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In cereals, the root system is mainly composed of post-embryonic shoot-borne roots, named crown roots. The CROWN ROOTLESS1 (CRL1) transcription factor, belonging to the ASYMMETRIC LEAVES2-LIKE/LATERAL ORGAN BOUNDARIES DOMAIN (ASL/LBD) family, is a key regulator of crown root initiation in rice (Oryza sativa). Here, we show that CRL1 can bind, both in vitro and in vivo, not only the LBD-box, a DNA sequence recognized by several ASL/LBD transcription factors, but also another not previously identified DNA motif that was named CRL1-box. Using rice protoplast transient transactivation assays and a set of previously identified CRL1-regulated genes, we confirm that CRL1 transactivates these genes if they possess at least a CRL1-box or an LBD-box in their promoters. In planta, ChIP-qPCR experiments targeting two of these genes that include both a CRL1- and an LBD-box in their promoter show that CRL1 binds preferentially to the LBD-box in these promoter contexts. CRISPR/Cas9-targeted mutation of these two CRL1-regulated genes, which encode a plant Rho GTPase (OsROP) and a basic helix–loop–helix transcription factor (OsbHLH044), show that both promote crown root development. Finally, we show that OsbHLH044 represses a regulatory module, uncovering how CRL1 regulates specific processes during crown root formation.

Genome editing tools have greatly facilitated the functional analysis of genes of interest by targeted mutagenesis. Many usable genome editing tools, including different site-specific nucleases and editor databases that allow single-nucleotide polymorphisms (SNPs) to be introduced at a given site, are now available. These tools can be used to generate high allelic diversity at a given locus to facilitate gene function studies, including examining the role of a specific protein domain or a single amino acid. We compared the effects, efficiencies and mutation types generated by our LbCPF1, SpCAS9 and base editor (BECAS9) constructs for the OsCAO1 gene. SpCAS9 and LbCPF1 have similar efficiencies in generating mutations but differ in the types of mutations induced, with the majority of changes being single-nucleotide insertions and short deletions for SpCAS9 and LbCPF1, respectively. The proportions of heterozygotes also differed, representing a majority in our LbCPF1, while with SpCAS9, we obtained a large number of biallelic mutants. Finally, we demonstrated that it is possible to specifically introduce stop codons using the BECAS9 with an acceptable efficiency of approximately 20%. Based on these results, a rational choice among these three alternatives may be made depending on the type of mutation that one wishes to introduce, the three systems being complementary. SpCAS9 remains the best choice to generate KO mutations in primary transformants, while if the desired gene mutation interferes with regeneration or viability, the use of our LbCPF1 construction will be preferred, because it produces mainly heterozygotes. LbCPF1 has been described in other studies as being as effective as SpCAS9 in generating homozygous and biallelic mutations. It will remain to be clarified in the future, whether the different LbCFP1 constructions have different efficiencies and determine the origin of these differences. Finally, if one wishes to specifically introduce stop codons, BECA

Genome editing tools have greatly facilitated the functional analysis of genes of interest by targeted mutagenesis. Many usable genome editing tools, including different site-specific nucleases and editor databases that allow single-nucleotide polymorphisms (SNPs) to be introduced at a given site, are now available. These tools can be used to generate high allelic diversity at a given locus to facilitate gene function studies, including examining the role of a specific protein domain or a single amino acid. We compared the effects, efficiencies and mutation types generated by our LbCPF1, SpCAS9 and base editor (BECAS9) constructs for the OsCAO1 gene. SpCAS9 and LbCPF1 have similar efficiencies in generating mutations but differ in the types of mutations induced, with the majority of changes being single-nucleotide insertions and short deletions for SpCAS9 and LbCPF1, respectively. The proportions of heterozygotes also differed, representing a majority in our LbCPF1, while with SpCAS9, we obtained a large number of biallelic mutants. Finally, we demonstrated that it is possible to specifically introduce stop codons using the BECAS9 with an acceptable efficiency of approximately 20%. Based on these results, a rational choice among these three alternatives may be made depending on the type of mutation that one wishes to introduce, the three systems being complementary. SpCAS9 remains the best choice to generate KO mutations in primary transformants, while if the desired gene mutation interferes with regeneration or viability, the use of our LbCPF1 construction will be preferred, because it produces mainly heterozygotes. LbCPF1 has been described in other studies as being as effective as SpCAS9 in generating homozygous and biallelic mutations. It will remain to be clarified in the future, whether the different LbCFP1 constructions have different efficiencies and determine the origin of these differences. Finally, if one wishes to specifically introduce stop codons, BECA

