Tiziana Pandolfini

Auxin and nitric oxide control indeterminate nodule formation

BackgroundRhizobia symbionts elicit root nodule formation in leguminous plants. Nodule development requires local accumulation of auxin. Both plants and rhizobia synthesise auxin. We have addressed the effects of bacterial auxin (IAA) on nodulation by using Sinorhizobium meliloti and Rhizobium leguminosarum bacteria genetically engineered for increased auxin synthesis.ResultsIAA-overproducing S. meliloti increased nodulation in Medicago species, whilst the increased auxin synthesis of R. leguminosarum had no effect on nodulation in Phaseolus vulgaris, a legume bearing determinate nodules. Indeterminate legumes (Medicago species) bearing IAA-overproducing nodules showed an enhanced lateral root development, a process known to be regulated by both IAA and nitric oxide (NO). Higher NO levels were detected in indeterminate nodules of Medicago plants formed by the IAA-overproducing rhizobia. The specific NO scavenger cPTIO markedly reduced nodulation induced by wild type and IAA-overproducing strains.ConclusionThe data hereby presented demonstrate that auxin synthesised by rhizobia and nitric oxide positively affect indeterminate nodule formation and, together with the observation of increased expression of an auxin efflux carrier in roots bearing nodules with higher IAA and NO content, support a model of nodule formation that involves auxin transport regulation and NO synthesis.

The defH9-iaaM auxin-synthesizing gene increases plant fecundity and fruit production in strawberry and raspberry

BackgroundThe DefH9-iaaM gene fusion which is expressed specifically in placenta/ovules and promotes auxin-synthesis confers parthenocarpic fruit development to eggplant, tomato and tobacco. Transgenic DefH9-iaaM eggplants and tomatoes show increased fruit production due mainly to an improved fruit set. However, the weight of the fruits is also frequently increased.ResultsDefH9-iaaM strawberry and raspberry plants grown under standard cultivation conditions show a significant increase in fruit number and size and fruit yield. In all three Rosaceae species tested, Fragaria vesca, Fragaria x ananassa and Rubus idaeus, DefH9-iaaM plants have an increased number of flowers per inflorescence and an increased number of inflorescences per plant. This results in an increased number of fruits per plant. Moreover, the weight and size of transgenic fruits was also increased. The increase in fruit yield was approximately 180% in cultivated strawberry, 140% in wild strawberry, and 100% in raspberry. The DefH9-iaaM gene is expressed in the flower buds of all three species. The total IAA (auxin) content of young flower buds of strawberry and raspberry expressing the DefH9-iaaM gene is increased in comparison to untransformed flower buds. The DefH9-iaaM gene promotes parthenocarpy in emasculated flowers of both strawberry and raspberry.ConclusionsThe DefH9-iaaM gene is expressed and biologically active in Rosaceae. The DefH9-iaaM gene can be used, under cultivation conditions that allow pollination and fertilization, to increase fruit productivity significantly in Rosaceae species. The finding that the DefH9-iaaM auxin-synthesizing gene increases the number of inflorescences per plant and the number of flowers per inflorescence indicates that auxin plays a role in plant fecundity in these three perennial Rosaceae species.

Genetic engineering of parthenocarpic fruit development in tomato

Parthenocarpy was engineered in two genotypes of Lycopersicon esculentum Mill. by using the DefH9-iaaM chimeric gene. The parthenocarpic trait consists of fruit set and growth in the absence of fertilization. Seedless parthenocarpic fruits were obtained from emasculated flowers, and fruits with seeds from pollinated flowers. All parthenocarpic tomato plants analysed expressed the DefH9-iaaM gene during flower development. The fruit set percentage of emasculated transgenic flowers was similar to that of control plants. In 7 out of 8 independent transgenic plants, the fresh weight of fruits derived from pollinated or emasculated flowers did not significantly differ from that of fruits obtained by pollination of the control plants. The pH of the parthenocarpic fruit was generally unaffected and the soluble solid concentration was either unchanged or increased. Thus, the DefH9-iaaM gene is a genetic tool that might be used to improve tomato productivity.

Genetic transformation of Vitis vinifera via organogenesis

BackgroundEfficient transformation and regeneration methods are a priority for successful application of genetic engineering to vegetative propagated plants such as grape. The current methods for the production of transgenic grape plants are based on Agrobacterium-mediated transformation followed by regeneration from embryogenic callus. However, grape embryogenic calli are laborious to establish and the phenotype of the regenerated plants can be altered.ResultsTransgenic grape plants (V. vinifera, table-grape cultivars Silcora and Thompson Seedless) were produced using a method based on regeneration via organogenesis. In vitro proliferating shoots were cultured in the presence of increasing concentrations of N6-benzyl adenine. The apical dome of the shoot was removed at each transplantation which, after three months, produced meristematic bulk tissue characterized by a strong capacity to differentiate adventitious shoots. Slices prepared from the meristematic bulk were used for Agrobacterium-mediated transformation of grape plants with the gene DefH9-iaaM. After rooting on kanamycin containing media and greenhouse acclimatization, transgenic plants were transferred to the field. At the end of the first year of field cultivation, DefH9-iaaM grape plants were phenotypically homogeneous and did not show any morphological alterations in vegetative growth. The expression of DefH9-iaaM gene was detected in transgenic flower buds of both cultivars.ConclusionsThe phenotypic homogeneity of the regenerated plants highlights the validity of this method for both propagation and genetic transformation of table grape cultivars. Expression of the DefH9-iaaM gene takes place in young flower buds of transgenic plants from both grape cultivars.

