53
Towards a functional analysis of the major factors involved in the reproductive barrier between Asian and African cultivated species of rice Andrés GUTIÉRREZ January 22, 2016
Towards a functional analysis of the major factors involved in the reproductive barrier between
Asian and African cultivated species of rice
Andrés GUTIÉRREZ
January 22, 2016
2
Model plant for genetics and genomics studies
- Diploid species: 2n = 24, AA - Genome size small: 390 Mb- Short growth duration- Efficient genetic transformation- Extensive genomic resources:
High-quality reference sequence, dense molecular maps, mutant libraries
Introduction
Rice (Oryza sativa): One of the most important crops in the world
Oryza sativa complex (AA-genome type)
Source: FAO, 2004
3
Many traits of agronomical interest (After Ali et al., 2010)
Introduction
Using AA genome species of rice to discover genes of importance
Cultivated species: O. sativa and O. glaberrima
4
Introduction
Oryza glaberrima: A source for the improvement of Oryza sativa
• Diploid species: 2n = 24, AA
• Genome sequenced
• Resistance or tolerance to abiotic and biotic stresses
• Traits have been combined with high yield O. sativa acc.
Strong reproductive barrier hamper his full utilization Source: archive.gramene.org
5
Pre-mating isolation
Prevent the copulation and fusion of gametes of interspecific crosses
Post-mating isolation
After mating has occurred => prevent fertilization or formation of zygotes hybrids
Introduction
• Habitat Isolation• Temporal Isolation• Behavioral Isolation• Mechanical Isolation• Gametic Mortality
Introduction
There are two basic categories of reproductive barriers
6
Pre-zygotic Isolation Mechanisms
Pollen–pistil interactions
Post-zygotic Isolation Mechanism
• Gametic incompatibility• Zygote dies after fertilization• Hybrid inviability• Hybrid breakdown• Hybrid Sterility
Arrest of the development of young zygotes
Introduction
Post-mating isolation
Hybrid Sterility
Inability to form functional gametes in a hybrid due to disturbances in sex-cell development or in meiosis, caused by
incompatible genetic constitution
Introduction
What is the importance of studying hybrid sterility?
For understanding biology of reproduction
When, where and how action of genes involved in reproduction take place
For studying evolution
Hybrid sterility is one of the mechanisms of reproductive barrier between species
For breeding Sterility decreases yield
Hybrid sterility genes prevent the introgression of useful genes
8
• This phenomenon is one of the strongest post-zygotic reproductive barriers in Oryza species
• Use of O. glaberrima in breeding is limited • Hybrid sterility prevent the introgression of useful genes
Introduction
Hybrid sterility between O. sativa x O. glaberrima
O. sativa (Asian rice, AA genome)
O. glaberrima (African rice, AA genome)
F1 hybridTotally male sterilePartially female sterile
9
Introduction
Hybrid sterility between O. sativa x O. glaberrima
Several loci causing hybrid sterility O. sativa x O. glaberrima => S1 locus exert the strong effect
Epistatic interactions between these loci and S1
Garavito et al., 2010
10
• Genetics: - S1 locus is the main factor of hybrid sterility between O. sativa X O. glaberrima (Sano, 1990)- S1 is a complex formed by three linked loci S1A, S1 and S1B
• Cytology: - Abnormalities in gametophytes
Pollen semi-sterility
Normal embryo sac Abnormal embryo sac
Koide et al., 2008
Introduction
Hybrid sterility between O. sativa x O. glaberrima
- The effect of S1 is Universal (Tao, 2010)
Garavito et al., 2010Guyot et al., 2011
11
• Female factor in a 27.8 kbp region nested in the male factor region
• Both male and female gamete elimination are probably controled by the same factor(s)
Garavito et al.,2010
Introduction
Fine mapping of the S1 locus
Garavito et al.,2010
• Model of gamete elimination
12
Guyot et al., 2011Garavito et al.,2010
Nip
ponb
are
(849
kpb
)
CG14 (813 kpb) S1S1A S1B
O. sativa (Nipponbare)
O. glaberrima (CG14)
ENOD-93 ENOD-93 Ribos_biog F-box ENOD-93
ENOD-93 ENOD-93 Ribos_biog F-box ENOD-93
S1
F-box F-box_dup
F-box
S1A
Introduction
Structural genomics
S1B remains to be elucidated
Introduction
The F-box is a protein motif of approximately 50 amino acids that functions as a site of protein-protein interaction
Lechner et al. 2006 Current Opinion in Plant Biology. 9:631-638
14
Introduction
F-box proteins are involved in self-incompatibility and floral organ determination
15
Introduction F-box as candidate for S1 & S1A
F-box proteins in rice
F-box at S1 locus
RiceXpro DB
Jain et al., 2007
Classification of 687 F-box proteins based on their domain architecture