Background: The DEFECTIVE IN OUTER CELL LAYER SPECIFICATION 1 (DOCS1) gene belongs to the Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) subfamily. It has been discovered few years ago in Oryza sativa (rice) in a screen to isolate mutants with defects in sensitivity to aluminum. The c68 (docs1–1) mutant possessed a nonsense mutation in the C-terminal part of the DOCS1 kinase domain. Findings: We have generated a new loss-of-function mutation in the DOCS1 gene (docs1–2) using the CRISPR-Cas9 technology. This new loss-of-function mutant and docs1–1 present similar phenotypes suggesting the original docs1–1 was a null allele. Besides the aluminum sensitivity phenotype, both docs1 mutants shared also several root phenotypes described previously: less root hairs and mixed identities of the outer cell layers. Moreover, our new results suggest that DOCS1 could also play a role in root cap development. We hypothesized these docs1 root phenotypes may affect gravity responses. As expected, in seedlings, the early gravitropic response was delayed. Furthermore, at adult stage, the root gravitropic set angle of docs1 mutants was also affected since docs1 mutant plants displayed larger root cone angles. Conclusions: All these observations add new insights into the DOCS1 gene function in gravitropic responses at several stages of plant development.

Background: The DEFECTIVE IN OUTER CELL LAYER SPECIFICATION 1 (DOCS1) gene belongs to the Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) subfamily. It has been discovered few years ago in Oryza sativa (rice) in a screen to isolate mutants with defects in sensitivity to aluminum. The c68 (docs1–1) mutant possessed a nonsense mutation in the C-terminal part of the DOCS1 kinase domain. Findings: We have generated a new loss-of-function mutation in the DOCS1 gene (docs1–2) using the CRISPR-Cas9 technology. This new loss-of-function mutant and docs1–1 present similar phenotypes suggesting the original docs1–1 was a null allele. Besides the aluminum sensitivity phenotype, both docs1 mutants shared also several root phenotypes described previously: less root hairs and mixed identities of the outer cell layers. Moreover, our new results suggest that DOCS1 could also play a role in root cap development. We hypothesized these docs1 root phenotypes may affect gravity responses. As expected, in seedlings, the early gravitropic response was delayed. Furthermore, at adult stage, the root gravitropic set angle of docs1 mutants was also affected since docs1 mutant plants displayed larger root cone angles. Conclusions: All these observations add new insights into the DOCS1 gene function in gravitropic responses at several stages of plant development.

Functional analyses of MADS-box transcription factors in plants have unraveled their role in major developmental programs (e.g; flowering and floral organ identity), as well as in stress-related developmental processes such as abscission, fruit ripening and senescence. Over-expression of the OsMADS26 gene in rice (Oryza sativa) has revealed a possible function related to stress response (Lee et al., 2008b). Here we show that OsMADS26 down-regulated plants exhibit enhanced resistance against two major rice pathogens, Magnaporthe oryzae and Xanthomonas oryzae. Despite this enhanced resistance to biotic stresses, OsMADS26 down-regulated plants also displayed enhanced tolerance to water deficit. These phenotypes were observed both in controlled and field conditions. Interestingly, alteration of OsMADS26 expression has no strong impact on plant development. Gene expression profiling revealed that a majority of genes miss-regulated in over-expresser and down-regulated OsMADS26 lines compared to control plants are associated to biotic or abiotic stress response. Altogether, our data indicate that OsMADS26 acts as an upstream regulator of stress-associated genes and thereby as a hub to modulate the response to various stresses in the rice plant. (Résumé d'auteur)

Functional analyses of MADS-box transcription factors in plants have unraveled their role in major developmental programs (e.g; flowering and floral organ identity), as well as in stress-related developmental processes such as abscission, fruit ripening and senescence. Over-expression of the OsMADS26 gene in rice (Oryza sativa) has revealed a possible function related to stress response (Lee et al., 2008b). Here we show that OsMADS26 down-regulated plants exhibit enhanced resistance against two major rice pathogens, Magnaporthe oryzae and Xanthomonas oryzae. Despite this enhanced resistance to biotic stresses, OsMADS26 down-regulated plants also displayed enhanced tolerance to water deficit. These phenotypes were observed both in controlled and field conditions. Interestingly, alteration of OsMADS26 expression has no strong impact on plant development. Gene expression profiling revealed that a majority of genes miss-regulated in over-expresser and down-regulated OsMADS26 lines compared to control plants are associated to biotic or abiotic stress response. Altogether, our data indicate that OsMADS26 acts as an upstream regulator of stress-associated genes and thereby as a hub to modulate the response to various stresses in the rice plant.

