Abdelhafid Bendahmane

Vice Director
bendahm@evry.inra.fr
+33 1 60 87 45 02

ksu-inra
Abdelhafid Bendahmane Portrait

Crop Genomics
Projects:
Cloning of Vat and Pmw loci in melon
Cloning of Rfo locus
Cloning of virus resistance genes
Cloning of two sex determination genes
Tilling:
Tomato TILLING platform: TOMATILL
TILLING platform: TOMATILL, PETILL
Rapeseed TILLING platform: RAPTILL
This page:
Publications
Reviewed Papers
Book Chapters
Patents

Scientific Career

1992 Master degree, Paris XI University, Orsay, France
1996 PhD, University of East Anglia, Norwich, UK.
1997 Post-doc at the Sainsbury Laboratory, JIC, Norwich, UK
1998 Senior post-doc at the Sainsbury Laboratory, JIC, Norwich, UK
1999 INRA Research Scientist, URGV, Evry, France
2005 NRA Director of Research, URGV, Evry, France
2010 URGV Vice Director

 

Publications

2014   2013 2012   2011   2010   2009   2008   2007   2006   2005   2004   2003   2002   2000   1999   1995  

 

2014

Development and evaluation of a cucumber TILLING population.

BMC Res Notes. 2014 Nov 26;7(1):846 PMID: 25425033
Fraenkel R, Kovalski I, Troadec C, Bendahmane A, Perl-Treves R.

BACKGROUND:
Ordered collections of mutants serve as invaluable tools in biological research. TILLING (Targeting Induced Local Lesions IN Genomes) provides an efficient method to discover, in mutagenized populations, the possible phenotypes controlled by gene sequences whose function is unknown. This method can replace transgenic techniques for the functional validation of cloned genes, especially in the case of transformation-recalcitrant plants such as cucumber.
RESULTS:
We report the development of a TILLING cucumber population, generated by EMS mutagenesis in the Poinsett76 genetic background. The population was evaluated by screening for morphological mutations, and a range of developmental, pigmentation and spontaneous lesion mutants were observed. Suitability for detecting single nucleotide polymorphism in selected genes has been tested by screening a sample of amplicons, with detection rate of 1 SNP in ~1 Mbp.

CONCLUSION: The population described in this Research Note represents a useful asset in cucumber research, to be exploited for forward genetic screens and functional genomics purposes.


First TILLING Platform in Cucurbita pepo: A New Mutant Resource for Gene Function and Crop Improvement.

PLoS One. 2014 Nov 11;9(11):e112743. doi: 10.1371/journal.pone.0112743. eCollection 2014. PMID: 25386735
Nelly Vicente-Dólera, Christelle Troadec, Manuel Moya, Mercedes del Río-Celestino, Teresa Pomares-Viciana, Abdelhafid Bendahmane, Belén Picó, Belén Román, Pedro Gómez

Although the availability of genetic and genomic resources for Cucurbita pepo has increased significantly, functional genomic resources are still limited for this crop. In this direction, we have developed a high throughput reverse genetic tool: the first TILLING (Targeting Induced Local Lesions IN Genomes) resource for this species. Additionally, we have used this resource to demonstrate that the previous EMS mutant population we developed has the highest mutation density compared with other cucurbits mutant populations. The overall mutation density in this first C. pepo TILLING platform was estimated to be 1/133 Kb by screening five additional genes. In total, 58 mutations confirmed by sequencing were identified in the five targeted genes, thirteen of which were predicted to have an impact on the function of the protein. The genotype/phenotype correlation was studied in a peroxidase gene, revealing that the phenotype of seedling homozygous for one of the isolated mutant alleles was albino. These results indicate that the TILLING approach in this species was successful at providing new mutations and can address the major challenge of linking sequence information to biological function and also the identification of novel variation for crop breeding.


The Vat Locus Encodes for a CC-NBS-LRR Protein that Confers Resistance to Aphis gossypii Infestation and A. gossypii-Mediated Virus Resistance.

Plant J. 2014 Oct 6. doi: 10.1111/tpj.12690 PMID: 25283874
Dogimont C, Chovelon V, Pauquet J, Boualem A, Bendahmane A.

Aphis gossypii is a polyphagous sucking aphid and a vector for many viruses. In Cucumis melo, a dominant locus, Vat, confers a high level of resistance to Aphis gossypii infestation and to viruses transmitted by this vector. To investigate the mechanism underlying this double resistance, we first genetically dissected the Vat locus. We delimited the double resistance to a single gene that encodes for a CC-NBS-LRR protein type. To validate the genetic data, transgenic lines expressing the Vat gene were generated and assessed for the double resistance. In this analysis, Vat-transgenic plants were resistant to A. gossypii infestation as well as A. gossypii-mediated virus transmission. When the plants were infected mechanically, virus infection occurred on both transgenic and non-transgenic control plants. These results confirmed that the cloned CC-NBS-LRR gene mediates both resistance to aphid infestation and virus infection using A. gossypii as a vector. This resistance also invokes a separate recognition and response phases in which the recognition phase involves the interaction of an elicitor molecule from the aphid and Vat from the plant. The response phase is not specific and blocks both aphid infestation and virus infection. Sequence analysis of Vat alleles suggests a major role of an unusual conserved LRR repeat in the recognition of A. gossypii. This article is protected by copyright. All rights reserved.


Development of a Cucumis sativus TILLinG platform for forward and reverse genetics.

PLoS One. 2014 May 16;9(5):e97963. doi: 10.1371/journal.pone.0097963. eCollection 2014. PMID: 24835852
Boualem A, Fleurier S, Troadec C, Audigier P, Kumar AP, Chatterjee M, Alsadon AA, Sadder MT, Wahb-Allah MA, Al-Doss AA, Bendahmane A.

BACKGROUND:
Cucumber (Cucumis sativus) belongs to the Cucurbitaceae family that includes more than 800 species. The cucumber genome has been recently sequenced and annotated. Transcriptomics and genome sequencing of many plant genomes are providing information on candidate genes potentially related to agronomically important traits. To accelerate functional characterization of these genes in cucumber we have generated an EMS mutant population that can be used as a TILLinG platform for reverse genetics.
PRINCIPAL FINDINGS:
A population of 3,331 M2 mutant seed families was generated using two EMS concentrations (0.5% and 0.75%). Genomic DNA was extracted from M2 families and eight-fold pooled for mutation detection by ENDO1 nuclease. To assess the quality of the mutant collection, we screened for induced mutations in five genes and identified 26 mutations. The average mutation rate was calculated as 1/1147 Kb giving rise to approximately 320 mutations per genome. We focused our characterization on three missense mutations, G33C, S238F and S249F identified in the CsACS2 sex determination gene. Protein modeling and crystallography studies predicted that mutation at G33 may affect the protein function, whereas mutations at S238 and S249 may not impair the protein function. As predicted, detailed phenotypic evaluation showed that the S238F and the S249F mutant lines had no sexual phenotype. In contrast, plants homozygous for the G33C mutation showed a complete sexual transition from monoecy to andromonoecy. This result demonstrates that TILLinG is a valuable tool for functional validation of gene function in crops recalcitrant to transgenic transformation.
CONCLUSIONS:
We have developed a cucumber mutant population that can be used as an efficient reverse genetics tool. The cucumber TILLinG collection as well as the previously described melon TILLinG collection will prove to be a valuable resource for both fundamental research and the identification of agronomically-important genes for crop improvement in cucurbits in general.


Genomics of sex determination.

Curr Opin Plant Biol. 2014 Apr;18C:110-116. doi: 10.1016/j.pbi.2014.02.012. Epub 2014 Mar 30. PMID: 24682067
Zhang J, Boualem A, Bendahmane A, Ming R.

Sex determination is a major switch in the evolutionary history of angiosperm, resulting 11% monoecious and dioecious species. The genomic sequences of papaya sex chromosomes unveiled the molecular basis of recombination suppression in the sex determination region, and candidate genes for sex determination. Identification and analyses of sex determination genes in cucurbits and maize demonstrated conservation of sex determination mechanism in one lineage and divergence between the two systems. Epigenetic control and hormonal influence of sex determination were elucidated in both plants and animals. Intensive investigation of potential sex determination genes in model species will improve our understanding of sex determination gene network. Such network will in turn accelerate the identification of sex determination genes in dioecious species with sex chromosomes, which are burdensome due to no recombination in sex determining regions. The sex determination genes in dioecious species are crucial for understanding the origin of dioecy and sex chromosomes, particularly in their early stage of evolution.
Copyright © 2014 Elsevier Ltd. All rights reserved.


Molecular and functional characterization of CpACS27A gene reveals its involvement in monoecy instability and other associated traits in squash (Cucurbita pepo L.).

Planta. 2014 Mar 5. PMID 24595516
Martínez C, Manzano S, Megías Z, Barrera A, Boualem A, Garrido D, Bendahmane A, Jamilena M.

A number of Cucurbita pepo genotypes showing instable monoecy or partial andromonoecy, i.e. an incomplete conversion of female into bisexual flowers, have been detected. Given that in melon and cucumber andromonoecy is the result of reduction of ethylene production in female floral buds, caused by mutations in the ethylene biosynthesis genes CmACS7 and CsACS2; we have cloned and characterized two related C. pepo genes, CpACS27A and CpACS27B. The molecular structure of CpACS27A and its specific expression in the carpels of female flowers during earlier stages of flower development suggests that this gene is the Cucurbita ortholog of CmACS7 and CsACS2. CpACS27B is likely to be a paralogous pseudogene since it has not been found to be expressed in any of the analyzed tissues. CpACS27A was sequenced in Bolognese (Bog) and Vegetable Spaghetti (Veg), two monoecious inbred lines whose F2 was segregating for partial andromonoecy. The Bog allele of CpACS27A carried a missense mutation that resulted in a substitution of the conserved serine residue in position 176 by an alanine. Segregation analysis indicated that this mutant variant is necessary but not sufficient to confer the andromonoecious phenotype in squash. In concordance with its involvement in stamen arrest, a reduction in CpACS27A expression has been found in bisexual flower buds at earlier stages of development. This reduction in CpACS27A expression was concomitant with a downregulation of other ethylene biosynthesis and signaling genes during earlier and later stages of ovary development. The role of CpACS27A is discussed regarding the regulation of ethylene biosynthesis and signaling genes in the control of andromonoecy-associated traits, such as the delayed maturation of corolla and stigma as well as the parthenocarpic development of the fruit.


2013

PT-Flax (phenotyping and TILLinG of flax): development of a flax (Linum usitatissimum L.) mutant population and TILLinG platform for forward and reverse genetics.

BMC Plant Biol. 2013 Oct 15;13(1):159. PMID: 24128060
Chantreau M, Grec S, Gutierrez L, Dalmais M, Pineau C, Demailly H, Paysant-Leroux C, Tavernier R, Trouvé JP, Chatterjee M, Guillot X, Brunaud V, Chabbert B, van Wuytswinkel O, Bendahmane A, Thomasset B, Hawkins S.

BACKGROUND:
Flax (Linum usitatissimum L.) is an economically important fiber and oil crop that has been grown for thousands of years. The genome has been recently sequenced and transcriptomics are providing information on candidate genes potentially related to agronomically-important traits. In order to accelerate functional characterization of these genes we have generated a flax EMS mutant population that can be used as a TILLinG (Targeting Induced Local Lesions in Genomes) platform for forward and reverse genetics.
RESULTS:
A population of 4,894 M2 mutant seed families was generated using 3 different EMS concentrations (0.3%, 0.6% and 0.75%) and used to produce M2 plants for subsequent phenotyping and DNA extraction. 10,839 viable M2 plants (4,033 families) were obtained and 1,552 families (38.5%) showed a visual developmental phenotype (stem size and diameter, plant architecture, flower-related). The majority of these families showed more than one phenotype. Mutant phenotype data are organised in a database and can be accessed and searched at UTILLdb (http://urgv.evry.inra.fr/UTILLdb). Preliminary screens were also performed for atypical fiber and seed phenotypes. Genomic DNA was extracted from 3,515 M2 families and eight-fold pooled for subsequent mutant detection by ENDO1 nuclease mis-match cleavage. In order to validate the collection for reverse genetics, DNA pools were screened for two genes coding enzymes of the lignin biosynthesis pathway: Coumarate-3-Hydroxylase (C3H) and Cinnamyl Alcohol Dehydrogenase (CAD). We identified 79 and 76 mutations in the C3H and CAD genes, respectively. The average mutation rate was calculated as 1/41Kb giving rise to approximately 9,000 mutations per genome. Thirty-five out of the 52 flax cad mutant families containing missense or codon stop mutations showed the typical orange-brown xylem phenotype observed in CAD down-regulated/mutant plants in other species.
CONCLUSIONS:
We have developed a flax mutant population that can be used as an efficient forward and reverse genetics tool. The collection has an extremely high mutation rate that enables the detection of large numbers of independant mutant families by screening a comparatively low number of M2 families. The population will prove to be a valuable resource for both fundamental research and the identification of agronomically-important genes for crop improvement in flax.


A TILLING Platform for Functional Genomics in Brachypodium distachyon.

Figure 2

PLoS One. 2013 Jun 19;8(6):e65503. Print 2013. PMID: 23840336
Dalmais M, Antelme S, Ho-Yue-Kuang S, Wang Y, Darracq O, d'Yvoire MB, Cézard L, Légée F, Blondet E, Oria N, Troadec C, Brunaud V, Jouanin L, Höfte H, Bendahmane A, Lapierre C, Sibout R.
URGV, Unité de Recherche en Génomique Végétale, Université d'Evry Val d'Essonne, INRA, Evry, France.

