GENE EXPRESSION PROFILE OF PATHOGENICITY FACTORS IN Xylella fastidiosa.R. HERNANDEZ-MARTINEZ, C. K. Dumenyo, D. A. Cooksey.

Department of Plant Pathology, University of California, Riverside, CA 92521.

GENE EXPRESSION PROFILE OF PATHOGENICITY FACTORS IN Xylella fastidiosa.R. HERNANDEZ-MARTINEZ, C. K. Dumenyo, D. A. Cooksey.

Department of Plant Pathology, University of California, Riverside, CA 92521.

ABSTRACT

Xylella fastidiosa (Xf) strains cause several economically important plant diseases including, Pierce's disease on grape (PD), citrus variegated chlorosis (CVC), and oleander leaf scorch (OLS). Although the whole genomic sequence of the CVC strain has been published, no functional characterization of Xfgenes has been performed. To identify genes involved in pathogenicity in the PD strain, the sequence of the CVC strain was used to select open reading frames specifying putative pathogenicity and virulence factors. DNA fragments of these genes were obtained by PCR amplification from the genome of the PD strain I03. In this study, we have constructed macroarrays for the analysis of the expression profile of select candidate pathogenicity genes of Xf and to study their expression in PD3 medium. We have show that these genes are expressed to varying degrees ranging from none to very high. These arrays will be used to analyze the gene expression profile of different Xylella strains in planta and in vitro in order to understand bacterial adaptation to varied environmental conditions thus providing an insight into the disease process.

ABSTRACT

Xylella fastidiosa (Xf) strains cause several economically important plant diseases including, Pierce's disease on grape (PD), citrus variegated chlorosis (CVC), and oleander leaf scorch (OLS). Although the whole genomic sequence of the CVC strain has been published, no functional characterization of Xfgenes has been performed. To identify genes involved in pathogenicity in the PD strain, the sequence of the CVC strain was used to select open reading frames specifying putative pathogenicity and virulence factors. DNA fragments of these genes were obtained by PCR amplification from the genome of the PD strain I03. In this study, we have constructed macroarrays for the analysis of the expression profile of select candidate pathogenicity genes of Xf and to study their expression in PD3 medium. We have show that these genes are expressed to varying degrees ranging from none to very high. These arrays will be used to analyze the gene expression profile of different Xylella strains in planta and in vitro in order to understand bacterial adaptation to varied environmental conditions thus providing an insight into the disease process.

Select candidate genes from the genome

Design primers to amplify the coding region of the gene (s) with a product size of 200 bp-664bp

PCR Amplify the target region from the Xf genomic DNA template and gel analysis of the product.

Purify the amplified DNA with PCR cleanup kit (Millipore Corp.)

Design the array layout by randomization. Each gene had at least three replicates

Spot the DNA onto Nylon (Amersham) membrane

Hybridize membranes with 33P-labeled cDNA from the total RNA of Xf strain I03 for 72 hr.

Wash membrane, expose, develop and analyze the data

Extract total RNA from Xf strain I03 grown in PD3 medium for 12 days.

FLOW CHART OF THE STEPS FOLLOWED IN THIS STUDY

Select candidate genes from the genome

Design primers to amplify the coding region of the gene (s) with a product size of 200 bp-664bp

PCR Amplify the target region from the Xf genomic DNA template and gel analysis of the product.

Purify the amplified DNA with PCR cleanup kit (Millipore Corp.)

Design the array layout by randomization. Each gene had at least three replicates

Spot the DNA onto Nylon (Amersham) membrane

Hybridize membranes with 33P-labeled cDNA from the total RNA of Xf strain I03 for 72 hr.

Wash membrane, expose, develop and analyze the data

Extract total RNA from Xf strain I03 grown in PD3 medium for 12 days.

