Comparative TMT Proteomic Analysis Unveils Unique Insights into Helicoverpa armigera (Hübner) Resistance in Cajanus scarabaeoides (L.) Thouars.

Pigeonpea [Cajanus cajan (L.) Millspaugh] is an economically important legume playing a crucial role in the semi-arid tropics. Pigeonpea is susceptible to Helicoverpa armigera (Hübner), which causes devastating yield losses. This pest is developing resistance to many commercially available insecticides. Therefore, crop wild relatives of pigeonpea, are being considered as potential sources of genes to expand the genetic base of cultivated pigeonpea to improve traits such as host plant resistance to pests and pathogens. Quantitative proteomic analysis was conducted using the tandem mass tag platform to identify differentially abundant proteins between IBS 3471 and ICPL 87 tolerant accession and susceptible variety to H. armigera, respectively. Leaf proteome were analysed at the vegetative and flowering/podding growth stages. H. armigera tolerance in IBS 3471 appeared to be related to enhanced defence responses, such as changes in secondary metabolite precursors, antioxidants, and the phenylpropanoid pathway. The development of larvae fed on an artificial diet with IBS 3471 lyophilised leaves showed similar inhibition with those fed on an artificial diet with quercetin concentrations with 32 mg/25 g of artificial diet. DAB staining (3,3'-diaminobenzidine) revealed a rapid accumulation of reactive oxygen species in IBS 3471. We conclude that IBS 3471 is an ideal candidate for improving the genetic base of cultivated pigeonpea, including traits for host plant resistance.

Efficacy of a cry1Ab Gene for Control of Maruca vitrata (Lepidoptera: Crambidae) in Cowpea (Fabales: Fabaceae).

Cowpea [Vigna unguiculata (L) Walp.] is an important staple legume in the diet of many households in sub-Saharan Africa. Its production, however, is negatively impacted by many insect pests including bean pod borer, Maruca vitrata F., which can cause 20-80% yield loss. Several genetically engineered cowpea events that contain a cry1Ab gene from Bacillus thuringiensis (Bt) for resistance against M. vitrata were evaluated in Nigeria, Burkina Faso, and Ghana (West Africa), where cowpea is commonly grown. As part of the regulatory safety package, these efficacy data were developed and evaluated by in-country scientists. The Bt-cowpea lines were planted in confined field trials under Insect-proof netting and artificially infested with up to 500 M. vitrata larvae per plant during bud formation and flowering periods. Bt-cowpea lines provided nearly complete pod and seed protection and in most cases resulted in significantly increased seed yield over non-Bt control lines. An integrated pest management strategy that includes use of Bt-cowpea augmented with minimal insecticide treatment for protection against other insects is recommended to control pod borer to enhance cowpea production. The insect resistance management plan is based on the high-dose refuge strategy where non-Bt-cowpea and natural refuges are expected to provide M. vitrata susceptible to Cry1Ab protein. In addition, there will be a limited release of this product until a two-toxin cowpea pyramid is released. Other than South African genetically engineered crops, Bt-cowpea is the first genetically engineered food crop developed by the public sector and approved for release in sub-Saharan Africa.

An Improved Transformation System for Cowpea (Vigna unguiculata L. Walp) via Sonication and a Kanamycin-Geneticin Selection Regime.

An improved cowpea transformation method utilizing Agrobacterium-mediated gene delivery to explants derived from the cotyledonary nodes of imbibed cowpea seed is described. The explants were regenerated following a sonication procedure and a stringent selection comprising alternating regimes of kanamycin and geneticin. The method was reproducible and led to the recovery of independent fertile transgenic plants in the greenhouse at a level of about one per cent of starting explants. A transgene encoding an insecticidal protein from Bacillus thuringiensis was used to demonstrate the efficacy of the system.

Resistance of αAI-1 transgenic chickpea (Cicer arietinum) and cowpea (Vigna unguiculata) dry grains to bruchid beetles (Coleoptera: Chrysomelidae).

Dry grain legume seeds possessing αAI-1, an α-amylase inhibitor from common bean (Phaseolus vulgaris), under the control of a cotyledon-specific promoter have been shown to be highly resistant to several important bruchid pest species. One transgenic chickpea and four cowpea lines expressing αAI-1, their respective controls, as well as nine conventional chickpea cultivars were assessed for their resistance to the bruchids Acanthoscelides obtectus (Say), Callosobruchus chinensis L. and Callosobruchus maculatus F. All transgenic lines were highly resistant to both Callosobruchus species. A. obtectus, known to be tolerant to αAI-1, was able to develop in all transgenic lines. While the cotyledons of all non-transgenic cultivars were highly susceptible to all bruchids, C. chinensis and C. maculatus larvae suffered from significantly increased mortality rates inside transgenic seeds. The main factor responsible for the partial resistance in the non-transgenic cultivars was deduced to reside in the seed coat. The αAI-1 present in seeds of transgenic chickpea and cowpea lines significantly increases their resistance to two important bruchid pest species (C. chinensis and C. maculatus) essentially to immunity. To control αAI-1 tolerant bruchid species such as A. obtectus and to avoid the development of resistance to αAI-1, varieties carrying this transgene should be protected with additional control measures.