The new model plant for temperate grasses, Brachypodium distachyon offers great potential as a tool for functional genomics. We have established a sodium azide-induced mutant collection and a TILLING platform, called "BRACHYTIL", for the inbred line Bd21-3. The TILLING collection consists of DNA isolated from 5530 different families. Phenotypes were reported and organized in a phenotypic tree that is freely available online. The tilling platform was validated by the isolation of mutants for seven genes belonging to multigene families of the lignin biosynthesis pathway. In particular, a large allelic series for BdCOMT6, a caffeic acid O-methyl transferase was identified. Some mutants show lower lignin content when compared to wild-type plants as well as a typical decrease of syringyl units, a hallmark of COMT-deficient plants. The mutation rate was estimated at one mutation per 396 kb, or an average of 680 mutations per line. The collection was also used to assess the Genetically Effective Cell Number that was shown to be at least equal to 4 cells in Brachypodium distachyon. The mutant population and the TILLING platform should greatly facilitate functional genomics approaches in this model organism.

Images from this publication

Simultaneous scalar and cross-phase modulation instabilities in highly birefringent photonic crystal fiber.

Opt Express. 2013 Apr 8;21(7):8437-43. doi: 10.1364/OE.21.008437. PMID: 23571933
Kudlinski A, Bendahmane A, Labat D, Virally S, Murray RT, Kelleher EJ, Mussot A.

We report the experimental observation of scalar and cross-phase modulation instabilities by pumping a highly birefringent photonic crystal fiber in the normal dispersion regime at 45° to its principal polarization axes. Five sideband pairs (two scalar and three vector ones) are observed simultaneously in the spontaneous regime, four of which have a large frequency shift from the pump, in the range 79-93 THz. These results are in excellent agreement with phase-matching arguments and numerical simulations.


SMART - Sunflower Mutant population And Reverse genetic Tool for crop improvement.

BMC Plant Biol. 2013 Mar 5;13:38. doi: 10.1186/1471-2229-13-38. PMID: 23496999
Kumar AP, Boualem A, Bhattacharya A, Parikh S, Desai N, Zambelli A, Leon A, Chatterjee M, Bendahmane A.

BACKGROUND:
Sunflower (Helianthus annuus L.) is an important oilseed crop grown widely in various areas of the world. Classical genetic studies have been extensively undertaken for the improvement of this particular oilseed crop. Pertaining to this endeavor, we developed a "chemically induced mutated genetic resource for detecting SNP by TILLING" in sunflower to create new traits.
RESULTS:
To optimize the EMS mutagenesis, we first conducted a "kill curve" analysis with a range of EMS dose from 0.5% to 3%. Based on the observed germination rate, a 50% survival rate i.e. LD50, treatment with 0.6% EMS for 8 hours was chosen to generate 5,000 M2 populations, out of which, 4,763 M3 plants with fertile seed set. Phenotypic characterization of the 5,000 M2 mutagenised lines were undertaken to assess the mutagenesis quality and to identify traits of interest. In the M2 population, about 1.1% of the plants showed phenotypic variations. The sunflower TILLING platform was setup using Endo-1-nuclease as mismatch detection system coupled with an eight fold DNA pooling strategy. As proof-of-concept, we screened the M2 population for induced mutations in two genes related to fatty acid biosynthesis, FatA an acyl-ACP thioesterase and SAD the stearoyl-ACP desaturase and identified a total of 26 mutations.
CONCLUSION:
Based on the TILLING of FatA and SAD genes, we calculated the overall mutation rate to one mutation every 480 kb, similar to other report for this crop so far. As sunflower is a plant model for seed oil biosynthesis, we anticipate that the developed genetic resource will be a useful tool to identify novel traits for sunflower crop improvement.


2012

A conserved molecular basis for photoperiod adaptation in two temperate legumes.

Proc Natl Acad Sci U S A. 2012 Dec 18;109(51):21158-63. doi: 10.1073/pnas.1207943110. Epub 2012 Dec 3., PMID 23213200
Weller JL, Liew LC, Hecht VF, Rajandran V, Laurie RE, Ridge S, Wenden B, Vander Schoor JK, Jaminon O, Blassiau C, Dalmais M, Rameau C, Bendahmane A, Macknight RC, Lejeune-Hénaut I.

Legumes were among the first plant species to be domesticated, and accompanied cereals in expansion of agriculture from the Fertile Crescent into diverse environments across the Mediterranean basin, Europe, Central Asia, and the Indian subcontinent. Although several recent studies have outlined the molecular basis for domestication and eco-geographic adaptation in the two main cereals from this region, wheat and barley, similar questions remain largely unexplored in their legume counterparts. Here we identify two major loci controlling differences in photoperiod response between wild and domesticated pea, and show that one of these, HIGH RESPONSE TO PHOTOPERIOD (HR), is an ortholog of EARLY FLOWERING 3 (ELF3), a gene involved in circadian clock function. We found that a significant proportion of flowering time variation in global pea germplasm is controlled by HR, with a single, widespread functional variant conferring altered circadian rhythms and the reduced photoperiod response associated with the spring habit. We also present evidence that ELF3 has a similar role in lentil, another major legume crop, with a distinct functional variant contributing to reduced photoperiod response in cultivars widely deployed in short-season environments. Our results identify the factor likely to have permitted the successful prehistoric expansion of legume cultivation to Northern Europe, and define a conserved genetic basis for major adaptive changes in flowering phenology and growth habit in an important crop group.


A role for an endosperm-localized subtilase in the control of seed size in legumes.

New Phytol. 2012 Nov;196(3):738-51. doi: 10.1111/j.1469-8137.2012.04296.x. Epub 2012 Sep 17. PMID: 22985172
D'Erfurth I, Le Signor C, Aubert G, Sanchez M, Vernoud V, Darchy B, Lherminier J, Bourion V, Bouteiller N, Bendahmane A, Buitink J, Prosperi JM, Thompson R, Burstin J, Gallardo K.

Here, we report a subtilase gene (SBT1.1) specifically expressed in the endosperm of Medicago truncatula and Pisum sativum seeds during development, which is located at a chromosomal position coinciding with a seed weight quantitative trait locus (QTL). Association studies between SBT1.1 polymorphisms and seed weights in ecotype collections provided further evidence for linkage disequilibrium between the SBT1.1 locus and a seed weight locus. To investigate the possible contribution of SBT1.1 to the control of seed weight, a search for TILLING (Targeting Induced Local Lesions in Genomes) mutants was performed. An inspection of seed phenotype revealed a decreased weight and area of the sbt1.1 mutant seeds, thus inferring a role of SBT1.1 in the control of seed size in the forage and grain legume species. Microscopic analyses of the embryo, representing the major part of the seed, revealed a reduced number of cells in the MtP330S mutant, but no significant variation in cell size. SBT1.1 is therefore most likely to be involved in the control of cotyledon cell number, rather than cell expansion, during seed development. This raises the hypothesis of a role of SBT1.1 in the regulation of seed size by providing molecules that can act as signals to control cell division within the embryo.
© 2012 INRA. New Phytologist © 2012 New Phytologist Trust.


Synchronization of the flowering transition by the tomato TERMINATING FLOWER gene.

Nat Genet. 2012 Nov 11;44(12):1393-8. doi: 10.1038/ng.2465. Epub 2012 Nov 11. PMID: 23143603

The transition to flowering is a major determinant of plant architecture, and variation in the timing of flowering can have profound effects on inflorescence architecture, flower production and yield. Here, we show that the tomato mutant terminating flower (tmf) flowers early and converts the multiflowered inflorescence into a solitary flower as a result of precocious activation of a conserved floral specification complex encoded by ANANTHA (AN) and FALSIFLORA (FA). Without TMF, the coordinated flowering process is disrupted, causing floral identity genes, such as AN and members of the SEPALLATA (SEP) family, to activate precociously, while the expression of flowering transition genes, such as FRUITFULL (FUL), is delayed. Indeed, driving AN expression precociously is sufficient to cause early flowering, and this expression transforms multiflowered inflorescences into normal solitary flowers resembling those of the Solanaceae species petunia and tobacco. Thus, by timing AN activation, TMF synchronizes flower formation with the gradual reproductive transition, which, in turn, has a key role in determining simple versus complex inflorescences.


Characterisation of alleles of tomato light signalling genes generated by TILLING.

Phytochemistry. 2012 May 15. PMID: 22595361
Jones MO, Piron-Prunier F, Marcel F, Piednoir-Barbeau E, Alsadon AA, Wahb-Allah MA, Al-Doss AA, Bowler C, Bramley PM, Fraser PD, Bendahmane A.

Targeting Induced Local Lesions IN Genomes (TILLING) combines chemical mutagenesis with high throughput screening to allow the generation of alleles of selected genes. In this study, TILLING has been applied to produce a series of mutations in genes encoding essential components of the tomato light signal transduction pathway in an attempt to enhance fruit nutritional quality. Point mutations to DEETIOLATED1 (DET1), which is responsible for the high pigment2 (hp2) tomato mutant, resulted in elevated levels of both carotenoid and phenylpropanoid phytonutrients in ripe fruit, whilst immature fruit showed increased chlorophyll content, photosynthetic capacity and altered fruit morphology. Furthermore, genotypes with mutations to the UV-DAMAGED DNA BINDING PROTEIN 1 (DDB1), COP1 and COP1like were also characterised. These genotypes largely did not display phenotypes characteristic of mutation to light signalling components but their characterisation has enabled interrogation of structure function relationships of the mutated genes.


Biosynthesis of the halogenated auxin, 4-chloroindole-3-acetic acid.

Plant Physiol. 2012 May 9. PMID: 22573801
Tivendale ND, Davidson SE, Davies NW, Smith JA, Dalmais M, Bendahmane AI, Quittenden LJ, Sutton L, Bala RK, Le Signor C, Thompson R, Horne J, Reid JB, Ross JJ.

Seeds of several agriculturally important legumes are rich sources of the only halogenated plant hormone, 4-chloroindole-3-acetic acid. However, the biosynthesis of this auxin is poorly understood. Here we show that in Pisum sativum (pea) seeds, 4-chloroindole-3-acetic acid is synthesized via the novel intermediate 4-chloroindole-3-pyruvic acid, which is produced from 4-chlorotryptophan by two aminotransferases, PsTAR1 and PsTAR2. We characterize a tar2 mutant, obtained by TILLING, the seeds of which contain dramatically reduced 4-chloroindole-3-acetic acid levels as they mature. We also show that the widespread auxin, indole-3-acetic acid, is synthesized by a parallel pathway in pea.
Free Full Text


VEGETATIVE1 is essential for development of the compound inflorescence in pea.

Nat Commun. 2012 Apr 24;3:797. doi: 10.1038/ncomms1801. PMID: 22531182
Berbel A, Ferrándiz C, Hecht V, Dalmais M, Lund OS, Sussmilch FC, Taylor SA, Bendahmane A, Ellis TH, Beltrán JP, Weller JL, Madueño F.
1] Instituto de Biología Molecular y Celular de Plantas, Consejo Superior de Investigaciones Científicas-Universidad Politécnica de Valencia, Valencia 46022, Spain. [2].

Unravelling the basis of variation in inflorescence architecture is important to understanding how the huge diversity in plant form has been generated. Inflorescences are divided between simple, as in Arabidopsis, with flowers directly formed at the main primary inflorescence axis, and compound, as in legumes, where they are formed at secondary or even higher order axes. The formation of secondary inflorescences predicts a novel genetic function in the development of the compound inflorescences. Here we show that in pea this function is controlled by VEGETATIVE1 (VEG1), whose mutation replaces secondary inflorescences by vegetative branches. We identify VEG1 as an AGL79-like MADS-box gene that specifies secondary inflorescence meristem identity. VEG1 misexpression in meristem identity mutants causes ectopic secondary inflorescence formation, suggesting a model for compound inflorescence development based on antagonistic interactions between VEG1 and genes conferring primary inflorescence and floral identity. Our study defines a novel mechanism to generate inflorescence complexity.


[Sex determination in cucurbits].

Biol Aujourdhui. 2012;206(1):57-62. Epub 2012 Apr 3. PMID: 22463996
[Article in French]
Foucart C, Boualem A, Lasseur B, Eleblu J, Fahraj I, Bendahmane A.
Source
INRA-CNRS, UMR1165, Unité de REcherche en Génomique Végétale, 91057 Évry Cedex, France.

Sex determination in plants leads to the development of unisexual flowers from an originally bisexual floral meristem. Cucurbits are not only species of agronomic interest but they also represent model species for the study of plant sex determination, because of their ability to harbor different sexual types. Such sexual forms are controlled by the identity of the alleles at the following loci: andromonoecious (a) and gynoecious (g) in melon, or androecious (a), Female (F), and Monoecious (M) in cucumber. We firstly showed that the andromonoecious a gene in melon encodes for an ACC synthase (CmACS7) and demonstrated that andromonoecy results from a mutation in the active site of the enzyme. Expression of the active enzyme inhibits the development of the male organs and is not required for carpel development. Because the a gene in melon and M gene in cucumber control the same sexual transition, monoecy to andromonoecy, we isolated the andromonoecy M gene in cucumber using a candidate gene approach in combination with genetic and biochemical analysis. We demonstrated the co-segregation of CsACS2, a close ortholog of CmACS7, with the M locus, and showed that the cucumber andromonoecious phenotype is also due to a loss of ACS enzymatic activity. CsACS2 is expressed specifically in carpel primordia of female flowers and should play a similar role to that of CmACS7 in melon in the inhibition of stamina development. Finally, we also showed that the transition from male to female flowers in the gynoecious lines results from epigenetic changes in the promoter of a C(2)H (2) zinc-finger transcription factor, CmWIP1. This epigenetic change is elicited by the insertion of a DNA transposon, which causes the spreading of DNA methylation to the CmWIP1 promoter. Expression of CmWIP1 leads to carpel abortion, resulting in the development of unisexual male flowers. From all these results, we built a model in which CmACS7 and CmWIP1 interact to control the development of male, female and hermaphrodite flowers in melon.
© Société de Biologie, 2012.


