further assessments of the large diversity within this species

(1)

15

Xanthomonas albilineans is the causal agent of leaf scald, a lethal disease of sugarcane

Leaf scald symptoms, including white foliar stripes

and bleaching caused by albicidin that blocks chloroplast differentiation

The complete genome of Xanthomonas albilineans provides insights into pathogenicity of this sugarcane pathogen and allows

further assessments of the large diversity within this species

M. Muller P. Rott P. Rott

white foliar stripes bleaching necrosis

Xanthomonas albilineans, unlike other xanthomonads, is a xylem- limited pathogen which:

INTRODUCTION and OBJECTIVES

10 genetic groups identified by Pulsed Field Gel Electrophoresis (PFGE) [3]

Xanthomonas albilineans strains show a high genetic diversity:

(1) Pieretti et al. 2009. BMC Genomics, 10:616.

(2) Marguerettaz et al. 2010. Molecular Plant-Microbe Interactions, 24:246-259.

(3) Davis et al. 1997. Phytopathology,87:316-324.

REFERENCES

produces the toxin albicidin, a potent DNA gyrase inhibitor

possesses a T3SS of the SPI-1 family [1-2]

dendrogram

MLSA PFGE

group RFLP *

haplotype

experienced a genome erosion, lacks the gum gene cluster (xanthan) and the T3SS of the Hrp injectisome families [1]

All strains involved in disease outbreaks since the late 1980s and reported in several locations belong to the same genetic group, called PFGE-B. We used the genome sequence of strain GPE PC73 to describe specific pathogenicity-related features of X. albilineans and to further investigate the large diversity of this species.

X. albilineans encodes 5 cell wall degrading enzymes which harbour a long linker and a cellulose binding domain (CBD) at their C-termini which suggests that X. albilineans is adapted to the utilization of cell breakdown products as a carbon source:

D

Y S ?1

T

G ?2 N N Y N N

?1S T ?2TE TETE

META-B:

XALc_1058 XALc_1057 XALc_1056 XALc_1055

IS

13 14 15 16 1 2 3 4 5 6 7 8 9 101112

S S N

Dab T V F Dab?1?3 YTE

META-A:

XALc_1550 XALc_1551

1 2

DaT StaM AraC

³ ⁷ ⁸ ⁹ ^{10 11}¹²

HpgT DpgC DpgB DpgA DpgD

ABC MbtH

NRPS

4 5 6

NRPS

XALc_1066 XALc_1065 XALc_1064 XALc_1063 XALc_1062 XALc_1061 XALc_1060 XALc_1059 XALc_1054 XALc_1052

XALc_1053

IS

IS META-C: ^?4^F^Dab^N ^A^?3^Q^TE

XALc_0772

1 2 3 4 5 6 7

XALB1:

albicidin biosynthesis gene cluster (3 NRPS genes previously described)

SSH clones

selected for analysis of genetic diversity X. albilineans encodes 10 large non ribosomal peptide synthetases genes (NRPS) grouped in 4 loci:

Large inter-strain variability in X. albilineans was confirmed using multi-locus sequence analysis (MLSA), spacer analysis of one clustered regularly interspaced short palindromic repeats (CRISPR1) and analysis of the diversity of suppression subtractive hybridization (SSH) markers

(1) CIRAD, UMR BGPI, F-34398 Montpellier Cedex 5, France. (2) Génoscope, Centre national de séquençage, F-91057 Evry Cedex, France. (3) INRA, UMR LIPM, F-31326 Castanet-Tolosan Cedex, France. (4) IRD, UMR RPB, F-34394 Montpellier cedex 5, France. (5) INRA, UMR PaVé, F-49071 Beaucouzé, France. (6) CNRS, UMR LIPM, F-31326 Castanet-Tolosan Cedex, France. (7) Agrocampus Ouest centre d'Angers, UMR PaVé, F-49071 Beaucouzé, France. (8) Université Paul Sabatier, UMR LIPM, F-31326 Castanet-Tolosan Cedex, France. (9) University of Florida, Plant Pathology Department, Gainesville 32605, U.S.A.

