CRISPR-associated sequence diversity provides new insights into evolution of Xanthomonas albilineans

(1)

CRISPR-1 s

pacer distri

bution

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:

10 genetic groups identified by Pulsed Field Gel Electrophoresis (PFGE) [4], and phylogenetic relationship between strains etablished by Multi Locus Sequence Analysis

Xanthomonas albilineans exhibits a large intra-species diversity, with:

[1] Pieretti et al. 2012. BMC Genomics, 13:658. [2] Pieretti et al. 2009. BMC Genomics, 10:616. [3] Marguerettaz et al. 2011. MPMI, 24:246-259. [4] 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 (Salmonella pathogenicity

island-1) family usually found in animal pathogens [1-2-3].

Experienced a genome erosion, lacks both the gum gene

cluster and the type III secretion system (T3SS) of the Hrp

(Hypersensitive response and pathogenicity) injectisome

families [1-2-3].

CRISPR-associated sequence diversity provides new

insights into evolution of

Xanthomonas albilineans

.

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

(1) CIRAD, UMR BGPI, F-34398 Montpellier Cedex 5, France.

(2) INRA, UMR LIPM, F-31326 Castanet-Tolosan Cedex, France.

contact: pieretti@cirad.fr

2 allelic forms of the T3SS SPI-1 xipD gene [3].

2 distinct and polymorphic Clustered Regularly Interspaced Short Palindromic Repeats/cas associated genes (CRISPR/cas systems) :

CRISPR-1

and

CRISPR-2

.

A high polymorphism of the CRISPR-spacers is observed between strains spanning the diversity of X. albilineans.

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-A PFGE- ?

P

F

G

E

group

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

Date & Origin of

PFGE-B strains

2003 Guadeloupe 1962 Martinique 1957 Martinique 2003 Guadeloupe 2003 Guadeloupe 2007 USA/Florida 54

MLSA relationship

XaS3 (outgroup) 1 2 3 4 5 6 7 8 1 -6 7 8 9 10 11 12 13 14 15 16

Locus CRISPR-1 absent

2 4 5 6 7 8 9 10 11 12 13 14 15 3 1 24 25 26 27 28 16 17 18 19 20 21 22 23 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 24 25 26 27 28 1 -3 1 2 3 4 5 6 7 8 9 10 11 13 14 15 16 17 18 20 21 22 24 25 26 23 15 12 27 28 29 31 34 30 36 35 -1 2 3 4 5 6 7 8 9 10 11 13 14 15 16 17 18 20 21 22 24 25 26 23 ₁₅ 12 27 28 29 31 34 30 36 35 -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-A PFGE- ?

P

F

G

E

group

allele

xipD

PFGE-B 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 54

Date & Origin

of strains

2003 Guadeloupe 1962 Martinique 1989 Martinique 1957 Martinique 2003 Guadeloupe 2003 Guadeloupe 2007 USA/Florida 1995 Reunion Island 1995 Reunion Island 1995 Reunion island 1962 Sri Lanka 2011 Gabon 1989 Burkina Faso 1980 Burkina Faso 1993 USA/Florida ? USA/? 1961 Fiji

1993 Papua New Guinea

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 59 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 59 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 1 2 3 4 5 9 10 11 12 -18 19 20 21 22 23 _- _- _- _- _- 3 2 1 1 2 3 4 5 6 7 8 9 10 11 -13 14 15 16 17 18 -20 21 22 -24 25 26 1 2 3 4 5 6 7 8 9 10 11 -13 14 15 16 17 18 -20 21 22 -24 25 26 1 2 3 4 5 6 7 8 9 10 11 -13 14 15 16 17 18 -20 21 22 -24 25 26 1 2 3 4 5 6 7 8 9 10 11 -13 14 15 16 17 18 -20 21 22 -24 25 26 1 2 3 4 5 6 7 8 9 10 11 -13 14 15 16 17 18 -20 21 22 -24 25 26 1 2 3 4 5 6 7 8 9 10 11 -13 14 15 16 17 18 -20 21 22 -24 25 26 1 2 11 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 1 2 11 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

clustalw2_alignement_du_24_04_2013_phy_phyml_tree.txt Mon Apr 29 10:37:21 2013 Page 1 of 1

PNG130 MTQ058 FIJ080 USA048 Xa23R1 HVO005 HVO082 GAB266 REU209 LKA070 REU174 REU173 MTQ032 TWN052 BRA115 XaFL07-1 GPE PC73 GPE PC86 GPE PC17 MTQ078 XaS3 0.01 MTQ078* GPE PC17 GPE PC86 GPE PC73 XaFL07-1 MTQ032 REU173* REU174 LAK070 REU209 GAB266 HVO082 HVO005 Xa23R1 USA048 FIJ080 MTQ058* PNG130