Functional analyses of MADS-box transcription factors in plants have unraveled their role in major developmental programs (e.g; flowering and floral organ identity), as well as in stress-related developmental processes such as abscission, fruit ripening and senescence. Over-expression of the OsMADS26 gene in rice (Oryza sativa) has revealed a possible function related to stress response (Lee et al., 2008b). Here we show that OsMADS26 down-regulated plants exhibit enhanced resistance against two major rice pathogens, Magnaporthe oryzae and Xanthomonas oryzae. Despite this enhanced resistance to biotic stresses, OsMADS26 down-regulated plants also displayed enhanced tolerance to water deficit. These phenotypes were observed both in controlled and field conditions. Interestingly, alteration of OsMADS26 expression has no strong impact on plant development. Gene expression profiling revealed that a majority of genes miss-regulated in over-expresser and down-regulated OsMADS26 lines compared to control plants are associated to biotic or abiotic stress response. Altogether, our data indicate that OsMADS26 acts as an upstream regulator of stress-associated genes and thereby as a hub to modulate the response to various stresses in the rice plant.

Background: In rice, the major part of the post-embryonic root system is made of stem-derived roots named crown roots (CR). Among the few characterized rice mutants affected in root development, crown rootless1 mutant is unable to initiate crown root primordia. CROWN ROOTLESS1 (CRL1) is induced by auxin and encodes an AS2/LOBdomain transcription factor that acts upstream of the gene regulatory network controlling CR development. Results: To identify genes involved in CR development, we compared global gene expression profile in stem bases of crl1 mutant and wild-type (WT) plants. Our analysis revealed that 250 and 236 genes are down- and upregulated respectively in the crl1 mutant. Auxin induces CRL1 expression and consequently it is expected that auxin also alters the expression of genes that are early regulated by CRL1. To identify genes under the early control of CRL1, we monitored the expression kinetics of a selected subset of genes, mainly chosen among those exhibiting differential expression, in crl1 and WT following exogenous auxin treatment. This analysis revealed that most of these genes, mainly related to hormone, water and nutrient, development and homeostasis, were likely not regulated directly by CRL1. We hypothesized that the differential expression for these genes observed in the crl1 mutant is likely a consequence of the absence of CR formation. Otherwise, three CRL1-dependent auxin-responsive genes: FSM (FLATENNED SHOOT MERISTEM)/FAS1 (FASCIATA1), GTE4 (GENERAL TRANSCRIPTION FACTOR GROUP E4) and MAP (MICROTUBULE-ASSOCIATED PROTEIN) were identified. FSM/FAS1 and GTE4 are known in rice and Arabidopsis to be involved in the maintenance of root meristem through chromatin remodelling and cell cycle regulation respectively. Conclusion: Our data showed that the differential regulation of most genes in crl1 versus WT may be an indirect consequence of CRL1 inactivation resulting from the absence of CR in the crl1 mutant. Nevertheless some genes, FAS1/FSM, G

Background: In rice, the major part of the post-embryonic root system is made of stem-derived roots named crown roots (CR). Among the few characterized rice mutants affected in root development, crown rootless1 mutant is unable to initiate crown root primordia. CROWN ROOTLESS1 (CRL1) is induced by auxin and encodes an AS2/LOBdomain transcription factor that acts upstream of the gene regulatory network controlling CR development. Results: To identify genes involved in CR development, we compared global gene expression profile in stem bases of crl1 mutant and wild-type (WT) plants. Our analysis revealed that 250 and 236 genes are down- and upregulated respectively in the crl1 mutant. Auxin induces CRL1 expression and consequently it is expected that auxin also alters the expression of genes that are early regulated by CRL1. To identify genes under the early control of CRL1, we monitored the expression kinetics of a selected subset of genes, mainly chosen among those exhibiting differential expression, in crl1 and WT following exogenous auxin treatment. This analysis revealed that most of these genes, mainly related to hormone, water and nutrient, development and homeostasis, were likely not regulated directly by CRL1. We hypothesized that the differential expression for these genes observed in the crl1 mutant is likely a consequence of the absence of CR formation. Otherwise, three CRL1-dependent auxin-responsive genes: FSM (FLATENNED SHOOT MERISTEM)/FAS1 (FASCIATA1), GTE4 (GENERAL TRANSCRIPTION FACTOR GROUP E4) and MAP (MICROTUBULE-ASSOCIATED PROTEIN) were identified. FSM/FAS1 and GTE4 are known in rice and Arabidopsis to be involved in the maintenance of root meristem through chromatin remodelling and cell cycle regulation respectively. Conclusion: Our data showed that the differential regulation of most genes in crl1 versus WT may be an indirect consequence of CRL1 inactivation resulting from the absence of CR in the crl1 mutant. Nevertheless some genes, FAS1/FSM, G