FLOW CHART OF THE STEPS FOLLOWED IN THIS STUDY

Strain and RNA Extraction

Total RNA was extracted from strain I03 which was isolated from a symptomatic,PD affected grapevine in the Temecula Valley of California

Bacterial cells were inoculated into 25 ml of PD3 medium and grown for 12 days at28°C on a shaker. RNA was extracted by hot phenol extraction method.

Genomic DNA was eliminated from the RNA preparation by digestion with RNase-Free DNase I (Epicentre) for 1 hour. RNA quality was evaluated on 1% denaturingagarose gels and concentration was determined by absorbance at 260 nm

Strain and RNA Extraction

Total RNA was extracted from strain I03 which was isolated from a symptomatic,PD affected grapevine in the Temecula Valley of California

Bacterial cells were inoculated into 25 ml of PD3 medium and grown for 12 days at28°C on a shaker. RNA was extracted by hot phenol extraction method.

Genomic DNA was eliminated from the RNA preparation by digestion with RNase-Free DNase I (Epicentre) for 1 hour. RNA quality was evaluated on 1% denaturingagarose gels and concentration was determined by absorbance at 260 nm

The selected ORFs wereblasted against the draftsequences of Oleander(Ann1) and Almond(Dixon) strains availableat the DOE-Joint GenomeInstitute web site Theprimers were designedusing the sequence ofDixon since it is theclosest to PD strainsphylogenetically

Construction of Macroarrays

DNA macroarrays were constructed with ORFs selected from the Xylella fastidiosaCVC genome sequence. The genes for the arrays were selected to include putativevirulence and pathogenicity genes based on the sequence annotations and functionsdescribed previously in other organisms. Other genes were also included as controls. 

Internal regions of the selected genes were amplified by PCR with products rangingbetween 200 to 600 bp in length. Product purification was done using PCR96

Cleanup kit (Millipore Corp.)

Agarose gel of purified PCR fragments to be spotted on the membrane.

The selected ORFs wereblasted against the draftsequences of Oleander(Ann1) and Almond(Dixon) strains availableat the DOE-Joint GenomeInstitute web site Theprimers were designedusing the sequence ofDixon since it is theclosest to PD strainsphylogenetically

Construction of Macroarrays

DNA macroarrays were constructed with ORFs selected from the Xylella fastidiosaCVC genome sequence. The genes for the arrays were selected to include putativevirulence and pathogenicity genes based on the sequence annotations and functionsdescribed previously in other organisms. Other genes were also included as controls. 

Internal regions of the selected genes were amplified by PCR with products rangingbetween 200 to 600 bp in length. Product purification was done using PCR96

Cleanup kit (Millipore Corp.)

Agarose gel of purified PCR fragments to be spotted on the membrane.

Purified products were transferred to 384-well microtiter plates for arraying. Twenty ng of total Xf genomic DNA, Lambda DNA and water were included as controls.

Purified PCR products were arrayed in triplicate onto positively charged 11 x15 cm nylon membranes (Hybond-Amersham) using a manual 384-pin replicator (V&P Scientific).

DNA on the spotted membranes was denatured with 0. 4 M NaOH, neutralized with 3X SSPE, UV cross-linked for 7 min and boiled in 0.1% of SDS for 5min.

384-well microtiter plate and 384-pin replication used for spotting DNA into membranes.

Purified products were transferred to 384-well microtiter plates for arraying. Twenty ng of total Xf genomic DNA, Lambda DNA and water were included as controls.

Purified PCR products were arrayed in triplicate onto positively charged 11 x15 cm nylon membranes (Hybond-Amersham) using a manual 384-pin replicator (V&P Scientific).

DNA on the spotted membranes was denatured with 0. 4 M NaOH, neutralized with 3X SSPE, UV cross-linked for 7 min and boiled in 0.1% of SDS for 5min.

384-well microtiter plate and 384-pin replication used for spotting DNA into membranes.

