Development of transgenic rice pure lines with enhanced resistance to rice brown planthopper

Summary Mature seed-derived callus from an elite Chinese japonica rice cv. Ewan 5 was cotransformed with two plasmids, pWRG1515 and pRSSGNAl, containing the selectable marker hygromycin phosphotransferase gene (hpt), the reporter β-glucuronidase gene (gusA) and the snowdrop (Galanthus nivalis) lectin gene (gna) via particle bombardment. Thirty-five independent transgenic rice plants were regenerated from 177 bombarded calluses. Eighty-three percent of the transgenic plants contained all three genes, as revealed by Southern blot analysis. Western blot analysis revealed that 23 out of 29 gna-containing transgenic plants expressed Galanthus nivalis agglutinin (GNA) (79%) at various levels, with the highest expression being approximately 0.5% of total soluble protein. Genetic analysis confirmed Mendelian segregation of all three transgenes (gna, hpt and gusA) in the R2 progeny. Amongst the R2 generation two independent homozygous lines were identified that expressed all three transgenes. Insect bioassay and feeding tests showed that these homozygous lines had significant inhibition to rice brown planthopper (Nilaparvata lugens, BPH) by decreasing the survival, overall fecundity of BPH, retarding development, and decreasing the feeding of BPH. These BPH-resistant lines have been incorporated into a rice insect resistance breeding program. This is the first report that homozygous transgenic rice lines expressing GNA, developed by genetic transformation and through genetic analysis-based selection, conferred enhanced resistance to BPH.     

Enhancement of resistance to aphids by introducing the snowdrop lectin genegna into maize plants

In order to enhance the resistance to pests, transgenic maize (Zea mays L.) plants from elite inbred lines containing the gene encoding snowdrop lectin (Galanthus nivalis L. agglutinin; GNA) under control of a phloem-specific promoter were generated through theAgrobacterium tumefaciens- mediated method. The toxicity of GNA-expressing plants to aphids has also been studied. The independently derived plants were subjected to molecular analyses. Polymerase chain reaction (PCR) and Southern blot analyses confirmed that thegna gene was integrated into maize genome and inherited to the following generations. The typical Mendelian patterns of inheritance occurred in most cases. The level of GNA expression at 0.13%-0.28% of total soluble protein was observed in different transgenic plants. The progeny of nine GNA-expressing independent transformants that were derived separately from the elite inbred lines DH4866, DH9942, and 8902, were selected for examination of resistance to aphids. These plants synthesized GNA at levels above 0.22% total soluble protein, and enhanced resistance to aphids was demonstrated by exposing the plants to corn leaf aphid (Rhopalosiphum maidis Fitch) under greenhouse conditions. The nymph production was significantly reduced by 46.9% on GNA-expressing plants. Field evaluation of the transgenic plants supported the results from the inoculation trial. After a series of artificial self-crosses, some homozygous transgenic maize lines expressing GNA were obtained. In the present study, we have obtained new insect-resistant maize material for further breeding work.     

Influence of plant development and environment on transgene expression in potato and consequences for insect resistance

Clonal replicates of different transformed potato plants expressing transgene constructs containing the constitutive Cauliflower Mosaic Virus (CaMV) 35S promoter, and sequences encoding the plant defensive proteins snowdrop lectin (Galanthus nivalis agglutinin; GNA), and bean chitinase (BCH) were propagated in tissue culture. Plants were grown to maturity, at first under controlled environmental conditions, and later in the glasshouse. For a given transgene product, protein accumulation was found to vary between the different lines of clonal replicates (where each line was derived from a single primary transformant plant), as expected. However, variability was also found to exist within each line of clonal replicates, comparable to the variation of mean expression levels observed between the different clonal lines. Levels of GNA, accumulated in different parts of a transgenic potato plant, also showed variation but to a lesser extent than plant–plant variation in expression. With the majority of the clonal lines investigated, accumulation of the transgene product was found to increase as the potato plant developed, with maximum levels found in mature plants. The variation in accumulation of GNA among transgenic plants within a line of clonal replicates was exploited to demonstrate that the enhanced resistance towards larvae of the tomato moth, Lacanobia oleracea L., caused by expression of this protein in potato, was directly correlated with the level of GNA present in the plants, and that conditions under which the plants were grown affect the levels of GNA expression and subsequent levels of insect resistance.     

