Fine-tuning CRISPR/Cas9 gene editing in common bean (Phaseolus vulgaris L.) using a hairy root transformation system and in silico prediction models.

The stable transformation of common bean is a challenging and time-consuming process. Although CRISPR/Cas9 has revolutionized gene editing with its high efficiency and specificity, the performance of the system can be affected by multiple factors, such as sgRNA specificity and effectiveness, and the choice of promoter used to drive Cas9 expression. The use of a hairy root transformation system to initially check the efficiency of sgRNAs and the impact of different promoters could speed up this process and increase the chances of success. We initially tested three different transformation methods to induce hairy roots and selected a preferred method suitable for a variety of different common bean genotypes. This method involved inoculating a severed radicle with Rhizobium rhizogenes K599 and was fast, had a high transformation frequency of 42-48%, and resulted in numerous hairy roots. This method was further used for the transformation of explants using R. rhizogenes harboring different CRISPR/Cas9 constructs and evaluated the on-target activity of sgRNAs targeting raffinose family oligosaccharides biosynthetic genes and the impact of different promoters driving Cas9 on the gene editing efficiency. Additionally, we evaluated the reliability of the in silico tools, CRISPOR, CRISPR RGEN, and inDelphi to predict the sgRNA efficiencies and resulting mutations. Our results showed that the hairy root transformation system allows for rapid evaluation of multiple sgRNAs and promoters. We also identified several highly efficient sgRNAs that induced frameshift mutations at rates of up to 70% when a parsley ubiquitin promoter was driving Cas9 expression, providing valuable information for the selection of the most effective sgRNAs and promoters for future transformation experiments. Although most of the computational models used to predict the sgRNA efficiency did not match the in planta results, the Lindel model proved to be the most reliable for P. vulgaris, accurately predicting the sgRNA efficiency and the type of induced mutation in most hairy roots. Furthermore, the inDelphi algorithm could correctly predict deletions and single nucleotide insertions resulting from DNA double-strand breaks in common bean. These results offer promising implications for enhancing precise editing in plants because they provide the possibility of predicting repair outcomes.

Impact of drought and salt stress on galactinol and raffinose family oligosaccharides in common bean (Phaseolus vulgaris).

Due to climate change, farmers will face more extreme weather conditions and hence will need crops that are better adapted to these challenges. The raffinose family oligosaccharides (RFOs) could play a role in the tolerance of crops towards abiotic stress. To investigate this, we determined for the first time the importance of galactinol and RFOs in the roots and leaves of common bean under drought and salt stress conditions. Initially, the physiological characteristics of common bean under agronomically relevant abiotic stress conditions were investigated by measuring the growth rate, transpiration rate, chlorophyll concentration and membrane stability, allowing to establish relevant sampling points. Subsequently, the differential gene expression profiles of the galactinol and RFO biosynthetic genes and the amount of galactinol and RFO molecules were measured in the primary leaves and roots of Phaseolus vulgaris cv. CIAP7247F at these sampling points, using RT-qPCR and HPAEC-PAD, respectively. Under drought stress, the genes galactinol synthase 1, galactinol synthase 3 and stachyose synthase were significantly upregulated in the leaves and had a high transcript level in comparison with the other galactinol and RFO biosynthetic genes. This was in accordance with the significantly higher amount of galactinol and raffinose detected in the leaves. Under salt stress, raffinose was also present in a significantly higher quantity in the leaves. In the roots, transcript levels of the RFO biosynthetic genes were generally low and no galactinol, raffinose or stachyose could be detected. These results suggest that in the leaves, both galactinol and raffinose could play a role in the protection of common bean against abiotic stresses. Especially, the isoform galactinol synthase 3 could have a specific role during drought stress and forms an interesting candidate to improve the abiotic stress resistance of common bean or other plant species.

Genome-Wide Classification and Phylogenetic Analyses of the GDSL-Type Esterase/Lipase (GELP) Family in Flowering Plants.