Expression of self-complementary hairpin RNA under the control of the rolC promoter confers systemic disease resistance to plum pox virus without preventing local infection

BackgroundHomology-dependent selective degradation of RNA, or post-transcriptional gene silencing (PTGS), is involved in several biological phenomena, including adaptative defense mechanisms against plant viruses. Small interfering RNAs mediate the selective degradation of target RNA by guiding a multicomponent RNAse. Expression of self-complementary hairpin RNAs within two complementary regions separated by an intron elicits PTGS with high efficiency. Plum pox virus (PPV) is the etiological agent of sharka disease in Drupaceae, although it can also be transmitted to herbaceous species (e.g. Nicotiana benthamiana). Once inside the plant, PPV is transmitted via plasmodesmata from cell to cell, and at longer distances, via phloem. The rolC promoter drives expression in phloem cells. RolC expression is absent in both epidermal and mesophyll cells. The aim of the present study was to confer systemic disease resistance without preventing local viral infection.ResultsIn the ihprolC-PP197 gene (intron hair pin rolC PPV 197), a 197 bp sequence homologous to the PPV RNA genome (from base 134 to 330) was placed as two inverted repeats separated by the DNA sequence of the rolA intron. This hairpin construct is under the control of the rolC promoter.N. benthamiana plants transgenic for the ihprolC-PP197 gene contain siRNAs homologous to the 197 bp sequence. The transgenic progeny of ihprolC-PP197 plants are resistant to PPV systemic infection. Local infection is unaffected. Most (80%) transgenic plants are virus free and symptomless. Some plants (20%) contain virus in uninoculated apical leaves; however they show only mild symptoms of leaf mottling. PPV systemic resistance cosegregates with the ihprolC-PP197 transgene and was observed in progeny plants of all independent transgenic lines analyzed. SiRNAs of 23–25 nt homologous to the PPV sequence used in the ihprolC-PP197 construct were detected in transgenic plants before and after inoculation. Transitivity of siRNAs was observed in transgenic plants 6 weeks after viral inoculation.ConclusionsThe ihprolC-PP197 transgene confers systemic resistance to PPV disease in N. benthamiana. Local infection is unaffected. This transgene and/or similar constructs could be used to confer PPV resistance to fruit trees where systemic disease causes economic damage.

Genetically modified parthenocarpic eggplants: improved fruit productivity under both greenhouse and open field cultivation.

BackgroundParthenocarpy, or fruit development in the absence of fertilization, has been genetically engineered in eggplant and in other horticultural species by using the DefH9-iaaM gene. The iaaM gene codes for tryptophan monoxygenase and confers auxin synthesis, while the DefH9 controlling regions drive expression of the gene specifically in the ovules and placenta. A previous greenhouse trial for winter production of genetically engineered (GM) parthenocarpic eggplants demonstrated a significant increase (an average of 33% increase) in fruit production concomitant with a reduction in cultivation costs.ResultsGM parthenocarpic eggplants have been evaluated in three field trials. Two greenhouse spring trials have shown that these plants outyielded the corresponding untransformed genotypes, while a summer trial has shown that improved fruit productivity in GM eggplants can also be achieved in open field cultivation. Since the fruits were always seedless, the quality of GM eggplant fruits was improved as well. RT-PCR analysis demonstrated that the DefH9-iaaM gene is expressed during late stages of fruit development.ConclusionsThe DefH9-iaaM parthenocarpic gene is a biotechnological tool that enhances the agronomic value of all eggplant genotypes tested. The main advantages of DefH9-iaaM eggplants are: i) improved fruit productivity (at least 30–35%) under both greenhouse and open field cultivation; ii) production of good quality (marketable) fruits during different types of cultivation; iii) seedless fruit with improved quality. Such advantages have been achieved without the use of either male or female sterility genes.

Aucsia gene silencing causes parthenocarpic fruit development in tomato.