Improved <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of GNA transgenic sugarcane

Six plasmids carrying a snowdrop lectin (Galanthus nivalis agglutinin, GNA) and one of three selection markers were successfully transferred into two sugarcane cultivars (FN81–745 and Badila) via Agrobacterium-mediated transformation. Agrobacterium strains LBA4404, EHA105 and A281 that harboured a super-binary vector were used for sugarcane transformation. The use of the hygromycin (Hyg) resistance gene (hpt II), phosphinothrincin (PPT) resistance gene (bar) or G418 resistance gene (npt II) as a screenable marker facilitated the initial selection of GNA transgenic sugarcane callus with different efficiencies and helped the rapid segregation of individual transformation events. All the three selective marker genes were controlled by CaMV 35S promoter, while GNA gene was controlled by promoter of RSs-1 (rice sucrose synthase-1) or Ubi (maize ubiquitin). Factors important to successful transformation mediated by Agrobacterium tumefaciens were optimized, which included concentration of A. tumefaciens, medium composition, co-cultivated methods with plant tissue, strain virulence and different selective marker genes. An efficient protocol for sugarcane transformation mediated by A. tumefaciens was established. The GNA gene has been integrated into sugarcane genome as demonstrated by PCR and Southern dot blotting detections. The preliminary results from bioassay demonstrated a significant resistance of the transgenic sugarcane plants to woolly aphid (Ceratovacuna lanigera Zehnther) indicating thus the possibility for obtaining a transgenic sugarcane cultivar with resistance to woolly aphid.     

Expression of snowdrop lectin in transgenic tobacco plants results in added protection against aphids

The range of sap-sucking insect pests to which GNA, (the mannose specific lectin from snowdrops (Galanthus nivalis) has been shown to be insecticidal in artificial diets has been extended to include the peach potato aphid (Myzus persicae). A gene construct for constitutive expression of GNA from the CaMV35S gene promoter has been introduced into tobacco plants. A transgenic tobacco line which expresses high levels of GNA has been shown to have enhanced resistance toM. persicae in leaf disc and whole plant bioassays,demonstrating the potential for extending transgenic plant technology to the control of sap-sucking insect pests.     

Enhanced insect resistance in Thai rice varieties generated by particle bombardment

We used particle bombardment to transform two elite Thai rice varieties, Khao Dawk Mali 105 (KDML105) and Supanburi 60 (SP60), with the snowdrop lectin gene gna (Galanthus nivalis agglutinin). This gene confers resistance to sap-sucking insects such as the brown planthopper (BPH; Nilaparvata lugens), which is one of the most damaging pests of rice. Traditionally, KDML105 and SP60 have been regarded as recalcitrant to transformation, and this is the first account of successful gene transfer to these varieties. By molecular analysis, we confirmed the recovery of over thirty gna-transgenic lines. GNA protein expression was characterised by western blot analysis, and we achieved expression levels of up to 0.25% total soluble protein. GNA-producing R₁ transgenic plants were significantly more resistant to BPH than control plants (P<0.0001), with 37% and 42% reduction in nymphal survival for constitutive and phloem-specific expression, respectively. Transferring the gna gene to these superior rice varieties thus represents a major step forward for crop improvement in Thailand, and should help to reduce the damage caused by rice pests, and hence increase yields for this vital domestic and export market.     

Transgenic GNA Expressing Potato Plants Augment the Beneficial Biocontrol of Lacanobia Oleracea (Lepidoptera: Noctuidae) by the parasitoid Eulophus Pennicornis (Hymenoptera; Eulophidae)

