Characterization of a resistance locus to coffee leaf rust

228 Pages·2011·5.58 MB·French

UNIVERSITE MONTPELLIER II
SCIENCES ET TECHINIQUES DU LANGUEDOC

THESE
Présentée pour l‘obtention du titre de

Docteur en Sciences de l’Université Montpellier II

Formation Doctorale : Biologie Intégrative des Plantes
Ecole Doctorale : SIBAGHE – Systèmes Intégrés en Biologie, Agronomie,
Géosciences, Hydro sciences et Environnement

par
Alessandra Ferreira RIBAS

Characterization of a resistance locus to coffee leaf rust (Hemileia
vastratrix) in coffee trees: genomic organization, diversity and
development of tools for functional gene validation

Soutenue publiquement devant le jury composé de,

Mme Françoise ADAN-BLONDON (INRA), Rapporteur
Mme Valérie GEFFROY (INRA), Examinatrice
M. Emmanuel DOUZERY (UM2), Examinateur
M. Gilles PILATE (INRA), Rapporteur
M. Hervé ETIENNE (CIRAD), Co-Directeur de thèse
M. Philippe LASHERMES (IRD), Co-Directeur de thèse
ABREVIATIONS

BAC Bacterial Artificial Chromosome
6-BA: 6-benzylaminopurine
CaMV: cauliflower mosaic virus
CC Coiled-Coil
CGA candidate gene approach
CG candidate gene
CLR Coffee Leaf Rust
CP medium: callus proliferation medium
DNA Deoxyribonucleic acid
2,4-D: 2,4-dichlorophenoxyacetic acid
ECP medium: embryogenic callus production medium
ETI Effector-Triggered Immunity
ETS Effector-Triggered Susceptibility
GFP Green Fluorescent Protein
HR Hypersensitive Response
HPTII hygromycin phosphotransferase II
i.e. id est
Ka non-synonymous substitutions
Ks synonymous substitutions
LRR Leucine-Rich Repeats
LTR Long Terminal Repeat
M medium: maturation medium
MAMP Microbe-Associated Molecular Pattern
MAS market assisted selection
NBS Nucleotide-Binding site
NPTII Neomycin Phosphotransferase III
OD: optical density
PAMP Pathogen-Associated Molecular Pattern
PCR Polymerase Chain Reaction
PEMs: proembryogenic masses
PTI PAMP-Triggered Immunity
QTL Quantitative-Trait Locus
R: regeneration medium
RLK Receptor-Like Kinase
RLP Receptor-Like Protein
SD: standard deviation
SNP single nucleotide polymorphism
TAIR: the Arabidopsis information resources
TIR Toll and Interleukin-1 Receptor
SUMMARY