Unintended changes in protein expression revealed by proteomic analysis of seeds from transgenic pea expressing a bean alpha-amylase inhibitor gene.

Seeds of genetically modified (GM) peas (Pisum sativum L.) expressing the gene for alpha-amylase inhibitor-1 (alphaAI1) from the common bean (Phaseolus vulgaris L. cv. Tendergreen) exhibit resistance to the pea weevil (Bruchus pisorum). A proteomic analysis was carried out to compare seeds from GM pea lines expressing the bean alphaAI1 protein and the corresponding alphaAI1-free segregating lines and non-GM parental line to identify unintended alterations to the proteome of GM peas due to the introduction of the gene for alphaAI1. Proteomic analysis showed that in addition to the presence of alphaAI1, 33 other proteins were differentially accumulated in the alphaAI1-expressing GM lines compared with their non-GM parental line and these were grouped into five expression classes. Among these 33 proteins, only three were found to be associated with the expression of alphaAI1 in the GM pea lines. The accumulation of the remaining 30 proteins appears to be associated with Agrobacterium-mediated transformation events. Sixteen proteins were identified after MALDI-TOF-TOF analysis. About 56% of the identified proteins with altered accumulation in the GM pea were storage proteins including legumin, vicilin or convicilin, phaseolin, cupin and valosin-containing protein. Two proteins were uniquely expressed in the alphaAI1-expressing GM lines and one new protein was present in both the alphaAI1-expressing GM lines and their alphaAI1-free segregating lines, suggesting that both transgenesis and transformation events led to demonstrable changes in the proteomes of the GM lines tested.

Identification of cis-localization elements of the maize 10-kDa delta-zein and their use in targeting RNAs to specific cortical endoplasmic reticulum subdomains.

The RNAs for the storage proteins of rice (Oryza sativa), prolamines and glutelins, which are stored as inclusions in the lumen of the endoplasmic reticulum (ER) and storage vacuoles, respectively, are targeted by specific cis-localization elements to distinct subdomains of the cortical ER. Glutelin RNA has one or more cis-localization elements (zip codes) at the 3' end of the RNA, whereas prolamine has two cis-elements; one located in the 5' end of the coding sequence and a second residing in the 3'-untranslated region (UTR). We had earlier demonstrated that the RNAs for the maize zeins ('prolamine' class) are localized to the spherical protein body ER (PB-ER) in developing maize endosperm. As the PB-ER localization of the 10-kDa delta-zein RNA is maintained in developing rice seeds, we determined the number and proximate location of their cis-localization elements by expressing GFP fusions containing various zein RNA sequences in transgenic rice and analyzing their spatial distribution on the cortical ER by in situ RT-PCR and confocal microscopy. Four putative cis-localization elements were identified; three in the coding sequences and one in the 3'-UTR. Two of these zip codes are required for restricted localization to the PB-ER. Using RNA targeting determinants we show, by mis-targeting the storage protein RNAs from their normal destination on the cortical ER, that the coded proteins are redirected from their normal site of deposition. Targeting of RNA to distinct cortical ER subdomains may be the underlying basis for the variable use of the ER lumen or storage vacuole as the final storage deposition site of storage proteins among flowering plant species.

Transgenic peas expressing an alpha-amylase inhibitor gene from beans show altered expression and modification of endogenous proteins.

Differential in-gel electrophoresis showed contrasting effects of the transgenic expression of an alpha-amylase inhibitor from beans on the proteomes of two pea cultivars. One cultivar showed minor changes relative to its non-transgenic parent with only one protein changing by more than about twofold. Changes in the abundance of certain endogenous proteins in the other cultivar were of greater number and magnitude with some endogenous proteins undetected while some new protein spots appeared in the transgenic proteome. The sets of proteins with altered expression were generally different between the two cultivars. Some of the proteins that were differentially expressed were identified by MS. Most were seed storage globulins, which are sited together with the transgenic product. Some of the changes may be due to alterations in expression levels but there were also changes due to post-translational processing.

Bean alpha-amylase inhibitors in transgenic peas inhibit development of pea weevil larvae.