Isoprenoid biosynthesis is required for miRNA function and affects membrane association of ARGONAUTE 1 in Arabidopsis.

Proc Natl Acad Sci U S A. 2012 Jan 12. PMID: 22247288
Brodersen P, Sakvarelidze-Achard L, Schaller H, Khafif M, Schott G, Bendahmane A, Voinnet O.

Plant and metazoan microRNAs (miRNAs) guide ARGONAUTE (AGO) protein complexes to regulate expression of complementary RNAs via base pairing. In the plant Arabidopsis thaliana, the main miRNA effector is AGO1, but few other factors required for miRNA activity are known. Here, we isolate the genes defined by the previously described miRNA action deficient (mad) mutants, mad3 and mad4. Both genes encode enzymes involved in isoprenoid biosynthesis. MAD3 encodes 3-hydroxy-3-methylglutaryl CoA reductase (HMG1), which functions in the initial C(5) building block biogenesis that precedes isoprenoid metabolism. HMG1 is a key regulatory enzyme that controls the amounts of isoprenoid end products. MAD4 encodes sterol C-8 isomerase (HYDRA1) that acts downstream in dedicated sterol biosynthesis. Using yeast complementation assays and in planta application of lovastatin, a competitive inhibitor of HMG1, we show that defects in HMG1 catalytic activity are sufficient to inhibit miRNA activity. Many isoprenoid derivatives are indispensable structural and signaling components, and especially sterols are essential membrane constituents. Accordingly, we provide evidence that AGO1 is a peripheral membrane protein. Moreover, specific hypomorphic mutant alleles of AGO1 display compromised membrane association and AGO1-membrane interaction is reduced upon knockdown of HMG1/MAD3. These results suggest a possible basis for the requirement of isoprenoid biosynthesis for the activity of plant miRNAs, and unravel mechanistic features shared with their metazoan counterparts.


2011

Genomic approach to study floral development genes in Rosa sp.

PLoS One. 2011;6(12):e28455. Epub 2011 Dec 14. PMID: 22194838
Dubois A, Remay A, Raymond O, Balzergue S, Chauvet A, Maene M, Pécrix Y, Yang SH, Jeauffre J, Thouroude T, Boltz V, Martin-Magniette ML, Janczarski S, Legeai F, Renou JP, Vergne P, Le Bris M, Foucher F, Bendahmane M.

Cultivated for centuries, the varieties of rose have been selected based on a number of flower traits. Understanding the genetic and molecular basis that contributes to these traits will impact on future improvements for this economically important ornamental plant. In this study, we used scanning electron microscopy and sections of meristems and flowers to establish a precise morphological calendar from early rose flower development stages to senescing flowers. Global gene expression was investigated from floral meristem initiation up to flower senescence in three rose genotypes exhibiting contrasted floral traits including continuous versus once flowering and simple versus double flower architecture, using a newly developed Affymetrix microarray (Rosa1_Affyarray) tool containing sequences representing 4765 unigenes expressed during flower development. Data analyses permitted the identification of genes associated with floral transition, floral organs initiation up to flower senescence. Quantitative real time PCR analyses validated the mRNA accumulation changes observed in microarray hybridizations for a selection of 24 genes expressed at either high or low levels. Our data describe the early flower development stages in Rosa sp, the production of a rose microarray and demonstrate its usefulness and reliability to study gene expression during extensive development phases, from the vegetative meristem to the senescent flower.


Identification and characterization of tomato mutants affected in the Rx-mediated resistance to PVX isolates.

Mol Plant Microbe Interact. 2011 Nov 16. PMID: 22088194
Sturbois BN, Dubrana-Ourabah MP, Gombert J, Lasseur B, Macquet A, Faure C, Bendahmane A, Baur S I, Candresse T.

Five tomato mutants affected in the Rx-mediated resistance against Potato virus X (PVX) were identified by screening a mutagenized population derived from a transgenic, Rx1-expressing Micro-Tom line. Contrary to their parental line, they failed to develop lethal systemic necrosis upon infection with the virulent PVX-KH2 isolate. Sequence analysis and quantitative RT-PCR experiments indicated that the mutants are not affected in the Rx1 transgene or in the Hsp90, RanGap1 and RanGap2, Rar1 and Sgt1 genes. Inoculation with the PVX-CP4 avirulent isolate demonstrated that the Rx1 resistance was still effective in the mutants. In contrast, the virulent PVX-KH2 isolate accumulation was readily detectable in all mutants, which could further be separated in two groups depending on their ability to restrict the accumulation of PVX-RR, a mutant affected at two key positions for Rx1 elicitor activity. Lastly, transient expression of the viral CP elicitor indicated that the various mutants have retained the ability to mount an Rx1-mediated hypersensitive response. Taken together, the results obtained are consistent with a modification of the specificity or of the intensity of the Rx1-mediated response. The five Micro-Tom mutants should provide very valuable resources for the identification of novel tomato genes affecting the functioning of the Rx gene. Key words: Tomato, extreme resistance, HR, R gene, Rx, PVX.


The pea TCP transcription factor PsBRC1 acts downstream of strigolactones to control shoot branching.

Plant Physiol. 2011 Nov 1. PMID: 22045922
Braun N, de Saint Germain A, Pillot JP, Boutet-Mercey S, Li X, Antoniadi I, Dalmais M, Maia-Grondard A, Lesignor C, Bouteiller N, Luo D, Bendahmane A, Turnbull C, Rameau C.

The function of PsBRC1, the pea (Pisum sativum L.) homolog of the maize TEOSINTE BRANCHED1 (TB1) and the Arabidopsis BRANCHED1 (AtBRC1) transcription factors was investigated. The pea Psbrc1 mutant displays an increased shoot branching phenotype, is able to synthesize strigolactone (SL) and does not respond to SL application. The level of pleiotropy of the SL deficient rms1 mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes. The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally upregulated by direct application of the synthetic SL GR24 and downregulated by the cytokinin (CK) 6-benzylaminopurine (BAP). The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth. However the Psbrc1 mutant responds to BAP application and decapitation by increasing axillary bud length implicating a PsBRC1-independent component of the CK response in sustained bud growth. In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside (ZR) in the xylem sap nor a strong increase in RMS1 transcript levels suggesting that the RMS2-dependent feedback is not activated in this mutant. Surprisingly the double rms1 Psbrc1 mutant displays a strong increase of numbers of branches at cotyledonary nodes whereas branching at upper nodes is not significantly higher than the branching in rms1. This phenotype implicates a localized regulation of branching at these nodes specific to pea.


Shoot Branching and Leaf Dissection in Tomato Are Regulated by Homologous Gene Modules.

Plant Cell. 2011 Oct 28. PMID: 22039213
Busch BL, Schmitz G, Rossmann S, Piron F, Ding J, Bendahmane A, Theres K.

Aerial plant architecture is predominantly determined by shoot branching and leaf morphology, which are governed by apparently unrelated developmental processes, axillary meristem formation, and leaf dissection. Here, we show that in tomato (Solanum lycopersicum), these processes share essential functions in boundary establishment. Potato leaf (C), a key regulator of leaf dissection, was identified to be the closest paralog of the shoot branching regulator Blind (Bl). Comparative genomics revealed that these two R2R3 MYB genes are orthologs of the Arabidopsis thaliana branching regulator REGULATOR OF AXILLARY MERISTEMS1 (RAX1). Expression studies and complementation analyses indicate that these genes have undergone sub- or neofunctionalization due to promoter differentiation. C acts in a pathway independent of other identified leaf dissection regulators. Furthermore, the known leaf complexity regulator Goblet (Gob) is crucial for axillary meristem initiation and acts in parallel to C and Bl. Finally, RNA in situ hybridization revealed that the branching regulator Lateral suppressor (Ls) is also expressed in leaves. All four boundary genes, C, Bl, Gob, and Ls, may act by suppressing growth, as indicated by gain-of-function plants. Thus, leaf architecture and shoot architecture rely on a conserved mechanism of boundary formation preceding the initiation of leaflets and axillary meristems.


Towards a TILLING platform for functional genomics in Piel de Sapo melons.

BMC Res Notes. 2011 Aug 11;4:289. PMID: 21834982
González M, Xu M, Esteras C, Roig C, Monforte AJ, Troadec C, Pujol M, Nuez F, Bendahmane A, Garcia-Mas J, Picó B.
Institute for the Conservation and Breeding of Agricultural Biodiversity (COMAV-UPV), Universitat Politècnica de València, Camino de Vera s/n, 46022 Valencia, Spain. MPICOSI@BTC.UPV.ES

ABSTRACT:
BACKGROUND:
The availability of genetic and genomic resources for melon has increased significantly, but functional genomics resources are still limited for this crop. TILLING is a powerful reverse genetics approach that can be utilized to generate novel mutations in candidate genes. A TILLING resource is available for cantalupensis melons, but not for inodorus melons, the other main commercial group.
RESULTS:
A new ethyl methanesulfonate-mutagenized (EMS) melon population was generated for the first time in an andromonoecious non-climacteric inodorus Piel de Sapo genetic background. Diverse mutant phenotypes in seedlings, vines and fruits were observed, some of which were of possible commercial interest. The population was first screened for mutations in three target genes involved in disease resistance and fruit quality (Cm-PDS, Cm-eIF4E and Cm-eIFI(iso)4E). The same genes were also tilled in the available monoecious and climacteric cantalupensis EMS melon population. The overall mutation density in this first Piel de Sapo TILLING platform was estimated to be 1 mutation/1.5 Mb by screening four additional genes (Cm-ACO1, Cm-NOR, Cm-DET1 and Cm-DHS). Thirty-three point mutations were found for the seven gene targets, six of which were predicted to have an impact on the function of the protein. The genotype/phenotype correlation was demonstrated for a loss-of-function mutation in the Phytoene desaturase gene, which is involved in carotenoid biosynthesis.
CONCLUSIONS:
The TILLING approach was successful at providing new mutations in the genetic background of Piel de Sapo in most of the analyzed genes, even in genes for which natural variation is extremely low. This new resource will facilitate reverse genetics studies in non-climacteric melons, contributing materially to future genomic and breeding studies.


Soybean cyst nematode resistance in soybean is independent of the Rhg4 locus LRR-RLK gen

Funct Integr Genomics. 2011 May 4 PMID: 21541782
Liu X, Liu S, Jamai A, Bendahmane A, Lightfoot DA, Mitchum MG, Meksem K.
Division of Plant Sciences and Bond Life Sciences Center, University of Missouri, Columbia, MO, 65211, USA.

Abstract
To test the function of candidate genes in soybean for resistance to the soybean cyst nematode (SCN), a large collection of EMS-mutants from the SCN-resistant soybean cultivar "Forrest" was developed for Targeting Induced Local Lesions IN Genomes (TILLING). Additionally, due to the complexity of the soybean genome, an integrated set of genomic and genetic analysis tools was employed to complement the TILLING approach. The efficiency of this integrated set of tools was tested using a candidate soybean gene for resistance to SCN, encoding a leucine-rich repeat receptor-like kinase (LRR-RLK) that was identified by map-based cloning at the Rhg4 locus. The Rhg4 locus is one of the major quantitative trait loci controlling soybean resistance against SCN race 3 (HG type 0) in cv. Forrest, but the gene(s) sequence for resistance remains to be determined. Using TILLING, a Forrest mutant containing a nonsense mutation in the LRR domain of the candidate resistance protein was identified and confirmed; however, the SCN-resistant phenotype of the mutant was not altered. Haplotyping and EcoTILLING of recombinant inbred lines along with complementation analysis corroborated the TILLING result and ruled out the possibility of functional redundancy by a second copy of the LRR-RLK gene identified in the soybean genome. This study validates the use of TILLING, in combination with an integrated set of genomic tools, as an efficient means of testing candidate genes for SCN resistance in soybean.


Role of tomato BRANCHED1-like genes in the control of shoot branching.

Plant J. 2011 May 7. doi: 10.1111/j.1365-313X.2011.04629.x. PMID: 21554455
Martín-Trillo M, Grandío EG, Serra F, Marcel F, Rodríguez-Buey ML, Schmitz G, Theres K, Bendahmane A, Dopazo H, Cubas P.
Departamento de Genética Molecular de Plantas, Centro Nacional de Biotecnología/CSIC, Campus Universidad Autónoma de Madrid, 28049 Madrid, Spain Evolutionary Genomics Unit. Bioinformatics and Genomics Department. Centro de Investigación Príncipe Felipe,Valencia, Spain. Unité de Recherche en Génomique Végétale (URGV) UMR INRA 1165, CNRS 8114-UEVE. 2, Rue Gaston Crémieux - CP 5708 - 91000 Evry Cedex, France. Department of Plant Production, College of Food and Agricultural Sciences, King Saud University, Riyadh 11451, Saudi Arabia Max Planck Institute for Plant Breeding Research, Carl-von-Linne'-Weg 10, D-50829 Cologne, Germany

Abstract In angiosperms, shoot branching greatly determines overall plant architecture and affects fundamental aspects of plant life. Branching patterns are determined by genetic pathways widely conserved across angiosperms. In Arabidopsis thaliana (Brassicaceae, Rosidae) BRANCHED1 (BRC1) plays a central role in this process, acting locally to arrest axillary bud growth. In tomato (Solanum lycopersicum, Solanaceae, Asteridae) we have identified two BRC1-like paralogs, SlBRC1a and SlBRC1b. These genes are expressed in arrested axillary buds and both are down-regulated upon bud activation, although SlBRC1a is transcribed at much lower levels than SlBRC1b. Alternative splicing of SlBRC1a renders two transcripts encoding two BRC1-like proteins with different C-t domains due to a 3'-terminal frameshift. The phenotype of loss-of-function lines suggests that SlBRC1b has retained the ancestral role of BRC1 in shoot branch suppression. We have isolated the BRC1a and BRC1b genes of other Solanum species and have studied their evolution rates across the lineages. These studies indicate that, after duplication of an ancestral BRC1-like gene, BRC1b genes continued evolving under a strong purifying selection, consistent with the conserved function of SlBRC1b in shoot branching control. In contrast, the coding sequences of Solanum BRC1a genes have evolved at a higher evolution rate. Branch-site tests indicate that this does not reflect relaxation but rather positive selective pressure for adaptation.
Copyright © 2011 Blackwell Publishing Ltd and the Society for Experimental Biology.


