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Yue JJ, Hong CY, Wei P, Tsai YC, Lin CS (2020) How to start your monocot CRISPR/Cas project: plasmid design, efficiency detection, and offspring analysis. Rice 13:9.
cslin The breakthrough CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9-mediated genome-editing technology has led to great progress in monocot research; however, several factors need to be considered for the efficient implementation of this technology. To generate genome-edited crops, single guide (sg)RNA and Cas9 DNA are delivered into plant cells and expressed, and the predicted position is targeted. Analyses of successful targeted mutations have revealed that the expression levels, expression timing, and variants of both sgRNA and Cas9 need to be sophisticatedly regulated; therefore, the promoters of these genes and the target site positions are the key factors for genome-editing efficiency. Currently, various vectors and online tools are available to aid sgRNA design. Furthermore, to reduce the sequence limitation of the protospacer adjacent motif (PAM) and for other purposes, many Cas protein variants and base editors can be used in plants. Before the stable transformation of a plant, the evaluation of vectors and target sites is therefore very important. Moreover, the delivery of Cas9-sgRNA ribonucleoproteins (RNPs) is one strategy that can be used to prevent transgene issues with the expression of sgRNA and Cas proteins. RNPs can be used to efficiently generate transgene-free genome-edited crops that can reduce transgene issues related to the generation of genetically modified organisms. In this review, we introduce new techniques for genome editing and identifying marker-free genome-edited mutants in monocot crops. Four topics are covered: the design and construction of plasmids for genome editing in monocots; alternatives to SpCas9; protoplasts and CRISPR; and screening for marker-free CRISPR/Cas9-induced mutants. We have aimed to encompass a full spectrum of information for genome editing in monocot crops.


S. Kailasam, S. Singh, M.-J. Liu, C.-C. Lin and K.-C. Yeh* (2020) A HemK class glutamine-methyltransferase is involved in the termination of translation and essential for iron homeostasis in Arabidopsis. New Phytologist doi.org/10.1111/nph.16440
kcyeh Fe-deficiency induced transcriptional cascades controls Fe transport and utilization is important for plant to maintain homeostasis of this important element executing the biological function. So far, we know that many genes participating in these processes are transcriptionally controlled by Fe-status. By a genetical approach, we identified a novel component, HemK-like methyltransferase, that is involved in the termination of translation. With defect of this gene, plants lose ion homeostasis and are highly sensitive to Fe deficiency and possess other developmental flaws. We detected that many Fe-deficiency-inducible transcripts exhibit ribosome stalling implying translational failings in the mutant. In this study, a check-point that fine tunes the peptide release for growth and development in plants is uncovered. Our report sheds light on mechanisms of translation termination and its importance in preserving both molecular and physiological responses to Fe-starvation.


