Donaldo Meynard

Highly efficient production and characterization of T-DNA plants for rice ( Oryza sativa L.) functional genomics

Abstract We investigated the potential of an improved Agrobacterium tumefaciens-mediated transformation procedure of japonica rice (Oryza sativa L.) for generating large numbers of T-DNA plants that are required for functional analysis of this model genome. Using a T-DNA construct bearing the hygromycin resistance (hpt), green fluorescent protein (gfp) and β-glucuronidase (gusA) genes, each individually driven by a CaMV 35S promoter, we established a highly efficient seed-embryo callus transformation procedure that results both in a high frequency (75–95%) of co-cultured calli yielding resistant cell lines and the generation of multiple (10 to more than 20) resistant cell lines per co-cultured callus. Efficiencies ranged from four to ten independent transformants per co-cultivated callus in various japonica cultivars. We further analysed the T-DNA integration patterns within a population of more than 200 transgenic plants. In the three cultivars studied, 30–40% of the T0 plants were found to have integrated a single T-DNA copy. Analyses of segregation for hygromycin resistance in T1 progenies showed that 30–50% of the lines harbouring multiple T-DNA insertions exhibited hpt gene silencing, whereas only 10% of lines harbouring a single T-DNA insertion was prone to silencing. Most of the lines silenced for hpt also exhibited apparent silencing of the gus and gfp genes borne by the T-DNA. The genomic regions flanking the left border of T-DNA insertion points were recovered in 477 plants and sequenced. Adapter-ligation Polymerase chain reaction analysis proved to be an efficient and reliable method to identify these sequences. By homology search, 77 T-DNA insertion sites were localized on BAC/PAC rice Nipponbare sequences. The influence of the organization of T-DNA integration on subsequent identification of T-DNA insertion sites and gene expression detection systems is discussed.

Multiple mechanisms of nitrate sensing by Arabidopsis nitrate transceptor NRT1.1

In Arabidopsis the plasma membrane nitrate transceptor (transporter/receptor) NRT1.1 governs many physiological and developmental responses to nitrate. Alongside facilitating nitrate uptake, NRT1.1 regulates the expression levels of many nitrate assimilation pathway genes, modulates root system architecture, relieves seed dormancy and protects plants from ammonium toxicity. Here, we assess the functional and phenotypic consequences of point mutations in two key residues of NRT1.1 (P492 and T101). We show that the point mutations differentially affect several of the NRT1.1-dependent responses to nitrate, namely the repression of lateral root development at low nitrate concentrations, and the short-term upregulation of the nitrate-uptake gene NRT2.1, and its longer-term downregulation, at high nitrate concentrations. We also show that these mutations have differential effects on genome-wide gene expression. Our findings indicate that NRT1.1 activates four separate signalling mechanisms, which have independent structural bases in the protein. In particular, we present evidence to suggest that the phosphorylated and non-phosphorylated forms of NRT1.1 at T101 have distinct signalling functions, and that the nitrate-dependent regulation of root development depends on the phosphorylated form. Our findings add to the evidence that NRT1.1 is able to trigger independent signalling pathways in Arabidopsis in response to different environmental conditions.

A Novel Two T-DNA Binary Vector Allows Efficient Generation of Marker-free Transgenic Plants in Three Elite Cultivars of Rice (Oryza sativa L.)