4. To investigate if the F-box plays a role in the development of male gametophytes
Objective
To study the nature of the S1 locus
1. To characterize the sterile phenotype and to precise the cellular stage where gamete development defect occurs
3. To evaluate the temporal and spatial expression of the F-box candidate genes
2. To identify patterns of sequence divergence of the F-box candidate orthologous genes
5. To verify the hypothesis that F-box is actually S1
17
- O. sativa: Caiapo- O. glaberrima: MG12- Introgression line: L229 BC3DH from Caiapo x MG12 (S1
g Introgressed)
CSSLs introgression Lines
S1 region _O. glaberrima
Chr. 6S1Ag S1
g S1Bg
L229 BC3DH
O. sativa L229 O. glaberrima
Materials and Methods
Isolation of the S1 region
Gutierrez et al., 2010
18
229 line (BC3DH) X O. sativa (Caiapo)
Chr. 6 S1Ag S1g S1Bg S1As S1
s S1Bs
• Panicle, pollen and embryo sac evaluation • Cytology observations
• Gene expression analysis
S1Ag
S1As S1Bs
BC4F1
S1g S1Bg
S1s
Materials and Methods
Genetic stocks
19Gutierrez et al., 2015 (submitted)
Materials and Methods
Evaluation at different stages of O. sativa, O.glaberrima & L229_F1
Femalegametophyte development
Malegametophyte development
4. To investigate if the F-box plays a role in the development of male gametophytes
Objective
3. To evaluate the temporal and spatial expression of the F-box candidate genes
2. To identify patterns of sequence divergence of the F-box candidate orthologous genes
5. To verify the hypothesis that F-box is actually S1
Panicle, pollen grains and embryo sac fertility evaluation
Histology analysis
1. To characterize the sterile phenotype and to precise the cellular stage where gamete development defect occurs
1. To characterize the sterile phenotype and to precise the cellular stage where gamete development defect occurs
21Caiapo MG12 L229-F10%
1000%
2000%
3000%
4000%
5000%
6000%
7000%
8000%
9000%
10000%
11000%
Panicle Fertility
Pollen grain fertility
DAPI
Pollen germination
Embryo sac fertility
O. sativa L229-F1 O. glaberrima
Results
Panicle Fertility
Reduced fertility in the L229-F1
22
L229-F1O. sativa
Caiapo MG12 L229-F10%
1000%
2000%
3000%
4000%
5000%
6000%
7000%
8000%
9000%
10000%
11000%
Panicle Fertility
Pollen grain fertility
DAPI
Pollen germination
Embryo sac fertility
Results
Pollen grain fertility
23
MspMP
AP
Msp
MP
Results
Male gametophyte development in O. sativa and L229_F1
Pollen grain abortion in the hybrid seems to occur at the early microspore stage
Meiosis
O. sativa
L229_F1
Early Microspore Mitosis Maturation
24Caiapo MG12 L229-F10%
1000%
2000%
3000%
4000%
5000%
6000%
7000%
8000%
9000%
10000%
11000%
Panicle Fertility
Pollen grain fertility
DAPI
Pollen germination
Embryo sac fertility
O. sativa L229-F1
Results
Embryo sac fertility
43.75% embryo sacs were aborted in the L229-F1
25
dm
fm
dm
Results
Female gametophyte development in O. sativa and L229_F1
Embryo sac abortion in the hybrid seems to occur after completion of meiosis
MMC Meiosis Maturation
O. sativa
L229_F1
Conclusion
L229_F1 showed around 50% of pollen and embryo sac sterility and 78% of panicle sterility
Pollen grain abortion in L229_F1 occur at the early microspore stage
Embryo sac abortion in L229_F1 occur after completion of meiosis
Precise determination of abnormalities
4. To investigate if the F-box plays a role in the development of male gametophytes
Objective
3. To evaluate the temporal and spatial expression of the F-box candidate genes
2. To identify patterns of sequence divergence of the F-box candidate orthologous genes
5. To verify the hypothesis that F-box is actually S1
2. To identify patterns of sequence divergence of the F-box candidate orthologous genes
1. To characterize the sterile phenotype and to precise the cellular stage where gamete development defect occurs
Sequence comparison and gene structure of orthologous F-box alleles
Results
F-box at S1 locus is an FBL (F-box and LRR) OsFBL-185
Pairwise % IdentityGenomic: 84.0%
CDS: 98.1%
29
Results
F-box at S1 locus (OsFBL-185)
OsFBL-185 lacks an Arginine at the 15th position of the first exon
F-box domain
LRR domain
O. sativaO. glaberrima
O. sativaO. glaberrima
O. sativaO. glaberrima
O. sativaO. glaberrima
O. sativaO. glaberrima
O. sativaO. glaberrima
O. sativaO. glaberrima
O. sativaO. glaberrima
O. sativaO. glaberrima
30
Results
F-box at S1A locus is an FBL (F-box and LRR) OsFBL-184
Pairwise % IdentityGenomic: 94.4%
CDS: 96.9%
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Results
F-box at S1A locus (OsFBL-184)
Two amino acid conversion in the F-box domain and two in the LRR domain
F-box domain
