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Zhiguo Zhu, Xiaojun Kang, Vai S Lor, David Weiss, Neil Olszewski
Gibberellin (GA) is a plant hormone that regulates important growth processes including germination, flowering, and fruit development. DELLA proteins inhibit GA responses. GA signaling increases through GA triggered destruction of the DELLA protein. Several mutants with reduced stature due to mutations that stabilize the DELLA protein are known. Many of these mutations cause deletions within the DELLA domain. Here we report several new dwarfing alleles of the tomato DELLA protein, PROCERA, generated using the CRISPR/Cas9 system to create intragenic suppressors of two pro loss-of-function alleles...
October 18, 2018: Plant Biotechnology Journal
Zhen Wang, Zai Zheng, Li Song, Dong Liu
Plants have evolved an array of adaptive responses to cope with phosphate (Pi) starvation. These responses are mainly controlled at the transcriptional level. In Arabidopsis, PHR1, a member of the MYB-CC transcription factor family, is a key component of the central regulatory system controlling plant transcriptional responses to Pi starvation. Its homologs in the MYB-CC family, PHL1 (PHR1-LIKE 1), PHL2, and perhaps also PHL3, act redundantly with PHR1 to regulate plant Pi starvation responses. The functions of PHR1's closest homolog in this family, PHL4, however, have not been characterized due to the lack of its null mutant...
2018: Frontiers in Plant Science
S Zoledowska, A Motyka-Pomagruk, W Sledz, A Mengoni, E Lojkowska
BACKGROUND: Pectobacterium parmentieri is a newly established species within the plant pathogenic family Pectobacteriaceae. Bacteria belonging to this species are causative agents of diseases in economically important crops (e.g. potato) in a wide range of different environmental conditions, encountered in Europe, North America, Africa, and New Zealand. Severe disease symptoms result from the activity of P. parmentieri virulence factors, such as plant cell wall degrading enzymes. Interestingly, we observe significant phenotypic differences among P...
October 16, 2018: BMC Genomics
Bastian Minkenberg, Jianwei Zhang, Kabin Xie, Yinong Yang
Since the beginning of CRISPR/Cas9's use for genome-editing, reports of off-targets have caused concerns among users. Previous studies identified three main types of off-targets. The first type of off-targets are sequences at other 5'-NGG-3' protospacer adjacent motifs (PAMs) that are identical or have a small number of mismatches or substitutions (Fu et al., 2013; Tsai et al., 2015). The second type of off-targets are sequences at other 5'-NGG-3' PAMs that have insertions or deletions compared to the gRNA spacer or the targeted DNA (Lin et al...
October 16, 2018: Plant Biotechnology Journal
Wenfeng Chen, Michelle Werdann, Yong Zhang
Tools that allow inducible and reversible depletion of target proteins are critical for biological studies. The plant-derived auxin-inducible degradation system (AID) enables the degradation of target proteins tagged with the AID motif. This system has been recently employed in mammalian cells as well as in C. elegans and Drosophila. To test the utility of the AID approach in the nervous system, we used circadian locomotor rhythms as a model and applied the AID method to temporally and spatially degrade PERIOD (PER), a critical pacemaker protein in Drosophila...
October 15, 2018: FEBS Journal
Carmen Martín-Pizarro, David Posé
Over the last few years, a series of tools for genome editing have been developed, allowing the introduction of precise changes into plant genomes. These have included Zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas9, which is so far the most successful and commonly used approach for targeted and stable editing of DNA, due to its ease of use and low cost. CRISPR/Cas9 is now being widely used as a new plant breeding technique to improve commercially relevant crop species...
2018: Frontiers in Plant Science
Long Lin, Wenwu Ye, Jiawei Wu, Mingrun Xuan, Yufei Li, Jian Gao, Yonglin Wang, Yan Wang, Suomeng Dong, Yuanchao Wang
Transcriptional regulation is critical for plant pathogen development and virulence. MADS-box transcription factors belong to a highly conserved transcriptional regulator family in eukaryotic organisms that are involved in various important biological processes. Only one predicted MADS-box gene, PsMAD1 , was identified in Phytophthora sojae , which was highly expressed during the sporangia and infection stages. To investigate its function, we generated PsMAD1 knockout mutants using the CRISPR/Cas9 system. Compared with the wild-type strain, the mutants showed no changes in vegetative growth, oospore production, or no differences in sensitivity to various abiotic stresses...
2018: Frontiers in Microbiology
Wanwan Liang, Solveig van Wersch, Meixuezi Tong, Xin Li
Intracellular nucleotide-binding (NB) and leucine-rich repeat (NLR) proteins function as immune receptors to recognize effectors from pathogens. They often guard host proteins that are the direct targets of those effectors. Recent findings revealed that a typical NLR sometimes cooperates with another atypical NLR for effector recognition. Here, by using CRISPR/Cas9 gene editing method, knockout analysis and biochemical assays, we uncovered differential pairings of typical Toll Interleukin1 receptor (TIR) type NLR (TNL) receptor SOC3 with atypical truncated TIR-NB (TN) proteins CHS1 or TN2 to guard the homeostasis of the E3 ligase SAUL1...
October 14, 2018: New Phytologist
Haixia Zhao, Liangsheng Wang, Fang-Jie Zhao, Longhua Wu, Anna Liu, Wenzhong Xu
Sedum plumbizincicola is able to hyperaccumulate cadmium (Cd), a nonessential and highly toxic metal, in the above-ground tissues, but the mechanisms for its Cd hypertolerance are not fully understood. Here, we show that the heavy metal ATPase 1 (SpHMA1) of S. plumbizincicola plays an important role in chloroplast Cd detoxification. Compared with the HMA1 ortholog in the Cd nonhyperaccumulating ecotype of Sedum alfredii, the expression of SpHMA1 in the leaves of S. plumbizincicola was >200 times higher. Heterologous expression of SpHMA1 in Saccharomyces cerevisiae increased Cd sensitivity and Cd transport activity in the yeast cells...