GDSL-type esterase/lipase (GELP) enzymes have key functions in plants, such as developmental processes, anther and pollen development, and responses to biotic and abiotic stresses. Genes that encode GELP belong to a complex and large gene family, ranging from tens to more than hundreds of members per plant species. To facilitate functional transfer between them, we conducted a genome-wide classification of GELP in 46 plant species. First, we applied an iterative phylogenetic method using a selected set of representative angiosperm genomes (three monocots and five dicots) and identified 10 main clusters, subdivided into 44 orthogroups (OGs). An expert curation for gene structures, orthogroup composition, and functional annotation was made based on a literature review. Then, using the HMM profiles as seeds, we expanded the classification to 46 plant species. Our results revealed the variable evolutionary dynamics between OGs in which some expanded, mostly through tandem duplications, while others were maintained as single copies. Among these, dicot-specific clusters and specific amplifications in monocots and wheat were characterized. This approach, by combining manual curation and automatic identification, was effective in characterizing a large gene family, allowing the establishment of a classification framework for gene function transfer and a better understanding of the evolutionary history of GELP.

The Hidden Potential of High-Throughput RNA-Seq Re-Analysis, a Case Study for DHDPS, Key Enzyme of the Aspartate-Derived Lysine Biosynthesis Pathway and Its Role in Abiotic and Biotic Stress Responses in Soybean.

DHDPS is a key enzyme in the aspartate-derived lysine biosynthesis pathway and an evident object of study for biofortification strategies in plants. DHDPS isoforms with novel regulatory properties in Medicago truncatula were demonstrated earlier and hypothesized to be involved in abiotic and biotic stress responses. Here, we present a phylogenetic analysis of the DHPDS gene family in land plants which establishes the existence of a legume-specific class of DHDPS, termed DHDPS B-type, distinguishable from the DHDPS A-type commonly present in all land plants. The G. max genome comprises two A-type DHDPS genes (Gm.DHDPS-A1; Glyma.09G268200, Gm.DHDPS-A2; Glyma.18G221700) and one B-type (Gm.DHDPS-B; Glyma.03G022300). To further investigate the expression pattern of the G. max DHDPS isozymes in different plant tissues and under various stress conditions, 461 RNA-seq experiments were exploited and re-analyzed covering two expression atlases, 13 abiotic and 5 biotic stress studies. Gm.DHDPS-B is seen almost exclusively expressed in roots and nodules in addition to old cotyledons or senescent leaves while both DHDPS A-types are expressed constitutively in all tissues analyzed with the highest expression in mature seeds. Furthermore, Gm.DHDPS-B expression is significantly upregulated in some but not all stress responses including salt stress, flooding, ethylene or infection with Phytophthora sojae and coincides with downregulation of DHDPS A-types. In conclusion, we demonstrate the potential of an in-depth RNA-seq re-analysis for the guidance of future experiments and to expand on current knowledge.

Transcriptome-Guided Identification of Pectin Methyl-Esterase-Related Enzymes and Novel Molecular Processes Effectuating the Hard-to-Cook Defect in Common Bean (Phaseolus vulgaris L.).

The hard-to-cook defect in common beans is dictated by the ability to achieve cell separation during cooking. Hydrolysis of pectin methyl-esters by the pectin methyl-esterase (PME) enzyme influences cell separation. However, the contributions of the PME enzyme and the cell wall to the hard-to-cook defect have not been studied using molecular tools. We compared relevant molecular processes in fast- and slow-cooking bean varieties to understand the mechanisms underpinning the hard-to-cook defect. A PME spectrophotometric assay showed minor differences in enzyme activity between varieties. Meanwhile, a PME HMMER search in the P. vulgaris genome unveiled 113 genes encoding PMEs and PME inhibitors (PMEIs). Through RNA sequencing, we compared the gene expression of the PME-related genes in both varieties during seed development. A PME (Phvul010g080300) and PMEI gene (Phvul005g007600) showed the highest expression in the fast- and slow-cooking beans, respectively. We further identified 2132 differentially expressed genes (DEGs). Genes encoding cell-wall-related enzymes, mainly glycosylphosphatidylinositol mannosyltransferase, xyloglucan O-acetyltransferase, pectinesterase, and callose synthase, ranked among the top DEGs, indicating novel relations to the hard-to-cook defect. Gene ontology mapping revealed hydrolase activity and protein phosphorylation as functional categories with the most abundant upregulated DEGs in the slow-cooking bean. Additionally, the cell periphery contained 8% of the DEGs upregulated in the slow-cooking bean. This study provides new insights into the role of pectin methyl-esterase-related genes and novel cell wall processes in the occurrence of the hard-to-cook defect.