The effect of expressing the gene encoding snowdrop lectin (Galanthus nivalis agglutinin, GNA) in transgenic potato plants, on parasitism of the phytophagous insect pest Lacanobia oleracea by the gregarious ectoparasitoid Eulophus pennicornis, was investigated in glasshouse trials. Expression of GNA (approx. 1.0% total soluble protein) by transgenic plants significantly reduced the level of pest damage, thus confirming previous studies. Furthermore, the presence of the parasitoid significantly reduced the levels of damage incurred either by the transgenic or control plants when compared to those plants grown in the absence of the parasitoid. For the GNA expressing plants the presence of the parasitoid resulted in further reductions (ca. 21%) in the level of damage caused by the pest species. The ability of the wasp to parasitise and subsequently develop on the pest larvae was not altered by the presence of GNA in the diet of the host. E. pennicornis progeny that developed on L. oleracea reared on GNA expressing plants showed no significant alteration in fecundity when compared with wasps that had developed on hosts fed on control potato plants, although mean size and longevity of female parasitoids was significantly reduced. The number of F ₂ progeny produced by parasitoids derived from hosts fed on GNA-expressing plants was not significantly different to those produced by parasitoids from hosts fed control plants. Results from the present study demonstrate that the use of transgenic plants expressing insecticidal proteins can be compatible with the deployment of beneficial insects and that the two factors may interact in a positive manner.     

Related lectins from snowdrop and maize differ in their carbohydrate-binding specificity

Searches in an EST database from maize revealed the expression of a protein related to the Galanthus nivalis (GNA) agglutinin, referred to as GNA_maize. Heterologous expression of GNA_maize in Pichia pastoris allowed characterization of the first nucleocytoplasmic GNA homolog from plants. GNA_maize is a tetrameric protein which shares 64% sequence similarity with GNA. Glycan microarray analyses revealed important differences in the specificity. Unlike GNA, which binds strongly to high-mannose N-glycans, the lectin from maize reacts almost exclusively with more complex glycans. Interestingly, GNA_maize prefers complex glycans containing β1-2 GlcNAc residues. The obvious difference in carbohydrate-binding properties is accompanied by a 100-fold reduced anti-HIV activity. Although the sequences of GNA and GNA_maize are clearly related they show only 28% sequence identity. Our results indicate that gene divergence within the family of GNA-related lectins leads to changes in carbohydrate-binding specificity, as shown on N-glycan arrays.     

Functional GNA expressed in Escherichia coli with high efficiency and its effect on Ceratovacuna lanigera Zehntner

The mannose-specific GNA (Galanthus nivalis agglutinin, snowdrop lectin) are the resistant proteins with many bioactivities. Snowdrop lectin is different with plant organs and development periods in lectin species, content, and bioactivities. It is an effective and cheap way to obtain much active GNA through overexpression of GNA gene in Escherichia coli. Constructs encoding mature GNA fused with an N-terminal pelB signal sequence protein (PelB) were expressed in E. coli with high efficiency. Recombinant protein productivity was higher than values published before. The insecticidal activity of purified recombinant proteins was assayed on feeding sugarcane wooly aphid (Ceratovacuna lanigera Zehntner), as well as spraying on sugarcane plants infected by aphids. The insecticidal activity was found to be comparable to native GNA. Oral delivery has obvious positive implications for crop protection against insect pests since peptides can be present in, or sprayed on, plant tissues susceptible to damage. A highly efficient expression of functional recombinant GNA would decrease the cost of GNA and promote its wide use, especially to give crop protection in the field.     

Wheat containing snowdrop lectin (GNA) does not affect infection of the cereal aphid <Emphasis Type="Italic">Metopolophium</Emphasis><Emphasis Type="Italic">dirhodum</Emphasis> by the fungal natural enemy<Emphasis Type="Italic">Pandora neoaphidis</Emphasis>

Studies were carried out to determine if susceptibility of the cereal aphid Metopolophium dirhodum to the fungus Pandora neoaphidis was affected by wheat expressing snowdrop lectin (GNA). Aphid infection did not differ significantly between the transgenic GNA and non-transformed lines (91 and 82%, respectively). Fecundity also did not differ between aphids on the two lines, and was ca. 18 nymphs adult⁻¹. Time to infection was ca. 5 days for M. dirhodum on both lines in two of three assays. Our results indicate that wheat expressing GNA would not compromise the efficacy of P. neoaphidis as a biocontrol agent.     

