The −689/+197 region of the maize protease inhibitor gene directs high level,wound-inducible expression of the cry1B gene which protects transgenic rice plants from stemborer attack

To investigate the activity of the regulatory region of the maize (Zea mays L.) proteinase inhibitor (mpi) gene, we transferred into rice (Oryza sativa L.) plants the –689/+197 (C1) fragment of the mpi genomic clone fused to either theuidA gene or a synthetic Bacillus thuringiensis cry1B gene. Although uidA and cry1B encode very different proteins consistent results were obtained from their respective histochemical and fluorometric and immunoblot detections in T₃ transgenic rice lines. In response to mechanical wounding, a 4–5 fold increase in GUS activity and a Cry1B accumulation reaching 0.1–0.2% of total soluble proteins were observed from basal and undetectable levels respectively in leaf tissue. The establishment of the time-course of wound response in both systems revealed a maximum induction level 12–16 h after treatment. From both systems we also deduced that the C1 region is not active in pollen and seed endosperm. Three independent transformation events expressing cry1B under the control of the C1 region exhibited protection against striped stem borer damage and showed 100% mortality of second instar larvae 8 days after release. These results illustrate the first evidence that wound-inducible expression of a Bacillus thuringiensis endotoxin gene affords full protection to transgenic rice plants.     

CRISPR/Cas9-mediated efficient targeted mutagenesis has the potential to accelerate the domestication of <Emphasis Type="Italic">Coffea canephora</Emphasis>

Genome editing, which is an unprecedented technological breakthrough, has provided a valuable means of creating targeted mutations in plant genomes. In this study, we developed a genomic web tool to identify all gRNA target sequences in the coffee genome, along with potential off-targets. In all, 8,145,748 CRISPR guides were identified in the draft genome of Coffea canephora corresponding to 5,338,568 different sequences and, of these, 4,655,458 were single, and 514,591 were covering exons. The proof of concept was established by targeting the phytoene desaturase gene (CcPDS) using the Agrobacterium tumefaciens transformation technique and somatic embryogenesis as the plant regeneration method. An analysis of the RNA-guided genome-editing events showed that 22.8% of the regenerated plants were heterozygous mutants and 7.6% were homozygous mutants. Mutation efficiency at the target site was estimated to be 30.4%. We demonstrated that genome editing by the CRISPR/Cas9 method is an efficient and reliable way of knocking out genes of agronomic interest in the coffee tree, opening up the way for coffee molecular breeding. Our results also showed that the use of somatic embryogenesis, as the method for regenerating genome-edited plants, could restrict the choice of targeted genes to those that are not essential to the embryo development and germination steps.     

The mariner Mos1 transposase produced in tobacco is active in vitro

The mariner-like transposon Mos1 is used for insertional mutagenesis and transgenesis in different animals (insects, nematodes), but has never been used in plants. In this paper, the transposition activity of Mos1 was tested in Nicotiana tabacum, but no transposition event was detected. In an attempt to understand the absence of in planta transposition, Mos1 transposase (MOS1) was produced and purified from transgenic tobacco (HMNtMOS1). HMNtMOS1 was able to perform all transposition reaction steps in vitro: binding to ITR, excision and integration of the same pseudo-transposon used in in planta transposition assays. The in vitro transposition reaction was not inhibited by tobacco nuclear proteins, and did not depend on the temperature used for plant growth. Several hypotheses are proposed that could explain the inhibition of HMNtMOS1 activity in planta.     

Efficient microprojectile bombardment-mediated transformation of rice using gene cassettes

This study was aimed at determining whether gene cassettes (promoter-coding sequence-terminator) can be efficiently used in microprojectile acceleration-mediated co-transformation of rice in the place of whole plasmids, and to what extent their use influences the integration and expression of the co-transferred gene of interest. Two non-linked marker genes (yfp and hph) were co-introduced by microprojectile bombardment into cells of embryogenic calli in three separate experiments. Three different DNA structures were compared for their ability to transiently and stably transform rice cells: supercoiled or linearized whole-plasmid DNA, gene cassette DNA and single-stranded gene cassette DNA coated with Escherichia coli single-stranded binding (SSB) proteins. Our results demonstrate that microprojectile bombardment-mediated transformation of rice using gene cassettes is possible without significantly reducing transformation efficiency in comparison to the use of whole-plasmid DNA. Furthermore, no obvious difference in transgene integration pattern and inheritance was observed among plants transformed with gene cassettes compared to those transformed with the whole plasmid, except that concatemerization of molecules prior to integration was rarely observed in gene cassette transformants. Received: 4 April 2001 / Accepted: 13 August 2001     

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.     