Yuh Tzean, Ho-Hsiung Chang, Tsui-Chin Tu, Bo-Han Hou, Ho-Ming Chen, Yi-Shu Chiu, Wei-Yi Chou, Li Chang, and Hsin-Hung Yeh* (2020) Engineering Plant Resistance to Tomato Yellow Leaf Curl Thailand Virus Using a Phloem-Specific Promoter Expressing Hairpin RNA https://apsjournals.apsnet.org/doi/10.1094/MPMI-06-19-0158-R (Selected for publication in MPMI Focus Issue on Cell Biology of Virus-Plant and Virus-Vector Interaction and commentary in the same issue).
hyeh Transgenic approaches employing RNA interference (RNAi) strategies have been successfully applied to generate desired traits in plants; however, variations between RNAi transgenic siblings and the ability to quickly apply RNAi resistance to diverse cultivars remain challenging. In this study, we assessed the promoter activity of a cauliflower mosaic virus 35S promoter (35S) and a phloem-specific promoter derived from rice tungro bacilliform virus (RTBV) and their efficacy to drive RNAi against the endogenous glutamate-1-semialdehyde aminotransferase gene (GSA) that acts as a RNAi marker, through chlorophyll synthesis inhibition, and against tomato yellow leaf curl Thailand virus (TYLCTHV), a begomovirus (family Geminiviridae) reported to be the prevalent cause of tomato yellow leaf curl disease (TYLCD) in Taiwan. Transgenic Nicotiana benthamiana expressing hairpin RNA of GSA driven by either the 35S or RTBV promoter revealed that RTBV::hpGSA induced stronger silencing along the vein and more uniformed silencing phenotype among its siblings than 35S::hpGSA. Analysis of transgenic N. benthamiana, 35S::hpTYLCTHV, and RTBV::hpTYLCTHV revealed that, although 35S::hpTYLCTHV generated a higher abundance of small RNA than RTBV::hpTYLCTHV, RTBV::hpTYLCTHV transgenic plants conferred better TYLCTHV resistance than 35S::hpTYLCTHV. Grafting of wild-type (WT) scions to TYLCTHV RNAi rootstocks allowed transferable TYLCTHV resistance to the scion. A TYLCTHV-inoculation assay showed that noninfected WT scions were only observed when grafted to RTBV::hpTYLCTHV rootstocks but not 35S::hpTYLCTHV nor WT rootstocks. Together, our findings demonstrate an approach that may be widely applied to efficiently confer TYLCD resistance.


Yamada M, Han X, Benfey PN (2020) RGF1 controls root meristem size through ROS signaling. >Nature. 2020 Jan;577(7788):85-88. doi: 10.1038/s41586-019-1819-6. Epub 2019 Dec 4.
M The stem cell niche and the size of the root meristem in plants are maintained by intercellular interactions and signalling networks involving a peptide hormone, root meristem growth factor 1 (RGF1). Understanding how RGF1 regulates the development of the root meristem is essential for understanding stem cell function. Although five receptors for RGF1 have been identified, the downstream signalling mechanism remains unknown. Here we report a series of signalling events that follow RGF1 activity. We find that the RGF1-receptor pathway controls the distribution of reactive oxygen species (ROS) along the developmental zones of the Arabidopsis root. We identify a previously uncharacterized transcription factor, RGF1-INDUCIBLE TRANSCRIPTION FACTOR 1 (RITF1) , that has a central role in mediating RGF1 signalling. Manipulating RITF1 expression leads to the redistribution of ROS along the root developmental zones. Changes in ROS distribution in turn enhance the stability of the PLETHORA2 protein, a master regulator of root stem cells. Our results thus clearly depict a signalling cascade that is initiated by RGF1, linking this peptide to mechanisms that regulate ROS.


Yang, Shu-Yi, Lu, Wen-Chien, Ko, Swee-Suak, Sun, Ching-Mei, Hung, Jo-Chi and Chiou, Tzyy-Jen* (2020) uORF and Phosphate-Regulated Expression of Rice OsNLA1 Controls Phosphate Transport and Reproduction. Plant Physiology: pp.01101.02019
tjc Rice (Oryza sativa) OsNLA1 has been proposed to play a crucial role in regulating phosphate (Pi) acquisition in roots, similar to that of Arabidopsis AtNLA. However, unlike AtNLA, OsNLA1 is not a target of miR827, a Pi starvation-induced miRNA. It is, therefore, of interest to know whether the expression of OsNLA1 depends on Pi supply and how it is regulated. In this study, we provide evidence that OsNLA1 controls Pi acquisition by directing the degradation of several OsPHT1 Pi transporters (i.e., OsPT1/2/4/7/8/12). We further show that OsNLA1 has an additional function in reproduction and uncover the mechanism of its expression regulation. Analysis of mRNA levels, promoter-GUS (β-glucuronidase) activity and protoplast transient expression showed that the expression of OsNLA1.1, the most abundant transcript variant, is upregulated in response to increasing Pi supply. The OsNLA1 promoter region was found to contain an upstream open reading frame (uORF) that is required for Pi-responsive expression regulation. OsNLA1 promoter activity was observed in roots, ligules, leaves, sheaths, pollen grains, and surrounding the vascular tissues of anthers, suggesting that OsNLA1 is important throughout the development of rice. Disruption of OsNLA1 resulted in increased Pi uptake from roots as well as impaired pollen development and reduced grain production. In summary, our study reveals that Pi-induced OsNLA1 expression regulated by a unique mechanism functions in Pi acquisition, Pi translocation, and reproductive success.