Transgenic tobacco plants containing <Emphasis Type="Italic">Bt</Emphasis> and <Emphasis Type="Italic">GNA</Emphasis> genes

A new plant expression vector (pBSbtCry1Ac-GNA) containing two insect resistant genes, a synthetic chimeric gene SbtCry1Ac encoding the insecticidal protein CrylAc and a gene GNA encoding snowdrop lectin (Galanthus nivalis agglutinin) was constructed. Transgenic tobacco plants containing these two genes were obtained through Agrobacterium-mediated transformation of tobacco leaf discs. Results from PCR detection and genomic DNA Southern blot analysis indicated that both SbtCrylAc gene and GNA gene were integrated into the genome of these plants. Results of Western blot analysis indicated that these two proteins were expressed in the analyzed plants. Bioassays of Myzus persicae and Helicoverpa assulta on detached leaves of transformed tobacco plants were carried out. The average aphid inhibition rate of these plants tested at 12 d post-infestation was 71.9 %. The average H. assulta mortality of these plants tested at 6 d post-infestation was up to 89.8 %. The kanamycin resistance of the T₁ progeny of these transgenic plants was analyzed and a typical 3:1 segregation was observed.     

Use of the rice sucrose synthase-1 promoter to direct phloem-specific expression of {beta}-glucuronidase and snowdrop lectin genes in transgenic tobacco plants

The promoter region from the rice sucrose synthase-1 gene (RSs1)was fused with coding sequences for ß-glucuronidase(GUS) and snowdrop (Galanthus nivalis) lectin (GNA). Tobaccoplants were transformed with these chimaenc genes in order todetermine the expression pattern directed by the RSs1 promoter.Histochemical and immunochemical assays demonstrated that theexpression of both GUS and GNA was restricted to phloem tissue,and was not observed in any other tissues. This phloem-specificexpression pattern was consistent in stem, leaf and root, andin different transgenic plants. Chimaeric genes of RSs 1-GUSand RSs1 GNA were stably inherited in T1 plants. In addition,GNA was detected by immunological assay in the honeydew producedby peach potato aphids (Myzus persicae) feeding on RSs1-GNAtransgenic tobacco plants. This provided direct evidence thatGNA was not only expressed in the phloem tissue, but was alsopresent in the phloem sap of transgenic tobacco plants. TheRSs1 promoter can thus be used to direct expression of an insecticidalprotein, such as GNA, in transgenic plants to control phloemsap-feeding insect pests. Key words: Rice sucrose synthase-1 promoter, phloemspecific, transgenic plants, ß-glucuronidase, Galanthus nivalis agglutinin, gene expression     

Transgenic plants expressing the AaIT/GNA fusion protein show increased resistance and toxicity to both chewing and sucking pests

The adoption of pest‐resistant transgenic plants to reduce yield losses and decrease pesticide use has been successful. To achieve the goal of controlling both chewing and sucking pests in a given transgenic plant, we generated transgenic tobacco, Arabidopsis, and rice plants expressing the fusion protein, AaIT/GNA, in which an insecticidal scorpion venom neurotoxin (Androctonus australis toxin, AaIT) is fused to snowdrop lectin (Galanthus nivalis agglutinin, GNA). Compared with transgenic tobacco and Arabidopsis plants expressing AaIT or GNA, transgenic plants expressing AaIT/GNA exhibited increased resistance and toxicity to one chewing pest, the cotton bollworm, Helicoverpa armigera. Transgenic tobacco and rice plants expressing AaIT/GNA showed increased resistance and toxicity to two sucking pests, the whitefly, Bemisia tabaci, and the rice brown planthopper, Nilaparvata lugens, respectively. Moreover, in the field, transgenic rice plants expressing AaIT/GNA exhibited a significant improvement in grain yield when infested with N. lugens. This study shows that expressing the AaIT/GNA fusion protein in transgenic plants can be a useful approach for controlling pests, particularly sucking pests which are not susceptible to the toxin in Bt crops.     

