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RNAi-mediated gene silencing reveals involvement of Arabidopsis chromatin-related genes in Agrobacterium-mediated root transformation.

ABSTRACT: We investigated the effect of RNAi-mediated gene silencing of 109 Arabidopsis thaliana chromatin-related genes (termed "chromatin genes" hereafter) on Agrobacterium-mediated root transformation. Each of the RNAi lines contains a single- or low-copy-number insertion of a hairpin construction that silences the endogenous copy of the target gene. We used three standard transient and stable transformation assays to screen 340 independent RNAi lines, representing 109 target genes, for the rat (resistant to Agrobacterium transformation) phenotype. Transformation frequency was not affected by silencing 85 of these genes. Silencing of 24 genes resulted in either a weak or strong rat phenotype. The rat mutants fell into three general groups: (i) severely dwarfed plants exhibiting a strong rat phenotype (CHC1); (ii) developmentally normal plants showing a reduced response to three transformation assays (HAG3, HDT1, HDA15, CHR1, HAC1, HON5, HDT2, GTE2, GTE4, GTE7, HDA19, HAF1, NFA2, NFA3, SGA1, and SGB2); or (iii) varying response among the three transformation assays (DMT1, DMT2, DMT4, SDG1, SDG15, SDG22, and SDG29). A direct molecular assay indicated that SGA1, HDT1, and HDT2 are important for T-DNA integration into the host genome in Arabidopsis roots.

SUBMITTER: Crane YM

PROVIDER: S-EPMC1986629 | biostudies-literature | 2007 Sep

REPOSITORIES: biostudies-literature

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RNAi-mediated gene silencing reveals involvement of Arabidopsis chromatin-related genes in Agrobacterium-mediated root transformation.

Crane Yan Ma YM Gelvin Stanton B SB

Proceedings of the National Academy of Sciences of the United States of America 20070907 38

We investigated the effect of RNAi-mediated gene silencing of 109 Arabidopsis thaliana chromatin-related genes (termed "chromatin genes" hereafter) on Agrobacterium-mediated root transformation. Each of the RNAi lines contains a single- or low-copy-number insertion of a hairpin construction that silences the endogenous copy of the target gene. We used three standard transient and stable transformation assays to screen 340 independent RNAi lines, representing 109 target genes, for the rat (resist ...[more]

PMID: 17827277

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Project description:BackgroundCKX genes encode cytokinin dehydrogenase enzymes (CKX), which metabolize cytokinins in plants and influence developmental processes. The genes are expressed in different tissues and organs during development; however, their exact role in barley is poorly understood. It has already been proven that RNA interference (RNAi)-based silencing of HvCKX1 decreased the CKX level, especially in those organs which showed the highest expression, i.e. developing kernels and roots, leading to higher plant productivity and higher mass of the roots [1]. The same type of RNAi construct was applied to silence HvCKX2 and analyze the function of the gene. Two cultivars of barley were transformed with the same silencing and selection cassettes by two different methods: biolistic and via Agrobacterium.ResultsThe mean Agrobacterium-mediated transformation efficiency of Golden Promise was 3.47% (±2.82). The transcript level of HvCKX2 in segregating progeny of T(1) lines was decreased to 34%. The reduction of the transcript in Agrobacterium-derived plants resulted in decreased CKX activity in the developing and developed leaves as well as in 7 DAP (days after pollination) spikes. The final phenotypic effect was increased productivity of T(0) plants and T(1) lines. Higher productivity was the result of the higher number of seeds and higher grain yield. It was also correlated with the higher 1000 grain weight, increased (by 7.5%) height of the plants and higher (from 0.5 to 2) numbers of spikes. The transformation efficiency of Golden Promise after biolistic transformation was more than twice as low compared to Agrobacterium. The transcript level in segregating progeny of T(1) lines was decreased to 24%. Otherwise, the enzyme activity found in the leaves of the lines after biolistic transformation, especially in cv. Golden Promise, was very high, exceeding the relative level of the control lines. These unbalanced ratios of the transcript level and the activity of the CKX enzyme negatively affected kernel germination or anther development and as a consequence setting the seeds. The final phenotypic effect was the decreased productivity of T(0) plants and T(1) lines obtained via the biolistic silencing of HvCKX2.ConclusionThe phenotypic result, which was higher productivity of silenced lines obtained via Agrobacterium, confirms the hypothesis that spatial and temporal differences in expression contributed to functional differentiation. The applicability of Agrobacterium-mediated transformation for gene silencing of developmentally regulated genes, like HvCKX2, was proven. Otherwise low productivity and disturbances in plant development of biolistic-silenced lines documented the unsuitability of the method. The possible reasons are discussed.

Project description:BackgroundAgrobacterium-mediated genetic transformation is a widely used and efficient technique for gene functional research in crop breeding and plant biology. While in some plant species, including soybean, genetic transformation is still recalcitrant and time-consuming, hampering the high-throughput functional analysis of soybean genes. Thus we pursue to develop a rapid, simple, and highly efficient hairy root system induced by Agrobacterium rhizogenes (A. rhizogenes) to analyze soybean gene function.ResultsIn this report, a rapid, simple, and highly efficient hairy root transformation system for soybean was described. Only sixteen days were required for the whole workflow and the system was suitable for various soybean genotypes, with an average transformation frequency of 58-64%. Higher transformation frequency was observed when wounded cotyledons from 1-day-germination seeds were inoculated and co-cultivated with A. rhizogenes in 1/2 B5 (Gamborg' B-5) medium. The addition of herbicide selection to root production medium increased the transformation frequency to 69%. To test the applicability of the hairy root system for gene functional analysis, we evaluated the protein expression and subcellular localization in transformed hairy roots. Transgenic hairy roots exhibited significantly increased GFP fluorescence and appropriate protein subcellular localization. Protein-protein interactions by BiFC (Bimolecular Fluorescent Complimentary) were also explored using the hairy root system. Fluorescence observations showed that protein interactions could be observed in the root cells. Additionally, hairy root transformation allowed soybean target sgRNA screening for CRISPR/Cas9 gene editing. Therefore, the protocol here enables high-throughput functional characterization of candidate genes in soybean.ConclusionA rapid, simple, and highly efficient A. rhizogenes-mediated hairy root transformation system was established for soybean gene functional analysis, including protein expression, subcellular localization, protein-protein interactions and gene editing system evaluation.

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RNAi-mediated gene silencing reveals involvement of Arabidopsis chromatin-related genes in Agrobacterium-mediated root transformation.

Publications

RNAi-mediated gene silencing reveals involvement of Arabidopsis chromatin-related genes in Agrobacterium-mediated root transformation.

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