PD0956 V8-like Glu-specific endopeptidase down
PD1299 Conserved Domains-polyvinylalcohol dehydrogenase down
PD0521 unknown down
PD0657 unknown down
PD0743 unknown down
PD0955 unknown down
PD0911 phage-related proteins, unknown function down
PD0912 phage-related proteins, unknown function down
PD0917 phage-related proteins, unknown function down
PD0924 phage-related proteins, unknown function down
PD0925 phage-related proteins, unknown function down
PD0930 phage-related proteins, unknown function down
Overlap genes in regulatory pathways
RsmA
Heat shock: DnaJ, HspA
Heat shock Metabolism: 34 genes
Unknown: 12 genes
Type II-SecB
RpmD
DnaK, GrpE,ClpB ppa
SecB Unknown
AlgU GacA Pilus- fimbrial
Posttranscription: RsmA, Hfq Attachment
CvaC Attachment: GumC, XadA
Attachment: AlgS, AlgS AlgR, Fimbrial Hsf
MucD, MopB, OmpW Unknown
Toxin: CvaC Toxin: CvaC
Heat shock: ClpS, ClpA Unknown
Metabolism: 12 genes Metabolism: 9 genes
Unknown: 9 genes Unknown: 10 genes

8. Differential gene expression of Xylella
fastidiosa in different xylem fluids
• Aggregation and biofilm formation of Xf were
differentially influenced by the xylem fluid of citrus vs.
grape
• Grape fluid stimulated the expression of genes
predicted to be involved in virulence, attachment,
biofilm formation, and twitching motility of Xf within
xylem
• Citrus may be resistant or tolerant to the PD strain of
Xf, in part, because citrus xylem fluid does not
support the induction of a number of virulence genes,
or has substances that repress expression.
Growth of PD strain of Xylella fastidiosa in
xylem fluid of grape and citrus

9. Effect of xylem fluid on X. fastidiosa
aggregation
Differential expression of genes of X. fastidiosa in grape fluid compared with citrus fluid
Expression
Gene ID NAME HYPOTHETICAL FUNCTION Grape/Citrus P Value
in Grape
PD1905 xrvA Virulence regulator 1.8 1.2E-03 Higher
PD2040 acvB Virulence protein 1.9 3.4E-03 Higher
PD0066 hsq RNA-binding protein 2.7 1.5E-03 Higher
PD1984 gacA Two-component regulator 1.8 3.0E-04 Higher
PD1284 algU RNA polymerase sigma-H factor 1.8 1.6E-03 Higher
PD1276 algH Transcriptional regulator 1.6 1.5E-03 Higher
PD0268 agmR Transcriptional regulator (luxr/uhpa family) 1.9 5.2E-03 Higher
PD1738 gcvR Transcriptional regulator 1.6 2.4E-03 Higher
PD1920 colS Two-component system, sensor protein 1.6 1.4E-03 Higher
PD0019 fimT Pre-pilin like leader sequence 2.0 1.8E-03 Higher
PD0062 - Fimbrial subunit precursor 2.6 1.1E-03 Higher
PD0846 pilI Pilus biogenesis protein 1.8 4.5E-03 Higher
PD1147 pilT Twitching motility protein 1.6 3.2E-03 Higher
PD1148 pilU Twitching motility protein 1.8 1.5E-03 Higher
PD1611 pilY1 Fimbrial assembly protein 2.3 2.1E-04 Higher

10. Differential expression of genes of X. fastidiosa in grape fluid compared with citrus fluid
Expression in
Gene ID NAME HYPOTHETICAL FUNCTION Grape/Citrus P Value
Grape
PD0312 - Outer membrane protein precursor 2.5 1.2E-03 Higher
PD0731 xadA Outer membrane protein 2.3 4.5E-03 Higher
PD0744 hsf Surface protein 1.7 3.4E-03 Higher
PD0757 pcp Peptidoglycan-associated outer membrane 1.6 1.7E-03 Higher
lipoprotein precursor
PD0182 secD Protein-export membrane protein 2.1 1.4E-03 Higher
PD0246 secG Protein-export membrane protein 2.4 1.2E-03 Higher
PD1280 hspA Low molecular weight heat shock protein 2.3 1.8E-03 Higher
PD0472 clpP ATP-dependent Clp protease proteolytic subunit 1.6 1.1E-03 Higher
PD0859 msrA Peptide methionine sulfoxide reductase 1.6 2.1E-03 Higher
PD1536 cutA Periplasmic divalent cation tolerance protein 1.8 1.6E-03 Higher
PD1475 ccmA Heme ABC transporter ATP-binding protein 1.8 4.1E-03 Higher
PD2031 - Hypothetical protein 2.5 1.2E-03 Higher
PD0008 - Hypothetical protein 1.8 1.2E-03 Higher
PD1741 - Hypothetical protein 2.0 1.7E-04 Higher
PD1485 pglA Polygalacturonase precursor -1.7 1.8E-03 Lower
PD0143 - Hemolysin III protein -1.6 1.7E-03 Lower
RT-PCR validation of macroarray data
Fig. 3. Reverse transcription polymerase chain reaction (RT-PCR) of differentially expressed
genes of X. fastidiosa in xylem fluid of citrus and grape. Water was the control.

11. Benefits of identifying regulatory
pathways in Xylella fastidiosa
• Regulatory pathways as targets for control
strategies
• Identification of virulence genes as targets
for control strategies
• Understanding host range determination