A pilot binary vector was constructed to assess the potential of the 2 T-DNA system for generating selectable marker-free progeny plants in three elite rice cultivars (ZhongZuo321, Ariete and Khao Dawk Mali 105) known to exhibit contrasting amenabilities to transformation. The first T-DNA of the vector, delimited by Agrobacterium tumefaciens borders, contains the hygromycin phosphotransferase (hpt) selectable gene and the green fluorescent protein (gfp) reporter gene while the second T-DNA, delimited by Agrobacterium rhizogenes borders, bears the phosphinothricin acetyl transferase (bar) gene, featuring the gene of interest. 82–90% of the hygromycin-resistant primary transformants exhibited tolerance to ammonium glufosinate mediated by the bar gene suggesting very high co-transformation frequency in the three cultivars. All of the regenerated plants were analyzed by Southern blot which confirmed co-integration of the T-DNAs at frequencies consistent with those of co-expression and allowed determination of copy number for each gene as well as detection of two different vector backbone fragments extending between the two T-DNAs. Hygromycin susceptible, ammonium glufosinate tolerant phenotypes represented 14.4, 17.4 and 14.3% of the plants in T1 progenies of ZZ321, Ariete and KDML105 primary transformants, respectively. We developed a statistical model for deducing from the observed copy number of each T-DNA in T0 plants and phenotypic segregations in T1 progenies the most likely constitution and linkage of the T-DNA integration locus. Statistical analysis identified in 40 out of 42 lines a most likely linkage configuration theoretically allowing genetic separation of the two T-DNA types and out segregation of the T-DNA bearing the bar gene. Overall, though improvements of the technology would be beneficial, the 2 T-DNA system appeared to be a useful approach to generate selectable marker-free rice plants with a consistent frequency among cultivars.

Inducibility by pathogen attack and developmental regulation of the rice Ltp1 gene

Using a genomic clone encoding a rice lipid transfer protein, LTP1, we analysed the activity of the 5′ region of the Ltp1 gene in transgenic rice (Oryza sativaL.) during plant development and under pathogen attack. The −1176/+13, −556/+13 and −284/+13 regions of the promoter were fused upstream from the uidA reporter gene and nos 3′ polyadenylation signal, resulting in the pΔ1176Gus, pΔ556Gus and pΔ284Gus constructs which were transferred to rice by microprojectile bombardment. Histochemical and fluorometric GUS assays and in situ detection of uidA transcripts in transgenic homozygous lines harbouring the pΔ1176Gus construct demonstrated that the Ltp1 promoter is preferentially active in aerial vegetative and reproductive organs and that both specificity and level of expression are regulated during organ development. In leaf sheath, GUS activity which is initially strictly localized in the epidermis of growing tissue, becomes restricted to the vascular system in mature tissues. In expanded leaf blade, expression of the uidA gene was restricted to the cutting level suggesting inducibility by wounding. Strong activity was detected in lemma and palea, sterile glumes, and immature anther walls and microspores but not in female reproductive organs. No GUS activity was detected during seed embryo maturation whereas the uidA gene was strongly expressed at early stages of somatic embryogenesis in scutellum tissue. The Ltp1 transcripts were found to strongly accumulate in response to inoculation with the fungal agent of the blast disease, Magnaporthe grisea,in two rice cultivars exhibiting compatible or incompatible host-pathogen interactions. Analysis of pΔ1176Gus leaf samples inoculated with the blast fungus demonstrated that the Ltp1promoter is induced in all cell types of tissues surrounding the lesion and notably in stomata guard cells. In plants harbouring the Ltp1 promoter deletion construct pΔ556Gus, activity was solely detected in the vascular system of mature leaves whereas no uidA gene expression was observed in pΔ284Gus plants. These observations are consistent with the proposed role of LTP1 in strenghtening of structural barriers and organ protection against mechanical disruption and pathogen attack.

Identification of an active LTR retrotransposon in rice.

Transposable elements are ubiquitous components of plant genomes. When active, these mobile elements can induce changes in the genome at both the structural and functional levels. Availability of the complete genome sequence for several model plant species provides the opportunity to study TEs in plants at an unprecedented scale. In the case of rice, annotation of the genomic sequence of the variety Nipponbare has revealed that TE-related sequences form more than 25% of its genome. However, most of the elements found are inactive, either because of structural alterations or because they are the target of various silencing pathways. In this paper, we propose a new post-genomic strategy aimed at identifying active TEs. Our approach relies on transcript profiling of TE-related sequences using a tiling microarray. We applied it to a particular class of TEs, the LTR retrotransposons. A transcript profiling assay of rice calli led to identification of a new transpositionally active family, named Lullaby. We provide a complete structural description of this element. We also show that it has recently been active in planta in rice, and discuss its phylogenetic relationships with Tos17, the only other active LTR retrotransposon described so far in the species.