DNA Macroarray Hybridization

After an hour of prehybridization, the membrane was hybridized at 45 °C in DIG Easy Hyb solution (Roche) in a rotisserie oven with the 33P-labeled cDNAprobes for 72 hr. Hybridized membranes were washed 5 min using 3X SSC, 0. 1% SDS at room temperature followed by 3X SSC, 0. 1% SDS at 45 °C.

After washing, membranes were exposed to a phosphor storage screen (Kodak) for 48 h and image data were captured on the Molecular Dynamics phosphor imager (Biorad). Data were obtained using the Quantity one®software (Biorad) and transferred to Microsoft Excel for further analysis

Autoradiograph of nylon filter macroarray probed with label cDNA from Xf.

DNA Macroarray Hybridization

After an hour of prehybridization, the membrane was hybridized at 45 °C in DIG Easy Hyb solution (Roche) in a rotisserie oven with the 33P-labeled cDNAprobes for 72 hr. Hybridized membranes were washed 5 min using 3X SSC, 0. 1% SDS at room temperature followed by 3X SSC, 0. 1% SDS at 45 °C.

After washing, membranes were exposed to a phosphor storage screen (Kodak) for 48 h and image data were captured on the Molecular Dynamics phosphor imager (Biorad). Data were obtained using the Quantity one®software (Biorad) and transferred to Microsoft Excel for further analysis

Autoradiograph of nylon filter macroarray probed with label cDNA from Xf.

CONCLUSIONS

1. Conditions used in the experiment allowed the detection of gene expression levels in Xf.2. Some Xylella genes express in PD3 medium and others do not 3. Genes that express do so at different levels ranging from very high to very low.4. Most of the highly expressed genes have functions in cell surface properties.

CONCLUSIONS

1. Conditions used in the experiment allowed the detection of gene expression levels in Xf.2. Some Xylella genes express in PD3 medium and others do not 3. Genes that express do so at different levels ranging from very high to very low.4. Most of the highly expressed genes have functions in cell surface properties.

At the moment there is no known cure or treatment for any of the diseases caused by Xf. The recent appearance of the more efficient glassy-winged sharpshooter insect vector in California has turned the problem of Pierce’s disease of grapes into a major one.

A couple of years ago, the genomic sequence of the CVC strain of Xf was published and the draft sequences of almond, grape and oleander strains are also available for BLAST (http://www.igi.doe.gov/JGI_microbial/html/index.html, http://onsona.lbi.ic.unicamo.br/world/xf-grape/blast/blast.html). The genomic sequence reveals numerous novel candidate pathogenicity factors such as extracellular polysaccharide gum, hydrolytic enzymes and bacterial surface factors. Up till now, non of the predicted pathogenicity genes has been tested experimentally because of the lack of molecular tools for the genetic analysis of the bacterium.

Our ultimate goal is to identify the pathogenicity genes of this bacterium and to elucidate the mechanisms by which Xf causes disease in its hosts, especially grape. As a first step towards this goal, we have developed macroarrays to study the expression of select candidate pathogenicity genes in the grape strains of the bacterium based on the available Xf genomic sequences.

Xylella fastidiosa (Xf) is a fastidious, xylem-limited, non-flagellated, insect-transmitted Gram-negative plant pathogenic bacterium. Various strains of the bacterium cause many diseases on over twenty plant hosts including Pierce’s disease (PD) of grapes, oleander leaf scorch (OLS), almond leaf scorch (ALS) and citrus variegated chlorosis (CVC).

Although the exact mechanisms of pathogenicity are not known, it has been suggested that, at least in some hosts, the growth and aggregation of the bacterium clog up the plants’ vascular systems leading to wilting and death

INTRODUCTION

Devastation of grapevine by Pierce’s disease

At the moment there is no known cure or treatment for any of the diseases caused by Xf. The recent appearance of the more efficient glassy-winged sharpshooter insect vector in California has turned the problem of Pierce’s disease of grapes into a major one.