Identification and Expression Analysis of the Genes Involved in the Raffinose Family Oligosaccharides Pathway of Phaseolus vulgaris and Glycine max.

Raffinose family oligosaccharides (RFO) play an important role in plants but are also considered to be antinutritional factors. A profound understanding of the galactinol and RFO biosynthetic gene families and the expression patterns of the individual genes is a prerequisite for the sustainable reduction of the RFO content in the seeds, without compromising normal plant development and functioning. In this paper, an overview of the annotation and genetic structure of all galactinol- and RFO biosynthesis genes is given for soybean and common bean. In common bean, three galactinol synthase genes, two raffinose synthase genes and one stachyose synthase gene were identified for the first time. To discover the expression patterns of these genes in different tissues, two expression atlases have been created through re-analysis of publicly available RNA-seq data. De novo expression analysis through an RNA-seq study during seed development of three varieties of common bean gave more insight into the expression patterns of these genes during the seed development. The results of the expression analysis suggest that different classes of galactinol- and RFO synthase genes have tissue-specific expression patterns in soybean and common bean. With the obtained knowledge, important galactinol- and RFO synthase genes that specifically play a key role in the accumulation of RFOs in the seeds are identified. These candidate genes may play a pivotal role in reducing the RFO content in the seeds of important legumes which could improve the nutritional quality of these beans and would solve the discomforts associated with their consumption.

Dehydrin ERD14 activates glutathione transferase Phi9 in Arabidopsis thaliana under osmotic stress.

BACKGROUND: Fully intrinsically disordered plant dehydrin ERD14 can protect enzymes via its chaperone-like activity, but it was not formally linked with enzymes of the plant redox system yet. This is of particular interest, as the level of H2O2 in Arabidopsis plants increases during osmotic stress, which can be counteracted by overexpression of ERD14. METHODS: The proteomic mass-spectrometry analysis of stressed plants was performed to find the candidates affected by ERD14. With cross-linking, microscale thermophoresis, and active-site titration kinetics, the interaction and influence of ERD14 on the function of two target proteins: glutathione transferase Phi9 and catalase was examined. RESULTS: Under osmotic stress, redox enzymes, specifically the glutathione transferase Phi enzymes, are upregulated. Using microscale thermophoresis, we showed that ERD14 directly interacts with GSTF9 with a KD of ~25 µM. ERD14 activates the inactive GSTF9 molecules, protects GSTF9 from oxidation, and can also increases the activity of the enzyme. Aside from GSTF9, we found that ERD14 can also interact with catalase, an important cellular H2O2 scavenging enzyme, with a KD of ~0.13 µM, and protects it from dehydration-induced loss of activity. CONCLUSIONS: We propose that fully intrinsically disordered dehydrin ERD14 might protect and even activate redox enzymes, helping plants to survive oxidative stress under dehydration conditions. GENERAL SIGNIFICANCE: ERD14 has a direct effect on the activity of redox enzymes.

New Phenotypes of Potato Co-induced by Mismatch Repair Deficiency and Somatic Hybridization.