H.-F. Chang, S.-L. Wang*, D.-C. Lee, S. S.-Y. Hsiao, Y. Hashimoto, and K.-C. Yeh* (2020) Assessment of indium toxicity to the model plant Arabidopsis. Journal of Hazardous Materials doi.org/10.1016/j.jhazmat.2019.121983
kcyeh The use of indium in semiconductor products has increased markedly in recent years. The release of indium into the ecosystem is inevitable. Under such circumstances, effective and accurate assessment of indium risk is important. An indispensable aspect of indium risk assessment is to understand the interactions of indium with plants, which are fundamental components of all ecosystems. Physiological responses of Arabidopsis thaliana exposed to indium were investigated by monitoring toxic effects, accumulation and speciation in plants. It was found that indium jeopardized phosphate uptake and translocation by inhibiting the accumulation of phosphate transporters PHOSPHATE TRANSPORTER1 (PHT1;1/4), responsible for phosphate uptake, and PHOSPHATE1 (PHO1), responsible for phosphate xylem loading.


Hsu CT, Cheng YJ, Yuan YH, Hung WF, Cheng QW, Wu FH, Lee LY, Gelvin SB, Lin CS. (2019) Application of Cas12a and nCas9-activation-induced cytidine deaminase for genome editing and as a non-sexual strategy to generate homozygous/multiplex edited plants in the allotetraploid genome of tobacco. Plant Mol Biol. 2019 Nov;101(4-5):355-371.
cslin Protoplast transfection and regeneration systems are useful platforms for CRISPR/Cas mutagenesis and genome editing. In this study, we demonstrate the use of Cpf1 (Cas12a) and nCas9-activation-induced cytidine deaminase (nCas9-Target-AID) systems to mutagenize Nicotiana tabacum protoplasts and to regenerate plants harboring the resulting mutations. We analyzed 20 progeny plants of Cas12a-mediated phytoene desaturase (PDS) mutagenized regenerants, as well as regenerants from wild-type protoplasts, and confirmed that their genotypes were inherited in a Mendelian manner. We used a Cas9 nickase (nCas9)-cytidine deaminase to conduct C to T editing of the Ethylene receptor 1 (ETR1) gene in tobacco protoplasts and obtained edited regenerates. It is difficult to obtain homozygous edits of polyploid genomes when the editing efficiency is low. A second round of mutagenesis of partially edited regenerants (a two-step transfection protocol) allowed us to derive ETR1 fully edited regenerants without the need for sexual reproduction. We applied three different Cas systems (SaCas9, Cas12a, and nCas9-Traget AID) using either a one-step or a two-step transfection platform to obtain triply mutated and/or edited tobacco regenerants. Our results indicate that these three Cas systems can function simultaneously within a single cell.