Transgenic rice plants expressing the snowdrop lectin gene (<Emphasis Type="Italic">gna</Emphasis>) exhibit high-level resistance to the whitebacked planthopper (<Emphasis Type="Italic">Sogatella furcifera</Emphasis>)

Transgenic rice plants, expressing snowdrop lectin [Galanthus nivalis agglutinin (GNA)], obtained by Agrobacterium-mediated genetic transformation, were evaluated for resistance against the insect, the whitebacked planthopper (WBPH). The transgene gna was driven by the phloem-specific, rice-sucrose synthase promoter RSs1, and the bar was driven by the CaMV 35S promoter. In our previous study, the transgenic status of these lines was confirmed by Southern, Northern and Western blot analyses. Both the transgenes, gna and bar, were stably inherited and co-segregated into progenies in T₁ to T₅ generations. Insect bioassays on transgenic plants revealed the potent entomotoxic effects of GNA on the WBPH. Also, significant decreases were observed in the survival, development and fecundity of the insects fed on transgenic plants. Furthermore, intact GNA was detected in the total proteins of WBPHs fed on these plants. Western blot analysis revealed stable and consistent expression of GNA throughout the growth and development of transgenic plants. Transgenic lines expressing GNA exhibited high-level resistance against the WBPH. As reported earlier, these transgenics also showed substantial resistance against the brown planthopper and green leafhopper .     

Linear transgene constructs lacking vector backbone sequences generate transgenic rice plants which accumulate higher levels of proteins conferring insect resistance

Biolistic transformation was used to introduce genes encoding the insecticidal proteins snowdrop lectin (Galanthus nivalis agglutinin; GNA) and cry1Ac Bt toxin (-endotoxin from Bacillus thuringiensis) into elite rice (Oryza sativa) cultivars. Plant transformation was carried out in parallel experiments simultaneously by using either whole plasmids containing suitable gene constructs, or the corresponding minimal gene cassettes, which were linear DNA fragments lacking vector sequences excised from the plasmids. Both transformation methods generated similar numbers of independent transformation events. Selected R₀ clonal plant lines were further characterised for presence and expression of transgenes. Co-transformation of the unselected genes (cry1Ac and gna) with the selectable marker (hpt) was at least as efficient for transformation with minimal gene cassettes as with whole plasmid DNA, and higher levels of accumulation of the insecticidal gene products GNA and cry1Ac were observed in plants resulting from minimal gene cassette transformation. Insect bioassays with major pests of rice showed that transgenic plants expressing gna showed enhanced resistance to brown planthopper (Nilaparvata lugens), and plants expressing cry1Ac were protected against attack by striped stem borer (Chilo suppressalis). Expression of both transgenes gave protection against both pests, but did not increase protection against either pest significantly over the levels observed in plants containing a single insecticidal transgene.     

Large-scale production and purification of recombinant Galanthus nivalis agglutinin (GNA) expressed in the methylotrophic yeast Pichia pastoris

The gene coding for agglutinin from Galanthus nivalis (GNA) was expressed in, and secreted by, the methylotrophic yeast, Pichia pastoris. Transformants of P. pastoris were selected and a process to produce and purify gram quantities of recombinant GNA was developed. GNA was secreted at approximately 80 mg l^–1 at the 200 l scale and was purified to 95% homogeneity using hydrophobic interaction chromatography. The recombinant protein was similar to the protein synthesised in plant with respect to structure and biological activity.     

The effect of genetic transformations for pest resistance on foliar solanidine-based glycoalkaloids of potato (Solatium tuberosuni)

Foliage of potato cv. Desiree was harvested from glasshouse‐cultivated plants of five experimental transgenic lines expressing three different insecticidal proteins (snowdrop lectin, Galanthus nivalis agglutinin (GNA); jackbean lectin, Concanavalin A (Con A), cowpea trypsin inhibitor; (CpTi)), tissue‐cultured control plants and standard control (non‐tissue cultured) plants. The foliage was subdivided into stems, upper, middle and lower leaves and analysed separately by HPLC for the solanidine‐based glycoalkaloids a‐solanine and a‐chaconine. The results demonstrate that one or more stages in the plant transformation process (i.e. insecticidal‐ and marker‐gene insertions, gene expression and tissue culture) resulted in a lower level of leaf glycoalkaloids than that found in either the tissue‐cultured controls or standard controls, based on the selected potato lines transformed for insecticidal protein expression. However, the distribution of glycoalkaloids throughout the plant foliage was unaffected by genetic transformation and tissue culture, with the highest glycoalkaloid levels being observed in the top third of the plant. The importance of investigating unexpected effects of genetic engineering on plant secondary metabolism is discussed from an ecological viewpoint.     