As plants are sessile they need a very efficient system for repairing damage done by external or internal mutagens to their DNA. Mismatch repair (MMR) is one of the systems that maintain genome integrity and prevent homeologous recombination. In all eukaryotes mismatches are recognized by evolutionary conserved MSH proteins often acting as heterodimers, the constant component of which is MSH2. Changes affecting the function of MSH2 gene may induce a 'mutator' phenotype and microsatellite instability (MSI), as is demonstrated in MSH2 knock-out and silenced lines of Arabidopsis thaliana. The goal of this study was to screen for 'mutator' phenotypes in somatic hybrids between potato cvs. 'Delikat' and 'Désirée' and MMR deficient Solanum chacoense transformed using antisense (AS) or dominant negative mutant (DN) AtMSH2 genes. The results demonstrate that first generation fusion hybrids have a range of morphological abnormalities caused by uniparental MMR deficiency; these mutant phenotypes include: dwarf or gigantic plants; bushiness; curled, small, large or abnormal leaves; a deterioration in chloroplast structure; small deep-purple tubers and early dehiscent flowers. Forty percent of the viable somatic hybrids planted in a greenhouse, (10 out of 25 genotypes) had mutant phenotypes accompanied by MSI. The majority of the hybrids with 'mutator' phenotypes cultured on media containing kanamycin developed roots so sustaining the presence of selectable marker gene nptII, from the initial constructs. Here for the first time, MMR deficiency combined with somatic hybridization, are used to induce new phenotypes in plants, which supports the role of MMR deficiency in increasing introgressions between two related species.

Biotechnological Approaches for Biomass and Cardenolide Production in Digitalis purpurea L.

Digitalis purpurea L. is one of the main economically viable sources of cardenolides (cardiac glycosides) for the pharmaceutical industry. Nevertheless, production of cardenolides in plants grown by traditional agriculture is not always an efficient process and can be affected by biotic and abiotic factors. This chapter provides two biotechnology strategies for biomass and cardenolide production in D. purpurea. Firstly, we report biomass production using a temporary immersion system (TIS), combined with cardenolide extraction and quantification. Secondly, an efficient protocol for genetic transformation via Agrobacterium tumefaciens is provided. These strategies can be used independently or combined in order to increase the content of cardiac glycosides in D. purpurea and to unravel biosynthetic pathways associated to cardiac glycoside production.

Optimization of a gas chromatography-mass spectrometry method with methyl chloroformate derivatization for quantification of amino acids in plant tissue.

Rapid, easy and reliable quantification of amino acids is crucial in research on plant amino acid metabolism and nutritional improvement of crops via enrichment of essential amino acids. A recently reported analysis method, based on solid phase extraction (SPE), derivatization with methyl chloroformate and gas chromatography-mass spectrometry was optimized and tested on three-week-old Arabidopsis thaliana leaf tissues. Optimization of the SPE cleanup yielded recovery rates of minimum 95% for all amino acids (except arginine). Variations in accuracy and precision did not exceed 12.5%, except for cysteine, histidine and tryptophane, which were excluded from analysis. Quantification of overlapping peaks for isoleucine/threonine and proline/asparagine was possible by selection of two specific fragment ions for each amino acid. Of the 16 selected amino acids, 14 were quantified successfully in at least 75% of the samples, while methionine and tyrosine were only quantifiable in 6% and 42%, respectively. A case study on the aspartate super pathway confirmed the applicability of the optimized method on wild type and genetically modified plants: external supplementation of methionine or lysine yielded a 146-fold or 27-fold increase in the respective absolute amino acid levels compared with the control treatment. Induced expression of dhdps-r1 (a mutated lysine biosynthesis gene encoding a feedback insensitive enzyme) caused an 83-fold increase in absolute lysine levels.

CENH3-GFP: a visual marker for gametophytic and somatic ploidy determination in Arabidopsis thaliana.

BACKGROUND: The in vivo determination of the cell-specific chromosome number provides a valuable tool in several aspects of plant research. However, current techniques to determine the endosystemic ploidy level do not allow non-destructive, cell-specific chromosome quantification. Particularly in the gametophytic cell lineages, which are physically encapsulated in the reproductive organ structures, direct in vivo ploidy determination has been proven very challenging. Using Arabidopsis thaliana as a model, we here assess the applicability of recombinant CENH3-GFP reporters for the labeling of the cell's chromocenters and for the monitoring of the gametophytic and somatic chromosome number in vivo. RESULTS: By modulating expression of a CENH3-GFP reporter cassette using different promoters, we isolated two reporter lines that allow for a clear and highly specific labeling of centromeric chromosome regions in somatic and gametophytic cells respectively. Using polyploid plant series and reproductive mutants, we demonstrate that the pWOX2-CENH3-GFP recombinant fusion protein allows for the determination of the gametophytic chromosome number in both male and female gametophytic cells, and additionally labels centromeric regions in early embryo development. Somatic centromere labeling through p35S-CENH3-GFP shows a maximum of ten centromeric dots in young dividing tissues, reflecting the diploid chromosome number (2x = 10), and reveals a progressive decrease in GFP foci frequency throughout plant development. Moreover, using chemical and genetic induction of endomitosis, we demonstrate that CENH3-mediated chromosome labeling provides an easy and valuable tool for the detection and characterization of endomitotic polyploidization events. CONCLUSIONS: This study demonstrates that the introgression of the pWOX2-CENH3-GFP reporter construct in Arabidopsis thaliana provides an easy and reliable methodology for determining the chromosome number in developing male and female gametes, and during early embryo development. Somatically expressed CENH3-GFP reporters, on the other hand, constitute a valuable tool to quickly determine the basic somatic ploidy level in young seedlings at the individual cell level and to detect and to quantify endomitotic polyploidization events in a non-destructive, microscopy-based manner.

