Lucia Landi

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 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 Defense Genes in Strawberry Fruits Treated with Different Resistance Inducers.

The expression of 18 defense genes in strawberry fruit treated with elicitors: chitosan, BTH, and COA, at 0.5, 6, 24, and 48 h post-treatment was analyzed. The genes were up-regulated differentially, according to the elicitor. Chitosan and COA treatments promoted the expression of key phenylpropanoid pathway genes, for synthesis of lignin and flavonoids; only those associated with flavonoid metabolism were up-regulated by BTH. The calcium-dependent protein kinase, endo-β 1,4-glucanase, ascorbate peroxidase, and glutathione-S-transferase genes were up-regulated by BTH. The K+ channel, polygalacturonase, polygalacturonase-inhibiting protein, and β-1,3-glucanase, increased in response to all tested elicitors. The enzyme activities of phenylalanine ammonia lyase, β-1,3-glucanase, Chitinase, and guaiacol peroxidase supported the gene expression results. Similarity of gene expression was >72% between chitosan and COA treatments, while BTH showed lower similarity (38%) with the other elicitors. This study suggests the relationship between the composition of the elicitors and a specific pattern of induced defense genes.

Auxin Synthesis-Encoding Transgene Enhances Grape Fecundity

Grape (Vitis vinifera) yield is largely dependent on the fecundity of the cultivar. The average number of inflorescences per shoot (i.e. shoot fruitfulness) is a trait related to fecundity of each grapevine. Berry number and weight per bunch are other features affecting grape yield. An ovule-specific auxin-synthesizing (DefH9-iaaM) transgene that increases the indole-3-acetic acid content of grape transgenic berries was transformed into cultivars Silcora and Thompson Seedless, which differ in the average number of inflorescences per shoots. Thompson Seedless naturally has very low shoot fruitfulness, whereas Silcora has medium shoot fruitfulness. The average number of inflorescences per shoot in DefH9-iaaM Thompson Seedless was doubled compared to its wild-type control. Berry number per bunch was increased in both transgenic cultivars. The quality and nutritional value of transgenic berries were substantially equivalent to their control fruits. The data presented indicate that auxin enhances fecundity in grapes, thus enabling to increase yield with lower production costs.

Seasonal Variation of Defense-Related Gene Expression in Leaves from Bois noir Affected and Recovered Grapevines

Although Bois noir is one of the main phytoplasma diseases of grapevine, the gene expression and enzyme activities that underlie physiological changes occurring in symptomatic and recovered (with spontaneous or induced symptom remission) plants are mostly unknown. Bois noir symptomatic leaves (September 2006, 2007) and symptomless leaves from infected symptomatic plants (September 2007) of Sangiovese (moderately susceptible) and Chardonnay (highly susceptible) cultivars were collected. Moreover, leaves from infected symptomless plants of both cultivars were harvested in June 2007. Leaves from recovered plants were also collected in the same periods. In recovered plants of both cultivars, class III chitinase and almost every time phenylalanine ammonia-lyase and chalcone synthase expression were increased for all collection periods. In symptomatic leaves of both cultivars, the expressions of the same genes were up-regulated and also those of β-1,3-glucanase and flavanone 3-hydroxylase. The activities of chitinase, phenylalanine ammonia-lyase, β-1,3-glucanase, and superoxide dismutase generally correlated with gene expression. For the moderately susceptible Sangiovese, the defense genes were generally up-regulated in both symptomatic and symptomless leaves (for all collection periods). This behavior was not observed in the highly susceptible Chardonnay, in which changes in gene expression were linked to evident symptom display. Therefore, the physiological response of the plants to this pathogen infection appear to be the reason for the resistance of the cultivar to the disease.

ROLC strawberry plant adaptability, productivity, and tolerance to soil-borne disease and mycorrhizal interactions.

The potential to improve strawberry cultivation was assessed regarding the use the rolC genes from Agrobacterium rhizogenes that can confer higher levels of free cytokinins. Strawberry (cv. Calypso) rolC lines were produced by genetic transformation of Agrobacterium tumefaciens. Yield and fruit quality of the control and transgenic lines were measured under open-field conditions. The effects of the transgenic rolC lines depended on gene copy number: rolC lines with one (Line A) or two gene (Line B) copies showed 30% greater yields than controls, due to 20% more fruit per plant and an increased fruit weight. Line A also differed in terms of the highest fruit quality, due to 10.5% increased soluble solids and 12.7% higher acidity. Moreover, cv. Calypso rolC lines A and B had increased tolerance to greenhouse infection by Phytophthora cactorum. Conversely, for all of these characters, Line F (five rolC copies) was not significantly different from the control line. The same lines were also used to examine their symbiosis with root arbuscular mycorrhizal fungi (AMF) using vital and non-vital staining of roots collected at different stages of plant growth. Control and rolC plants showed similar intensities of AMF infection according to plant phenology and/or physiology. Furthermore, possible horizontal gene transfer of the rolC gene was tested for the AMF spores by PCR, with all AMF samples negative using rolC primers. The use of the rolC gene should be considered for the improvements provided in productivity, fruit quality and disease resistance of cultivated strawberry that show no effects on soil microorganisms.

Natural phytoplasma infection of four phloem-feeding Auchenorrhyncha across vineyard agroecosystems in central-eastern Italy.