Analysis of expressed sequence tags generated from full-length enriched cDNA libraries of melon.

BMC Genomics. 2011 May 20;12(1):252. PMID: 21599934
Clepet C, Joobeur T, Zheng Y, Jublot D, Huang M, Truniger V, Boualem A, Hernandez-Gonzalez ME, Dolcet-Sanjuan R, Portnoy V, Mascarell-Creus A, Cano-Delgado AI, Katzir N, Bendahmane A, Giovannoni JJ, Aranda MA, Garcia-Mas J, Fei Z.

ABSTRACT:
BACKGROUND: Melon (Cucumis melo), an economically important vegetable crop, belongs to the Cucurbitaceae family which includes several other important crops such as watermelon, cucumber, and pumpkin. It has served as a model system for sex determination and vascular biology studies. However, genomic resources currently available for melon are limited.
RESULTS: We constructed eleven full-length enriched and four standard cDNA libraries from fruits, flowers, leaves, roots, cotyledons, and calluses of four different melon genotypes, and generated 71,577 and 22,179 ESTs from full-length enriched and standard cDNA libraries, respectively. These ESTs, together with ~35,000 ESTs available in public domains, were assembled into 24,444 unigenes, which were extensively annotated by comparing their sequences to different protein and functional domain databases, assigning them Gene Ontology (GO) terms, and mapping them onto metabolic pathways. Comparative analysis of melon unigenes and other plant genomes revealed that 75% to 85% of melon unigenes had homologs in other dicot plants, while approximately 70% had homologs in monocot plants. The analysis also identified 6,972 gene families that were conserved across dicot and monocot plants, and 181, 1,192, and 220 gene families specific to fleshy fruit-bearing plants, the Cucurbitaceae family, and melon, respectively. Digital expression analysis identified a total of 175 tissue-specific genes, which provides a valuable gene sequence resource for future genomics and functional studies. Furthermore, we identified 4,068 simple sequence repeats (SSRs) and 3,073 single nucleotide polymorphisms (SNPs) in the melon EST collection. Finally, we obtained a total of 1,382 melon full-length transcripts through the analysis of full-length enriched cDNA clones that were sequenced from both ends. Analysis of these full-length transcripts indicated that sizes of melon 5' and 3' UTRs were similar to those of tomato, but longer than many other dicot plants. Codon usages of melon full-length transcripts were largely similar to those of Arabidopsis coding sequences.
CONCLUSION: The collection of melon ESTs generated from full-length enriched and standard cDNA libraries is expected to play significant roles in annotating the melon genome. The ESTs and associated analysis results will be useful resources for gene discovery, functional analysis, marker-assisted breeding of melon and closely related species, comparative genomic studies and for gaining insights into gene expression patterns.


Sex chromosomes in land plants.

Annu Rev Plant Biol. 2011 Jun 2;62:485-514. PMID: 21526970

Ming R, Bendahmane A, Renner SS.

Abstract
Sex chromosomes in land plants can evolve as a consequence of close linkage between the two sex determination genes with complementary dominance required to establish stable dioecious populations, and they are found in at least 48 species across 20 families. The sex chromosomes in hepatics, mosses, and gymnosperms are morphologically heteromorphic. In angiosperms, heteromorphic sex chromosomes are found in at least 19 species from 4 families, while homomorphic sex chromosomes occur in 20 species from 13 families. The prevalence of the XY system found in 44 out of 48 species may reflect the predominance of the evolutionary pathway from gynodioecy towards dioecy. All dioecious species have the potential to evolve sex chromosomes, and reversions back from dioecy to various forms of monoecy, gynodioecy, or androdioecy have also occurred. Such reversals may occur especially during the early stages of sex chromosome evolution before the lethality of the YY (or WW) genotype is established.


2010

Engineering melon plants with improved fruit shelf life using the TILLING approach.

PLoS One. 2010 Dec 30;5(12):e15776. PMID: 21209891

Dahmani-Mardas F, Troadec C, Boualem A, Lévêque S, Alsadon AA, Aldoss AA, Dogimont C, Bendahmane A.

Fig 1

BACKGROUND: Fruit ripening and softening are key traits that have an effect on food supply, fruit nutritional value and consequently, human health. Since ethylene induces ripening of climacteric fruit, it is one of the main targets to control fruit over ripening that leads to fruit softening and deterioration. The characterization of the ethylene pathway in Arabidopsis and tomato identified key genes that control fruit ripening.
METHODOLOGY/PRINCIPAL FINDINGS: To engineer melon fruit with improved shelf-life, we conducted a translational research experiment. We set up a TILLING platform in a monoecious and climacteric melon line, cloned genes that control ethylene production and screened for induced mutations that lead to fruits with enhanced shelf life. Two missense mutations, L124F and G194D, of the ethylene biosynthetic enzyme, ACC oxidase 1, were identified and the mutant plants were characterized with respect to fruit maturation. The L124F mutation is a conservative mutation occurring away from the enzyme active site and thus was predicted to not affect ethylene production and thus fruit ripening. In contrast, G194D modification occurs in a highly conserved amino acid position predicted, by crystallographic analysis, to affect the enzymatic activity. Phenotypic analysis of the G194D mutant fruit showed complete delayed ripening and yellowing with improved shelf life and, as predicted, the L124F mutation did not have an effect.
CONCLUSIONS/SIGNIFICANCE: We constructed a mutant collection of 4023 melon M2 families. Based on the TILLING of 11 genes, we calculated the overall mutation rate of one mutation every 573 kb and identified 8 alleles per tilled kilobase. We also identified a TILLING mutant with enhanced fruit shelf life. This work demonstrates the effectiveness of TILLING as a reverse genetics tool to improve crop species. As cucurbits are model species in different areas of plant biology, we anticipate that the developed tool will be widely exploited by the scientific community.


Identification of Mendel's white flower character.

PLoS One. 2010 Oct 11;5(10):e13230. PMID: 20949001
Hellens RP, Moreau C, Lin-Wang K, Schwinn KE, Thomson SJ, Fiers MW, Frew TJ, Murray SR, Hofer JM, Jacobs JM, Davies KM, Allan AC, Bendahmane A, Coyne CJ, Timmerman-Vaughan GM, Ellis TH.

Fig 3

Abstract
BACKGROUND: The genetic regulation of flower color has been widely studied, notably as a character used by Mendel and his predecessors in the study of inheritance in pea.
METHODOLOGY/PRINCIPAL FINDINGS: We used the genome sequence of model legumes, together with their known synteny to the pea genome to identify candidate genes for the A and A2 loci in pea. We then used a combination of genetic mapping, fast neutron mutant analysis, allelic diversity, transcript quantification and transient expression complementation studies to confirm the identity of the candidates.
CONCLUSIONS/SIGNIFICANCE: We have identified the pea genes A and A2. A is the factor determining anthocyanin pigmentation in pea that was used by Gregor Mendel 150 years ago in his study of inheritance. The A gene encodes a bHLH transcription factor. The white flowered mutant allele most likely used by Mendel is a simple G to A transition in a splice donor site that leads to a mis-spliced mRNA with a premature stop codon, and we have identified a second rare mutant allele. The A2 gene encodes a WD40 protein that is part of an evolutionarily conserved regulatory complex.


An Induced Mutation in Tomato eIF4E Leads to Immunity to Two Potyviruses

PLoS ONE 5(6): e11313. doi:10.1371/journal.pone.0011313 June 25, 2010 PMID: 20593023
Florence Piron, Maryse Nicolaï, Silvia Minoïa, Elodie Piednoir, André Moretti, Aurélie Salgues, Dani Zamir, Carole Caranta, Abdelhafid Bendahmane

Background
The characterization of natural recessive resistance genes and Arabidopsis virus-resistant mutants have implicated translation initiation factors of the eIF4E and eIF4G families as susceptibility factors required for virus infection and resistance function.
Methodology/Principal Findings
To investigate further the role of translation initiation factors in virus resistance we set up a TILLING platform in tomato, cloned genes encoding for translation initiation factors eIF4E and eIF4G and screened for induced mutations that lead to virus resistance. A splicing mutant of the eukaryotic translation initiation factor, S.l_eIF4E1 G1485A, was identified and characterized with respect to cap binding activity and resistance spectrum. Molecular analysis of the transcript of the mutant form showed that both the second and the third exons were miss-spliced, leading to a truncated mRNA. The resulting truncated eIF4E1 protein is also impaired in cap-binding activity. The mutant line had no growth defect, likely because of functional redundancy with others eIF4E isoforms. When infected with different potyviruses, the mutant line was immune to two strains of Potato virus Y and Pepper mottle virus and susceptible to Tobacco each virus.
Conclusions/Significance
Mutation analysis of translation initiation factors shows that translation initiation factors of the eIF4E family are determinants of plant susceptibility to RNA viruses and viruses have adopted strategies to use different isoforms. This work also demonstrates the effectiveness of TILLING as a reverse genetics tool to improve crop species. We have also developed a complete tool that can be used for both forward and reverse genetics in tomato, for both basic science and crop improvement. By opening it to the community, we hope to fulfill the expectations of both crop breeders and scientists who are using tomato as their model of study.


Host plant resistance to aphids in cultivated crops: genetic and molecular bases, and interactions with aphid populations.

C R Biol. 2010 Jun-Jul;333(6-7):566-73. Epub 2010 May 15. PMID: 20541167
Dogimont C, Bendahmane A, Chovelon V, Boissot N.

Host plant resistance is an efficient and environmentally friendly means of controlling insects, including aphids, but resistant-breaking biotypes have occurred in several plant-aphid systems. Our review of the genetic and molecular bases of aphid resistance in crop species emphasizes the limited number of aphid resistance genes and alleles. Inheritance of aphid resistance may be monogenic (dominant or recessive genes) or polygenic. Two dominant, aphid resistance genes have been isolated to date. They both encode NBS-LRR proteins involved in the specific recognition of aphids. Strategies to ensure aphid resistance effectiveness and durability are discussed. Innovative research activities are proposed. Copyright 2010 Académie des sciences. Published by Elsevier SAS. All rights reserved.


A blessing in disguise: Transposable elements are more than parasites.

Epigenetics. 2010 Jul 22;5(5). PMID: 20458177
Martin A, Bendahmane A.

Transposable elements (TEs) are various DNA fragments inserted throughout genomes, which are able to move or duplicate themselves. Recent advances in genomics have placed them back at the center of genome dynamics. One of the emerging observations, especially in plants, is the importance of interactions between TEs and genes to generate or to participate in relevant functions essential for development, adaptation and/or life cycle. A recent publication illustrates the influence of TEs epigenetic control on the expression of a neighboring gene crucial for reproduction. Different reports lately showed that a fundamental mechanism such as imprinting is likely to be closely linked to the dynamics of TEs epigenetic control. Here we discuss and bring together these and others recent findings, to underline that the cis-vicinity or the trans-relation between TEs and genes could bring unexpected but positive outcomes.
Epigenetics, Volume 5, Issue 5, July 1, 2010


Multiple Coat Protein Mutations Abolish Recognition of Pepino mosaic potexvirus (PepMV) by the Potato Rx Resistance Gene in Transgenic Tomatoes.

Mol Plant Microbe Interact. 2010 Apr;23(4):376-83. PMID: 20192825
Candresse T, Marais A, Faure C, Dubrana MP, Gombert J, Bendahmane A.

Despite the fact that Pepino mosaic virus (PepMV) and Potato virus X (PVX) share less than 40% identity in their coat proteins (CP), the known PVX elicitor of Rx, transgenic tomato (cv. Microtom) plants expressing a functional potato Rx resistance gene showed resistance toward PepMV. However, in a low percentage of plants, PepMV accumulation was observed and back inoculation experiments demonstrated that these plants contained resistance-breaking PepMV variants. Sequencing of the CP gene of these variants showed the accumulation of mutations in the amino acid 41 to 125 region the CP, whereas no mutations were observed in the nonevolved isolates. Agroinfiltration-mediated transient expression of the mutant CP demonstrated that they had a greatly attenuated or abolished ability to induce a hypersensitive reaction in Rx-expressing Nicotiana benthamiana leaves. The transient expression of truncated forms of the PepMV CP allowed the identification of a minimal elicitor domain (amino acids 30 to 136). These results demonstrate that the Rx-based sensing system is able to recognize the PepMV CP but, contrary to the situation with PVX, for which only two closely spaced resistance-breaking mutations are known, many mutations over a significant stretch of the PepMV CP allow escape from recognition by Rx.