PNG130 MTQ058 FIJ080 USA048 Xa23R1 HVO005 HVO082 GAB266 REU209 LKA070 REU174 REU173 MTQ032 TWN052 BRA115 XaFL07-1 GPE PC73 GPE PC86 GPE PC17 MTQ078 XaS3 0.01 -14 15 16 17 18 19 20 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 BLZ102 PFGE-B 1992 Belize BLZ103 PFGE-B 1992 Belize BLZ105 PFGE-B 1992 Belize

USA128 PFGE-B 1993 USA/Florida

USA133 PFGE-B 1993 USA/Florida

USA156 PFGE-B 1993 USA/Texas

TWN052 PFGE-B 1988 Taïwan JPN88 PFGE-B 1990 Japan 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 68 66 42 100 99 95 99 95 100

PNG130 MTQ058 FIJ080 USA048 Xa23R1 HVO005 HVO082 GAB266 REU209 LKA070 REU174 REU173 MTQ032 TWN052 BRA115 XaFL07-1 GPE PC73 GPE PC86 GPE PC17 MTQ078 XaS3 0.01 MTQ078* GPE PC17 GPE PC86 GPE PC73 XaFL07-1 MTQ032 REU173* REU174 LAK070 REU209 GAB266 HVO082 HVO005 Xa23R1 USA048 FIJ080 MTQ058* PNG130

KNA93 PFGE-B 1990 St Kitts

BRA51 PFGE-B 1988 Brazil

GLP10 PFGE-B ? Guadeloupe

GLP12 PFGE-B ? Guadeloupe

BRB101 PFGE-B 1992 Barbados

PNG130 MTQ058 FIJ080 USA048 Xa23R1 HVO005 HVO082 GAB266 REU209 LKA070 REU174 REU173 MTQ032 TWN052 BRA115 XaFL07-1 GPE PC73 GPE PC86 GPE PC17 MTQ078 XaS3 0.01 1 -3 1 -3 4 5 6 7 8 9 10 11 12 13 14 18 19 -21 -28 30 29 17 5 6 7 8 9 -28 30 29 - - 16 -5 6 7 8 9 10 11 12 13 14 18 19 20 21 22 23 24 25 26 27 28 30 31 29 17 - 15 1 2 3 4 5 6 7 8 9 10 11 12 14 13

MLSA relationship

Within the non-ubiquitous

CRISPR-1

locus, a high variability of CRISPR-spacers is observed, not only between phylogenetically distant strains but also

between closely related strains. The spacers’ polymorphism among strains belonging to the single PFGE-B group suggests a relationship between this

variability and the geographical origin of the strains studied. It could also allow to better understand the nature of outbreaks of leaf scald disease of

su-garcane that occurred in several susu-garcane production regions of the world since the late 1980’s.

The

CRISPR-2

spacer polymorphism allows a distribution of X. albilineans strains into eight distinct CRISPR-2 clades. Interestingly, this distribution is

congruent with the distribution of the two allelic forms of gene xipD as well as with the MLSA tree, suggesting occurrence of different evolution histories

of X. albilineans.

Strain with *: strain unsequenced (no draft available). Spacers are represented by colored and

numbered squares. For each CRISPR system, squares with same color and same number are identical.

CRISPR-2 s

pacer distri

bution

- : lost spacer

1 2 3 4 5 7 8 9 10 -13 14 15 16 17 18 20 21 22 24 25 26 23 ₁₅ 12 27 28 29 31 34 30 36 35 _{- -} 6 1 2 3 4 5 7 8 9 10 -13 14 15 16 17 18 20 21 22 24 25 26 23 15 12 27 28 29 31 34 30 36 35 - - 6 Asia North America Central America South America & Caribbean 1 2 3 4 5 6 7 8 9 10 11 13 14 15 -- - - 12 28 29 31 34 30 36 35 - - 27 - 16 1 2 3 4 5 6 7 8 9 10 11 13 14 15 16 17 22 24 25 26 23 12 27 28 29 31 34 30 36 35 - - 21 20 ₁₅ 18

CRISPR’s distal end unknown (unsequenced spacers)

Comments:

16 17 18 19 20 21 22 23

CRISPR’s distal end unknown (unsequenced spacers)

CRISPR’s distal end unknown (unsequenced spacers) CRISPR’s distal end unknown

(unsequenced spacers) XaS3 (outgroup) 68 66 42 100 99 95 99 95 100 -31 31 32