LRR domain
O. sativaO. glaberrima
O. sativaO. glaberrima
O. sativaO. glaberrima
O. sativaO. glaberrima
O. sativaO. glaberrima
O. sativaO. glaberrima
O. sativaO. glaberrima
O. sativaO. glaberrima
O. sativaO. glaberrima
Conclusion
There are important structural changes between the orthologous genes (S1 and S1A)
=> OsFBL-185 lacks an Arginine at the 15th position of the first exon
=> Two amino acid conversion in the F-box domain and two in the LRR domain in OsFBL-184
4. To investigate if the F-box plays a role in the development of male gametophytes
Objective
3. To evaluate the temporal and spatial expression of the F-box candidate genes
2. To identify patterns of sequence divergence of the F-box candidate orthologous genes
5. To verify the hypothesis that F-box is actually S1
3. To evaluate the temporal and spatial expression of the F-box candidate genes
1. To characterize the sterile phenotype and to precise the cellular stage where gamete development defect occurs
qPCR analysis
in situ hybridization
34Highest expression in the hybrid at critical meiosis/mitosis stages
Results
Expression analysis of F-box at S1 locus
O. sativa
O. glaberrima
S1A S1
<<<
< 2 cm 2 - 5 cm 6 - 9 cm 10 - 13 cm
14 - 17 cm
> 18 cm0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
Rel
ativ
e ex
pres
sion
< 2 cm 2 - 5 cm 6 - 9 cm 10 - 13 cm
14 - 17 cm
> 18 cm0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Rel
ativ
e ex
pres
sion
MaturationMMC Meiosis Mitosis I - II Mitosis III
PMC Meiosis Microspore formation
Mitosis I Mitosis II - Maturation
Floral organs differentiation
Spikelet Palea-Lemma-Glumes
Pistil Stamen0
0.2
0.4
0.6
0.8
1
1.2 CaiapoBC4F1
MaturationMMC Meiosis Mitosis I - II Mitosis III
PMC Meiosis Microspore formation
Mitosis I Mitosis II - Maturation
Floral organs differentiation
Spikelet Palea-Lemma-Glumes
Pistil Stamen0
0.2
0.4
0.6
0.8
1
1.2MG12BC4F1
35Specifically expressed in the embryo sac and pollen grains in development
Results
Spatial expression of F-box at S1 locus in female and male gametophytes
Caiapo L229_BC4F1 MG12
Anti-sense probe
Sense probeAnti-sense probe Sense probe
Caiapo L229_BC4F1
LNA probe_O. sativa
Caiapo
MG12
36
< 2 cm 2 - 5 cm 6 - 9 cm 10 - 13 cm
14 - 17 cm
> 18 cm0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
Rel
ativ
e ex
pres
sion
Highest expression in the hybrid at floral organ differentiation and meiosis/mitosis stages
Results
Expression analysis of F-box at S1A locus
S1A S1
O. sativa
O. glaberrima
< 2 cm 2 - 5 cm 6 - 9 cm 10 - 13 cm
14 - 17 cm
> 18 cm0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
Rel
ativ
e ex
pres
sion
MaturationMMC Meiosis Mitosis I - II Mitosis III
PMC Meiosis Microspore formation
Mitosis I Mitosis II - Maturation
Floral organs differentiation
MaturationMMC Meiosis Mitosis I - II Mitosis III
PMC Meiosis Microspore formation
Mitosis I Mitosis II - Maturation
Floral organs differentiation
Spikelet Palea-Lemma-Glumes
Pistil Stamen0
0.5
1
1.5
2
2.5
Caiapo
BC4F1
Spikelet Palea-Lemma-Glumes
Pistil Stamen0
0.2
0.4
0.6
0.8
1
1.2
1.4
MG12
BC4F1
Conclusion
The F-box genes of S1 and S1A in the hybrid showed highest expression at Critical meiosis/mitosis stages
The F-box OsFBL-185 is specifically expressed in the immature embryo sac and pollen grains
4. To investigate if the F-box plays a role in the development of male gametophytes
Objective
3. To evaluate the temporal and spatial expression of the F-box candidate genes
2. To identify patterns of sequence divergence of the F-box candidate orthologous genes
5. To verify the hypothesis that F-box is actually S1
4. To investigate if the F-box plays a role in the development of male gametophytes
1. To characterize the sterile phenotype and to precise the cellular stage where gamete development defect occurs
Knock-out of the F-box OsFBL-185 using the CRISPR/Cas9 system
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Expression vector pOsUbi-Cas9Cas9/sgRNA complex
Materials and Methods
Functional validation of OsFBL-185 through CRISPR-Cas9 systemProkaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages
sgRNA_S1-1
sgRNA_S1-2 sgRNA_S1-3
Selected sgRNA target sites of OsFBL-185
40
Results
Functional validation of OsFBL-185 through CRISPR-Cas9 system
Transformed plants in the T0 generation
A total of 79 plants were obtained
Vegetative phenotype similar to WT
Pollen fertility of 72 lines was observedWT Transgenic
Pollen fertility of 72 lines was observed
41
Results
Functional validation of OsFBL-185 through CRISPR-Cas9 system
sgRNA_S1-1 sgRNA_S1-2 sgRNA_S1-3 T7 assay
From 72 lines observed:56 showed a deletion in the OsFBL-185 gene.