Liao, Ya-Yun, Li, Jia-Ling, Pan, Rong-Long* and Chiou, Tzyy-Jen* (2019) Structure-Function Analysis Reveals Amino Acid Residues of Arabidopsis Phosphate Transporter AtPHT1;1 Crucial for Its Activity. Frontiers in Plant Science 10:1158
tjc Phosphorus (P), an essential plant macronutrient, is acquired in the form of inorganic phosphate (Pi) by transporters located at the plasma membrane of root cells. To decipher the Pi transport mechanism, Arabidopsis thaliana Pi transporter 1;1 (AtPHT1;1), the most predominantly H+-coupled Pi co-transporter in the root, was selected for structure-function analysis. We first predicted its secondary and tertiary structures on the basis of the Piriformospora indica Pi transporter (PiPT) and identified 28 amino acid residues potentially engaged in the activity of AtPHT1;1. We then mutagenized these residues into alanine and expressed them in the yeast pam2 mutant defective in high-affinity Pi transporters and Arabidopsis pht1;1 mutant, respectively, for functional complementation validation. We further incorporated the functional characterization and structure analyses to propose a mechanistic model for the function of AtPHT1;1. We showed that D35, D38, R134 and D144, implicated in H+ transfer across the membrane, and Y312 and N421, involved in initial interaction and translocation of Pi, are all essential for its transport activity. When Pi enters the binding pocket, the two aromatic moieties of Y145 and F169 and the hydrogen bonds generated from Q172, W304, Y312, D308, and K449 can build a scaffold to stabilize the structure. Subsequent interaction between Pi and the positive residue of K449 facilitates its release. Furthermore, D38, D93, R134, D144, D212, R216, R233, D367, K373, and E504 may form internal electrostatic interactions for structure ensemble and adaptability. This study offers a comprehensive model for elucidating the transport mechanism of a plant Pi transporter.


Hsing‐Yi Cho, Elena Loreti, Ming‐Che Shih, Pierdomenico Perata (2019) Energy and Sugar Signaling during Hypoxia. New Phytologist https://doi.org/10.1111/nph.16326
MCS The major consequence of hypoxia is a dramatic reduction in energy production. At the onset of hypoxia, both oxygen and ATP availability decrease. Oxygen and energy sensing therefore converge to induce an adaptive response at both the transcriptional and translational levels. Oxygen sensing results in stabilization of the transcription factors that activate hypoxia-response genes, including enzymes required for efficient sugar metabolism, allowing plants to produce enough energy to ensure survival. The translation of the resulting mRNAs is mediated by SnRK1, acting as an energy sensor. However, as soon as the sugar availability decreases, a homeostatic mechanism, detecting sugar starvation, dampens the hypoxia-dependent transcription to reduce energy consumption and preserves carbon reserves for regrowth when oxygen availability is restored.


Ying-Lan Chen, Wei-Hung Chang, Chi-Ying Lee and Yet-Ran Chen* (2019) An Improved Scoring Method for the Identification of Endogenous Peptides based on Mascot MS/MS Ion Search. Analyst. 2019 Apr 23;144(9):3045-3055. doi: 10.1039/c8an02141d.
Yet-Ran Chen To identify the endogenous peptides using MS/MS analysis and searching against a polypeptide sequence database, a non-enzyme specific (NES) search considering all of the possible proteolytic cleavages is required. However, the use of a NES search generates more false positive hits than an enzyme specific search, and therefore has lower identification performance. In this study, the use of the sub-ranked matches for improving the identification performance of Mascot NES search was investigated and a new scoring method was developed that considered the contribution of all sub-ranked random match probabilities, named contribution score (CS). The CS showed the highest identification sensitivity using Mascot NES search with a full protein database when compared to the use of the Mascot first ranked score and delta score (DS). The confident peptides identified by DS and CS were showed to be complementary. When applied to plant endogenous peptide identification, the identification numbers of tomato endogenous peptides using DS and CS were 176.3% and 184.2%, respectively, higher than the use of the first ranked score of Mascot. The combination of DS and CS identified 200.0% and 8.6% more tomato endogenous peptides than the use of Mascot and DS, respectively. This method by combining the CS and DS can significantly improve the identification performance of endogenous peptides without complex computational steps and is also able to improve the identification performance of enzyme specific search. In addition to the application on the plant peptidomics analysis, this method may be applied to the improvement of peptidomics studies in different species. A web interface for calculating the DS and CS based on Mascot search results was developed herein.


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