Thirty years of transgenic research in plants.

In 1983, the first transgenic tissues and plants were generated by means of disarmed Agrobacterium tumefaciens strains, in which the oncogenes had been replaced by antibiotic resistance markers. Hence, this Special Issue of The International Journal of Developmental Biology celebrates 30 years of transgenic research in plants! Eminent scientists working in the field of plant transformation or plant biotechnology have contributed to this publication and reviewed the state of the art of their particular subdomain or summarized the importance of transgenic research in the discovery of new mechanisms and the establishment of an entirely new field, such as epigenetics.

From the tumor-inducing principle to plant biotechnology and its importance for society.

This dialogue was held between the Guest Editors of the Special Issue on "Plant Transgenesis" of the Int. J. Dev. Biol. and Marc Van Montagu. Research in the group of Marc Van Montagu and Jeff Schell in the 1970s was essential to reveal how the phytopathogenic bacterium Agrobacterium tumefaciens transfers DNA to host plants to cause crown gall disease. Knowledge of the molecular mechanism underlying gene transfer, subsequently led to the development of plant transgene technology, an indispensable tool in fundamental plant research and plant improvement. In the early 1980s, Marc Van Montagu founded a start-up company, Plant Genetic Systems, which successfully developed insect-resistant plants, herbicide-tolerant plants and a hybrid seed production system based on nuclear male sterility. Even before the first transgenic plant had been produced, Marc Van Montagu realized that the less developed countries might benefit most from plant biotechnology and throughout his subsequent career, this remained a focus of his efforts. After becoming emeritus professor, he founded the Institute of Plant Biotechnology Outreach (IPBO), which aims to raise awareness of the major role that plant biotechnology can play in sustainable agricultural systems, especially in less developed countries. Marc Van Montagu has been honored with many prizes and awards, the most recent being the prestigious World Food Prize 2013. In this paper, we look to the past and present of plant biotechnology and to the promises this technology holds for the future, on the basis of the personal perspective of Marc Van Montagu.

Transgenic plants: from first successes to future applications.

This dialogue was held between the Guest Editors of the Special Issue on "Plant Transgenesis" of the Int. J. Dev. Biol. and Marc De Block. He was one of the first scientists worldwide to obtain transgenic plants transformed with the chimeric selectable marker genes encoding neomycin phosphotransferase and bialaphos that confer resistance against the antibiotic kanamycin and the herbicide Basta®/glufosinate, respectively at the Department of Genetics of Ghent University and, later on, at the spin-off company, Plant Genetic Systems. Today, these two genes are still the most frequently utilized markers in transgene technology. Marc De Block chose to work on the improvement of crops in an industrial environment to help realize the production of superior seeds or products. He was part of the team that developed the male sterility/restorer system in canola (Brassica napus var. napus) that led to the first hybrid lines to be commercialized as successful products of transgene technology. In more than 30 years of research, he developed transformation procedures for numerous crops, designed histochemical, biochemical and physiological assays to monitor plant performance, and made original and innovative contributions to plant biology. Presently, he considers transgenic research part of the toolbox for plant improvement and essential for basic plant research.

T-DNA transfer and T-DNA integration efficiencies upon Arabidopsis thaliana root explant cocultivation and floral dip transformation.