Expression of the maize proteinase inhibitor (mpi) gene in rice plants enhances resistance against the striped stem borer (Chilo suppressalis): effects on larval growth and insect gut proteinases

The maize proteinase inhibitor (mpi) gene was introduced into two elite japonica rice varieties. Both constitutive expression of the mpi gene driven by the maize ubiquitin 1 promoter and wound-inducible expression of the mpi gene driven by its own promoter resulted in the accumulation of MPI protein in the transgenic plants. No effect on plant phenotype was observed in mpi-expressing lines. The stability of transgene expression through successive generations of mpi rice lines (up to the T(4) generation) and the production of functional MPI protein were confirmed. Expression of the mpi gene in rice enhanced resistance to the striped stem borer (Chilo suppressalis), one of the most important pests of rice. In addition, transgenic mpi plants were evaluated in terms of their effects on the growth of C. suppressalis larvae and the insect digestive proteolytic system. An important dose-dependent reduction of larval weight of C. suppressalis larvae fed on mpi rice, compared with larvae fed on untransformed rice plants, was observed. Analysis of the digestive proteolytic activity from the gut of C. suppressalis demonstrated that larvae adapted to mpi transgene expression by increasing the complement of digestive proteolytic activity: the serine and cysteine endoproteinases as well as the exopeptidases leucine aminopeptidase and carboxypeptidases A and B. However, the induction of such proteolytic activity did not prevent the deleterious effects of MPI on larval growth. The introduction of the mpi gene into rice plants can thus be considered as a promising strategy to protect rice plants against striped stem borer.     

Bt rice harbouring cry genes controlled by a constitutive or wound-inducible promoter: protection and transgene expression under Mediterranean field conditions

Seven homozygous transgenic lines of two European commercial cultivars of rice (Ariete (A) and Senia (S)), harbouring the cry1B or cry1Aa Bacillus thuringiensis (Bt) delta-endotoxin genes, were field evaluated for protection from striped stem borer (SSB) (Chilo suppressalis) damage during the 2001 and 2002 summer crop seasons in the Delta de l'Ebre region, Spain. The plant codon-optimized toxin gene was placed under the control of the promoter of either the constitutive ubi1 gene or the wound-inducible mpi gene from maize. Stable, high-level, insecticidal protein accumulation was observed throughout root, leaf and seed tissues of field-grown plants harbouring the cry1B (lines A64.1, A33.1, A3.4 and S98.9) or cry1Aa (lines S05.1 and A19.14) genes under the control of the ubi1 promoter. Conversely, no toxin was detected in unwounded vegetative tissues of the A9.1 line harbouring the cry1B gene controlled by the mpi promoter, indicating that natural environmental stresses did not trigger the activity of the wound-inducible promoter. However, the toxin accumulated at 0.2% total soluble proteins in A9.1 sheath tissue exhibiting brown lesions resulting from SSB damage. The agronomical traits and performance of the transgenic lines were generally comparable with parental controls, except in the two lines accumulating Cry1Aa, which exhibited a high frequency of plants non-true to type. Natural infestation was assisted with manual infestations of L2/L3 SSB larvae in border control plants surrounding the experimental plots, which served as a reservoir for the second-cycle SSB population. The observation of damage (brown lesions and dead hearts) during the crop season and dissection of plants at harvest stage revealed a range of protection amongst the transgenic lines, which was highly consistent with the level of toxin accumulation and with previous experience in greenhouse assays. Lines A3.4 and S05.1 were found to exhibit stable and full protection against SSB attacks, mediated by the accumulation of Cry1B and Cry1Aa toxin, respectively, which was comparable with that afforded by the spraying of chemical insecticides on control plants. The wound-induced A9.1 line exhibited a satisfactory level of protection, with a notably low level of penetration of SSB larvae in the stems, but higher external symptoms than constitutive lines, probably due to the time lag to benefit from the protective effect of Cry1B.     

Transposon-mediated generation of T-DNA- and marker-free rice plants expressing a Bt endotoxin gene

Transposon-mediated repositioning of transgenes is an attractive strategy to generate plants that are free of selectable markers and T-DNA inserts. By using a minimal number of transformation events a large number of transgene insertions in the genome can be obtained so as to benefit from position effects in the genome that can contribute to higher levels of expression. We constructed a Bacillus thuringiensis synthetic cry1B gene expressed under control of the maize ubiquitin promoter between minimal terminal inverted repeats of the maize Ac-Ds transposon system, which was cloned in the 5' untranslated sequence of a gfp gene used as an excision marker. The T-DNA also harboured the Ac transposase gene driven by the CaMV 35S promoter and the hph gene conferring resistance to the antibiotic hygromycin. Sixty-eight independent rice (Oryza sativa L.) transformants were regenerated and molecularly analysed revealing excision and reinsertion of the Ds-cry1B element in 37% and 25% respectively of the transformation events. Five independent transformants harbouring 2–4 reinserted Ds-Cry1B copies were analysed in the T1 progeny, revealing 0.2 to 1.4 new transpositions per plant. Out segregation of the cry1B gene from the T-DNA insertion site was observed in 17 T1 plants, representing 10 independent repositioning events without selection. Western analysis of leaf protein extracts of these plants revealed detectable Cry1B in all the plants indicating efficient expression of the transgene reinsertions. Stability of position and expression of the cry1B transgene was further confirmed in T2 progeny of T-DNA-free T1 plants. New T-DNA-free repositioning events were also identified in T2 progenies of T1 plants heterozygous for the T-DNA. Furthermore, preliminary whole plant bioassay of T-DNA-free lines challenged with striped stem borer larvae suggested that they are protected against SSB attacks. These results indicate that transposon mediated relocation of the gene of interest is a powerful method for generating T-DNA integration site-free transgenic plants and exploiting favourable position effects in the plant genome.     

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. Received: 7 February 2000 / Revision received: 18 May 2000 / Accepted: 29 May 2000