Expression profile analysis of early fruit development in iaaM-parthenocarpic tomato plants

<p>Abstract</p> <p>Background</p> <p>Fruit normally develops from the ovary after pollination and fertilization. However, the ovary can also generate seedless fruit without fertilization by parthenocarpy. Parthenocarpic fruit development has been obtained in tomato (<it>Solanum lycopersicum</it>) by genetic modification using auxin-synthesising gene(s) (<it>DefH9-iaaM</it>; <it>DefH9-RI-iaaM</it>) expressed specifically in the placenta and ovules.</p> <p>Findings</p> <p>We have performed a cDNA Amplified Fragment Length Polymorphism (cDNA-AFLP) analysis on pre-anthesis tomato flower buds (0.5 cm long) collected from <it>DefH9-iaaM </it>and <it>DefH9-RI-iaaM </it>parthenocarpic and wild-type plants, with the aim to identify genes involved in very early phases of tomato fruit development. We detected 212 transcripts differentially expressed in auxin-ipersynthesising pre-anthesis flower buds, 65 of them (31%) have unknown function. Several differentially expressed genes show homology to genes involved in protein trafficking and protein degradation via proteasome. These processes are crucial for auxin cellular transport and signaling, respectively.</p> <p>Conclusion</p> <p>The data presented might contribute to elucidate the molecular basis of the fruiting process and to develop new methods to confer parthenocarpy to species of agronomic interest. In a recently published work, we have demonstrated that one of the genes identified in this screening, corresponding to #109 cDNA clone, regulates auxin-dependent fruit initiation and its suppression causes parthenocarpic fruit development in tomato.</p

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

BACKGROUND: Homology-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. RESULTS: In 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. CONCLUSIONS: The 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

Collective radioresistance of T47D breast carcinoma cells is mediated by a Syncytin-1 homologous protein

It is generally accepted that radiotherapy must target clonogenic cells, i.e., those cells in a tumour that have self-renewing potential. Focussing on isolated clonogenic cells, however, may lead to an underestimate or even to an outright neglect of the importance of biological mechanisms that regulate tumour cell sensitivity to radiation. We develop a new statistical and experimental approach to quantify the effects of radiation on cell populations as a whole. In our experiments, we change the proximity relationships of the cells by culturing them in wells with different shapes, and we find that the radiosensitivity of T47D human breast carcinoma cells in tight clusters is different from that of isolated cells. Molecular analyses show that T47D cells express a Syncytin-1 homologous protein (SyHP). We observe that SyHP translocates to the external surface of the plasma membrane of cells killed by radiation treatment. The data support the fundamental role of SyHP in the formation of intercellular cytoplasmic bridges and in the enhanced radioresistance of surviving cells. We conclude that complex and unexpected biological mechanisms of tumour radioresistance take place at the cell population level. These mechanisms may significantly bias our estimates of the radiosensitivity of breast carcinomas in vivo and thereby affect treatment plans, and they call for further investigations

The involvement of root-specific LTPs in the symbiotic interaction between Medicago truncatula and Sinorhizobium meliloti

Lipid transfer proteins (LTPs) are small basic proteins that constitute a large family characterized by the ability to transfer phospholipids between a donor and an acceptor membrane and can have many different roles in vivo. Recently it has been demonstrated that MtN5, a non specific LTP (ns-LTP) classified as type III (Wang et al., 2012), is involved in the symbiotic interaction between legumes and rhizobia (Pii et al. 2009, Pii et al., 2012). MtN5 is a nod factor responsive gene expressed at a very early phase of rhizobial symbiosis in the epidermis and root hairs and later in primordia and nodules. There are evidences that MtN5 positively regulates the nodulation process. Interestingly, two other putative type III ns-LTPs (Medtr3g055250 and Medtr7g052640) have been identified in Medicago truncatula genome. The aim of this study is to shed light on the role of these ns-LTPs in the symbiotic interaction between M. truncatula and Sinorhizobium meliloti

Plant cystine-knot peptides: pharmacological perspectives.

