the causal agent of leaf scald of sugarcane

(1)

27 26 19 x x x x x x x x 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 1

CRISPR-associated sequence diversity within Xanthomonas albilineans,

the causal agent of leaf scald of sugarcane

contact: pieretti@cirad.fr

clustalw2_i20120529_101306_0874_31921594_pg_phy_phyml_tree.txt Tue May 29 11:33:48 2012 Page 1 of 1

GPE_PC73 GPE_PC17 GPE_PC86 TWN052 XaFL07_1 BRA115 MTQ032 LKA070

GAB266 REU174draft HVO082 REU209 REU173 HVO005

PNG130 FIJ080

MTQ058

Xa23R1 USA048 XaS3

0.005

Analysis of the inter-strain variability of CRISPR-spacers in X. albilineans (CRISPR1 and CRISPR2) :

RESULTS

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 PFGE-B PFGE-A

PFGE- ?

clustalw2_i20120529_101306_0874_31921594_pg_phy_phyml_tree.txt Tue May 29 11:33:48 2012 Page 1 of 1

GPE_PC73 GPE_PC17 GPE_PC86 TWN052 XaFL07_1 BRA115 MTQ032 LKA070

GAB266 REU174draft HVO082 REU209 REU173 HVO005

PNG130 FIJ080

MTQ058

Xa23R1 USA048 XaS3

0.005

locus CRISPR1 absent

36 35 34 x x 31 30 29 28 27 26 25 24 23 22 21 20 15 18 17 16 15 14 13 12 x 10 9 8 7 6 5 4 3 2 1

36 35 34 x x 31 30 29 28 27 26 25 24 23 22 21 20 15 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1

36 35 34 x x 31 30 29 28 27 x x x x x x x x x x 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1

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

36 35 34 x x 31 30 29 28 27 26 25 24 23 22 21 20 15 18 17 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 4 x x 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

8 7 6 5 4 3 2 1

14 13 12 11 10 9 8 7 6 5 4 3 2 1

18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1

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 PFGE-B PFGE-A

PFGE- ?

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

M. Muller P. Rott P. Rott

white foliar stripes bleaching necrosis

Xanthomonas albilineans, unlike other xanthomonads, is a xylem- inviding 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. 2011. 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]

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. The genome of X. albilineans contains

two different CRISPR/cas systems (loci CRISPR1 and CRISPR2, respectively). Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are composed of repeats separated by spacers. Analysis of the variability of these spacers is curently used to perform diversity or epidemiological studies in bacteria.

(1) CIRAD, UMR BGPI, F-34398 Montpellier Cedex 5, France. (2) INRA, UMR LIPM, F-31326 Castanet-Tolosan Cedex, France.

Isabelle Pieretti1, Mélanie Marguerettaz1, Stéphanie Bolot2 , Sébastien Carrere2, Stéphane Cociancich1, Jérôme Gouzy2, Philippe Rott1, Monique Royer1.

CONCLUSIONS

CRISPR1 spacer distribution CRISPR1 spacer distribution PFGE group

PFGE group MLSA tree

MLSA tree

CRISPR2 spacer distribution CRISPR2 spacer distribution PFGE group

PFGE group MLSA tree

MLSA tree

24 23 22 21 20 19 x 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1

24 23 22 21 20 19 x 17 16 15 14 13 12 11 10 9 8 ? ? ? ? ? ? ?

24 23 22 21 20 19 x 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1

24 23 22 21 20 19 x 17 16 15 14 13 12 11 10 9 ? ? ? ? ? ? ? ?

24 23 22 21 20 19 x 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1

21 20 19 18 17 16 15 14 x x x x x x x x x x x x x

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

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

25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 x 4 x 2 1

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

22 21 20 19 18 17 x x x x 12 11 10 9 x x x x x 3 2 1

3 2 1

15 14 13 12 11 10 9 8 7 6 5 4 3 2 1

32 31 30 29 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 21 x x x x x x x x x x x x x x x x x x x x

59 °°° ³⁶ ³⁵ ³⁴ ³³ ³² ³¹ ³⁰ ²⁹ 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

52 °°° ³⁶ ³⁵ ³⁴ ³³ ³² ³¹ ³⁰ ²⁹ 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

A large inter-strain variability of the CRISPR-spacers was observed not only between phylogenetically distant strains, but also between closely related strains. The variability is congruent with MLSA phylogenetic analysis, and CRISPR markers appear more polymorphic and discriminent than PFGE markers. Interstingly, analysis of the CRISPR1-spacer variability appears as a promising

tool to study the phylogeny of PFGE-B strains and to better understand the occurence of the disease outbreaks since the late 1980s.

phylogenetic relationships between PFGE groups established by Multi Locus Sequence Analysis (MLSA) [2].

X

?

region with no spacer unsequenced region

100

84

100

45

76

84 100

96

100

84

100

45

76

84 100

96

27 26 27 x x x x x x x x x x x x x x x x x 9 8 7 6 5 4 3 1

29 28 27 26 25 24 23 22 21 20 19 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 1

36 35 34 13 16 31 30 29 28 27 26 25 24 23 22 ? ? ? ? ? ? ? ? ? ? 11 x 9 8 7 6 5 4 3 2 1

x

x French National Institute for Agricultural Research