LIST OF FIGURES .................................................................................................................... 7
LIST OF TABLES .................................................................................................................... 11
General Introduction ................................................................................................................ 13
CHAPTER I ............................................................................................................................... 14
LITERATURE REVIEW ......................................................................................................... 14
PART I ....................................................................................................................................... 15
1. Generalities about Coffee ................................................................................................. 15
1.1 Coffee consumption, cultivation and economical trade .................................................... 15
1.2 Coffee taxonomy ............................................................................................................... 16
1.3 Characteristics of cultivated coffee species ....................................................................... 19
1.4 Cytological aspects of Coffea ............................................................................................ 22
1.5 Speciation and genome features of C. arabica .................................................................. 22
1.6 Coffee arabica genetic diversity and breeding .................................................................. 23
1.7 Interspecific hybridization and coffee introgression ......................................................... 26
2. Coffee leaf rust: the main coffee disease ............................................................................. 28
2.1 The genus Hemileia ........................................................................................................... 28
2.2 Coffee leaf rust (CLR) symptoms ..................................................................................... 29
2.3 CLR epidemic ................................................................................................................... 31
2.4 Disease Management......................................................................................................... 32
2.4.1 Chemical control ........................................................................................................ 33
2.4.2 Biological control ....................................................................................................... 33
2.5.1 CLR resistance genes ................................................................................................. 35
2.5.2 Physiological races of Hemileia vastatrix .................................................................. 36
3. Identification of molecular markers associated to CLR .................................................... 39
3.1 Partial resistance to CLR ................................................................................................... 41
PART II ...................................................................................................................................... 42
1. Mechanisms of disease resistance in plants ..................................................................... 42
1.1 Non host resistance............................................................................................................ 42
1.1.1 PAMP/MAMP-triggered immunity (PTI/MTI) ......................................................... 44
1.2 Host resistance (Effector-Triggered Immunity (ETI)) ...................................................... 45
2. Modes of plant-pathogen interaction ............................................................................... 49
2.1 Direct Interaction .............................................................................................................. 49
2.2 The Guard Model (indirect R-Avr) interactions ................................................................ 50
2.3 Decoy model ..................................................................................................................... 51
3. Avirulence genes (effectors) .............................................................................................. 53
4. Resistance Genes (R genes) ............................................................................................... 54
4.1 Classes of Resistance Gene ............................................................................................... 58
4.1.1 RLP and RLK class of R genes .................................................................................. 58
4.2.1 The function of main domains in NB-LRR proteins .................................................. 61
5. How NBS-LRR proteins recognize pathogens and activate defense responses? ......... 66
6. Expression of R proteins ................................................................................................... 67
7. Plant-pathogen co evolution ............................................................................................. 68
7.1 Evolution of R genes at plant population level ................................................................. 68
7.2 Evolutionary patterns R genes clusters ............................................................................. 71
8. Wide genome analysis of NBS-LRR encoding genes ...................................................... 72
8.1 Organization of NBS-LRR genes into plant genomes ...................................................... 75
9. Mechanisms of R-genes diversification ........................................................................... 77
9.1 Duplication ........................................................................................................................ 79
9.1.1 Duplication by polyploidization ................................................................................. 79
9.2 Sequence recombination ................................................................................................... 81
9.3 Selection ............................................................................................................................ 82
9.4 Other mechanisms that contributed to generate R gene diversification ............................ 84
10. Patterns of R gene function .......................................................................................... 86
11. Comparative analysis of R genes ................................................................................. 88
PART III .................................................................................................................................... 91
1. Identification of candidate genes...................................................................................... 91
1.1 Identification of CG genes based on genomic synteny ..................................................... 91
1.2 Identification of CG using positional cloning strategy ..................................................... 93
2. Functional validation of CG ............................................................................................. 95
2.1 RNA interference .............................................................................................................. 95
2.2 Chemical mutagenesis ....................................................................................................... 96
2.3 Transformation by Agrobacterium .................................................................................... 97
3. Genetic transformation of woody plants ......................................................................... 97
3.1 Genetic transformation of coffee plants .......................................................................... 100
Objectives ................................................................................................................................. 103
CHAPTER II ........................................................................................................................... 105
Genetic and physical mapping of the S 3 region that confers resistance to leaf rust in
H
coffee tree (Coffea arabica L.) ................................................................................................ 106
ABSTRACT .......................................................................................................................... 106
INTRODUCTION ................................................................................................................. 106
MATERIALS AND METHODS .......................................................................................... 108
RESULTS ............................................................................................................................. 110
DISCUSSION ....................................................................................................................... 117
CHAPTER III .......................................................................................................................... 120
Organization and molecular evolution of a disease-resistance gene cluster in coffee trees
................................................................................................................................................... 121
ABSTRACT .......................................................................................................................... 121
BACKGROUND ................................................................................................................... 122
RESULTS ............................................................................................................................. 124
DISCUSSION ....................................................................................................................... 135
CONCLUSIONS ................................................................................................................... 138
MATERIALS AND METHODS .......................................................................................... 138
CHAPTER IV .......................................................................................................................... 144
Agrobacterium-mediated transformation of Coffea arabica (L.) is highly enhanced by using
long-term maintained embryogenic callus ............................................................................ 145
ABSTRACT .......................................................................................................................... 145
BACKGROUND ................................................................................................................... 146
RESULTS ............................................................................................................................. 148
DISCUSSION ....................................................................................................................... 160
CONCLUSIONS ................................................................................................................... 163
METHODS ........................................................................................................................... 163
CHAPTER V ........................................................................................................................... 168
Identification and functional validation of candidate genes to CLR .................................. 169
PART I. .................................................................................................................................... 170
Estimate of S 3-CNL gene members in the resistant cultivar ............................................ 170
H
PART II .................................................................................................................................... 175
Cloning S 3-CNL candidate genes to CLR resistance ......................................................... 175
H
PART III .................................................................................................................................. 178
Construction of expression vectors and functional gene validation ................................... 178
CHAPTER VI .......................................................................................................................... 187
General Discussion and Perspectives ..................................................................................... 188
References ................................................................................................................................ 198
LIST OF FIGURES

CHAPTER I

Figure I. 1 Major countries producers of coffee. Top ten coffee producers are shown in yellow ............. 16

Figure I. 2 Position of genus Coffea on the angiosperms phylogeny. Modified from:
(http://www.mobot.org/mobot/research/APweb) ............................................................................... 17

Figure I. 3 Typical coffee bean morphology: A- Endocarp hard (horny/crustaceous), B- Seeds (and
pyrenes) with a deep ventral groove. ................................................................................................. 18