Expression of a Bacillus thuringiensis cry1B synthetic gene protects Mediterranean rice against the striped stem borer.

Bacillus thuringiensis Cry1Ba endotoxin, which was shown to exhibit a tenfold lower lethal concentration 50 (LC50) than Cry1Ac in a Striped Stem Borer (SSB) diet incorporation assay. The 1.950-bp synthetic cry1B gene, possessing an overall GC content of 58 %, was cloned under the control of the maize ubiquitin promoter first intron and first exon regions. The resulting vector, designated as pUbi-cry1B, was transferred to two commercial Mediterranean cultivars of rice, Ariete and Senia, using microprojectile acceleration-mediated transformation. Thirty-two and 47 T0 events were generated in cvs. Ariete and Senia, respectively. Southern blot and immunoblot analyses allowed the identification of 7 Senia and 1 Ariete events harbouring both an intact gene cassette and expressing Cry1B at a level ranging from 0.01% to 0.4% of the total soluble proteins. Three Senia and 1 Ariete events were found to be protected against second instar SSB larvae in whole plant feeding assays, exhibiting 90–100% mortality 7 days after infestation. Spatial and temporal variation in transgene expression was further examined in resistant event 64 of cv. Ariete. Stable accumulation of Cry1B, representing 0.4% of the total soluble proteins, was observed over the T2 to T4 generations in leaf tissue 20, 40, 70 and 90 days after germination in both young and old leaves and in internodes. Ariete event 64 was found to be fully protected from attacks of third and fourth instar SSB larvae over subsequent generations.

Interaction between the GROWTH-REGULATING FACTOR and KNOTTED1-LIKE HOMEOBOX Families of Transcription Factors[W]

Members of the GRF family are conserved transcriptional regulators in both monocot and dicot plants. KNOTTED1-LIKE HOMEOBOX (KNOX) genes are important regulators of meristem function, and a complex network of transcription factors ensures tight control of their expression. Here, we show that members of the GROWTH-REGULATING FACTOR (GRF) family act as players in this network. A yeast (Saccharomyces cerevisiae) one-hybrid screen with the upstream sequence of the KNOX gene Oskn2 from rice (Oryza sativa) resulted in isolation of OsGRF3 and OsGRF10. Specific binding to a region in the untranslated leader sequence of Oskn2 was confirmed by yeast and in vitro binding assays. ProOskn2:β-glucuronidase reporter expression was down-regulated by OsGRF3 and OsGRF10 in vivo, suggesting that these proteins function as transcriptional repressors. Likewise, we found that the GRF protein BGRF1 from barley (Hordeum vulgare) could act as a repressor on an intron sequence in the KNOX gene Hooded/Barley Knotted3 (Bkn3) and that AtGRF4, AtGRF5, and AtGRF6 from Arabidopsis (Arabidopsis thaliana) could repress KNOTTED-LIKE FROM ARABIDOPSIS THALIANA2 (KNAT2) promoter activity. OsGRF overexpression phenotypes in rice were consistent with aberrant meristematic activity, showing reduced formation of tillers and internodes and extensive adventitious root/shoot formation on nodes. These effects were associated with down-regulation of endogenous Oskn2 expression by OsGRF3. Conversely, RNA interference silencing of OsGRF3, OsGRF4, and OsGRF5 resulted in dwarfism, delayed growth and inflorescence formation, and up-regulation of Oskn2. These data demonstrate conserved interactions between the GRF and KNOX families of transcription factors in both monocot and dicot plants.

The Triticum aestivum non-specific lipid transfer protein (TaLtp) gene family: comparative promoter activity of six TaLtp genes in transgenic rice