A new mutant genetic resource for tomato crop improvement by TILLING technology.

BMC Res Notes. 2010 Mar 12;3:69. PMID: 20222995
Minoia S, Petrozza A, D'Onofrio O, Piron F, Mosca G, Sozio G, Cellini F, Bendahmane A, Carriero F.

ABSTRACT: BACKGROUND: In the last decade, the availability of gene sequences of many plant species, including tomato, has encouraged the development of strategies that do not rely on genetic transformation techniques (GMOs) for imparting desired traits in crops. One of these new emerging technology is TILLING (Targeting Induced Local Lesions In Genomes), a reverse genetics tool, which is proving to be very valuable in creating new traits in different crop species. RESULTS: To apply TILLING to tomato, a new mutant collection was generated in the genetic background of the processing tomato cultivar Red Setter by treating seeds with two different ethylemethane sulfonate doses (0.7% and 1%). An associated phenotype database, LycoTILL, was developed and a TILLING platform was also established. The interactive and evolving database is available online to the community for phenotypic alteration inquiries. To validate the Red Setter TILLING platform, induced point mutations were searched in 7 tomato genes with the mismatch-specific ENDO1 nuclease. In total 9.5 kb of tomato genome were screened and 66 nucleotide substitutions were identified. The overall mutation density was estimated and it resulted to be 1/322 kb and 1/574 kb for the 1% EMS and 0.7% EMS treatment respectively. CONCLUSIONS: The mutation density estimated in our collection and its comparison with other TILLING populations demonstrate that the Red Setter genetic resource is suitable for use in high-throughput mutation discovery. The Red Setter TILLING platform is open to the research community and is publicly available via web for requesting mutation screening services.


2009

A transposon-induced epigenetic change leads to sex determination in melon.

Nature. 2009 Oct 22;461(7267):1135-8. PMID: 19847267
Antoine Martin (1), Christelle Troadec (1), Adnane Boualem (1), Mazen Rajab (1), Ronan Fernandez (1), Halima Morin (2), Michel Pitrat (3), Catherine Dogimont (3) & Abdelhafid Bendahmane (1)
(1) INRA-CNRS, UMR1165, Unité de Recherche en Génomique Végétale, 2 rue Gaston Crémieux, F-91057 Evry, France
(2) Plateforme de Cytologie et d'Imagerie Végétale, Institut Jean Pierre Bourgin, INRA, 78026 Versailles Cedex, France
(3) INRA, UR 1052, Unité de Génétique et d'Amélioration des Fruits et Légumes, BP 94, F-84143 Montfavet, France

Sex determination in plants leads to the development of unisexual flowers from an originally bisexual floral meristem. This mechanism results in the enhancement of outcrossing and promotes genetic variability, the consequences of which are advantageous to the evolution of a species. In melon, sexual forms are controlled by identity of the alleles at the andromonoecious (a) and gynoecious (g) loci. We previously showed that the a gene encodes an ethylene biosynthesis enzyme, CmACS-7, that represses stamen development in female flowers. Here we show that the transition from male to female flowers in gynoecious lines results from epigenetic changes in the promoter of a transcription factor, CmWIP1. This natural and heritable epigenetic change resulted from the insertion of a transposon, which is required for initiation and maintenance of the spreading of DNA methylation to the CmWIP1 promoter. Expression of CmWIP1 leads to carpel abortion, resulting in the development of unisexual male flowers. Moreover, we show that CmWIP1 indirectly represses the expression of the andromonoecious gene, CmACS-7, to allow stamen development. Together our data indicate a model in which CmACS-7 and CmWIP1 interact to control the development of male, female and hermaphrodite flowers in melon.


Non-synonymous single nucleotide polymorphisms in the watermelon eIF4E gene are closely associated with resistance to Zucchini yellow mosaic virus.

Theor Appl Genet. 2009 Oct 10. PMID: 19820912
Kai-Shu Ling (1), Karen R. Harris (1), Jenelle D. F. Meyer (2), Amnon Levi (1), Nihat Guner (3), Todd C. Wehner (3), Abdelhafid Bendahmane (4) and Michael J. Havey (2)
(1) U.S. Vegetable Laboratory, U.S. Department of Agriculture, Agriculture Research Service, Charleston, SC 29414, USA
(2) U.S. Department of Agriculture, Agriculture Research Service, Department of Horticulture, University of Wisconsin, Madison, WI, USA
(3) Department of Horticultural Science, North Carolina State University, Raleigh, NC 27695, USA
(4) INRA (Institut National de la Recherche Agronomique) – CNRS, UMR1165, Unité de Recherche en Génomique Végétale, 2 rue Gaston Crémieux, 91057 Evry, France

Abstract Zucchini yellow mosaic virus (ZYMV) is one of the most economically important potyviruses infecting cucurbit crops worldwide. Using a candidate gene approach, we cloned and sequenced eIF4E and eIF(iso)4E gene segments in watermelon. Analysis of the nucleotide sequences between the ZYMV-resistant watermelon plant introduction PI 595203 (Citrullus lanatus var. lanatus) and the ZYMV-susceptible watermelon cultivar ‘New Hampshire Midget’ (‘NHM’) showed the presence of single nucleotide polymorphisms (SNPs). Initial analysis of the identified SNPs in association studies indicated that SNPs in the eIF4E, but not eIF(iso)4E, were closely associated to the phenotype of ZYMV-resistance in 70 F2 and 114 BC1R progenies. Subsequently, we focused our efforts in obtaining the entire genomic sequence of watermelon eIF4E. Three SNPs were identified between PI 595203 and NHM. One of the SNPs (A241C) was in exon 1 and the other two SNPs (C309A and T554G) were in the first intron of the gene. SNP241 which resulted in an amino acid substitution (proline to threonine) was shown to be located in the critical cap recognition and binding area, similar to that of several plant species resistance to potyviruses. Analysis of a cleaved amplified polymorphism sequence (CAPS) marker derived from this SNP in F2 and BC1R populations demonstrated a cosegregation between the CAPS-2 marker and their ZYMV resistance or susceptibility phenotype. When we investigated whether such SNP mutation in the eIF4E was also conserved in several other PIs of C. lanatus var. citroides, we identified a different SNP (A171G) resulting in another amino acid substitution (D71G) from four ZYMV-resistant C. lanatus var. citroides (PI 244018, PI 482261, PI 482299, and PI 482322). Additional CAPS markers were also identified. Availability of all these CAPS markers will enable marker-aided breeding of watermelon for ZYMV resistance.
The GenBank accession numbers for the sequences reported in this paper are FJ184033–FJ184038.


A conserved ethylene biosynthesis enzyme leads to andromonoecy in two cucumis species.

PLoS One. 2009 Jul 3;4(7):e6144. PMID: 19578542
Adnane Boualem (1), Christelle Troadec (1), Irina Kovalski (2), Marie-Agnes Sari (3), Rafael Perl-Treves (2), Abdelhafid Bendahmane (1*)
(1) INRA-CNRS, UMR1165, Unité de Recherche en Génomique Végétale, Evry, France,
(2) The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel,
(3) CNRS, UMR 8601, Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, Université René Descartes, Paris, France

PMID: 19578542

Andromonoecy is a widespread sexual system in angiosperms, characterized by plants carrying both male and bisexual flowers. Monoecy is characterized by the presence of both male and female flowers on the same plant. In cucumber, these sexual forms are controlled by the identity of the alleles at the M locus. In melon, we recently showed that the transition from monoecy to andromonoecy result from a mutation in 1-aminocyclopropane-1-carboxylic acid synthase (ACS) gene, CmACS-7. To isolate the andromonoecy gene in cucumber we used a candidate gene approach in combination with genetical and biochemical analysis. We demonstrated co-segregation of CsACS2, a close homolog of CmACS-7, with the M locus. Sequence analysis of CsACS2 in cucumber accessions identified four CsACS2 isoforms, three in andromonoecious and one in monoecious lines. To determine whether the andromonoecious phenotype is due to a loss of ACS enzymatic activity, we expressed the four isoforms in Escherichia coli and assayed their activity in vitro. Like in melon, the isoforms from the andromonoecious lines showed reduced to no enzymatic activity and the isoform from the monoecious line was active. Consistent with this, the mutations leading andromonoecy were clustered in the active site of the enzyme. Based on this, we concluded that active CsACS2 enzyme leads to the development of female flowers in monoecious lines, whereas a reduction of enzymatic activity yields hermaphrodite flowers. Consistent with this, CsACS2, like CmACS-7 in melon, is expressed specifically in carpel primordia of buds determined to develop carpels. Following ACS expression, inter-organ communication is likely responsible for the inhibition of stamina development. In both melon and cucumber, flower unisexuality seems to be the ancestral situation, as the majority of Cucumis species are monoecious. Thus, the ancestor gene of CmACS-7/CsACS2 likely have controlled the stamen development before speciation of Cucumis sativus (cucumber) and Cucumis melo (melon) that have diverged over 40 My ago. The isolation of the genes for andromonoecy in Cucumis species provides a molecular basis for understanding how sexual systems arise and are maintained within and between species.


Phenotypic and fine genetic characterization of the D locus controlling fruit acidity in peach.

BMC Plant Biol. 2009 May 15;9(1):59. PMID: 19445673
Boudehri K (1), Bendahmane A (2), Cardinet G (1), Troadec C (2), Moing A (3, 4), Dirlewanger E. (1*)
(1) INRA, UR0419, Unité de Recherches sur les Espèces Fruitières, Centre de Bordeaux, BP 81, F-33140 Villenave d'Ornon, France
(2) INRA-CNRS, UMR1165 Unité de Recherche en Génomique Végétale (URGV), 2 rue Gaston Crémieux, F-91057 Evry, France
(3) INRA - UMR619 Fruit Biology, INRA, Université de Bordeaux 1, Université de Bordeaux 2, BP 81, F-33140 Villenave d’Ornon, France
(4) Metabolome-Fluxome Pole, IFR103 BVI, BP 81, F-33140 Villenave d’Ornon, France

ABSTRACT: BACKGROUND: Acidity is an essential component of the organoleptic quality of fleshy fruits. However, in these fruits, the physiological and molecular mechanisms that control fruit acidity remain unclear. In peach the D locus controls fruit acidity; low-acidity is determined by the dominant allele. Using a peach progeny of 208 F2 trees, the D locus was mapped to the proximal end of linkage group 5 and co-localized with major QTLs involved in the control of fruit pH, titratable acidity and organic acid concentration and small QTLs for sugar concentration. In order to investigate the molecular basis of fruit acidity in peach we initiated the map-based cloning of the D locus. RESULTS: In order to generate a high-resolution linkage map in the vicinity of the D locus, 1,024 AFLP primer combinations were screened using DNA of bulked acid and low-acid segregants. We also screened a segregating population of 1,718 individuals for chromosomal recombination events linked to the D locus and identified 308 individuals with recombination events close to D. Using these recombinant individuals we delimited the D locus to a genetic interval of 0.4 cM. We also constructed a peach BAC library of 52,000 clones with a mean insert size of 90 kb. The screening of the BAC library with markers tightly linked to D locus indicated that 1 cM corresponds to 250 kb at the vicinity of the D locus. CONCLUSIONS: In the present work we presented the first high-resolution genetic map of D locus in peach. We also constructed a peach BAC library of approximately 15x genome equivalent. This fine genetic and physical characterization of the D locus is the first step towards the isolation of the gene(s) underlying fruit acidity in peach.


Tendril-less Regulates Tendril Formation in Pea Leaves.

Plant Cell. 2009 Feb 10. [Epub ahead of print] PMID: 19208900
Hofer J, Turner L, Moreau C, Ambrose M, Isaac P, Butcher S, Weller J, Dupin A, Dalmais M, Le Signor C, Bendahmane A, Ellis N.
Department of Crop Genetics, John Ines Centre, Colney Lane, Norwich NR4 7UH, United Kingdom.

Figure 5. Expression Domains of Tl.

Tendrils are contact-sensitive, filamentous organs that permit climbing plants to tether to their taller neighbors. Tendrilled legume species are grown as field crops, where the tendrils contribute to the physical support of the crop prior to harvest. The homeotic tendril-less (tl) mutation in garden pea (Pisum sativum), identified almost a century ago, transforms tendrils into leaflets. In this study, we used a systematic marker screen of fast neutron-generated tl deletion mutants to identify Tl as a Class I homeodomain leucine zipper (HDZIP) transcription factor. We confirmed the tendril-less phenotype as loss of function by targeting induced local lesions in genomes (TILLING) in garden pea and by analysis of the tendril-less phenotype of the t mutant in sweet pea (Lathyrus odoratus). The conversion of tendrils into leaflets in both mutants demonstrates that the pea tendril is a modified leaflet, inhibited from completing laminar development by Tl. We provide evidence to show that lamina inhibition requires Unifoliata/LEAFY-mediated Tl expression in organs emerging in the distal region of the leaf primordium. Phylogenetic analyses show that Tl is an unusual Class I HDZIP protein and that tendrils evolved either once or twice in Papilionoid legumes. We suggest that tendrils arose in the Fabeae clade of Papilionoid legumes through acquisition of the Tl gene.


2008

Characterization of Raphanus sativus Pentatricopeptide Repeat Proteins Encoded by the Fertility Restorer Locus for Ogura Cytoplasmic Male Sterility.

Plant Cell. 2008 Dec 19. PMID: 19098270
Uyttewaal M, Arnal N, Quadrado M, Martin-Canadell A, Vrielynck N, Hiard S, Gherbi H, Bendahmane A, Budar F, Mireau H.
Institut National de la Recherche Agronomique, Station de Génétique et d'Amélioration des Plantes, 78026 Versailles, France;
Ecole Normale Supérieure, Laboratoire de Reproduction et Développement des Plantes, 69364 Lyon, France.

