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Functional analysis of cassiicolin, effector of the rubber
tree pathogen Corynespora cassiicola.
Sébastien Ribeiro, Marine Deon, Dinh Minh Tran, Mouman Soumahoro,
Aurélien Masson, André Clément-Demange, Valérie Pujade-Renaud
To cite this version:
Sébastien Ribeiro, Marine Deon, Dinh Minh Tran, Mouman Soumahoro, Aurélien Masson, et al..
Functional analysis of cassiicolin, effector of the rubber tree pathogen Corynespora cassiicola.. 12th
EFPP-10th SFP conference: Deepen Knowledge in Plant Pathology for Innovative Agro-Ecology, May
2017, Dunkerque, France. pp.A0. �hal-01605689�
Functional analysis of cassiicolin, effector of the
rubber tree pathogen
Corynespora cassiicola
Sébastien Ribeiro
1,2
, Marine Déon
1,2
, Dinh Minh Tran
2,3
, Mouman Soumahoro
4
, Aurélien Masson
5
, André Clément-Demange
2
, Valérie Pujade-Renaud
1,2
1
Université Clermont Auvergne (UCA), UMR 547 PIAF, BP 10448, F-63000 Clermont-Ferrand, France
2
CIRAD, UMR AGAP, F-34398 Montpellier, France
3
Rubber Research Institute of Vietnam, Ho Chi Minh City, Vietnam
4
Mouman Soumahoro, Société Africaine de Plantations d’Hévéa, Côte d’Ivoire
5
Société des Caoutchoucs de Grand-Béréby, Côte d’Ivoire
Objective
To determine the
importance of cassiicolin Cas1 for virulence of C. cassiicola,
by comparing the wild-type virulent CCP strain and the same strain deleted for the cassiicolin-encoding gene Cas1, in interaction with rubber tree.
Deletion of the Cas1 gene did not modify major
physiological functions in the CCP strain.
The growth rate, conidia production and percentage of germination
were compared in vitro between
CCP
and
ccpΔcas1
strains:
Strain
Growth rate
(mm/day)
Conidiation
(conidia/µl)
Germination (%)
CCP
4.13 ± 0.16a
413 ± 77a
71.75 ± 9a
ccpΔcas1
4.29 ± 0.08a
479.5 ± 42a
69.75 ± 6a
Comparison between CCP and ccpΔcas1
R
EFERENCES
1. B
RETON
, F. (2000) Journal of Natural Rubber Research 3, 115–128.
2. BARTHE, P. (2007) Journal of Molecular Biology 367, 89–1013.
3.
DE
L
AMOTTE
, F. (2007) Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences 849, 357–362.
4. D
ÉON
, M. (2012a) Plant science : an international journal of experimental plant biology 185–186, 227–237.
5. D
ÉON
, M. (2014) Fungal Biology 118, 32–47.
6. T
RAN
, D.M. (2016) PLOS ONE 11, e0162807. Public Library of Science.
Toxicity of ccpΔcas1 filtrate
Eighteen rubber clones were tested for their sensitivity to C. cassiicola exudates, by
conductivity measurement of the induced electrolyte leakage (%EL)
6
.
Hierarchical clustering =
Clones (CL) x Treatments (Tr)
Treatments:
Culture filtrates (strain name – toxin class)
Purified cassiicolin at various concentration (Tox1, 5 or 10)
Blanks (Tox0 or culture medium Cz)
Both CCP and ccpΔcas1 filtrates are toxic, although with different profiles:
CCP
is clustered in Tr2, with Tox5 and Tox10
ccpΔcas1
is clustered in Tr4, with Cas0 strains
Conclusion
Cassiicolin Cas1 is the major virulence factor involved in the
compatible interaction between CCP and rubber clones
, despite
the presence of other putative effectors causing filtrate toxicity.
Background
Rubber tree
(Hevea brasiliensis) is an economically important tropical tree that provides
natural rubber
. In Asia
and Africa, it is affected by the
Corynespora Leaf Fall (CLF) disease
caused by the
necrotrophic fungus
Corynespora cassiicola
. On the most susceptible rubber clones, C. cassiicola can cause massive defoliation leading
yield losses. Some pathogenic strains secrete a small protein toxin, the
cassiicolin
1,2,3
, which is transcriptionally
up-regulated during early stages of the disease
4
. Six isoforms were identified and used to classify the various strains
into
toxin classes
5
. However, C. cassiicola strains without cassiicolin gene (toxin class Cas0) may also produce toxic
exudates
6
, suggesting the existence of effectors other than cassiicolin.
susceptible clone
tolerant clone
Construction a Cas1 gene deletion mutant
Deletion mutant was obtained by replacing the Cas1 gene from a C. cassiicola highly
aggressive strain (
CCP
) with a hygromycin resistance cassette by homologous
recombination. The deletion mutant was named
ccpΔcas1
.
