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A simple and efficient micrografting method for stably transformed Nicotiana attenuata plants to examine shoot-root signaling.

ABSTRACT: To adjust their development to the environment, plants rely on specific signals that travel from shoot to root and vice versa. Here we describe an efficient micrografting protocol for Nicotiana attenuata, a useful tool for identifying these signals and understanding their functions. Additionally we analyzed transcript accumulation profiles of scions and rootstocks of grafts performed with wild-type and stably transformed N. attenuata. Our results are consistent with the source-to-sink movement of an sRNA silencing signal.

SUBMITTER: Fragoso V

PROVIDER: S-EPMC3207920 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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A simple and efficient micrografting method for stably transformed Nicotiana attenuata plants to examine shoot-root signaling.

Fragoso Variluska V Goddard Hannah H Baldwin Ian T IT Kim Sang-Gyu SG

Plant methods 20111020

To adjust their development to the environment, plants rely on specific signals that travel from shoot to root and vice versa. Here we describe an efficient micrografting protocol for Nicotiana attenuata, a useful tool for identifying these signals and understanding their functions. Additionally we analyzed transcript accumulation profiles of scions and rootstocks of grafts performed with wild-type and stably transformed N. attenuata. Our results are consistent with the source-to-sink movement o ...[more]

PMID: 22014154

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Project description:BackgroundGenetically modified plants are widely used in agriculture and increasingly in ecological research to enable the selective manipulation of plant traits in the field. Despite their broad usage, many aspects of unwanted transgene silencing throughout plant development are still poorly understood. A transgene can be epigenetically silenced by a process called RNA directed DNA methylation (RdDM), which can be seen as a heritable loss of gene expression. The spontaneous nature of transgene silencing has been widely reported, but patterns of acquirement remain still unclear.ResultsTransgenic wild tobacco plants (Nicotiana attenuata) expressing heterologous genes coding for antimicrobial peptides displayed an erratic and variable occurrence of transgene silencing. We focused on three independently transformed lines (PNA 1.2, PNA 10.1 and ICE 4.4) as they rapidly lost the expression of the resistance marker and down-regulated transgene expression by more than 200 fold after only one plant generation. Bisulfite sequencing indicated hypermethylation within the 35S and NOS promoters of these lines. To shed light on the progress of methylation establishment, we successively sampled leaf tissues from different stages during plant development and found a rapid increase in 35S promoter methylation during vegetative growth (up to 77% absolute increase within 45 days of growth). The levels of de novo methylation were inherited by the offspring without any visible discontinuation. A secondary callus regeneration step could interfere with the establishment of gene silencing and we found successfully restored transgene expression in the offspring of several regenerants.ConclusionsThe unpredictability of the gene silencing process requires a thorough selection and early detection of unstable plant lines. De novo methylation of the transgenes was acquired solely during vegetative development and did not require a generational change for its establishment or enhancement. A secondary callus regeneration step provides a convenient way to rescue transgene expression without causing undesirable morphological effects, which is essential for experiments that use transformed plants in the analysis of ecologically important traits.

Project description:BackgroundHerbivore feeding elicits dramatic increases in defenses, most of which require jasmonate (JA) signaling, and against which specialist herbivores are thought to be better adapted than generalist herbivores. Unbiased transcriptional analyses of how neonate larvae cope with these induced plant defenses are lacking.Methodology/principal findingsWe created cDNA microarrays for Manduca sexta and Heliothis virescens separately, by spotting normalized midgut-specific cDNA libraries created from larvae that fed for 24 hours on MeJA-elicited wild-type (WT) Nicotiana attenuata plants. These microarrays were hybridized with labeled probes from neonates that fed for 24 hours on WT and isogenic plants progressively silenced in JA-mediated defenses (N: nicotine; N/PI: N and trypsin protease inhibitors; JA: all JA-mediated defenses). H. virescens neonates regulated 16 times more genes than did M. sexta neonates when they fed on plants silenced in JA-mediated defenses, and for both species, the greater the number of defenses silenced in the host plant (JA > N/PI > N), the greater were the number of transcripts regulated in the larvae. M. sexta larvae tended to down-regulate while H. virescens larvae up- and down-regulated transcripts from the same functional categories of genes. M. sexta larvae regulated transcripts in a diet-specific manner, while H. virescens larvae regulated a similar suite of transcripts across all diet types.Conclusions/significanceThe observations are consistent with the expectation that specialists are better adapted than generalist herbivores to the defense responses elicited in their host plants by their feeding. While M. sexta larvae appear to be better adapted to N. attenuata's defenses, some of the elicited responses remain effective defenses against both herbivore species. The regulated genes provide novel insights into larval adaptations to N. attenuata's induced defenses, and represent potential targets for plant-mediated RNAi to falsify hypotheses about the process of adaptation.

Dataset Information

A simple and efficient micrografting method for stably transformed Nicotiana attenuata plants to examine shoot-root signaling.

Publications

A simple and efficient micrografting method for stably transformed Nicotiana attenuata plants to examine shoot-root signaling.

Similar Datasets

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