A couple of years ago, the genomic sequence of the CVC strain of Xf was published and the draft sequences of almond, grape and oleander strains are also available for BLAST (http://www.igi.doe.gov/JGI_microbial/html/index.html, http://onsona.lbi.ic.unicamo.br/world/xf-grape/blast/blast.html). The genomic sequence reveals numerous novel candidate pathogenicity factors such as extracellular polysaccharide gum, hydrolytic enzymes and bacterial surface factors. Up till now, non of the predicted pathogenicity genes has been tested experimentally because of the lack of molecular tools for the genetic analysis of the bacterium.

Our ultimate goal is to identify the pathogenicity genes of this bacterium and to elucidate the mechanisms by which Xf causes disease in its hosts, especially grape. As a first step towards this goal, we have developed macroarrays to study the expression of select candidate pathogenicity genes in the grape strains of the bacterium based on the available Xf genomic sequences.

Xylella fastidiosa (Xf) is a fastidious, xylem-limited, non-flagellated, insect-transmitted Gram-negative plant pathogenic bacterium. Various strains of the bacterium cause many diseases on over twenty plant hosts including Pierce’s disease (PD) of grapes, oleander leaf scorch (OLS), almond leaf scorch (ALS) and citrus variegated chlorosis (CVC).

Although the exact mechanisms of pathogenicity are not known, it has been suggested that, at least in some hosts, the growth and aggregation of the bacterium clog up the plants’ vascular systems leading to wilting and death

INTRODUCTION

Devastation of grapevine by Pierce’s disease

cDNA synthesis and labeling conditions

Radiolabelled cDNA probes were prepared using ORF-specific oligonucleotideprimers, AMV Reverse Transcripase (Promega), -33P dCTP and 20 µg of total RNAfrom I03 according to manufacturer recommendations. Following inactivation ofreverse transcriptase, cDNA probes were purified using Sephadex G-50 spin columns(Amersham Pharmacia).

cDNA synthesis and labeling conditions

Radiolabelled cDNA probes were prepared using ORF-specific oligonucleotideprimers, AMV Reverse Transcripase (Promega), -33P dCTP and 20 µg of total RNAfrom I03 according to manufacturer recommendations. Following inactivation ofreverse transcriptase, cDNA probes were purified using Sephadex G-50 spin columns(Amersham Pharmacia).