CCP
ccpΔcas1
hygromycin cassette
wild-type
CCP genome
292 pb
1 661 pb
469 pb
505 pb
5’ UTR Cas1
3’ UTR Cas1
trpC
promoter
hph gene
469 pb
5’ UTR Cas1
Cas1 gene
505 pb
3’ UTR Cas1
Cas1 promoter
469 pb
5’ UTR Cas1
3’ UTR Cas1
Cas1 promoter
505 pb
1 661 pb
trpC
promoter
hph gene
deletion mutant
ccpΔcas1 genome
%EL:
CCP
and
ccpΔcas1
strains were compared for their virulence on
detached leaves of three rubber clones, 4 days after application of
conidia (50 conidia/µl , 6 drops per leaves):
GT1
tolerant
PB217
susceptible
IRCA631
susceptible
Without cassiicolin Cas1, the CCP strain is almost
avirulent and doesn’t extend beyond the inoculation spot.
Pathogenicity of ccpΔcas1 strain
c
c
c
a
b
c
0
20
40
60
80
100
CCP
ccpΔcas1
Surface necrosed tissue (mm²)
CD1174
MDX624
IRCA41
FDR5665
RRIM600
FDR5240
GT1
FDR4575
FDR5788
RRIM926
RRIM901
IRCA19
IRCA303
PB217
PB254
MDX607
RRIC100
PB260
0
2000
4000
CCI6
–
Cas
0
CCAM
3
–
Cas
1
CCI13
–
Cas
0
CSRI5
–
Cas
0
T
ox
10
CCP
–
Cas1
T
ox
5
CCI403
–
Cas
0
CCI434
–
Cas
0
ccp
Δ
cas1
–
Cas0
CNIG
404
–
Cas
1
CCI501
–
Cas
1
CSB
16
–
Cas
5
TSB1
–
Cas
5
CIND3
–
Cas
0
CTHA3
–
Cas
0
CLN16
–
Cas
0
T
ox
1
T
ox
0
CZ
CCI416
–
Cas
5
CCI405
–
Cas
1
CCI402
–
Cas
2
CCI423
–
Cas
4
CCI449
–
Cas
0
0
2000
4000
6000
Mean/treatment
Me
an/
cl
one
54 123 83 119 110 62 69 28 46 35 62 58 83 53 38 49 32 44 21 32 40 33 26 45 26 27
56 156 84 107 103 58 48 53 85 51 81 56 86 32 45 64 36 41 23 32 27 22 20 25 39 23
36 24 80 64 22 82 43 63 37 24 21 22 22 39 30 51 38 48 35 24 17 22 23 20 23 18
48 109 68 98 80 78 52 47 16 25 34 42 37 39 48 85 34 61 52 27 32 18 24 49 22 19
48 71 76 87 85 56 62 56 27 55 19 38 39 67 56 65 44 55 37 40 24 23 26 22 38 24
42 65 32 33 35 36 29 49 50 65 60 57 54 26 40 25 28 30 25 17 28 42 59 60 45 65
71 68 75 89 80 77 63 73 64 75 73 80 78 78 65 77 68 56 45 57 70 67 76 67 91 57
134 179 136 223 202 139 144 96 249 212 160 139 166 88 108 90 81 77 79 68 75 109 115 102 135 167
111 188 194 154 163 201 166 147 82 100 121 164 170 82 83 78 61 70 57 34 41 63 74 87 89 106
118 186 176 206 193 197 161 163 154 152 133 98 111 96 111 127 91 79 72 25 52 80 64 78 65 78
84 136 102 126 145 136 95 93 149 158 89 118 104 41 44 51 46 60 32 21 38 55 57 57 68 74
84 118 92 96 92 199 133 143 180 153 121 111 96 56 38 56 31 49 91 22 27 36 32 50 34 39
79 172 85 95 103 95 99 74 123 129 40 68 46 108 113 90 143 60 94 64 55 25 27 30 26 20
72 79 92 85 78 177 123 114 128 99 31 28 44 78 92 82 87 78 82 33 32 31 38 25 44 32
82 115 90 120 75 222 181 176 65 73 23 34 29 146 99 109 84 68 103 38 31 49 21 24 35 32
71 179 81 91 143 185 82 88 40 73 38 38 47 65 42 80 100 74 75 45 36 23 33 35 39 31
79 115 128 116 154 230 80 140 58 90 48 69 63 105 64 90 57 79 51 31 27 28 47 50 20 33
79 153 144 152 152 177 109 132 88 90 29 31 52 120 90 96 60 71 79 16 39 15 16 19 18 19
124 101 114 112 134 97 96 91 92 66 69 74 73 67 76 62 61 58 35 38 41 43 47 48 48
Tr1
Tr2
Tr3 Tr4
Tr5
Tr6
CL1
CL2
CL3