Molecular cloning of the lectin and a lectin-related protein from common Solomon's seal (Polygonatum multiflorum)

The most prominent protein ofPolygonatum multiflorum (common Solomon's seal) rhizomes has been identified as a mannose-binding lectin. Analysis of the purified lectin demonstrated that it is a tetramer of four identical subunits of 14 kDa. Molecular cloning further revealed that the lectin from this typical Liliaceae species belongs to the superfamily of monocot mannose-binding proteins. Screening of cDNA libraries constructed with RNA isolated from buds, leaves and flowers ofP. multiflorum also yielded cDNA clones encoding a protein, which contains two tandemly arranged domains with an obvious sequence homology to the mannose-binding lectins. Molecular modelling of thePolygonatum lectin and lectin-related protein indicated that the three-dimensional structure of both proteins strongly resembles that of the snowdrop lectin. In addition, this approach suggested that the presumed carbohydrate-binding sites of the lectin can accommodate a mannose residue whereas most of the carbohydratebinding sites of the lectin-related protein cannot.Abbreviations GNA Galanthus nivalis agglutinin - HCA hydrophobic cluster analysis - LECPMA cDNA clone encoding PMA - PM30 30 kDa protein isolated fromPolygonatum multiforum - PMA Polygonatum multiflorum agglutinin - PMLRP Polygonatum multiflorum lectin-related protein     

Assessing the utilization of a carbohydrate food source and the impact of insecticidal proteins on larvae of the green lacewing, Chrysoperla carnea

A concern with the widespread use of insecticidal transgenic crops is their potential to adversely affect non-target organisms, including biological control agents such as larvae of the green lacewing, Chrysoperla carnea (Neuroptera: Chrysopidae). Since the insecticidal proteins expressed by the current transgenic plants are active only after ingestion, dietary bioassays are required to test direct effects on non-target organisms. After showing that C. carnea larvae utilize carbohydrate foods, we exposed them to insecticidal proteins dissolved in a sucrose solution. Feeding on snowdrop lectin (Galanthus nivalis agglutinin, GNA) as a model compound, the larvae were negatively affected in a number of life-table parameters. Interestingly, GNA caused a prolongation in first instar development, but had no effect on subsequent utilization of prey resulting in an increased weight of second instars. Comparable studies with avidin, a biotin-binding protein, revealed strong effects on C. carnea survival at the concentration tested. Despite the fact that the proteolytic digestion of C. carnea larvae is reported to be dominated by serine proteases, ingestion of soybean trypsin inhibitor (SBTI) did not cause any detrimental effects. Similarly, two Cry proteins derived from Bacillus thuringiensis (Cry1Ac and Cry1Ab) did not cause negative effects on C. carnea, what is consistent with earlier studies. The here presented bioassay provides a valuable tool to assess direct impacts of insecticidal proteins to C. carnea larvae and other predators that are known to feed on carbohydrate solutions.     

Expression of multiple insecticidal genes confers broad resistance against a range of different rice pests

We report the simultaneous introduction of three insecticidal genes (the Bt genes cry1Ac and cry2A, and the snowdrop lectin gene gna) into commercially important indica rice varieties M7 and Basmati 370, by particle bombardment. Transgenic plants expressed Cry1Ac, Cry2A and GNA at different levels, either singly or in combination at 0.03–1%, 0.01–0.5% and 0.01–2.5% of total soluble protein, respectively. The transgenes showed stable transmission and expression, and R₁ transgenic plants provided significant (p<0.01) protection against three of the most important insect pests of rice: rice leaf folder (Cnaphalocrocis medinalis), yellow stemborer (Scirpophaga incertulas) and brown planthopper (Nilaparvata lugens). The triple transformants showed significantly (p<0.05) higher resistance to these insects than plants expressing single transgenes. Bioassays using the triple-transgenic plants showed 100% eradication of the rice leaf folder and yellow stem borer, and 25% reduction in the survival of the brown planthopper. The greatest reduction in insect survival, and the greatest reduction in plant damage, occurred in plants expressing all three transgenes. This approach maximises the utility of gene transfer technology to introduce combinations of genes whose products disrupt different biochemical or physiological processes in the same insect, providing a multi-mechanism defence.