Cas9-assisted targeting of DNA fragments in complex genomes is viewed as an essential strategy to obtain high-quality and continuous sequence data. However, the purity of target loci selected by ...pulsed-field gel electrophoresis (PFGE) has so far been insufficient to assemble the sequence in one contig. Here, we describe the μLAS technology to capture and purify high molecular weight DNA. First, the technology is optimized to perform high sensitivity DNA profiling with a limit of detection of 20 fg/μl for 50 kb fragments and an analytical time of 50 min. Then, μLAS is operated to isolate a 31.5 kb locus cleaved by Cas9 in the genome of the plant Medicago truncatula. Target purification is validated on a Bacterial Artificial Chromosome plasmid, and subsequently carried out in whole genome with μLAS, PFGE or by combining these techniques. PacBio sequencing shows an enrichment factor of the target sequence of 84 with PFGE alone versus 892 by association of PFGE with μLAS. These performances allow us to sequence and assemble one contig of 29 441 bp with 99% sequence identity to the reference sequence.
Abstract
Cas9-assisted targeting of DNA fragments in complex genomes is viewed as an essential strategy to obtain high-quality and continuous sequence data. However, the purity of target loci ...selected by pulsed-field gel electrophoresis (PFGE) has so far been insufficient to assemble the sequence in one contig. Here, we describe the μLAS technology to capture and purify high molecular weight DNA. First, the technology is optimized to perform high sensitivity DNA profiling with a limit of detection of 20 fg/μl for 50 kb fragments and an analytical time of 50 min. Then, μLAS is operated to isolate a 31.5 kb locus cleaved by Cas9 in the genome of the plant Medicago truncatula. Target purification is validated on a Bacterial Artificial Chromosome plasmid, and subsequently carried out in whole genome with μLAS, PFGE or by combining these techniques. PacBio sequencing shows an enrichment factor of the target sequence of 84 with PFGE alone versus 892 by association of PFGE with μLAS. These performances allow us to sequence and assemble one contig of 29 441 bp with 99% sequence identity to the reference sequence.
Nod factors induce massive reprogramming of gene expression in the root epidermis, including the CRE1 cytokinin pathway which leads to both positive and negative regulation of nodulation.
Nod factors ...(
NF
s) are lipochitooligosaccharidic signal molecules produced by rhizobia, which play a key role in the rhizobium-legume symbiotic interaction. In this study, we analyzed the gene expression reprogramming induced by purified
NF
(4 and 24 h of treatment) in the root epidermis of the model legume
Medicago truncatula
. Tissue-specific transcriptome analysis was achieved by laser-capture microdissection coupled to high-depth RNA sequencing. The expression of 17,191 genes was detected in the epidermis, among which 1,070 were found to be regulated by
NF
addition, including previously characterized
NF
-induced marker genes. Many genes exhibited strong levels of transcriptional activation, sometimes only transiently at 4 h, indicating highly dynamic regulation. Expression reprogramming affected a variety of cellular processes, including perception, signaling, regulation of gene expression, as well as cell wall, cytoskeleton, transport, metabolism, and defense, with numerous
NF
-induced genes never identified before. Strikingly, early epidermal activation of cytokinin (
CK
) pathways was indicated, based on the induction of
CK
metabolic and signaling genes, including the CRE1 receptor essential to promote nodulation. These transcriptional activations were independently validated using promoter:β-glucuronidase fusions with the
MtCRE1
CK
receptor gene and a
CK
response reporter (
TWO COMPONENT SIGNALING SENSOR NEW
). A
CK
pretreatment reduced the
NF
induction of the
EARLY NODULIN11
(
ENOD11
) symbiotic marker, while a
CK
-degrading enzyme (CYTOKININ OXIDASE/DEHYDROGENASE3) ectopically expressed in the root epidermis led to increased
NF
induction of
ENOD11
and nodulation. Therefore,
CK
may play both positive and negative roles in
M. truncatula
nodulation.
Summary
A plant–microbe dual biological system was set up involving the model legume
M
edicago truncatula
and two bacteria, the soil‐borne root pathogen
R
alstonia solanacearum
and the beneficial ...symbiont
S
inorhizobium meliloti
.
Comparison of transcriptomes under symbiotic and pathogenic conditions highlighted the transcription factor
M
t
EFD
(Ethylene response Factor required for nodule Differentiation) as being upregulated in both interactions, together with a set of cytokinin‐related transcripts involved in metabolism, signaling and response.