In angiosperms, auxin phytohormones play a crucial regulatory role in fruit initiation. The expression of auxin biosynthesis genes in ovules and placenta results in uncoupling of tomato (Solanum lycopersicum) fruit development from fertilization with production of parthenocarpic fruits. We have identified two newly described genes, named Aucsia genes, which are differentially expressed in auxin-synthesis (DefH9-iaaM) parthenocarpic tomato flower buds. The two tomato Aucsia genes encode 53-amino-acid-long peptides. We show, by RNA interference-mediated gene suppression, that Aucsia genes are involved in both reproductive and vegetative plant development. Aucsia-silenced tomato plants exhibited auxin-related phenotypes such as parthenocarpic fruit development, leaf fusions, and reflexed leaves. Auxin-induced rhizogenesis in cotyledon explants and polar auxin transport in roots were reduced in Aucsia-silenced plants compared with wild-type plants. In addition, Aucsia-silenced plants showed an increased sensitivity to 1-naphthylphthalamic acid, an inhibitor of polar auxin transport. We further prove that total indole-3-acetic acid content was increased in preanthesis Aucsia-silenced flower buds. Thus, the data presented demonstrate that Aucsia genes encode a novel family of plant peptides that control fruit initiation and affect other auxin-related biological processes in tomato. Aucsia homologous genes are present in both chlorophytes and streptophytes, and the encoded peptides are distinguished by a 16-amino-acid-long (PYSGXSTLALVARXSA) AUCSIA motif, a lysine-rich carboxyl-terminal region, and a conserved tyrosine-based endocytic sorting motif.

Open field trial of genetically modified parthenocarpic tomato: seedlessness and fruit quality

BackgroundParthenocarpic tomato lines transgenic for the DefH9-RI-iaaM gene have been cultivated under open field conditions to address some aspects of the equivalence of genetically modified (GM) fruit in comparison to controls (non-GM).ResultsUnder open field cultivation conditions, two tomato lines (UC 82) transgenic for the DefH9-RI-iaaM gene produced parthenocarpic fruits. DefH9-RI-iaaM fruits were either seedless or contained very few seeds. GM fruit quality, with the exception of a higher β-carotene level, did not show any difference, neither technological (colour, firmness, dry matter, °Brix, pH) nor chemical (titratable acidity, organic acids, lycopene, tomatine, total polyphenols and antioxidant capacity – TEAC), when compared to that of fruits from control line. Highly significant differences in quality traits exist between the tomato F1 commercial hybrid Allflesh and the three UC 82 genotypes tested, regardless of whether or not they are GM. Total yield per plant did not differ between GM and parental line UC 82. Fruit number was increased in GM lines, and GM fruit weight was decreased.ConclusionThe use in the diet of fruits from a new line or variety introduces much greater changes than the consumption of GM fruits in comparison to its genetic background. Parthenocarpic fruits, produced under open field conditions, contained 10-fold less seeds than control fruits. Thus parthenocarpy caused by DefH9-RI-iaaM gene represents also a tool for mitigating GM seeds dispersal in the environment.

Seedless fruit production by hormonal regulation of fruit set.

Seed and fruit development are intimately related processes controlled by internal signals and environmental cues. The absence of seeds is usually appreciated by consumers and producers because it increases fruit quality and fruit shelf-life. One method to produce seedless fruit is to develop plants able to produce fruits independently from pollination and fertilization of the ovules. The onset of fruit growth is under the control of phytohormones. Recent genomic studies have greatly contributed to elucidate the role of phytohormones in regulating fruit initiation, providing at the same time genetic methods for introducing seedlessness in horticultural plants.

The Medicago truncatula N5 gene encoding a root-specific lipid transfer protein is required for the symbiotic interaction with Sinorhizobium meliloti.

The Medicago truncatula N5 gene is induced in roots after Sinorhizobium meliloti infection and it codes for a putative lipid transfer protein (LTP), a family of plant small proteins capable of binding and transferring lipids between membranes in vitro. Various biological roles for plant LTP in vivo have been proposed, including defense against pathogens and modulation of plant development. The aim of this study was to shed light on the role of MtN5 in the symbiotic interaction between M. truncatula and S. meliloti. MtN5 cDNA was cloned and the mature MtN5 protein expressed in Escherichia coli. The lipid binding capacity and antimicrobial activity of the recombinant MtN5 protein were tested in vitro. MtN5 showed the capacity to bind lysophospholipids and to inhibit M. truncatula pathogens and symbiont growth in vitro. Furthermore, MtN5 was upregulated in roots after infection with either the fungal pathogen Fusarium semitectum or the symbiont S. meliloti. Upon S. meliloti infection, MtN5 was induced starting from 1 day after inoculation (dpi). It reached the highest concentration at 3 dpi and it was localized in the mature nodules. MtN5-silenced roots were impaired in nodulation, showing a 50% of reduction in the number of nodules compared with control roots. On the other hand, transgenic roots overexpressing MtN5 developed threefold more nodules with respect to control roots. Here, we demonstrate that MtN5 possesses biochemical features typical of LTP and that it is required for the successful symbiotic association between M. truncatula and S. meliloti.