Isabelle Pieretti1, Stéphane Cociancich1, Valérie Barbe2, Sébastien Carrere3, Ralf Koebnik4, Patrice Champoiseau1, Arnaud Couloux2, Armelle Darrasse5, Jérôme Gouzy3, Marie-Agnès Jacques5, Emmanuelle Lauber6, Charles Manceau5, Sophie Mangenot2, Mélanie Marguerettaz1, Stéphane Poussier7, Béatrice Segurens2, Boris Szurek4, Valérie Verdier4, Matthieu Arlat8, Dean W Gabriel9, Philippe Rott1, Monique Royer1

contact: pieretti@cirad.fr

RESULTS

CONCLUSION AND PERSPECTIVES

5th European Conference on Prokaryotic and Fungal Genomics

September 18–21, 2011 Göttingen/Germany

*

: RFLP-HB profile was experimentally shown to be related to the presence and expression in these strains of the XamI methyltransferase (XALc_2634)

no data gene present gene absent Endoglucanase - EngXCA

XALc_2969 XALc_2967 Cellobiosidase - CbhA

XALc_0484 Cellulase - CelS

XALc_0865 Putative cell wall degrading enzyme

XALc_0874 134 152

CBD

62 96

26 CBD

CBD

CBD CBD Aa

Aa

X. albilineans is a highly distinctive xanthomonad harbouring specific cell wall degrading enzymes with long linkers and CBD suggesting its adaptation for the degradation of cell-wall breakdown products present in the xylem of sugarcane. NRPS genes are good candidates to produce small molecules potentially involved in interactions with host sugarcane cells and these molecules are currently being identified and characterized. MLSA analysis, coupled to analysis of SSH markers and spacer content in CRISPR1, revealed a large inter-strain variability in X. albilineans.

No pathogeniticy-related gene specific to PFGE-B strains was isolated by SSH. Further analysis of the XamI methyl- transferase will be necessary to investigate its putative role in pathogenicity of PFGE-B strains associated with sugarcane leaf scald disease outbreaks.

OUTGROUP HVO005 GPE PC73 XaFL07-1 MTQ078 GLP056 BRA115 TWN052 LKA070 REU173 REU174

HVO082 REU209

PNG130 Xa23RI MTQ058 FIJ080

PFGE-F

PFGE-H PFGE-G

PFGE-A PFGE-J PFGE-D PFGE-C

PFGE-I PFGE-E PFGE-D PFGE-B PFGE-B PFGE-B PFGE-B PFGE-B PFGE-B 84

52 99

99 67

98 86

100 95

97

HB HA7

HB HB2 HB2 HB1 HB3 HA4 HA6 HA6

HA2 HA2

HA10 HA1 HA9 HA8

Name Accession Linker size in amino acid (Aa)

CRISPR1

spacer distribution

5 4 3 2 1

5 4 3 2 1 35 34 X X 31 30 29 28 27 26 25 24 23 22 21 20 18 17 16 15 14 13 12 X 10 9 8 7 6

36 35 34 X X 31 30 29 28 27 26 25 24 23 22 ? ? ? ? 17 16 15 14 13 12 11 10 9 8 7 6

8 7 6 5 4 3 2 1

20 20 19 X X X X X X X X X X X X X X X X X X X X 9 8 7 6 3 4 3 X 1 36 35 34 X X 31 30 29 28 27 26 25 24 23 22 ? ? ? ? ? 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1

15 14 13 12 11 10 9 8 7 6 5 X X 2 1

28 27 26 25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 35 34 33 32 31 30 29 28 27 26 25 24 23 22 ? ? ? ? ? ? ? ? ? ? 11 X 9 8 7 6 5 4 3 2 1

4 3 2 4 3 2

4 3 2

21 20 19 24 18 17 16 15 14 13 12 10 9 ? ? 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 X 1

14 33 12 11 10 9 8 7 6 5 4 3 2 1 5 4 3 2 1 35 34 X X 31 30 29 28 27 26 25 24 23 22 21 20 15 18 17 16 15 14 13 12 10 9 8 7 6 4 3 2

5 4 3 2 1 35 34 X X 31 30 29 28 27 26 25 24 23 22 21 20 15 18 17 16 15 14 13 121110 9 8 7 6 4 3 2

11

28 27 26 25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1

locus CRISPR1 absent 36

36 36 36

?2

X :

? :region with no spacer unsequenced region The NRPS genes are represented by black arrows, length of arrows is not proportional to the length of genes. NRPS modules are represented by

circles. Large circles indicate complete NRPS modules (domains C, A and PCP). The amino acid predicted to be assembled by the corresponding module is indicated within each circle. The chronological order of amino acid incorporation is illustrated by the number above each circle. Small circles represent TE domains. Brown arrow: gene required for hydroxylation of tyrosine. Pink arrow: gene required for biosynthesis of Dab (2,4-Diaminobutyric acid). Orange arrows: genes required for biosynthesis of unknown substrates similar to Dpg (3,5-Dihydroxyphenyl-glycine). Brown circle: NRPS module predicted to be specific for hydroxytyrosine. Pink circle: NRPS module predicted to be specific to Dab. Yellow circle: NRPS module predicted to be specific of Dpg. Orange circles with ?1, ?2, ?3 and ?4 indicate four different unpredicted amino-acid substrates.

NRPS NRPS

NRPS NRPS NRPS

NRPS NRPS

I II III IV V VI VII VIII IX X