Cytoplasmic male sterility is a maternally inherited trait in higher plants that prevents the production of functional pollen. Ogura cytoplasmic male sterility in radish (Raphanus sativus) is regulated by the orf138 mitochondrial locus. Male fertility can be restored when orf138 accumulation is suppressed by the nuclear Rfo locus, which consists of three genes putatively encoding highly similar pentatricopeptide repeat proteins (PPR-A, -B, and -C). We produced transgenic rapeseed (Brassica napus) plants separately expressing PPR-A and PPR-B and demonstrated that both encoded proteins accumulated preferentially in the anthers of young flower buds. Immunodetection of ORF138 showed that, unlike PPR-B, PPR-A had no effect on the synthesis of the sterility protein. Moreover, immunolocalization experiments indicated that complete elimination of ORF138 from the tapetum of anthers correlated with the restoration of fertility. Thus, the primary role of PPR-B in restoring fertility is to inhibit ORF138 synthesis in the tapetum of young anthers. In situ hybridization experiments confirmed, at the cellular level, that PPR-B has no effect on the accumulation of orf138 mRNA. Lastly, immunoprecipitation experiments demonstrated that PPR-B, but not PPR-A, is associated with the orf138 RNA in vivo, linking restoration activity with the ability to directly or indirectly interact with the orf138 RNA. Together, our data support a role for PPR-B in the translational regulation of orf138 mRNA.


The rx gene confers resistance to a range of potexviruses in transgenic Nicotiana plants.

Mol Plant Microbe Interact. 2008 Sep;21(9):1154-64. PMID: 18700820
Baurès I, Candresse T, Leveau A, Bendahmane A, Sturbois B.

Rx-mediated resistance was analyzed in Rx-expressing transgenic Nicotiana plants. The infection outcome of nine Potato virus X isolates mutated at amino acid positions 121 and 127 of the coat protein (CP) confirmed the key role of these amino acids but provided a more complex picture than previously reported. In particular, in Rx-expressing Nicotiana spp., eliciting activity modulated by amino acid 121 was conditioned by the nature of amino acid 127. These results suggest that the specificity of recognition might be modulated by host factors that are somehow subtly modified between Rx-expressing potato and Rx-expressing transgenic Nicotiana plants. Moreover, the CP of three Potexviruses, Narcissus mosaic virus (NMV), White clover mosaic virus (WClMV), and Cymbidium mosaic virus (CymMV), are all recognized by the Rx-based machinery and able to trigger an Rx-dependant hypersensitive response. A smaller elicitor of 90 amino acids was identified in the CP of NMV and WClMV, which contains the previously identified key positions 121 and 127. This elicitor is only weakly conserved (approximately 40% identity) among the CP of the various recognized viruses, suggesting that the Rx molecular machinery targets a conserved structural element of the Potexvirus CP rather than a conserved amino acid motif.


A conserved mutation in an ethylene biosynthesis enzyme leads to andromonoecy in melons.

Science. 2008 Aug 8;321(5890):836-8. PMID: 18687965
Boualem A, Fergany M, Fernandez R, Troadec C, Martin A, Morin H, Sari MA, Collin F, Flowers JM, Pitrat M, Purugganan MD, Dogimont C, Bendahmane A.
INRA (Institut National de la Recherche Agronomique)-CNRS, UMR1165, URGV, Evry, France.

Andromonoecy is a widespread sexual system in angiosperms characterized by plants carrying both male and bisexual flowers. In melon, this sexual form is controlled by the identity of the alleles at the andromonoecious (a) locus. Cloning of the a gene reveals that andromonoecy results from a mutation in the active site of 1-aminocyclopropane-1-carboxylic acid synthase. Expression of the active enzyme inhibits the development of the male organs and is not required for carpel development. A causal single-nucleotide polymorphism associated with andromonoecy was identified, which suggests that the a allele has been under recent positive selection and may be linked to the evolution of this sexual system.


Mutation detection using ENDO1: application to disease diagnostics in humans and TILLING and Eco-TILLING in plants.

BMC Mol Biol. 2008 Apr 23;9(1):42 [Epub ahead of print] PMID: 18433472
Triques K, Piednoir E, Dalmais M, Schmidt J, Le Signor C, Sharkey M, Caboche M, Sturbois B, Bendahmane A.

ABSTRACT:
BACKGROUND: Enzymatic mutation detection methods are based on the cleavage of heteroduplex DNA by a mismatch-specific endonuclease at mismatch sites and the analysis of the digestion product on a DNA sequencer. Important limitations of these methods are the availability of a mismatch-specific endonuclease, their sensitivity in detecting one allele in pool of DNA, the cost of the analysis and the ease by which the technique could be implemented in a standard molecular biology laboratory.
RESULTS: The co-agroinfiltration of ENDO1 and p19 constructs into N. benthamiana leaves allowed high level of transient expression of a mismatch-specific and sensitive endonuclease, ENDO1 from Arabidopsis thaliana. We demonstrate the broad range of uses of the produced enzyme in detection of mutations. In human, we report the diagnosis of the G1691A mutation in Leiden factor-V gene associated with venous thrombosis and the fingerprinting of HIV-1 quasispecies in patients subjected to antiretroviral treatments. In plants, we report the use of ENDO1 system for detection of mutant alleles of Retinoblastoma-related gene by TILLING in Pisum sativum and discovery of natural sequence variations by Eco-TILLING in Arabidopsis thaliana.
CONCLUSIONS: We introduce a cost-effective tool based on a simplified purification protocol of a mismatch-specific and sensitive endonuclease, ENDO1. Especially, we report the successful applications of ENDO1 in mutation diagnostics in humans, fingerprinting of complex population of viruses, and in TILLING and Eco-TILLING in plants.


UTILLdb, a Pisum sativum in silico forward and reverse genetics tool

fig 3

Genome Biology 2008, 9:R43 doi:10.1186/gb-2008-9-2-r43 PMID:18302733
Marion Dalmais, Julien Schmidt, Christine Le Signor, Francoise Moussy, Judith Burstin, Vincent Savois, Gregoire Aubert, Veronique Brunaud, Yannick de Oliveira, Cecile Guichard, Richard Thompson and Abdelhafid Bendahmane
URGV, INRA, Unité Mixte de Recherche en Génétique et Ecophysiologie des Légumineuses (INRA-ENESAD), Domaine d'Epoisses, 21110 Bretenières,

The systematic characterisation of gene functions in species recalcitrant to Agrobacterium-based transformation, like Pisum sativum, remains a challenge. To develop a high throughput forward and reverse genetics tool in pea, we have constructed a reference EMS-mutant population and developed a database, UTILLdb, which contains phenotypic as well as sequence information on mutant genes. UTILLdb can be searched online for TILLING alleles, through the BLAST tool, or for phenotypic information about mutants by keywords.


Structure of two melon regions reveals high microsynteny with sequenced plant species.

Mol Genet Genomics. 2007 Dec;278(6):611-22. Epub 2007 Jul 31. PMID: 17665215
Deleu W, González V, Monfort A, Bendahmane A, Puigdomènech P, Arús P, Garcia-Mas J.
IRTA, Centre de Recerca en Agrigenòmica CSIC-IRTA-UAB, Carretera de Cabrils Km2, 08348, Cabrils (Barcelona), Spain, jordi.garcia@irta.es.

In this study, two melon bacterial artificial chromosome (BAC) clones have been sequenced and annotated. BAC 1-21-10 spans 92 kb and contains the nsv locus conferring resistance to the Melon Necrotic Spot Virus (MNSV) in melon linkage group 11. BAC 13J4 spans 98 kb and belongs to a BAC contig containing resistance gene homologues, extending a previous sequenced region of 117 kb in linkage group 4. Both regions have microsyntenic relationships to the model plant species Arabidopsis thaliana, and to Medicago truncatula and Populus trichocarpa. The network of synteny found between melon and each of the sequenced genomes reflects the polyploid structure of Arabidopsis, Populus, and Medicago genomes due to whole genome duplications (WGD). A detailed analysis revealed that both melon regions have a lower relative syntenic quality with Arabidopsis (eurosid II) than when compared to Populus and Medicago (eurosid I). Although phylogenetically Cucurbitales seem to be closer to Fabales than to Malphigiales, synteny was higher between both melon regions and Populus. Presented data imply that the recently completed Populus genome sequence could preferentially be used to obtain positional information in melon, based on microsynteny.


2007

Characterization of Arabidopsis thaliana mismatch specific endonucleases: application to mutation discovery by TILLING in pea.

Plant J. 2007 Sep;51(6):1116-25. Epub 2007 Jul 25. PMID: 17651368
Triques K, Sturbois B, Gallais S, Dalmais M, Chauvin S, Christian Clepet, Sébastien Aubourg, Rameau C, Michel Caboche, Abdel Ihafid Bendahmane
URGV, UMR INRA CNRS. Evry, France.

Scanning DNA sequences for mutations and polymorphisms has become one of the most challenging, often expensive and time-consuming obstacles in many molecular genetic applications, including reverse genetic and clinical diagnostic applications. Enzymatic mutation detection methods are based on the cleavage of heteroduplex DNA at the mismatch sites. These methods are often limited by the availability of a mismatch-specific endonuclease, their sensitivity in detecting one allele in a pool of DNA and their costs. Here, we present detailed biochemical analysis of five Arabidopsis putative mismatch-specific endonucleases. One of them, ENDO1, is presented as the first endonuclease that recognizes and cleaves all types of mismatches with high efficiency. We report on a very simple protocol for the expression and purification of ENDO1. The ENDO1 system could be exploited in a wide range of mutation diagnostic tools. In particular, we report the use of ENDO1 for discovery of point mutations in the gibberellin 3beta-hydrolase gene of Pisum sativum. Twenty-one independent mutants were isolated, five of these were characterized and two new mutations affecting internodes length were identified. To further evaluate the quality of the mutant population we screened for mutations in four other genes and identified 5-21 new alleles per target. Based on the frequency of the obtained alleles we concluded that the pea population described here would be suitable for use in a large reverse-genetics project.


EcoTILLING for the identification of allelic variants of melon eIF4E, a factor that controls virus susceptibility.

BMC Plant Biol. 2007 Jun 21;7:34. PMID: 17584936
Nieto C, Piron F, Dalmais M, Marco CF, Moriones E, Gómez-Guillamón ML, Truniger V, Gómez P, Garcia-Mas J, Aranda MA, Bendahmane A.
Centro de Edafología y Biología Aplicada del Segura-CSIC, Espinardo, Murcia, Spain. agr009@cebas.csic.es

BACKGROUND: Translation initiation factors of the 4E and 4G protein families mediate resistance to several RNA plant viruses in the natural diversity of crops. Particularly, a single point mutation in melon eukaryotic translation initiation factor 4E (eIF4E) controls resistance to Melon necrotic spot virus (MNSV) in melon. Identification of allelic variants within natural populations by EcoTILLING has become a rapid genotype discovery method. RESULTS: A collection of Cucumis spp. was characterised for susceptibility to MNSV and Cucumber vein yellowing virus (CVYV) and used for the implementation of EcoTILLING to identify new allelic variants of eIF4E. A high conservation of eIF4E exonic regions was found, with six polymorphic sites identified out of EcoTILLING 113 accessions. Sequencing of regions surrounding polymorphisms revealed that all of them corresponded to silent nucleotide changes and just one to a non-silent change correlating with MNSV resistance. Except for the MNSV case, no correlation was found between variation of eIF4E and virus resistance, suggesting the implication of different and/or additional genes in previously identified resistance phenotypes. We have also characterized a new allele of eIF4E from Cucumis zeyheri, a wild relative of melon. Functional analyses suggested that this new eIF4E allele might be responsible for resistance to MNSV. CONCLUSION: This study shows the applicability of EcoTILLING in Cucumis spp., but given the conservation of eIF4E, new candidate genes should probably be considered to identify new sources of resistance to plant viruses. Part of the methodology described here could alternatively be used in TILLING experiments that serve to generate new eIF4E alleles.


2006

An eIF4E allele confers resistance to an uncapped and non-polyadenylated RNA virus in melon.

Plant J. 2006 Nov;48(3):452-62. Epub 2006 Oct 5. PMID: 17026540
Nieto C, Morales M, Orjeda G, Clepet C., Monfort A, Sturbois B., Puigdomenech P, Pitrat M., Caboche M, Dogimont C., Garcia-Mas J, Aranda M., and A. Bendahmane (2006).
Centro de Edafología y Biología Aplicada del Segura (CEBAS)- CSIC, Apdo. correos 164, 30100 Espinardo, Murcia, Spain.