M
t
RR
4
(
R
esponse
R
egulator), a cytokinin primary response gene negatively regulating cytokinin signaling and known as a target of
M
t
EFD
in nodulation processes, was retrieved in this set of transcripts.
Refined studies of
M
t
EFD
and Mt
RR
4
expression during
M
. truncatula
and
R
. solanacearum
interaction indicated differential kinetics of induction and requirement of central regulators of bacterial pathogenicity,
H
rp
G
and
H
rp
B
. Similar to
M
t
RR
4
,
M
t
EFD
upregulation during the pathogenic interaction was dependent on cytokinin perception mediated by the
M
t
CRE
1
(Cytokinin REsponse 1) receptor.
The use of
M
. truncatula efd‐1
and
cre1‐1
mutants evidenced
M
t
EFD
and cytokinin perception as positive factors for bacterial wilt development. These factors therefore play an important role in both root nodulation and root disease development.
An integrative biology approach was conducted in
Medicago truncatula for: (i) unraveling the coordinated regulation of
NO
3
−
,
NH
4
+
and N
2 acquisition by legumes to fulfill the plant N demand; ...and (ii) modeling the emerging properties occurring at the whole plant level. Upon localized addition of a high level of mineral N, the three N acquisition pathways displayed similar systemic feedback repression to adjust N acquisition capacities to the plant N status. Genes associated to these responses were in contrast rather specific to the N source. Following an N deficit,
NO
3
−
fed plants maintained efficiently their N status through rapid functional and developmental up regulations while N
2 fed plants responded by long term plasticity of nodule development. Regulatory genes associated with various symbiotic stages were further identified. An ecophysiological model simulating relations between leaf area and roots N retrieval was developed and now furnishes an analysis grid to characterize a spontaneous or induced genetic variability for plant N nutrition.
To cite this article: C. Salon et al., C. R. Biologies 332 (2009).
Une approche de biologie intégrative a été conduite sur
Medicago truncatula dans le but : (i) de comprendre les processus coordonnés de régulation de l'acquisition de
NO
3
−
,
NH
4
+
et N
2 mis en place chez les légumineuses pour satisfaire la demande en azote de la plante ; et (ii) de modéliser les propriétés émergentes observées à l'échelle de la plante. En situation de disponibilité locale forte en azote minéral, les 3 voies d'acquisition d'azote sont identiquement réprimées de manière systémique, ce qui permet d'ajuster l'acquisition d'N au statut azoté de la plante. En revanche les gènes associés à ces réponses sont plutôt spécifiques de la source d'N. Dans les situations de déficit en N, les plantes alimentées avec du
NO
3
−
maintiennent efficacement leur statut azoté grâce à une réponse structurale et fonctionnelle leur permettant d'augmenter le prélèvement de N ; au contraire les plantes alimentées par la symbiose fixatrice d'azote ajustent leur statut azoté sur le long terme grâce à la plasticité du développement nodulaire. Des gènes de régulation associés aux différents stades de la symbiose ont été identifiés. Un modèle écophysiologique simulant le prélèvement d'N en relation avec la mise en place de la surface foliaire a été développé et fournit une grille d'analyse de la variabilité génétique naturelle ou induite pour la nutrition azotée des légumineuses.
Pour citer cet article : C. Salon et al., C. R. Biologies 332 (2009).
Carl von Linné (1707-1778), naturaliste suédois, dévoile son système en 1735. À l’époque de cette publication, il n’est pas un naturaliste reconnu. L’évocation de son simple nom ne confère pas à son ...oeuvre une autorité telle qu’elle puisse expliquer son succès. De plus, la diffusion de son travail crée de profondes polémiques. Dans ces conditions, par quelles stratégies Linné diffuse-t-il son système et s’impose-t-il lui-même dans la cohorte des naturalistes de renom? Cette question en appelle une autre : quelles sont les modalités du succès qu’il connaît en quelques années et jusqu’à quel point son oeuvre vise-t-elle et atteint-elle un large public de scientifiques, mais aussi d’amateurs? Nous étudierons ces questions pour le seul cas de la botanique, dans un ouvrage de 1751, la Philosophie botanique, qui reprend ou compile l’ensemble des principes développés par Linné dans ses ouvrages précédents.
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