Several transcription factors have been involved in rice response against biotic and abiotic stresses (Khong et al., 2008). In A. thaliana, the MADS box transcription factor AGL12 control cell division in root meristem and is involved in flowering transition (Tapia-Lopez et al., 2008). When over expressed in Catharanthus roseus cell suspension AGL12 promotes tissue like organization and alkaloid biosynthesis (Montiel et al., 2007). OsMADS26, the rice orthologous of AGL12 in A. thaliana was recently over expressed under the control of a DEX inducible promoter and described as a stress related gene Lee et al., 2008). Nevertheless its precise function remains unknown. QPCR analysis revealed that this gene is expressed both in leaves and roots and is upregulated in response to osmotic stress. In order to precise the function of OsMAD26 in stress response or in development, and in absence of insertion mutant, we had generated rice lines RNA interfered for this gene. Interfered lines were obtained with two GST tagging the 5' or the 3' end of the mRNA. Interfered lines were affected in their development (root, tillering, height, dry weight) and were delayed for their flowering time in comparison to control. Roots of interfered lines present a reduced geotropism. Interfered lines were not affected in comparison to control line in their availability to resist to drought stress. But interestingly, interfered lines present an increased resistance against Magnaeporthae grisea, Xanthomonas oryzae and the Rice Yellow Mottle Virus. This suggests that OsMADS26 is a negative regulator of biotic stress response and acts as a general activator of development. In order to identify the genes and the biological pathways regulated by OsMADS26 we have proceeded to a transcriptome comparative analysis of genes expressed in WT and RNA interfered lines. Data analysis is in progress. (Texte intégral)

Several transcription factors have been involved in rice response against biotic and abiotic stresses (Khong et al., 2008). In A. thaliana, the MADS box transcription factor AGL12 control cell division in root meristem and is involved in flowering transition (Tapia-Lopez et al., 2008). When over expressed in Catharanthus roseus cell suspension AGL12 promotes tissue like organization and alkaloid biosynthesis (Montiel et al., 2007). OsMADS26, the rice orthologous of AGL12 in A. thaliana was recently over expressed under the control of a DEX inducible promoter and described as a stress related gene Lee et al., 2008). Nevertheless its precise function remains unknown. QPCR analysis revealed that this gene is expressed both in leaves and roots and is upregulated in response to osmotic stress. In order to precise the function of OsMAD26 in stress response or in development, and in absence of insertion mutant, we had generated rice lines RNA interfered for this gene. Interfered lines were obtained with two GST tagging the 5' or the 3' end of the mRNA. Interfered lines were affected in their development (root, tillering, height, dry weight) and were delayed for their flowering time in comparison to control. Roots of interfered lines present a reduced geotropism. Interfered lines were not affected in comparison to control line in their availability to resist to drought stress. But interestingly, interfered lines present an increased resistance against Magnaeporthae grisea, Xanthomonas oryzae and the Rice Yellow Mottle Virus. This suggests that OsMADS26 is a negative regulator of biotic stress response and acts as a general activator of development. In order to identify the genes and the biological pathways regulated by OsMADS26 we have proceeded to a transcriptome comparative analysis of genes expressed in WT and RNA interfered lines. Data analysis is in progress. (Texte intégral)

The potential role of a molecular chaperone on the rate of production of extensively altered "Bacillus thuringiensis# Cry I C proteins was investigated. Analysis of the proteins produced by the recombinant #B. thuringiensis# strains showed that the truncated proteins were produced at a low rate. Expression of the 20-kDa protein gene from #B. thuringiensis# sp. #israelensis# in tandem with the truncated-crylC genes led to the production of a greater amount of proteins. The formation of inclusion bodies, however, did not occur even when the 20-kDa protein gene was expressed.

The potential role of a molecular chaperone on the rate of production of extensively altered "Bacillus thuringiensis# Cry I C proteins was investigated. Analysis of the proteins produced by the recombinant #B. thuringiensis# strains showed that the truncated proteins were produced at a low rate. Expression of the 20-kDa protein gene from #B. thuringiensis# sp. #israelensis# in tandem with the truncated-crylC genes led to the production of a greater amount of proteins. The formation of inclusion bodies, however, did not occur even when the 20-kDa protein gene was expressed.