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The characterization of natural recessive resistance genes and virus-resistant mutants of Arabidopsis have implicated translation initiation factors of the 4E family [eIF4E and eIF(iso)4E] as susceptibility factors required for virus multiplication and resistance expression. To date, viruses controlled by these genes mainly belong to the family Potyviridae. Melon necrotic spot virus (MNSV) belongs to the family Tombusviridae (genus Carmovirus) and is an uncapped and non-polyadenylated RNA virus. In melon, nsv-mediated resistance is a natural source of recessive resistance against all strains of MNSV except MNSV-264. Analyses of chimeras between non-resistance-breaking and resistance-breaking strains have shown that the avirulence determinant maps to the 3′-untranslated region (3′-UTR) of the viral genome. Using a combination of positional cloning and microsynteny analysis between Arabidopsis thaliana and melon, we genetically and physically delimited the nsv locus to a single bacterial artificial chromosome clone and identified the melon eukaryotic translation initiation factor 4E (Cm-eIF4E) as a candidate gene. Complementation analysis using a biolistic transient expression assay, confirmed Cm-eIF4E as the product of nsv. A single amino acid change at position 228 of the protein led to the resistance to MNSV. Protein expression and cap-binding analysis showed that Cm-eIF4E encoded by a resistant plant was not affected in it's cap-binding activity. The Agrobacterium-mediated transient expression of the susceptibility allele of Cm-eIF4E in Nicotiana benthamiana enhanced MNSV-264 accumulation. Based on these results, a model to explain melon resistance to MNSV is proposed. These data, and data from other authors, suggest that translation initiation factors of the eIF4E family are universal determinants of plant susceptibility to RNA viruses.


Physical delimitation of the pepper Bs3 resistance gene specifying recognitionof the AvrBs3 protein from Xanthomonas campestris pv. vesicatoria.

Theor Appl Genet. 2006 Sep;113(5):895-905. Epub 2006 Jul 28. PMID: 16874489
Jordan T, Romer P, Meyer A, Szczesny R, Pierre M, Piffanelli P, Bendahmane A., Bonas U, Lahaye T.
Institute of Genetics, Martin-Luther-Universität Halle-Wittenberg, 06099, Halle (Saale), Germany.

The pepper (Capsicum annuum) Bs3 gene confers resistance to avrBs3-expressing strains of the bacterial spot pathogen Xanthomonas campestris pv. vesicatoria. To physically delimit Bs3, a pepper YAC library was screened with two flanking DNA markers that are separated from Bs3 by 1.0 and 1.2 cM, respectively resulting in the identification of three YAC clones. Genetic mapping of the corresponding YACends revealed however, that these YACs do not cover Bs3 and subsequent screens with newly developed YACend markers failed to identify new YAC clones. Marker saturation at the Bs3 locus was carried out by amplified fragment length polymorphism (AFLP). The analysis of 1,024 primer combinations resulted in the identification of 47 new Bs3-linked AFLPs. High-resolution linkage mapping of Bs3 was accomplished by inspecting more than 4,000 F2 segregants resulting in a genetic resolution of 0.01 cM. Using tightly Bs3-linked YACend- and AFLP-derived markers we established a Bs3-spanning BAC contig and physically delimited the target gene within one BAC clone. The analysis of the Bs3-containing genomic region revealed substantial local variation in the correlation of genetic and physical distances.


2005

A physical map covering the nsv locus that confers resistance to Melon necroticspot virus in melon (Cucumis melo L.).

Theor Appl Genet. 2005 Sep;111(5):914-22. Epub 2005 Oct 18. PMID: 16052354
Morales M, Orjeda G, Nieto C, van Leeuwen H, Monfort A, Charpentier M, Caboche M, Arus P, Puigdomenech P, Aranda MA, Dogimont C, Bendahmane A,Garcia-Mas J.
Departament de Genètica Vegetal, Laboratori de Genètica Molecular Vegetal CSIC-IRTA, carretera de Cabrils s/n, 08348 Cabrils (Barcelona), Spain.

Melon necrotic spot virus (MNSV) is a member of the genus Carmovirus, which produces severe yield losses in melon and cucumber crops. The nsv gene is the only known natural source of resistance against MNSV in melon, and confers protection against all widespread strains of this virus. nsv has been previously mapped in melon linkage group 11, in a region spanning 5.9 cM, saturated with RAPD and AFLP markers. To identify the nsv gene by positional cloning, we started construction of a high-resolution map for this locus. On the basis of the two mapping populations, F(2) and BC1, which share the same resistant parent PI 161375 (nsv/nsv), and using more than 3,000 offspring, a high-resolution genetic map has been constructed in the region around the nsv locus, spanning 3.2 cM between CAPS markers M 29 and M 132. The availability of two melon BAC libraries allowed for screening and the identification of new markers closer to the resistance gene, by means of BAC-end sequencing and mapping. We constructed a BAC contig in this region and identified the marker 52 K 20 sp 6, which co-segregates with nsv in 408 F(2) and 2.727 BC1 individuals in both mapping populations. We also identified a single 100 kb BAC that physically contains the resistance gene and covers a genetic distance of 0.73 cM between both BAC ends. These are the basis for the isolation of the nsv recessive-resistance gene.


2004

Structural analysis of the eukaryotic initiation factor 4E gene controllingpotyvirus resistance in pepper: exploitation of a BAC library

Gene. 2004 Sep 1;338(2):209-16. PMID: 15315824
Ruffel S, Caranta C, Palloix A, Lefebvre V, Caboche M, Bendahmane A.
INRA, Plant Genomics Research Unit, 2 rue G. Cremieux, CP 5708, F-91057 Evry Cedex, France.

The pvr2 locus in pepper codes for a eukaryotic translation initiation factor 4E (eIF4E) gene that confers resistance to viruses belonging to the potyvirus genus. In this work, we describe the isolation and characterisation of the genomic sequence carrying the pvr2 locus. A Bacterial Artificial Chromosome (BAC) library that consisted of 239,232 clones with an average insert size of 123 kilobases (kb) was constructed from a Capsicum annuum line with the pvr2(+) allele for susceptibility to potato virus Y (PVY) and tobacco etch virus (TEV). Based on a polymerase chain reaction (PCR) screen with single-copy markers, three to seven positive BAC clones per markers were identified, indicating that the BAC library is suitable for pepper genome analysis. To determine the genomic organization of the pepper eIF4E gene, the library was screened with primers designed from the cDNA sequence and four positive BAC clones carrying the pvr2 locus were identified. A 7-kb DNA fragment containing the complete eIF4E gene was sub-cloned from the positive BAC clones and analysed. The eIF4E gene is organised into five exons and four introns and showed a strictly conserved exon/intron structure with eIF4E genes from Arabidopsis thaliana and rice. Moreover, the splice sites between plant exons 1/2 and 2/3 are conserved among eukaryotes including human, Drosophila and yeast. Several potential binding sites for MADS box transcription factors within the 5' flanking region of eIF4E genes from the three plant species were also predicted.


Advances in understanding recessive resistance to plant viruses

Molecular Plant Pathology, Volume 5 Issue 3 Page 223-233, May 2004
Diaz-pendon j, Truniger V., Nieto C., Garcia-mas J., Bendahmane A., Aranda M. (2004)

Recent work carried out to characterize recessive mutations which render experimental hosts non-permissive to viral infection (loss-of-susceptibility mutants) seems to be converging with new data on natural recessive resistance in crop species, and also with functional analyses of virus avirulence determinants. Perhaps the most well known examples are the studies that identified the eukaryotic translation initiation factors 4E(iso) (eIF(iso)4E) and 4E(eIF4E) as the host factors required for potyvirus multiplication within experimental and natural hosts, respectively, and the potyviral genome-linked protein (VPg) as the viral factor that directly interacts with eIF4E to promote potyvirus multiplication. The purpose of this paper is to review the available information on the characterization of loss-of-susceptibility mutants in experimental hosts, natural recessive resistances and virus avirulence factors, and also to comment on possible implications for the design of new sources of sustainable virus resistance.


2003

High throughput virus-induced gene silencing implicates heat shock protein 90in plant disease resistance

EMBO J. 2003 Nov 3;22(21):5690-9. PMID: 14592968
Lu R, Malcuit I, Moffett P, Ruiz MT, Peart J, Wu AJ, Rathjen JP, Bendahmane A, Day L, Baulcombe DC.
The Sainsbury Laboratory, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK.

Virus-induced gene silencing was used to assess the function of random Nicotiana benthamiana cDNAs in disease resistance. Out of 4992 cDNAs tested from a normalized library, there were 79 that suppressed a hypersensitive response (HR) associated with Pto-mediated resistance against Pseudomonas syringae. However, only six of these clones blocked the Pto-mediated suppression of P.syringae growth. The three clones giving the strongest loss of Pto resistance had inserts corresponding to HSP90 and also caused loss of Rx-mediated resistance against potato virus X and N-mediated tobacco mosaic virus resistance. The role of HSP90 as a cofactor of disease resistance is associated with stabilization of Rx protein levels and could be accounted for in part by SGT1 and other cofactors of disease resistance acting as co-chaperones. This approach illustrates the potential benefits and limitations of RNA silencing in forward screens of gene function in plants.


Characterization of a radish introgression carrying the Ogura fertility restorer gene Rfo in rapeseed, using the Arabidopsis genome sequence and radish genetic mapping.

Theor Appl Genet. 2003 Nov;107(8):1442-51. Epub 2003 Aug 27. PMID: 12942173
Giancola S, Marhadour S, Desloire S, Clouet V, Falentin-Guyomarc'h H, LalouiW, Falentin C, Pelletier G, Renard M, Bendahmane A, Delourme R, Budar F.
Station de Génétique et d'Amélioration des Plantes, INRA, Route de Saint-Cyr, 78026, Versailles cedex, France.

The radish Rfo gene restores male fertility in radish or rapeseed plants carrying Ogura cytoplasmic male-sterility. This system was first discovered in radish and was transferred to rapeseed for the production of F1 hybrid seeds. We aimed to identify the region of the Arabidopsis genome syntenic to the Rfo locus and to characterize the radish introgression in restored rapeseed. We used two methods: amplified consensus genetic markers (ACGMs) in restored rapeseed plants and construction of a precise genetic map around the Rfo gene in a segregating radish population. The use of ACGMs made it possible to detect radish orthologs of Arabidopsis genes in the restored rapeseed genome. We identified radish genes, linked to Rfo in rapeseed and whose orthologs in Arabidopsis are carried by chromosomes 1, 4 and 5. This indicates several breaks in colinearity between radish and Arabidopsis genomes in this region. We determined the positions of markers relative to each other and to the Rfo gene, using the progeny of a rapeseed plant with unstable meiotic transmission of the radish introgression. This enabled us to produce a schematic diagram of the radish introgression in rapeseed. Markers which could be mapped both on radish and restored rapeseed indicate that at least 50 cM of the radish genome is integrated in restored rapeseed. Using markers closely linked to the Rfo gene in rapeseed and radish, we identified a contig spanning six bacterial artificial chromosome (BAC) clones on Arabidopsis chromosome 1, which is likely to carry the orthologous Rfo gene.


Identification of the fertility restoration locus, Rfo, in radish, as a member of the pentatricopeptide-repeat protein family.

EMBO Rep. 2003 Jun;4(6):588-94. PMID: 12740605
Desloire S, Gherbi H, Laloui W, Marhadour S, Clouet V, Cattolico L, Falentin C, Giancola S, Renard M, Budar F, Small I, Caboche M, Delourme R and Bendahmane A (2003).
URGV, INRA, CNRS

Ogura cytoplasmic male sterility (CMS) in radish (Raphanus sativus) is caused by an aberrant mitochondrial gene, Orf138, that prevents the production of functional pollen without affecting female fertility. Rfo, a nuclear gene that restores male fertility, alters the expression of Orf138 at the post-transcriptional level. The Ogura CMS/Rfo two-component system is a useful model for investigating nuclear-cytoplasmic interactions, as well as the physiological basis of fertility restoration. Using a combination of positional cloning and microsynteny analysis of Arabidopsis thaliana and radish, we genetically and physically delimited the Rfo locus to a 15-kb DNA segment. Analysis of this segment shows that Rfo is a member of the pentatricopeptide repeat (PPR) family. In Arabidopsis, this family contains more than 450 members of unknown function, although most of them are predicted to be targeted to mitochondria and chloroplasts and are thought to have roles in organellar gene expression.


2002

A natural recessive resistance gene against potato virus Y in peppercorresponds to the eukaryotic initiation factor 4E (eIF4E)

Plant J. 2002 Dec;32(6):1067-75. PMID: 12492847
Ruffel S, Dussault MH, Palloix A, Moury B, Bendahmane A, Robaglia C, Caranta C.
Genetics and Breeding of Fruits and Vegetable, Dom St Maurice, BP94, F-84143 Montfavet.

We show here that the pvr2 locus in pepper, conferring recessive resistance against strains of potato virus Y (PVY), corresponds to a eukaryotic initiation factor 4E (eIF4E) gene. RFLP analysis on the PVY-susceptible and resistant pepper cultivars, using an eIF4E cDNA from tobacco as probe, revealed perfect map co-segregation between a polymorphism in the eIF4E gene and the pvr2 alleles, pvr2(1) (resistant to PVY-0) and pvr2(2) (resistant to PVY-0 and 1). The cloned pepper eIF4E cDNA encoded a 228 amino acid polypeptide with 70-86% nucleotide sequence identity with other plant eIF4Es. The sequences of eIF4E protein from two PVY-susceptible cultivars were identical and differed from the eIF4E sequences of the two PVY-resistant cultivars Yolo Y (YY) (pvr2(1)) and FloridaVR2 (F) (pvr2(2)) at two amino acids, a mutation common to both resistant genotypes and a second mutation specific to each. Complementation experiments were used to show that the eIF4E gene corresponds to pvr2. Thus, potato virus X-mediated transient expression of eIF4E from susceptible cultivar Yolo Wonder (YW) in the resistant genotype YY resulted in loss of resistance to subsequent PVY-0 inoculation and transient expression of eIF4E from YY (resistant to PVY-0; susceptible to PVY-1) rendered genotype F susceptible to PVY-1. Several lines of evidence indicate that interaction between the potyvirus genome-linked protein (VPg) and eIF4E are important for virus infectivity, suggesting that the recessive resistance could be due to incompatibility between the VPg and eIF4E in the resistant genotype.