The cystine-knot peptides are well represented in several plant species. The pharmacological interest for plant cystine-knot peptides derived from their broad biological activities. The mechanisms of action of plant cystine-knot peptides are still largely undiscovered, although eveidences indicate that they interfere with plasma membranes. In some cases, as tomato TCMPs, the cystine-knot peptides target human growth factor receptors either by acting as growth factor antagonist or by altering their signal transduction pathway. The possibility to identify specific molecular targets of plant cystine-knot peptides in human cells opens novel possibilities for the pharmacological use of these peptides besides their use as scaffold to develop stable disease molecular markers and therapeutic agent

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

BACKGROUND: Parthenocarpic 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). RESULTS: Under 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. CONCLUSION: The 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

Chapter 3: "Exogenous Application of RNAs as a Silencing Tool for Discovering Gene Function" - In the book "RNAi for plant improvement and protection"

RNA silencing is a powerful technique to unravel the function of genes by inhibiting gene expression at the post-transcriptional level. This technique is particularly appropriate for studying developmental processes such as fruit setting and growth that require a tight organ/tissue and time-specific regulation of target genes expression. Gene silencing in plants is usually achieved by the stable or transient expression of genetic constructs producing hairpin (hp) RNA or microRNA (miRNA). The use of exogenously applied small RNAs (sRNAs) and long doublestranded RNAs (dsRNAs) for transient gene silencing in whole plant and/or detached organs would allow a much higher number of genes to be analysed in a shorter time. The successful application of this technique requires efficient systems for sRNA delivery as well as methods to enhance RNA stability in plant cells

Fruit improvement using intragenesis and artificial microRNA

Genetic engineering methods based on the use of transgenes have been successfully adopted to improve crops. A novel all-native DNA gene technology consists of the creation of intragenic constructs by isolating genetic elements from a crop, rearranging them in vitro, and inserting them back into the plant. The ever-increasing genomic information and the elucidation of the molecular mechanisms that control fruit development could be exploited to confer the desired fruit phenotypes using endogenous DNA. The spatial/temporal regulation of genes can be modified by using appropriate endogenous regulatory elements, such as fruit-specific promoters. In addition, intragenic silencing can be employed to downregulate fruit-related genes. Here, we describe the available tools for intragenic manipulation of early phases of fleshy fruit initiation

A class of non-specific lipid transfer proteins of Medicago truncatula modulates the host response to rhizobia infection.

The establishment of the symbiotic interaction between leguminous plants and nitrogen fixing bacteria is a complex process that requires a molecular dialogue between the host and the bacteria during the root infection and the colonization of the nodule, where the bacteria fix nitrogen. The formation of a functional root nodule needs a spatial and temporal coordination of all these events. Rhizobia enter root hair via a tubular plant-derived structure, named infection thread (IT), and then they are released into the cortical layers, where the bacteria enclosed in a plant-derived membrane called symbiosome, enter nodule primordia cells. The formation of the ITs as well as the release of rhizobia in the nodule cells require the synthesis of new membranes and modifications of their lipid composition. Lipid transfer proteins (LTPs) are small basic secreted proteins, characterized by lipid-binding capacity and putatively involved in lipid trafficking. In plants, the non specific LTPs (nsLTPs) are a large group of proteins implicated in various processes; most of them possess antimicrobial activity and are likely involved in pathogen defence. Recently, two LTPs, AsE246 from Astragalus sinicus and MtN5 from Medicago truncatula, have been implicated in the host regulation of N-fixing symbiosis suggesting their participation in the de-novo formation and rearrangement of infection threads and symbiosome membranes (Lei et al. 2014; Santi et al., 2017). We have identified in M.truncatula genome two novel nsLTPs that cluster with MtN5 in the type III nsLTP group (Wang et al., 2012), an intermediate group between the two major nsLTP1 and nsLTP2 families. We have demonstrated that these LTPs are transcriptionally induced by rhizobia infection and they can modulate the host nodulation capacity

MINIPROTEINS AS CONTROLLING FACTORS IN REPRODUCTIVE DEVELOPMENT

Fruit set is dependent on successful fertilization and is subjected to many genetic factors as well as environmental conditions. Not only classical hormones and transcription factors orchestrate this process, also several miniproteins can participate in the flowering and fruit set. A recent study demonstrated the involvement of a class of Solanaceae-specific cystine-knot miniproteins, named TCMPs in the fruit set of Solanum lycopersicum (Molesini et al,2018). The tomato TCMP-1 and TCMP-2 share with the other cystineknot miniproteins a unique three-dimensional structure characterized by three intra-molecular disulfide bonds forming a cystine-knot. The expression pattern of TCMPs is tightly regulated during flower and fruit development. TCMP-1 is highly expressed in flower buds before anthesis whereas TCMP-2 is poorly expressed before anthesis and then, the expression level gradually increases reaching the maximum expression in the mature fruit. To investigate their function, the endogenous level of these genes was altered generating transgenic plants expressing the TCMP-1 coding sequence under the control of the TCMP-2 promoter (TCMP-2p:TCMP-1). The expression of the transgene caused an earlier fruit setting with no visible phenotypic effects on vegetative growth associated with an increased expression of TCMP-2 in pre-anthesis flower buds. This suggests to us a possible important involvement of these genes in the early stage of flower/fruit development