This glasshouse study used an improved larval measurement procedure to evaluate the impact of transgenic pea, Pisum sativum L., seeds expressing a-amylase inhibitor (AI)-1 or -2 proteins on pea weevil, Bruchus pisorum L. Seeds of transgenic 'Laura' and 'Greenfeast' peas expressing alpha-(AI)-1 reduced pea weevil survival by 93-98%. Larval mortality occurred at an early instar. Conversely, in nontransgenic cultivars, approximately 98-99% of the pea weevils emerged as adults. By measuring the head capsule size, we determined that larvae died at the first to early third instar in alpha-(AI)-1 transgenic peas, indicating that this inhibitor is highly effective in controlling this insect. By contrast, transgenic Laura and 'Dundale' expressing alpha-(AI)-2 did not affect pea weevil survival, but they did delay larval development. After 77 d of development, the head capsule size indicated that the larvae were still at the third instar stage in transgenic alpha-(AI)-2 peas, whereas adult bruchids had developed in the nontransgenic peas.

Genetic transformation of cowpea (Vigna unguiculata L.) and stable transmission of the transgenes to progeny.

Cowpeas are nutritious grains that provide the main source of protein, highly digestible energy and vitamins to some of the world's poorest people. The demand for cowpeas is high but yields remain critically low, largely because of insect pests. Cowpea germplasm contains little or no resistance to major insect pests and a gene technology approach to adding insect protection traits is now a high priority. We have adapted features of several legume and other transformation systems and reproducibly obtained transgenic cowpeas that obey Mendelian rules in transmitting the transgene to their progeny. Critical parameters in this transformation system include the choice of cotyledonary nodes from developing or mature seeds as explants and a tissue culture medium devoid of auxins in the early stages, but including the cytokinin BAP at low levels during shoot initiation and elongation. Addition of thiol-compounds during infection and co-culture with Agrobacterium and the choice of the bar gene for selection with phosphinothricin were also important. Transgenic cowpeas that transmit the transgenes to their progeny can be recovered at a rate of one fertile plant per thousand explants. These results pave the way for the introduction of new traits into cowpea and the first genes to be trialled will include those with potential to protect against insect pests.

Decreased accumulation of glutelin types in rice grains constitutively expressing a sunflower seed albumin gene.

Previous studies have shown differential accumulation of sulfur rich glutelins and sulfur poor prolamins in transgenic rice seeds expressing a sunflower seed albumin gene [Hagan, N.D., Upadhyaya, N., Tabe, L.M., Higgins, T.J., 2003. The redistribution of protein sulfur in transgenic rice expressing a gene for a foreign, sulfur-rich protein. Plant J 34, 1-11]. Here, we show, by two-dimensional electrophoresis, differential accumulation of three classes of glutelin proteins - type I, II and III - and a globulin, not previously resolved, in transgenic seeds grown under low and high sulfur nutrition. Several glutelin polypeptides were resolved and four identified as a type I glutelin, two type II glutelins and a type III glutelin. Although sulfur nutrition did not affect the accumulation of sunflower seed albumin, the levels of all four identified glutelins and the globulin were lower in mature seeds derived from transgenic plants grown under sulfur-optimum or sulfur limited conditions compared to non-transgenic rice seeds. The reduction of all four glutelin polypeptides and the globulin varied from 21% to 68%. The re-allocation of sulfur reserves from endogenous proteins to the sulfur sink in transgenic grain is suggestive of a transcriptional control of sulfur mobilization in plants.

Response to water deficit and high temperature of transgenic peas (Pisum sativum L.) containing a seed-specific alpha-amylase inhibitor and the subsequent effects on pea weevil (Bruchus pisorum L.) survival.

The effects of water deficit and high temperature on the production of alpha-amylase inhibitor 1 (alpha-AI-1) were studied in transgenic peas (Pisum sativum L.) that were developed to control the seed-feeding pea weevil (Bruchus pisorum L., Coleoptera: Bruchidae). Transgenic and non-transgenic plants were subjected to water-deficit and high-temperature treatments under controlled conditions in the glasshouse and growth cabinet, beginning 1 week after the first pods were formed. In the water-deficit treatments, the peas were either adequately watered (control) or water was withheld after first pod formation. The high-temperature experiments were performed in two growth cabinets, one maintained at 27/22 degrees C (control) and one at 32/27 degrees C day/night temperatures, with the vapour pressure deficit maintained at 1.3 kPa. The plants exposure to high temperatures and water deficit produced 27% and 79% fewer seeds, respectively, than the controls. In the transgenic peas the level of alpha-AI-1 as a percentage of total protein was not influenced by water stress, but was reduced on average by 36.3% (the range in two experiments was 11-50%) in the high-temperature treatment. Transgenic and non-transgenic pods of plants grown at 27/22 degrees C and 32/27 degrees C were inoculated with pea weevil eggs to evaluate whether the reduction in level of alpha-AI-1 in the transgenic pea seeds affected pea weevil development and survival. At the higher temperatures, 39% of adult pea weevil emerged, compared to 1.2% in the transgenic peas grown at the lower temperatures, indicating that high temperature reduced the protective capacity of the transgenic peas.