Figure I. 4 Flower morphology in coffee genera. A. Coffea sessiflora in subgenus Coffea has flowers
with long style, medium corolla tube, exserted anthers. B. Psilanthus mannii in subgenus Psilanthus
has flowers with short style, long corolla tube, encased anthers........................................................ 18

Figure I. 5 Coffea arabica tree with cherry beans. From: Leo Junior, Incaper ES .................................... 20

Figure I. 6 Genetic origin of allotetraploid C. arabica. Natural hybridization between C. eugenioides as
female parent and C. canephora as male parent originated the C. arabica species (Lashermes et al.,
1999). ................................................................................................................................................. 24

Figure I. 7 Triploid and tetraploid strategies of gene introgression from diploid coffee species into C.
arabica genome (Lashermes et al., 2000). ......................................................................................... 28

Figure I. 8 Urediniospores of Hemileia vastatrix. Photo from: Liberato JR & Silva MC (2006). Available
online: http://www.padil.gov.au ........................................................................................................ 29

Figure I. 9 Life cycle of Hemileia vastatrix. Drawing from: (www.apsnet.org/online/feature/edcenter). 30

Figure I. 10 Symptoms of coffee leaf rust. A - coffee plants highly infected showing drop of leaves. B
Detail of coffee leaf with yellow-orange powder urediniospores. Photo A from:
(www.apsnet.org/online/feature/edcenter) and photo B (www2.iict.pt/index). ................................. 31

Figure I. 11 World distribution of coffee rust. (Schieber & Zentmyer, 1984)........................................... 32

Figure I. 12 Linkage map based on analysis of the chromosome segments in coupling surrounding the
S 3 gene on the F2 population (Matari x S.288)(Mahé et al., 2008). ................................................ 40
H

Figure I. 13 Different types of plant-pathogen intecation. From: Hammond-Kosack & Kanyuka (2007).
........................................................................................................................................................... 43

Figure I. 14 Quadratic diagram illustrating the gene-for-gene interaction between host resistance (R)
genes and rust avirulence (Avr) genes using coffee-rust as model. ................................................... 45

Figure I. 15 The‗zigzag‘ model proposed to represent four phases of the plant immune system (Jones and
Dangl 2006). ...................................................................................................................................... 47

Figure I. 16 Simplified schematic representation of the plant immune system. From: Pieterse et al., 2009.
........................................................................................................................................................... 48

Figure I. 17 The three Modes of Pathogen Recognition (Caplan et al., 2008) .......................................... 50

Figure I. 18 Opposing selection forces on guarded effector targets in a plant population polymorphic for
R genes (van der Horn et al., 2008) ................................................................................................... 52

Figure I. 19 Comparisons of the Guard and Decoy Models (van der Horn et al., 2008) ........................... 53
Figure I. 20 Classes of R protein and their cellular location. Adptated from: Hammond-Kosack & Kostya
Kanyuka, 2007. .................................................................................................................................. 57

Figure I. 21 Schematic representation of a typical NB–LRR protein. (Lukasik and Takken 2009). ......... 60

Figure I. 22 Leucine-rich repeat (LRR) structure of the PvPGIP2 (Bent and Mackey, 2007). ................. 64

Figure I. 23 Bait and switch model of NB-LRR protein function (Collier and Moffett 2009). ................. 67

Figure I. 24 A general model for plant–pathogen coevolution with a multilayered plant defense system.
(Xiao et al., 2008). ............................................................................................................................. 69

Figure I. 25 Evolutionary pattern of R genes (Fredman and Baker, 2007). .............................................. 72

Figure I. 26 Neighbor-joining tree showing the family-specific amplification of NBS sequences. (a)
TNLs. (b) CNLs. (McHale et al., 2006). ............................................................................................ 75

Figure I. 27 Physical locations of Arabidopsis sequences that encode NBS proteins similar to Plant R
Genes. (Meyers et al., 2003). ............................................................................................................. 77

Figure I. 28 The mechanisms that contribute to diversity of R genes and their loci. Adapted from: Leister
et al. 2004. ......................................................................................................................................... 78

Figure I. 29 Three potential fates of duplicate gene pairs with multiple regulatory regions (Force et al.,
1999. .................................................................................................................................................. 80

Figure I. 30 Route towards the application of candidate genes (Salentijn et al 2007). .............................. 92

Figure I. 31 Steps of positional cloning (Bouchez et al., 2006) ................................................................ 94