SIGNAL: 0 -1000

SIGNAL: 1001 -2000

SIGNAL: MORE THAN 3000

SIGNAL BELOW 0

SIGNAL: 2001 – 3000

PCR PRODUCT SIZE

GENE NUMBER GENE NAME GENE FUNCTION

C0RRECTED MEAN

602 XF0007 None Hypothetical protein 1703

558 XF0028 fimT Pre-pilin like leader sequence 3

576 XF0032 pilY1 Pily1 gene product 24

575 XF0043 HI0441 Conserved hypothetical protein -81

599 XF0073 ffh Signal recognition particle protein 188

603 XF0077 mrkD Fimbrial adhesin precursor 1669

597 XF0081 fimD Outer membrane usher protein precursor 665

528 XF0083 F17A-A Fimbrial subunit precursor 25088

201 XF0125 csrA Carbon storage regulator 7004

370 XF0131 None Hypothetical protein 387

608 XF0132 copA Copper resistance protein A precursor 39

606 XF0175 None Hemolysin III protein 178

601 XF0225 secD Protein-export membrane protein 78

638 XF1851 None Serine protease 327

602 XF0285 hrA, degP or ptd Heat shock protein -6

603 XF0287 rpfB Regulator of pathogenicity factors 68

604 XF0290 rpfA Aconitase 989

601 XF0384 phur Outer membrane hemin receptor -102

598 XF0432 brk Brkb protein -23

604 XF0478 pilY1 Fimbrial assembly protein 4

365 XF0479 pilE Type IV pilin 15

640 XF0506 vapE Virulence-associated protein E -156

618 XF0562 tatC or mttB Sec-independent protein translocase 229

620 XF0591 none Virulence factor 152

322 XF0619cutA, cycY or cutA1 Periplasmic divalent cation tolerance protein 107

604 XF0620 dsbD C-type cytochrome biogenesis protein (Cu tolerance) 125

601 XF2759 frpC Hemolysin-type calcium binding protein -170

348 XF0677 pilZ Type 4 f imbriae assembly protein 5125

518 XF0720 higB Proteic killer active protein 2413

361 XF0749 xrvA Virulence regulator 14

608 XF0754 acvB Virulence protein 122

593 XF0781 estA Lipase/esterase 0

595 XF0810 engXCA Extracellular endoglucanase precursor 33

621 XF0818 engXCA Endo-1,4-beta-glucanase 393

609 XF0845 xylA Family 3 glycoside hydrolase -75

575 XF0858 surE Survival protein -11

636 XF0866 czcD Cobalt-zinc-cadmium resistance protein -32

682 XF0889 pspA Hemagglutinin-like secreted protein -37

589 XF0911 sspA, ssp or pog Stringent starvation protein A -38

595 XF0972 agmR or glpR Transcriptional regulator (luxr/uhpa family) 50

408 XF1013 b0817 Conserved hypothetical protein -211

482 XF1020 none Pathogenicity-related protein 731

304 XF1024 none Outer membrane protein H.8 precursor 1846

628 XF1114 rpfC Regulator of pathogenicity factors 27

613 XF1115 rpfF Regulator of pathogenicity factors 436

603 XF1182 act Lipase modulator 299

595 XF1253 lipP Lipase 225

598 XF1267 cbhA 1,4-beta-cellobiosidase -6

568 XF1341 cutC Copper homeostasis protein 738

601 XF1368 HI1201 Adenine-specif ic methylase 118

632 XF1379 y4wF Luciferase 941

620 XF1408 rpoN RNA polymerase sigma-54 factor 603

598 XF1424 chi Chitinase 826

377 XF1493 xrvA Virulence regulator 414

581 XF1516 uspA1 Surface-exposed outer membrane protein 15

595 XF1517 xpsE or pefE General secretory pathw ay protein E 223

600 XF1529 hsf Surface protein 466

430 XF1547 pcp or lpp Peptidoglycan-assoc.outer mem. Lipoprot. Prec. 4755

603 XF1623 mdoH Periplasmic glucan biosynthesis protein 219

594 XF1625 algZ Tw o-component system, sensor protein 4

586 XF1632 pilU Tw itching motility protein 680

600 XF1804 sphIM Site-specif ic DNA-methyltransferase 438

591 XF1913 mttC Type V secretory pathw ay protein 388

608 XF1954 pilI Pilus biogenesis protein 51

694 XF1987 vacB Vacb protein 213

558 XF2228 algH Transcriptional regulator 2241

450 XF2234 hspA Low molecular w eight heat shock protein 3474

608 XF2239 algU or algT RNA polymerase sigma-H factor 1274