T-DNA transfer and integration frequencies during Agrobacterium-mediated root explant cocultivation and floral dip transformations of Arabidopsis thaliana were analyzed with and without selection for transformation-competent cells. Based on the presence or absence of CRE recombinase activity without or with the CRE T-DNA being integrated, transient expression versus stable transformation was differentiated. During root explant cocultivation, continuous light enhanced the number of plant cells competent for interaction with Agrobacterium and thus the number of transient gene expression events. However, in transformation competent plant cells, continuous light did not further enhance cotransfer or cointegration frequencies. Upon selection for root transformants expressing a first T-DNA, 43-69 % of these transformants showed cotransfer of another non-selected T-DNA in two different light regimes. However, integration of the non-selected cotransferred T-DNA occurred only in 19-46 % of these transformants, indicating that T-DNA integration in regenerating root cells limits the transformation frequencies. After floral dip transformation, transient T-DNA expression without integration could not be detected, while stable T-DNA transformation occurred in 0.5-1.3 % of the T1 seedlings. Upon selection for floral dip transformants with a first T-DNA, 8-34 % of the transformants showed cotransfer of the other non-selected T-DNA and in 93-100 % of them, the T-DNA was also integrated. Therefore, a productive interaction between the agrobacteria and the female gametophyte, rather than the T-DNA integration process, restricts the floral dip transformation frequencies.

Excision of a selectable marker gene in transgenic banana using a Cre/lox system controlled by an embryo specific promoter.

Antibiotic and herbicide resistance genes have been used in transgene technology as powerful selection tools. Nonetheless, once transgenic events have been obtained their presence is no longer needed and can even be undesirable. In this work, we have developed a system to excise the selectable marker and the cre recombinase genes from transgenic banana cv. 'Grande Naine' (Musa AAA). To achieve this, the embryo specific REG-2 promoter was isolated from rice and its expression pattern in banana cell clumps, somatic embryos and regenerated plantlets was characterized by using a pREG2::uidA fusion construct. Subsequently, the REG-2 promoter was placed upstream of the cre gene, conferring Cre functionality in somatic embryos and recombination of lox sites resulting in excision of the selectable marker and cre genes. PCR analysis revealed that 41.7 % of the analysed transgenic plants were completely marker free, results that were thereafter confirmed by Southern blot hybridization. These results demonstrate the feasibility of using developmentally controlled promoters to mediate marker excision in banana. This system does not require any extra handling compared to the conventional transformation procedure and might be useful in other species regenerating through somatic embryogenesis.

Regulatory mechanisms after short- and long-term perturbed lysine biosynthesis in the aspartate pathway: the need for isogenes in Arabidopsis thaliana.

The aspartate-derived amino acid pathway in plants is an intensively studied metabolic pathway, because of the biosynthesis of the four essential amino acids lysine, threonine, isoleucine and methionine. The pathway is mainly controlled by the key regulatory enzymes aspartate kinase (AK; EC 2.7.2.4), homoserine dehydrogenase (HSDH; EC 1.1.1.3) and 4-hydroxy-tetrahydrodipicolinate synthase (EC 4.3.3.7), formerly referred to as dihydrodipicolinate synthase (DHDPS). They are encoded by isoenzyme families and it is not known why such families are evolutionarily maintained. To gain more insight into the specific roles and regulation of the isoenzymes, we inhibited DHDPS in Arabidopsis thaliana with the chemical compound (N,N-dimethylglycinatoboranyloxycarbonylmethyl)-dimethylamine-borane (DDAB) and compared the short-term effects on the biochemical and biomolecular level to the long-term adaptations in dhdps knockout mutants. We found that DHDPS2 plays a crucial role in controlling lysine biosynthesis, thereby stabilizing flux through the whole aspartate pathway. Moreover, DHDPS2 was also shown to influence the threonine level to a large extent. In addition, the lysine-sensitive AKs, AKLYS1 and AKLYS3 control the short- and long-term responses to perturbed lysine biosynthesis in Arabidopsis thaliana.

Glutamate metabolism in plant disease and defense: friend or foe?