CHAPTER II

Figure II. 1 Southern hybridisation of HindIII-digested DNA from BAC clones of the S 3 region by a
H
BAC-end marker probe (143-8C-r). Based on the polymorphism revealed, the positive clones were
classified in two groups that putatively correspond to the two Arabica subgenomes. ..................... 112

Figure II. 2 BAC clone contig around the S 3 gene locus, and the S 3 region on the physical map...... 114
H H

CHAPTER III

Figure III. 1 BAC clone contigs spanning the S 3 locus. BAC clones spanning the S 3 locus as
H H
previously contigued [46]. Sequenced BACs from the three genomes are indicated by gray boxes.
......................................................................................................................................................... 125

Figure III. 2 Organization of S 3-CNL members in three coffee genomes: ........................................... 126
H

Figure III. 3 Southern blot hybridization of genomic DNA of C. arabica: ............................................ 127

Figure III. 4 Southern blot hybridization of genomic DNA from diploid coffee species: ...................... 127

Figure III. 5 Evolution of the S 3 locus in coffee species: ..................................................................... 129
H

Figure III. 6 Alignment of the predicted amino acid sequences from S 3-CNL members. .................... 132
H

Figure III. 7 Nucleotide diversity among S 3-CNL members from C. canephora ................................. 134
H
Figure III. 8 Ka/Ks ratio in the solvent-exposed residue of S 3-CNL members:. .................................. 142
H

Figure III. 9 Ka/Ks ratio in the solvent-exposed residue of S 3-CNL_A2. ............................................ 143
H

CHAPTER IV

Figure IV. 1 GFP fluorescence in embryogenic callus cultures transformed with A. tumefaciens and in
derived somatic embryos. ................................................................................................................ 149

Figure IV. 2 Effect of 6-BA and 2,4-D concentrations in the proliferation medium on growth intensity
and transformation efficiency of coffee embryogenic callus cultures. ............................................ 151

Figure IV. 3 Morphological and histological aspect of the different callus phenotypes observed in
maintained embryogenic cultures assayed for coffee genetic transformation.................................. 153

Figure IV. 4 Transformation efficiency depending on the phenotype of maintained embryogenic callus
cultures. Transformation efficiency was assessed by observing GFP epifluorescence 6 weeks after
the end of co-cultivation. ................................................................................................................. 154

Figure IV. 5 Effect of the embryogenic callus culture age on A. tumefaciens-mediated transformation
efficiency in C. arabica. Transformation efficiency was assayed 6 weeks after the end of co-
cultivation by GFP epifluorescence. ................................................................................................ 155

Figure IV. 6. Histological aspect of coffee embryogenic callus cultures depending on the age of the
culture.. ............................................................................................................................................ 156

Figure IV. 7 Regeneration of transformed coffee plants from maintained embryogenic cultures. .......... 158

Figure IV. 8 PCR detection of the hygromycin gene in transgenic coffee plants. The plants were
produced from 7-month-old embryogenic callus cultures co-cultivated with A. tumefaciens strain
LBA1119 carrying out the pMDC32 binary vector.. ....................................................................... 159

Figure IV. 9 Southern blot analyses of transgenic coffee plants. ............................................................ 159

CHAPTER V

Figure V. 1 Southern blot patterns of C. arabica cultivars hybridized with S 3-CNL specific probe. ... 172
H

Figure V. 2 Schematic representation of hybridization pattern obtained with EcoRI in non-introgressed
and introgressed acession of C. arabica. ......................................................................................... 173

Figure V. 3 Construction of binary vectors used for plant transformation carrying out candidate R genes
against CLR. .................................................................................................................................... 179

Figure V. 4 Expression cassettes used to clone candidates genes............................................................ 180

Figure V. 5 Schema representing the coffee transformation experiment for CG functional validation. .. 182

Figure V. 6 Overview of the transformation method used to produce transgenic coffee plants. ............. 184

Figure V. 7 Transgenic coffee plants carrying out the candidate genes A2 not conferred resistance to
CLR.. ............................................................................................................................................... 185

Figure V. 8 Strategy used for constructing a pooled BAC library of S.795 resistant cultivar to coffee leaf
rust (http://cnrgv.toulouse.inra.fr/fr/services/non_gridded_bac_library). ........................................ 186
CHAPTER VI

Figure VI. 1 Identification of BAC clones containing the S 3 locus in the S.795 resistant cultivar of C.
H
arabica. A. BAC clones containing the S 3 locus; B. Pulse field of BACs digested with Not I
H
enzyme; C. BACs fingerprint digested by Eco RI enzymes. ........................................................... 190

Figure VI. 2 Compared time durations for producing transgenic coffee plants using leaf explants versus
cryopreserved calluses......................................................................................................................197