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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. 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. All these observations add new insights into the DOCS1 gene function in gravitropic responses at several stages of plant development.

In the framework of the Genoplante project, we have produced a library of rice (cv. Nipponbare) lines containing T-DNA and novel insertions of Tos 17 retrotransposon. Using a subset of 400 primary transformants harbouring the pC-4978 T-DNA construct, we attempted to establish a relationship between T-DNA organization, walk-PCR patterns of flanking regions and nature of the FSTs. Thorough Southern blot analyses revealed integration of an average of 2.2 T-DNA copies per plant, associated with an often-complex T-DNA organisation resulting from sequence rearrangements. Moreover, we observed that backbone vector sequences are often integrated in transformants harbouring multiple T-DNA copies. A highly efficient digestion-ligation walk-PCR protocol to amplify genomic sequences flanking both right and left T-DNA borders was developed for five restriction enzymes. Our study revealed that the analysis by walk-PCR with three enzymes enabled to amplify at least one Flanking Sequence Tag (FST) from 90% of the primary transformants. Comparative analysis of FST sequences obtained from both right and left TDNA borders revealed that in approximately 25% of cases novel FST sequences were obtained. In the same subset of 400 lines we estimated that on average insertion of 3.2 new copies of TOS17 had occurred per primary transformant. A novel highly efficient protocol of selective amplification of newly inserted copies of Tos17 retrotransposon was developed and enabled us to recover at least one sequence flanking a newly-transposed Tos17 copy in 70% the lines.Walk-PCR based protocols for both T-DNA and TOS17 enabled to obtain on average 70% of monoband and 30% of multiband products for every restriction enzyme we used. The development of a pipeline of bioanalysis of the sequences derived from multiband products, enabled us to maximize the efficiency of our walk-PCR based technology. The results of this pilot study will serve as a guideline to efficiently analyse the entire rice collection and help us to understand the mechanisms of integration of both T-DNA and TOS17 in the rice genome.

We investigated the diversity of the Tos17 retrotransposon in the Oryza genus using a combination of Southern hybridization with a reverse transcriptase (RT) probe and an adapter-PCR mediated amplification which allowed the sequencing of the genomic regions flanking Tos17 insertions. This analysis was carried out in a collection of 96 Oryza accessions covering the diversity of the genus and 246 accessions of a core collection of O. sativa representative of the diversity of the species. Our Southern hybridization results show that Tos17 is present in all the accessions of the A-genome species, except in the South American species O. glumaepatula and in the African species O. glaberrima and O. breviligulata. Surprisingly, a hybridization signal was observed in O. officinalis, O. minuta and O. latifolia. In O. sativa, the number of putative copies of Tos17 per accession ranged from 1-11. Moreover, multivariate analysis based on presence/absence of putative copies yielded varietal clusters consistent with the isozyme classification of rice. Adapter PCR amplification and sequencing of flanking regions of Tos17 insertions in Oryza species yielded 124 good sequences which were anchored on the rice genome sequence, overall revealing 71 insertions of Tos17 in most the Oryza species, which corresponded to 36 orthologous sites. In O. sativa the same approach yielded 461 good sequences, revealing 62 positions in the 6 varietal groups, representing 28 orthologous insertion sites. Comparison of these insertions among Oryza species in one hand and between cultivated and wild Oryza species on the other hand will be presented and discussed.

The objective of this project, funded by the Genomics initiative Génoplante, is to generate through Agrobacterium-mediated transformation a library of 50,000 insertion lines of rice cv. Nipponbare. In a 2 year time-frame, we produced more than 39,400 primary transformants, with an average efficiency of 4.2 independent transformation events per cocultured callus. Each line was found to harbour an average of 2.2 and 3.2 copies of the T-DNA and of new inserts of the endogenous ty1-copia retrotransposon Tos17 respectively. 89% of the 14,893 walk-PCR products of amplification of the regions flanking the left border of T-DNA inserts yielded a readable sequence. A first survey of 7,480 genomic sequences against the rice BAC/PAC sequences detected 5,791 hits (77%), consistent with the percentage of the rice genome sequenced at time of analysis (76%). 4,303 (56%) T-DNA insertion sites were assigned to at least one position on the rice genome. The T-DNA density averaged 17.9 insertions per Mb sequenced and ranged from 14 to 20 insertions per Mb across the 12 chromosomes. A more detailed examination of the distribution of T-DNA inserts along the Chromosome 1 showed that 668 (92.8%) of 720 T-DNA insertions are assigned to a unique location. The results also demonstrated that i. the centromeric region exhibits a lower insertion density whereas higher insertion density is observed in the subtelomeric regions, ii. T-DNA inserts are very rarely found in repetitive DNA, iii. T-DNA seems to preferentially integrate promoter and intron regions of the predicted rice genes. Frequencies of putative insertions in both chr1 genes predicted by TIGR annotation and transcription factors identified by the whole genome sequencing (Goff et al 2002) led to the same conclusion that about 7-10% of the rice gene complement is already covered by the 6,111 tagged lines characterized in the collection. (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.

Orf-1 and orf-2 are associated in operon with the cryIIA gene and their products appear to be essential for the crystallization of CryIIA most likely by acting as molecular chaperones. A novel strain of Bacillus thuriagiensis subsp. thompsoni named HnC produces during sporulation pyramidal crystals that are composed of two proteins of 38 kDa and 40 kDa. The two genes coding for the 38 kDa and 40 kDa proteins were isolated from HnC and placed under the control of the cryIIA promoter from NRD-12. Similary, these genes were cloned downstream from the cryIIA promoter, orf-1 and orf-2 in place of cryIIA. These constructs were transfered into a crystal-minus strain of B. thuringiensis subsp. Kurstaki HD-1 using the shuttle vector pHT 3101. Expression of the 38-kDa or the 40-kDa protein gene under the control of the CryIIA promoter, in HD-1 cry(-) cells resulted in low level of production of protein without formation of inclusion bodies. Bacteria expressing the two genes show the same characteristics. In contrary, simultaneous expression of each gene with orf-1 and orf-2 in HD-1cry(-) cells allowed accumulation of both 38 kDa or 40 kDa proteins in parasporal inclusion bodies visible under light microscopy. Analysis of these inclusions by SDS-PAGE and immunodetection demonstrated the presence of the 38 kDa and 40 kDa proteins which migrated in SDS-PAGE to the same position than crystal proteins from HnC. These results demonstrate that orf-1 and orf-2 can direct the crystallization of toxins with no homology to CryII proteins.