590 XF2370 gumB Gumb protein -90436 XF2392 lyc Autolytic lysozyme 261

598 XF2397 hlyB Toxin secretion ABC transporter ATP-binding protein 92

570 XF2407 none Bacteriocin 498

605 XF2420 mviN Virulence factor -25

280 XF1151rpsJ, rps10 or HI0776 30S ribosomal protein S10 15957

506 XF2455 ccmA Heme ABC transporter ATP-binding protein -37

580 XF2466 pglA Polygalacturonase precursor 144

625 XF2535 colS Tw o-component system, sensor protein 457

606 XF2538 pilC Fimbrial assembly protein -43

412 XF2539 None Fimbrial protein 3107

616 XF2544 pilB Pilus biogenesis protein 770

608 XF2545 pilR Tw o-component system, regulatory protein 155

591 XF2546 pilS Tw o-component system, sensor protein -50

584 XF2550 hecB Outer membrane hemolysin activator protein 394

598 XF2608 gacA Transcriptional regulator (luxr/uhpa family) 1317

246 XF2622 tapB Temperature acclimation protein B 47837

641 XF2625 htpX Heat shock protein 334

664 XF2679 acvB Virulence protein 28

598 XF2682 mdoG Periplasmic glucan biosynthesis protein 673

602 XF2708 egl Endo-1,4-beta-glucanase 602

602 XF2714 fucA1 Alpha-L-fucosidase 343

557 XF1940 msrA or pms Peptide methionine sulfoxide reductase 1844

589 XF2385 yegN Acrif lavin resistance protein D -69

536 XF1127 tldD Tldd protein 132

609 XF0960 Hypothetical protein 48

586 XF0833 cysB or HI1200 Transcriptional regulator (lysr family) -124

437 XF1490 none Transcriptional regulator (marr/emrr family) 90

586 XF1633 pilT Tw itching motility protein 1094

595 XF0122 lexa Lexa repressor 29

607 XF0223 tgt or vacC Queuine trna-ribosyltransferase 114

602 XF0286 sula Cell division inhibitor 43

366 XF0304 secG or HI0445 Protein-export membrane protein 236

614 XF0785 sac1 Sulfur deprivation response regulator 62

588 XF0837Imp, ostA or HI0730 Organic solvent tolerance precursor 16

596 XF0840 bga Beta-galactosidase 263

605 XF0962 gcvR Transcriptional regulator 932

568 XF1187 clpP or lopP ATP-dependent Clp protease proteolytic subunit 11101

600 XF1189lon, capR , deg, muc or LopA ATP-dependent serine proteinase La 4089

600 XF1858 exsb Transcriptional regulator 1029

430 XF2085 SCI30A.12c Transcriptional regulator (acrr family) -137

558 XF2165 tex Transcription-related protein 447

REFERENCES1. Davis, M. J.; A. H. Purcell and S. V. Thompson. 1980. Phytopathology. 70:425-429. 2. Simpson A. J. G.; Reinach F. C.; Arruda P.; et al. 2000. Nature 406: 151-1573. Dow, J. M.; Daniels, M. J. 2000. Yeast. 17 (4): 263-271.4. Lambais, M. R.; Goldman, M. H. S.; Camargo, L. E. A.; Goldman, G. H.  2000.  Current Opinion in Microbiology. 3 (5): 459-462. 5. Keen, N.T.; Dumenyo, C K.; Yang, C-H; Cooksey, D. A. 2000. Genome Biology. 1 (3): 1019.1-1019.4

ACKNOWLEDGEMENTSHamid Azad, Dept. Plant Pathology, UCR; James Borneman, Dept. Plant Pathology, UCR; Carmen Gispert, UC Cooperative ExtensionEd Raetz, Dept. Entomology/Plant Pathology, UCR; Heather Costa, Dept. Entomology; Blake Bextine, Dept. Entomology

REFERENCES1. Davis, M. J.; A. H. Purcell and S. V. Thompson. 1980. Phytopathology. 70:425-429. 2. Simpson A. J. G.; Reinach F. C.; Arruda P.; et al. 2000. Nature 406: 151-1573. Dow, J. M.; Daniels, M. J. 2000. Yeast. 17 (4): 263-271.4. Lambais, M. R.; Goldman, M. H. S.; Camargo, L. E. A.; Goldman, G. H.  2000.  Current Opinion in Microbiology. 3 (5): 459-462. 5. Keen, N.T.; Dumenyo, C K.; Yang, C-H; Cooksey, D. A. 2000. Genome Biology. 1 (3): 1019.1-1019.4

ACKNOWLEDGEMENTSHamid Azad, Dept. Plant Pathology, UCR; James Borneman, Dept. Plant Pathology, UCR; Carmen Gispert, UC Cooperative ExtensionEd Raetz, Dept. Entomology/Plant Pathology, UCR; Heather Costa, Dept. Entomology; Blake Bextine, Dept. Entomology