Plant non-specific lipid transfer proteins (nsLTPs) are encoded by a multigene family and support physiological functions, which remain unclear. We adapted an efficient ligation-mediated polymerase chain reaction (LM-PCR) procedure that enabled isolation of 22 novel TriticumaestivumnsLtp (TaLtp) genes encoding types 1 and 2 nsLTPs. A phylogenetic tree clustered the wheat nsLTPs into ten subfamilies comprising 1–7 members. We also studied the activity of four type 1 and two type 2 TaLtp gene promoters in transgenic rice using the β-Glucuronidase reporter gene. The activities of the six promoters displayed both overlapping and distinct features in rice. In vegetative organs, these promoters were active in leaves and root vascular tissues while no β-Glucuronidase (GUS) activity was detected in stems. In flowers, the GUS activity driven by the TaLtp7.2a, TaLtp9.1a, TaLtp9.2d, and TaLtp9.3e gene promoters was associated with vascular tissues in glumes and in the extremities of anther filaments whereas only the TaLtp9.4a gene promoter was active in anther epidermal cells. In developing grains, GUS activity and GUS immunolocalization data evidenced complex patterns of activity of the TaLtp7.1a, TaLtp9.2d, and TaLtp9.4a gene promoters in embryo scutellum and in the grain epicarp cell layer. In contrast, GUS activity driven by TaLtp7.2a, TaLtp9.1a, and TaLtp9.3e promoters was restricted to the vascular bundle of the embryo scutellum. This diversity of TaLtp gene promoter activity supports the hypothesis that the encoded TaLTPs possess distinct functions in planta.

Expression of the Aeluropus littoralis AlSAP gene in rice confers broad tolerance to abiotic stresses through maintenance of photosynthesis

The expression of AlSAP, in rice cv. Nipponbare, enhances plant tolerance to cold, drought and salt stresses. AlSAP lines showed 100% survival rate and set seeds while control plants did not recover from the cold treatment. Under a severe drought stress treatment (fraction of transpirable soil water down to 0.1), AlSAP lines exhibited enhanced Transpiration Efficiency (TE) and maintained a high A (Assimilation rate) value (22 µmol·m(-2)  s(-1) ) while these values dramatically decreased (A = 4 µmol·m(-2)  s(-1) ) in control plants which were subsequently unable to recover from the stress. Of noteworthy is that AlSAP rice plants yielded a similar and a 60% seed set under control and stress conditions respectively, with regard to wild-type (WT) plants grown under control conditions. This indicates that AlSAP expression imposes no yield penalty and allows seed production even following a severe drought stress at the vegetative stage. Furthermore, AlSAP rice was shown to accumulate transcripts of a pilot set of eight stress-related genes at a significantly higher level than WT plants, both under control and stressed conditions. The results suggest that AlSAP expression generates stress tolerance in plants through maintenance of the photosynthetic apparatus integrity and by stimulating an endogenous adaptive potential which is not effectively accomplished in WT plants.

Cross-Talk between the KNOX and Ethylene Pathways Is Mediated by Intron-Binding Transcription Factors in Barley

In the barley (Hordeum vulgare) Hooded (Kap) mutant, the duplication of a 305-bp intron sequence leads to the overexpression of the Barley knox3 (Bkn3) gene, resulting in the development of an extra flower in the spikelet. We used a one-hybrid screen to identify four proteins that bind the intron-located regulatory element (Kap intron-binding proteins). Three of these, Barley Ethylene Response Factor1 (BERF1), Barley Ethylene Insensitive Like1 (BEIL1), and Barley Growth Regulating Factor1 (BGRF1), were characterized and their in vitro DNA-binding capacities verified. Given the homology of BERF1 and BEIL1 to ethylene signaling proteins, we investigated if these factors might play a dual role in intron-mediated regulation and ethylene response. In transgenic rice (Oryza sativa), constitutive expression of the corresponding genes produced phenotypic alterations consistent with perturbations in ethylene levels and variations in the expression of a key gene of ethylene biosynthesis. In barley, ethylene treatment results in partial suppression of the Kap phenotype, accompanied by up-regulation of BERF1 and BEIL1 expression, followed by down-regulation of Bkn3 mRNA levels. In rice protoplasts, BEIL1 activates the expression of a reporter gene driven by the 305-bp intron element, while BERF1 can counteract this activation. Thus, BEIL1 and BERF1, likely in association with other Kap intron-binding proteins, should mediate the fine-tuning of Bkn3 expression by ethylene. We propose a hypothesis for the cross talk between the KNOX and ethylene pathways.