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Chapter 3: "Exogenous Application of RNAs as a Silencing Tool for Discovering Gene Function" - In the book "RNAi for plant improvement and protection"
No full textRNA 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
Study of the role of a putative acetylornithine deacetylase on fruit development in Arabidopsis thaliana
No full textThe aim of this work is to investigate the role of N-acetylornithine deacetylase (NAOD) in fruit development. To study its biological role, we have generated silenced lines by transforming Arabidopsis plants with a hairpin construct and searched for T-DNA null mutants. We obtained two silenced lines that showed approximately 90%reduction in NAOD transcript level in comparison with wild type. The phenotypical analysis of these lines revealed a reduced growth and a lower number of siliques in comparison with control plants. A T-DNA insertional line was obtained from the SALK collection. The expression of the gene was almost completely abolished in the homozygous mutant line
Involvement of Medicago truncatula lipid transfer protein N5 in epidermal phases of rhizobia-host interaction.
No full textThe early nodulin N5 of Medicago truncatula is a lipid transfer
protein that has been proven to positively regulate nodulation, although it displays in vitro inhibitory activity against Sinorhizobium meliloti. We utilized a RNAi-based hairpin construct to down-regulate MtN5 expression in M. truncatula hairy roots.
MtN5-silenced roots inoculated with rhizobia displayed an increased number of root hair curling events and a reduced number of invaded primordia and mature nodules
as compared to wild type roots. Nodule primordia formation appeared unaltered in MtN5-silenced roots. This phenotype was associated with the stimulation of ENOD11 expression, an early marker of infection, and with the down-regulation of Flotillin 4(FLOT4), a protein involved in rhizobia entry pathway
The non-specific lipid transfer protein N5 of Medicago truncatula is required for efficient nodulation during symbiosis with N-fixing rhizobia
No full textTransgenic adventitious hairy roots carrying a hairpin construct (MtN5hp) for MtN5 silencing are impaired in nodulation, showing a 50% of reduction in the number of nodules compared with inoculated control roots (Pii et al., 2009). This finding indicates that MtN5 is required in the roots for a successful establishment of the symbiotic interaction with S.meliloti. In order to further support the results obtained in transgenic composite plants, we have undertaken the stable genetic transformation of M. truncatula with A. tumefaciens bearing the MtN5hp construct. The analyses carried out on stable-transformed MtN5-silenced plants confirm the role of the lipid transfer protein N5 in root nodule formation
The involvement of Medicago truncatula non-specific lipid transfer protein N5 in the control of rhizobial infection.
No full textCysteine-rich proteins seem to play important regulatory roles in Medicago truncatula/Sinorhizobium meliloti symbiosis. In particular, a large family of nodule-specific cysteine-rich (NCR) peptides is crucial for the differentiation of nitrogen-fixing bacteroids.The Medicago truncatula N5 protein (MtN5) is currently the only reported non-specific lipid transfer protein necessaryfor successful rhizobial symbiosis; in addition, MtN5 shares several characteristics with NCR peptides: a smallsize, a conserved cysteine-rich motif, an N-terminal signal peptide for secretion and antimicrobial activity. UnlikeNCR peptides, MtN5 expression is not restricted to the root nodules and is induced during the early phases of symbiosisin root hairs and nodule primordia.Recently, MtN5 was determined to be involved in the regulation of root tissue invasion; while, it was dispensablefor nodule primordia formation. Here, we discuss the hypothesis that MtN5 participates in linking the progressionof bacterial invasion with restricting the competence of root hairs for infection
Study of Mtn5 transcriptional control and of its involvement in Medicago truncatula nodulation pathway
No full textThe symbiosis between legumes and rhizobia starts with an exchange of molecular signals between the two partners. In response to the plant-derived flavonoids, bacteria synthesize Nod Factors (NFs), which are able to induce a series of events, such as ion fluxes, root hair deformation and the expression of the early nodulin genes, that eventually lead to the formation of root nodules. We previously demonstrated that MtN5 is an early nodulin, required for the establishment of the symbiosis and also present in mature nodules. In order to investigate the role of MtN5 in root nodules induction pathway, its expression profile during the early stages of infection was studied. MtN5promoter::GUS fusion showed that the promoter was active in epidermis and root hairs a few hours after inoculation, whilst in mature nodules, GUS was observed in the distal zone.In a time course nodulation experiment, MtN5 showed to be co-expressed with early markers of rhizobia infection, such as RIP1, NIN and ENOD11, and resulted to be more precocious than ENOD20 and MtN6. In transgenic adventitious root silenced for MtN5 expression (MtN5hp roots), we observed that upon rhizobia infection the nodulin MtNIN was not induced, whilst ENOD11 was strongly upregulated with respect to control roots. Furthermore, in MtN5hp roots the expression of FLOT4, a nodulin gene known to be involved in the infection thread growth, was unaffected by the inoculation with symbiotic bacteria, in contrast with what observed in control roots