Plant glutamate metabolism (GM) plays a pivotal role in amino acid metabolism and orchestrates crucial metabolic functions, with key roles in plant defense against pathogens. These functions concern three major areas: nitrogen transportation via the glutamine synthetase and glutamine-oxoglutarate aminotransferase cycle, cellular redox regulation, and tricarboxylic acid cycle-dependent energy reprogramming. During interactions with pathogens, the host GM is markedly altered, leading to either a metabolic state, termed "endurance", in which cell viability is maintained, or to an opposite metabolic state, termed "evasion", in which the process of cell death is facilitated. It seems that endurance-natured modulations result in resistance to necrotrophic pathogens and susceptibility to biotrophs, whereas evasion-related reconfigurations lead to resistance to biotrophic pathogens but stimulate the infection by necrotrophs. Pathogens, however, have evolved strategies such as toxin secretion, hemibiotrophy, and selective amino acid utilization to exploit the plant GM to their own benefit. Collectively, alterations in the host GM in response to different pathogenic scenarios appear to function in two opposing ways, either backing the ongoing defense strategy to ultimately shape an efficient resistance response or being exploited by the pathogen to promote and facilitate infection.

Medicago truncatula dihydrodipicolinate synthase (DHDPS) enzymes display novel regulatory properties.

Lysine biosynthesis in plants is tightly regulated by feedback inhibition of the end product on the first enzyme of the lysine-specific branch, dihydrodipicolinate synthase (DHDPS). Three complete DHDPS coding sequences and one partial sequence were obtained in Medicago truncatula via inverse PCR. Analysis of the MtDHDPS sequences indicated the presence of isozymes (MtDHDPS2 and MtDHDPS3) with multiple amino acid substitutions on positions previously shown to be involved in feedback inhibition and of residues important for catalytic activity, possibly affecting the enzymatic properties of these isoforms. Sequences similar to MtDHDPS2 and 3 are present in Lotus japonicus and Glycine max, suggesting the existence of a specific conserved class of DHDPS genes within the Fabaceae family. The MtDHDPS genes were found by quantitative RT-PCR analysis to be expressed in an organ-specific manner in M. truncatula. All four MtDHDPS enzymes were expressed separately in Escherichia coli, revealing a strongly reduced sensitivity of the MtDHDPS2 protein to lysine feedback inhibition and a severely reduced activity of the MtDHDPS3 protein. Remarkably, MtDHDPS3 expression in Arabidopsis thaliana produced transgenic plants with a significantly increased threonine level, suggesting a dominant DHDPS inhibiting role of this isoform. This is supported by co-expression experiments in E. coli which indicate that AtDHDPS and MtDHDPS3 interact and may form hetero-oligomers with strongly reduced enzymatic activity. In conclusion, analysis of DHDPS in M. truncatula revealed the presence of unique isozymes displaying novel regulatory properties.

Heat shock induced excision of selectable marker genes in transgenic banana by the Cre-lox site-specific recombination system.

Selectable marker genes are indispensable for efficient production of transgenic events, but are no longer needed after the selection process and may cause public concern and technological problems. Although several gene excision systems exist, few have been optimized for vegetatively propagated crops. Using a Cre-loxP auto-excision strategy, we obtained transgenic banana plants cv. Grande Naine (Musa AAA) devoid of the marker gene used for selection. We used T-DNA vectors with the cre recombinase gene under control of a heat shock promoter and selectable marker gene cassettes placed between two loxP sites in direct orientation, and a gene of interest inserted outside of the loxP sites. Heat shock promoters pGmHSP17.6-L and pHSP18.2, from soybean and Arabidopsis respectively, were tested. A transient heat shock treatment of primary transgenic embryos was sufficient for inducing cre and excising cre and the marker genes. Excision efficiency, as determined by PCR and Southern hybridization was 59.7 and 40.0% for the GmHSP17.6-L and HSP18.2 promoters, respectively. Spontaneous excision was not observed in 50 plants derived from untreated transgenic embryos. To our knowledge this is the first report describing an efficient marker gene removal system for banana. The method described is simple and might be generally applicable for the production of marker-free transgenic plants of many crop species.