ABSTRACT The seasonal variations of grapevine yellow phytoplasma were investigated in four phloem-feeding planthopper and leafhopper species that are vectors of plant disease agents. In total, 1,148 wild specimens were collected from three vineyard agroecosystems in the Marche region (central-eastern Italy), from May to September 2008, and analyzed using polymerase chain reactionrestriction fragment-length polymorphism methods. Of 525 Euscelis lineolatus Brullé, 25.1% were positive for aster yellow phytoplasma (16SrI-C, 16SrI-B subgroups) and stolbur phytoplasma (16SrXII-A subgroup; Vergilbungskrankheit type I [VK-I]). Of 368 Hyalesthes obsoletus Signoret, 19.3% were positive for the 16SrXII-A subgroup (VK-I, VK-II; mainly according to their host plant). Of 146 Neoaliturus fenestratus (Herrich-Schäffer), 15.1% were positive for the 16SrI-C and 16SrI-B subgroups, and 7.3% of 109 Psammotettix alienus (Dahlbom) were positive for the 16SrI-B subgroup. The total inoculation efficiency in the feeding medium assays was 57.1% for P. alienus, 44.7% for E. lineolatus, 44.4% for N. fenestratus and 33.9% for H. obsoletus. All of the phytoplasma subgroups identified in the insect bodies were also detected in their feeding media. Detection of stolbur phytoplasma in E. lineolatus feeding media strengthens the hypothesis that it is a candidate vector of Bois noir disease causal agent. The phytoplasma subgroups detected in the Auchenorrhyncha species showed variations according to season and/or vineyard agroecosystem. This study highlights the different specificities of these phytoplasma-Auchenorrhyncha species relationships, and suggests a primary role of the entire vineyard agroecosystem in the epidemiology of grapevine yellow phytoplasma diseases.

Genetic Variability of Stolbur Phytoplasma in Hyalesthes obsoletus (Hemiptera: Cixiidae) and its Main Host Plants in Vineyard Agroecosystems

ABSTRACT Bois noir is an economically important grapevine yellows that is induced by ‘Candidatus Phytoplasma solani’ and principally vectored by the planthopper Hyalesthes obsoletus Signoret (Hemiptera: Cixiidae). This study explores the ‘Ca. P. solani’ genetic variability associated to the nettle—H. obsoletus and bindweed—H. obsoletus systems in vineyard agroecosystems of the central-eastern Italy. Molecular characterization of ‘Ca. P. solani’ isolates was carried out using polymerase chain reaction/ restriction fragment length polymorphism to investigate the nonribosomal vmp1 gene. Seven phytoplasma vmp-types were detected among the host plants- and insect-associated field-collected samples. The vmp1 gene showed the highest polymorphism in the bindweed—H. obsoletus system, according to restriction fragment length polymorphism analysis, which is in agreement with nucleotide sequence analysis. Five vmp-types were associated with H. obsoletus from bindweed, of which one was solely restricted to planthoppers, with one genotype also in planthoppers from nettle. Type V12 was the most prevalent in both planthoppers and bindweed. H. obsoletus from nettle harbored three vmp-types, of which V3 was predominant. V3 was the only type detected for nettle. Our data demonstrate that planthoppers might have acquired some ‘Ca. P. solani’ profiles from other plant hosts before landing on nettle or bindweed. Overall, the different vmp1 gene rearrangements observed in these two plant hosts-H. obsoletus systems might represent different adaptations of the pathogen to the two host plants. Molecular information about the complex of vmp-types provides useful data for better understanding of Bois noir epidemiology in vineyard agroecosystem.

Global Transcriptome Analysis and Identification of Differentially Expressed Genes in Strawberry after Preharvest Application of Benzothiadiazole and Chitosan

The use of resistance inducers is a novel strategy to elicit defense responses in strawberry fruit to protect against preharvest and postharvest decay. However, the mechanisms behind the specific resistance inducers are not completely understood. Here, global transcriptional changes in strawberry fruit were investigated using RNA-Seq technology. Preharvest, benzothiadiazole (BTH) and chitosan were applied to the plant canopy, and the fruit were harvested at 6, 12, and 24 h post-treatment. Overall, 5,062 and 5,210 differentially expressed genes (fold change ≥ 2) were identified in these fruits under the BTH and chitosan treatments, respectively, as compared to the control expression. About 80% of these genes were differentially expressed by both elicitors. Comprehensive functional enrichment analysis highlighted different gene modulation over time for transcripts associated with photosynthesis and heat-shock proteins, according to elicitor. Up-regulation of genes associated with reprogramming of protein metabolism was observed in fruit treated with both elicitors, which led to increased storage proteins. Several genes associated with the plant immune system, hormone metabolism, systemic acquired resistance, and biotic and abiotic stresses were differentially expressed in treated versus untreated plants. The RNA-Seq output was confirmed using RT-qPCR for 12 selected genes. This study demonstrates that these two elicitors affect cell networks associated with plant defenses in different ways, and suggests a role for chloroplasts as the primary target in this modulation of the plant defense responses, which actively communicate these signals through changes in redox status. The genes identified in this study represent markers to better elucidate plant/pathogen/resistance-inducer interactions, and to plan novel sustainable disease management strategies.