From these 56 lines:22 (39.3%) => partial or complete pollen
sterility34 (60.7%) => pollen fertile
42
Results
Functional validation of OsFBL-185 through CRISPR-Cas9 system
Deletion-Phenotype 1 Deletion-Phenotype 2
Fertile
Semi-sterile Sterile
OsFBL-185 seems to play a significant role in the development of male gametes
Pollen grain evaluation
4. To investigate if the F-box plays a role in the development of male gametophytes
Objective
3. To evaluate the temporal and spatial expression of the F-box candidate genes
2. To identify patterns of sequence divergence of the F-box candidate orthologous genes
5. To verify the hypothesis that F-box is actually S1
5. To verify the hypothesis that F-box is actually S1
1. To characterize the sterile phenotype and to precise the cellular stage where gamete development defect occurs
Functional complementation strategy
Results
Validation of OsFBL-185 through functional complementationGenetic transformation of Caiapo (O. sativa) with the F-box “S1
g” from O. glaberrima
Over-Express-O. glaberrima-CDS
Over-Express-O. sativa-CDS
45T0 generation
Results
Validation of OsFBL-185 through functional complementation
46
O. sativa Over_sat Over_glabPollen grain evaluation
Results
Validation of OsFBL-185 through functional complementation
O. sativa Over_sat Over_glab0.00
20.00
40.00
60.00
80.00
100.00
Failure in male gamete development in the O. sativa x O. glaberrima hybrid is due to allelic interaction between S1
g and S1s
Conclusion
OsFBL-185 seems to play a significant role in the development and viability of male gametes in rice => knock-out by CRISPR/Cas9 system
- Mutants with deletions showed a sterile phenotype
OsFBL-185 F-box is actually S1, the main sterility factor in the interspecific O. sativa x O. glaberrima hybrid
=> Functional complementation - Over expression of S1
g in O. sativa show a sterile phenotype like the natural hybrid
48
General Conclusion
OsFBL-185 is actually S1, the main sterility factor in the interspecific O. sativa x O. glaberrima hybrid
OsFBL-1850
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
Pollen grain sterility
49
Pollen abortion in the hybrid is possibly due to the alteration of a target protein anchored by OsFBL-185
OsFBL-185 may assemble into an active SCF complex which interact with an unknown target protein => Protein Degradation
50
Perspectives
Functional analysis: Validation of CRISPR and Complementation results=> Characterization of T1 plants by co-segregation analysis
What it is the function of the F-box OsFBL-185? Does this protein form an SCF Complex? What is the Target Protein?
=> Identification of protein subunits of the SCF complex => Bacterial 2 hybrid assays=> Protein expression and allelic imbalance
What are OsFBL-185 interactions? => Bimolecular fluorescence complementation (BiFC) assay
Elucidation of pathways at the molecular levelin O. sativa and the hybrid
51
Perspectives
Develop compatible interspecific bridgesUsing CRISPR/Cas9 by suppressing the expression of the S1
g allele and/or for large chromosomal deletions => S1 region
Identification of factors involved in female gamete development (S1A - S1B loci)=> RNA-seq and transcriptome profiles of recombinants lines around S1
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IRDMathias LorieuxHélène AdamLaurence AlbarFrançois Sabot Christine TranchantPierre LarmandeHélène PidonCecile MonatHarold ChrestinSophie Cheron Elise GrenonAnais RoudiereMyriam CollinStéphane JouannicAlain Ghesquière
CIRADEmmanuel GuiderdoniDonaldo Meynard Anne Cecile Meunier Jérôme Puig MumuAurore Vernet Martine Bes Julie Petit
Acknowledgments
CIATSilvio James CarabalíNatalia FrancoLady Arbelaez Alex Aguirre Marco BritoVictor LozanoPaul Chavarriaga Sandra Vidal Didier Marin
Thank you for your attention
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