AUCSIA An ancestral green plant miniprotein and the emergence of auxin transport
No full textAucsia is a green plant gene family. In Angiosperms, Aucsia genes control several aspects of auxin biology, including polar auxin transport. AUCSIA miniproteins are produced via splicing of three exons. The first two exons span the conserved AUCSIA motif, while the third exon(s) encodes the more variable carboxyterminal end. AUCSIA presence in green algae indicates that the AUCSIA gene family predated the emergence of land plants and the complex auxin biology of Angiosperms. In algae, however, AUCSIA might have been involved in a primitive auxin biology, when auxin was just a simple metabolite, probably noxious at high concentrations, and consequently pump out via the ancestral auxin exporters, i.e., ABCB1/19 homologous. This speculative scenario implies that in green algae AUCSIA is involved in controlling the ABCB-dependent efflux of noxious metabolites, including auxin. Such speculative hypothesis might be tested in living green algae
Molecular dissection of the role of auxin in fruit initiation
No full textFruit set and growth usually requires fertilization. Fruit set and development without fertilization is called parthenocarpy. Feeding auxin to virgin flowers induces fruit development without fertilization. Recent studies by Hua Wang et al. and Marc Goetz et al. have identified molecular events leading to fruit initiation in the absence of fertilization, showing that parthenocarpy can be achieved by altering different steps of the auxin signaling pathway. Thus, independent evidence indicates that auxin plays a key role in fruit initiation
Which role for Medicago truncatula non-specific lipid transfer proteins in rhizobial infection?
No full textLipid transfer proteins (LTPs) are secreted cysteine-rich proteins highly distributed among the plant kingdom, characterized by lipid-binding capacity and possessing antimicrobial activity. Several biological roles have been proposed for LTPs in plants, including response to biotic stress. In Medicago truncatula, the N5 LTP has been proved to be necessary for the establishment of a successful symbiosis with Sinorhizobium meliloti by limiting bacterial spread at the epidermal level and promoting nodule primordia invasion. We identified two novel MtLTPs, named LTP3 and LTP7 highly similar to MtN5 for the amino acid sequence and the cysteine pattern. Their putative promoter regulatory regions contain response elements related to rhizobial symbiosis. We demonstrated that the expression of these LTP genes is induced after infection, particularly in nodule primordia and mature nodules. LTP7-silencing impaired nodule formation: the number of nodules in MtLTP7—silenced roots was reduced by ~60% as compared with control roots. When roots were inoculated with mycorrhiza, we did not observe any change in LTP3 and LTP7 expression, suggesting that these LTPs specifically respond to the rhizobial symbiont. These data suggest that LTPs might represent a novel group of proteins involved in the regulation of the symbiosis. In this chapter, we propose several hypotheses about the mechanisms of action of these LTPs
Parthenocarpy in crops.
No full textFruit set and growth in the absence of fertilization (parthenocarpy) is a useful trait in plants grown for the value of their fruit. Auxins and gibberellins are widely used to spray flowers to chemically confer parthenocarpy. In recent years,
genetic modifications of either auxin or gibberellin biology have been used to confer parthenocarpy to tomato and other crops. Present knowledge indicates that parthenocarpy can be achieved by genetic modification of either auxin synthesis (iaaM), auxin sensitivity (rolB), auxin content (Aucsia) or auxin signal transduction (IAA9 or ARF8). Genetic modification of gibberellin signal transduction (DELLA) has also been shown to confer parthenocarpy. Available data, obtained under both
open field and protected cultivation, show that genetic parthenocarpy can be used to improve fruit production and/or fruit quality. The mechanisms, genetically modified to confer parthenocarpy, are active also in other plant organs. Observations
consistent with the Euanthial theory that envisages the fruit as a modified leaf predict that the mechanisms underlying fruit initiation have been recruited from molecular machineries present and controlling other plant developmental processes.
The flower/fruit represents the last evolutionary innovation of the green plant lineage, and yet genes (i.e. Aucsia) controlling fruit initiation are most likely older than 1 billion years being present in Prasinophytes (i.e. probable ancestors of Charophytes, which themselves are considered ancestors of all land plants)
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