Changes in methylation during progressive transcriptional silencing in transgenic subterranean clover.

A transgenic line of subterranean clover (Trifolium subterraneum) containing a gene for a sulphur-rich sunflower seed albumin (ssa gene) and a gene conferring tolerance to the herbicide phosphinothricin (bar gene) was previously shown to stably express these genes as far as the T3 generation. In subsequent generations there was a progressive decline in the level of expression of both of these genes such that, by the T7 generation, the plants were almost completely susceptible to the herbicide and the mean level of sunflower seed albumin was reduced to 10-30% of the level in the T2 and T3 generations. The decline in SSA protein correlated closely with a decline in the level of ssa RNA. In vitro transcription experiments with nuclei isolated from plants of the early and late generations showed that the reduced mRNA level was associated with a reduced level of transcription of the ssa transgene. Transcription of the bar transgene was also reduced in the late generations. Bisulphite sequencing analysis showed that the decline in expression of the ssa gene between T3 and subsequent generations correlated closely with increased CpG methylation in the promoter, but not in the coding region. Analysis of the bar gene promoter showed that high levels of CpG methylation preceded the first detectable decline in expression of the bar gene by one generation, suggesting that methylation was not the direct cause of transgene silencing in these plants.

Seed-specific overexpression of a potato sucrose transporter increases sucrose uptake and growth rates of developing pea cotyledons.

During the storage phase, cotyledons of developing pea seeds are nourished by nutrients released to the seed apoplasm by their maternal seed coats. Sucrose is transported into pea cotyledons by sucrose/H+ symport mediated by PsSUT1 and possibly other sucrose symporters. PsSUT1 is principally localised to plasma membranes of cotyledon epidermal and subepidermal transfer cells abutting the seed coat. We tested the hypothesis that endogenous sucrose/H+ symporter(s) regulate sucrose import into developing pea cotyledons. This was done by supplementing their transport activity with a potato sucrose symporter (StSUT1), selectively expressed in cotyledon storage parenchyma cells under control of a vicilin promoter. In segregating transgenic lines, enhanced [(14)C]sucrose influx into cotyledons above wild-type levels was found to be dependent on StSUT1 expression. The transgene significantly increased (approximately 2-fold) transport activity of cotyledon storage parenchyma tissues where it was selectively expressed. In contrast, sucrose influx into whole cotyledons through the endogenous epidermal transfer cell pathway was increased by only 23% in cotyledons expressing the transgene. A similar response was found for rates of biomass gain by intact cotyledons and by excised cotyledons cultured on a sucrose medium. These observations demonstrate that transport activities of sucrose symporters influence cotyledon growth rates. The attenuated effect of StSUT1 overexpression on sucrose and dry matter fluxes by whole cotyledons is consistent with a large proportion of sucrose being taken up at the cotyledonary surface. This indicates that the cellular location of sucrose transporter activity plays a key role in determining rates of sucrose import into cotyledons.

Fructan formation in transgenic white clover expressing a fructosyltransferase from Streptococcus salivarius.

White clover (Trifolium repens L.) is an important pasture legume that does not normally accumulate fructan as a storage carbohydrate. We have generated transgenic white clover plants that accumulate fructan, by expressing the fructosyltransferase (Ftf) enzyme from the bacterium Streptococcus salivarius under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Fructan accumulated in leaves, petioles, stolons, flowers, and roots of transgenic plants. Levels of fructan up to approximately 2% dry weight were measured in leaves. The fructan was of high molecular mass ( > 5000 kDa), typical of bacterial fructans. Ftf enzyme activity up to 120 nmol min-1 g-1 fresh weight was determined in leaf extracts of the transformed plants, and appeared to be stable throughout leaf development. Most transformed lines appeared normal, flowered and produced seed, but the growth rate of some transformed lines decreased. Photosynthetic carbon assimilation and levels of endogenous carbohydrates (hexoses, sucrose and starch) were not substantially changed in a clonal line with relatively low fructan. However, in a clonal line with relatively high fructan accumulation, plant growth was reduced, leaf photosynthesis was decreased by 60%, and carbohydrate contents were reduced. The results are discussed in the context of manipulating soluble carbohydrate composition in pasture species to improve nutritive quality for grazing animals.