Reviewed Papers

An eIF4E allele confers resistance against an uncapped and nonpolyadenylated RNA virus in melon

Plant Journal, 48 :452-62
Nieto CMorales M, Orjeda G, Clepet C., Monfort A, Sturbois B., Charpentier M, Puigdomenech P, Pitrat M., Caboche M, Dogimont C., Garcia-Mas J, Aranda M., Bendahmane A (2006)

High-resolution genetic mapping and physical delimitation of the pepper Bs3 resistance gene conferring recognition of the AvrBs3 protein from Xanthomonas campestris pv. Vesicatoria.

TAG 113 :895-905.
Jordan T., Römer P., Meyer A., Szczesny R., Pierre M., Staskawicz B., Piffanelli P., Bendahmane A., Bonas U., Lahaye T. (2006)

A physical map covering the nsv locus that confers resistance to Melon necrotic spot virus in melon (Cucumis melo L.)

TAG 111(5):914-22.
Morales M, Orjeda G, Nieto C, van Leeuwen H, Monfort A, Charpentier M, Caboche M, Arus P, Puigdomenech P, Aranda MA, Dogimont C, Bendahmane A, Garcia-Mas J. (2005)

Advances in understanding recessive resistance to plant viruses

Molecular Plant Pathology 5: 223–233
Diaz-pendon j, Truniger V., Nieto C., Garcia-mas J., Bendahmane A., Aranda M. (2004)

Structural analysis of the eukaryotic initiation factor 4E (eIF4E) gene controlling Potyvirus resistance in pepper: exploitation of a BAC library.

Gene, 338 : 209-216
Ruffel S., Caranta C., Palloix A., Lefebvre V., Caboche M., Bendahmane A. (2004)

High throughput virus-induced gene silencing identifies heat shock protein 90 as a cofactor of plant disease resistance

EMBO J. 22: 5690-5699.
R Lu, I Malcuit, P Moffett, T Ruiz, J Peart, A Wu, J Rathjen, A Bendahmane, L Day and D C. Baulcombe. (2003)

Characterization of a radish introgression carrying the ogura fertility restorer gene rfo in rapeseed, using the arabidopsis genome sequence and radish genetic mapping

TAG 107: 1442-1451
Giancola S., Marhadour S., Desloire S., Clouet V., Falentin-guyomarc’h H., Laloui W., Falentin C., Pelletier G., Renard M., Bendahmane A., Delourme R., and Budar F. (2003),

Identification of the fertility restoration locus, Rfo, in radish as a member of the pentatricopeptide-repeat protein family

EMBO report 4: 588-594
Desloire S, Gherbi H, Laloui W, Marhadour S, Clouet V, Falentin C, Giancola S, Budar F, Small I, Caboche M, Delourme R, Bendahmane A. (2003)

Constitutive gain-offunction mutants in a nucleotide binding site-leucine rich repeat protein encoded at the Rx locus of potato

Plant J. 2002 Oct;32(2):195-204. PMID: 12383085
Bendahmane A. Farnham G, Moffett P, and Baulcombe D.C. (2002)
The Sainsbury Laboratory, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK.

Rx in potato encodes a protein with a nucleotide binding site (NBS) and leucine-rich repeats (LRR) that confers resistance against Potato virus X. The NBS and LRR domains in Rx are present in many disease resistance proteins in plants and in regulators of apoptosis in animals. To investigate structure-function relationships of NBS-LRR proteins we exploited the potential of Rx to mediate a cell death response. With wild-type Rx cell death is elicited only in the presence of the viral coat protein. However, following random mutagenesis of Rx, we identified mutants in which cell death is activated in the absence of viral coat protein. Out of 2500 Rx clones tested there were seven constitutive gain-of-function mutants carrying eight independent mutations. The mutations encoded changes in the LRR or in conserved RNBS-D and MHD motifs of the NBS. Based on these findings we propose that there are inhibitory domains in the NBS and LRR. The constitutive gain-of-function phenotypes would be due to deletion or modification of these inhibitory domains. However activation of Rx is not simply release of negative regulation by the LRR and adjacent sequence because deleted forms of Rx that lack constitutive gain of function mutations are not active unless the protein is overexpressed.


2000

Agrobacterium transient expression system as a tool for disease resistance genes isolation: application to Rx2 locus in potato

Plant J. 2000 Jan;21(1):73-81. PMID: 10652152
Bendahmane A. Querci M, Kanyuka K, and Baulcombe D.C. (2000).
The Sainsbury Laboratory, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK.

Rx2 confers resistance against potato virus X (PVX). To clone Rx2, we developed a system based on Agrobacterium-mediated transient expression of candidate R genes in transgenic tobacco leaves expressing the PVX coat protein elicitor of Rx2-mediated resistance. Using this system, a potato gene eliciting HR specifically in the presence of the elicitor was identified. Based on genetical and functional analysis, it is concluded that the cloned gene is Rx2. The transient expression system is potentially adaptable to cloning of any other resistance gene. The Rx2 locus is on chromosome V of potato and the encoded protein is highly similar to the products of Rx1 and Rxh1 encoded on potato chromosome XII. Rxh1 has been shown elsewhere to encode a potato cyst nematode resistance gene Gpa2. All three proteins are in the leucine zipper-nucleotide binding site-leucine rich repeat class of resistance gene products. Rx1 and Rx2 are functionally identical and are almost identical in the C terminal region consistent with a role of the leucine rich repeats in recognition of the PVX coat protein. In the N terminal, half there are some regions where the Rx1 and Rx2 proteins are more similar to each other than to the Rxh1 protein. However, in other regions these proteins are more similar to Rxh1 than to each other. Based on this mosaic pattern of sequence similarity, we conclude that sequence exchange occurs repeatedly between genetically unlinked disease resistance genes through a process of gene conversion.


Homologues of a singleresistance- gene cluster in potato confer resistance to distinct pathogens: a virus and a nematode

Plant J. 2000 Sep;23(5):567-76. PMID: 10972883
Van der Vossen E., Rouppe van der Voort J., Kanyuka K., Bendahmane A., Sandbrink H., Baulcombe D.C., Bakker J., Stiekema W., Lankhorst R.K. (2000)
Plant Research International, Business unit Genomics, PO Box 16, 6700 AA, Wageningen, The Netherlands. e.a.g.vandervossen@plant.wag-ur.nl

The isolation of the nematode-resistance gene Gpa2 in potato is described, and it is demonstrated that highly homologous resistance genes of a single resistance-gene cluster can confer resistance to distinct pathogen species. Molecular analysis of the Gpa2 locus resulted in the identification of an R-gene cluster of four highly homologous genes in a region of approximately 115 kb. At least two of these genes are active: one corresponds to the previously isolated Rx1 gene that confers resistance to potato virus X, while the other corresponds to the Gpa2 gene that confers resistance to the potato cyst nematode Globodera pallida. The proteins encoded by the Gpa2 and the Rx1 genes share an overall homology of over 88% (amino-acid identity) and belong to the leucine-zipper, nucleotide-binding site, leucine-rich repeat (LZ-NBS-LRR)-containing class of plant resistance genes. From the sequence conservation between Gpa2 and Rx1 it is clear that there is a direct evolutionary relationship between the two proteins. Sequence diversity is concentrated in the LRR region and in the C-terminus. The putative effector domains are more conserved suggesting that, at least in this case, nematode and virus resistance cascades could share common components. These findings underline the potential of protein breeding for engineering new resistance specificities against plant pathogens in vitro.


1999

The Rx gene in potato controls separate virus resistance and cell death responses

Plant Cell. 1999 May;11(5):781-92. PMID: 10330465
Bendahmane A., Kanyuka K., Baulcombe D.C. (1999)
Sainsbury Laboratory, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, United Kingdom.

Rx-mediated extreme resistance against potato virus X in potato does not involve a necrotic hypersensitive response at the site of initial infection and thereby differs from the more usual type of disease resistance in plants. However, the Rx protein is structurally similar to products of disease resistance genes conferring the hypersensitive response. We show in both Nicotiana spp and potato that Rx has the potential to initiate a cell death response but that extreme resistance is separate and epistatic to necrosis. These data indicate that cell death and pathogen arrest are separate disease resistance responses in plants.


A zero background plasmid vector for BAC library construction

Biotechniques. 1999 Feb;26(2):228-30, 232. PMID: 10023529
Bendahmane A. (1999)
Sainsbury Laboratory, John Innes Centre, Colney, Norwich, England, UK. bendahm@bbsrc.ac.uk


A high resolution molecular map around the Rx locus of potato: Analysis of a complex locus in a tetraploid background

Theoretical and Applied Genetics 98:679-689.
Bendahmane A., Kanyuka K., Rouppe van der Voort J., Van der Vossen E., Baulcombe D.C. (1999)

Development of YLM, a codominant PCR marker closely linked to the Yd2 gene for resistance to barley yellow dwarf disease

Theoretical and Applied Genetics 97:1170-1177
Patridge N. G., Collins N. C., Bendahmane A. and Symons R. H. (1998)

New pathogen resistance gene cluster on chromosome XII of potato: Tight physical linkage of the nematode resistance gene Gpa2 and the virus resistance gene Rx

Mol Plant Microbe Interact 12:197-206.
Rouppe van der Voort J., Kanyuka K., Van der Vossen E., Bendahmane A., Lankhorst R.K., Stiekema W., Baulcombe D.C., Bakker J. (1998)

High resolution genetical and physical mapping of the Rx gene for extreme resistance to potato virus X in tetraploid potato

Theoretical and Applied Genetics 95:153-162
Bendahmane A., Kanyuka K., Baulcombe D.C. (1997)

Mapping of the nematode resistance locus Gpa2 in potato using a strategy based on comigrating AFLP markers

Theoretical and Applied Genetics 95: 874-880
Rouppe van der Voort J., Wolters P., Folkertsma R., Hutten R., van Zandvoort P., Vinke H., Kanyuka K., Bendahmane A., Jacobsen E., Janssen R., Bakker J. (1997)

1995

The coat protein of potato virus X is a strain-specific elicitor of Rx-mediated virus resistance in potato

Plant J. 1995 Dec;8(6):933-41. PMID: 8580963
Bendahmane A., Kohm B., Dedi C. and Baulcombe D. (1995)
Sainsbury Laboratory, Norwich Research Park, Colney, UK.

The Rx1 gene in potato confers extreme resistance to potato virus X (PVX). To investigate the mechanism and elicitation of Rx resistance, protoplasts of potato cv. Cara (Rx1 genotype) and Maris Bard (rx1 genotype) were inoculated with PVX and tobacco mosaic virus (TMV). At 24 h post-inoculation in Maris Bard protoplasts there was at least 100-fold more PVX RNA than in protoplasts of Cara. TMV RNA accumulated to the same level in both types of protoplast. However, when the TMV was inoculated together with PVX the accumulation of TMV RNA was suppressed in the Cara (Rx1 genotype) protoplasts to the same extent as PVX. The Rx1 resistance also suppressed accumulation of a recombinant TMV in which the coat protein gene was replaced with the coat protein gene of PVX. It is therefore concluded that Rx1-mediated resistance is elicited by the PVX coat protein, independently of any other proteins encoded by PVX. The domain of the coat protein with elicitor activity was localized by deletion and mutation analysis to the structural core of a nonvirion form of the coat protein.

Book Chapters

Genetic approaches to an Understanding of Specific Resistance Responses of Arabidopsis thaliana against phytopathogenic Pseudomonads

Advances in Molecular Genetics of Plant-Microbe Interactions. 2: 405-415, Kluwer Academic Publishers.
Dangl J., Debener T., Gerwin M., Kiedrowski S., Ritter C., Bendahmane A., Liedgens H., Lewald J. (1992)

Two paralogous genes of the GPA2 resistance locus confer both virus and nematode resistance to potato

Advances in Molecular Genetics of Plant-Microbe Interactions. 2: 428-432, Kluwer Academic Publishers.
Stiekema W., Van der Vossen E., Rouppe van der Voort J., Kanyuka K., Bendahmane A., Bakker J., Lankhorst R.K. (1999)

Génomique et caractérisation de gènes majeurs et QTL

Livre de génomique végétale, INRA.
Causse M, Lefebvre V, Bendahmane A (2003)

The key role of the eukaryotic initiation factor 4E (eIF4E) in plantpotyvirus interactions

Advances in Molecular Genetics of Plant-Microbe Interactions., Kluwer Academic Publishers.
Ruffel S, Dussault M.H, Duprat A, Palloix A, Moury B., Revers F.4, Bendahmane A., Robaglia C., Caranta C.- (2003)

Patents

Burget E, Pauquet J, Bendahmane A, Dogimont C, Pitrat M, Caboche M. (2003)
Aphis gosypii resistance gene. FR 0300287.

Bendahmanne A, Desloire S, Gherbi H, Laloui W, Caboche M, Budard F, Giancola S, Pelletier G, Delourme R, Marhadour S, Clouet V, Falentin C, Renard M. FR03/03224.

Ruffel S, Caranta C, Bendahmane A, Caboche M, Palloix A, Robaglia C. (2002)
Methods and DNA sequences used to produce plants resistant to potyviruses. PCT/FR03/00397, FR0201583

Sturbois B., Triques K., Gallais S., Caboche M., Bendahmane A. (2004)
Method for producing recombinant endonucleases having high specificity and sensitivity activity. PCT/FR04/02067

Triques K., Sturbois B., Aubourg S., Caboche M., Bendahmane A. (2004)
Method to identify and characterize plant endonucleases having specificity and sensitivity activity, and use of said endonucleases for mutation diagnostic. (FR 65629/D22388)