October 12, 2018: Plant, Cell & Environment
Lu Long, Dan-Dan Guo, Wei Gao, Wen-Wen Yang, Li-Pan Hou, Xiao-Nan Ma, Yu-Chen Miao, Jose Ramon Botella, Chun-Peng Song
Background: When developing CRISPR/Cas9 systems for crops, it is crucial to invest time characterizing the genome editing efficiency of the CRISPR/Cas9 cassettes, especially if the transformation system is difficult or time-consuming. Cotton is an important crop for the production of fiber, oil, and biofuel. However, the cotton stable transformation is usually performed using Agrobacterium tumefaciens taking between 8 and 12 months to generate T0 plants. Furthermore, cotton is a heterotetraploid and targeted mutagenesis is considered to be difficult as many genes are duplicated in this complex genome...
2018: Plant Methods
Erika Varkonyi-Gasic, Tianchi Wang, Charlotte Voogd, Subin Jeon, Revel Sm Drummond, Andrew P Gleave, Andrew C Allan
Annualization of woody perennials has the potential to revolutionize the breeding and production of fruit crops and rapidly improve horticultural species. Kiwifruit (Actinidia chinensis) is a recently domesticated fruit crop with a short history of breeding and tremendous potential for improvement. Previously, multiple kiwifruit CENTRORADIALIS (CEN)-like genes have been identified as potential repressors of flowering. In this study, CRISPR/Cas9- mediated manipulation enabled functional analysis of kiwifruit CEN-like genes AcCEN4 and AcCEN...
October 10, 2018: Plant Biotechnology Journal
Fei Ma, Hui Qi, Yufei Hu, Qianru Jiang, Li-Guang Zhang, Peng Xue, Fu-Quan Yang, Rui Wang, Yan Ju, Hidenobu Uchida, Quan Zhang, Sodmergen Sodmergen
Maintaining the appropriate number of mitochondrial DNA (mtDNA) molecules is crucial for supporting mitochondrial metabolism and function in both plant and animal cells. For example, a substantial decrease in mtDNA levels occurs as a key part of pollen development. The molecular mechanisms regulating mtDNA copy number are largely unclear, particularly with regard to those that reduce mtDNA levels. Here, we identified and purified a 20 kD endonuclease, M20, from maize (Zea mays) pollen mitochondria. We found M20 to be an H-N-H/N nuclease that degrades linear and circular DNA in the presence of Mg2+ or Mn2+...
October 9, 2018: Plant Physiology
Jianying Li, Hakim Manghwar, Lin Sun, Pengcheng Wang, Guanying Wang, Hanyan Sheng, Jie Zhang, Hao Liu, Lei Qin, Hangping Rui, Bo Li, Keith Lindsey, Henry Daniell, Shuangxia Jin, Xianlong Zhang
The CRISPR/Cas9 system has been extensively applied for crop improvement. However, our understanding of Cas9 specificity is very limited in Cas9-edited plants. To identify on- and off-target mutation in an edited crop, we described whole genome sequencing (WGS) of 14 Cas9-edited cotton plants targeted to three genes, and 3 negative (Ne) control and 3 wild-type (WT) plants. In total, 4,188 - 6,404 unique single nucleotide polymorphisms (SNPs) and 312 - 745 insertions/deletions (indels) were detected in 14 Cas9-edited plants compared with WT, negative and cotton reference genome sequences...
October 6, 2018: Plant Biotechnology Journal
Manal Tashkandi, Zahir Ali, Fatimah Aljedaani, Ashwag Shami, Magdy M Mahfouz
CRISPR/Cas systems confer molecular immunity against phages and conjugative plasmids in prokaryotes. Recently, CRISPR/Cas9 systems have been used to confer interference against eukaryotic viruses. Here, we engineered Nicotiana benthamiana and tomato (Solanum lycopersicum) plants with the CRISPR/Cas9 system to confer immunity against the Tomato yellow leaf curl virus (TYLCV). Targeting the TYLCV genome with Cas9-single guide RNA at the sequences encoding the coat protein (CP) or replicase (Rep) resulted in efficient virus interference, as evidenced by low accumulation of the TYLCV DNA genome in the transgenic plants...
October 5, 2018: Plant Signaling & Behavior
Zachary H Lemmon, Nathan T Reem, Justin Dalrymple, Sebastian Soyk, Kerry E Swartwood, Daniel Rodriguez-Leal, Joyce Van Eck, Zachary B Lippman
Genome editing holds great promise for increasing crop productivity, and there is particular interest in advancing breeding in orphan crops, which are often burdened by undesirable characteristics resembling wild relatives. We developed genomic resources and efficient transformation in the orphan Solanaceae crop 'groundcherry' (Physalis pruinosa) and used clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease (Cas9) (CRISPR-Cas9) to mutate orthologues of tomato domestication and improvement genes that control plant architecture, flower production and fruit size, thereby improving these major productivity traits...
October 2018: Nature Plants
Wen Chen, Guoqiang Zhang, Jing Li, Xuan Zhang, Shulan Huang, Shuanglin Xiang, Xiang Hu, Changning Liu
The CRISPR/Cas9 system, as a revolutionary genome editing tool for all areas of molecular biology, provides new opportunities for research on lncRNA's function. However, designing a CRISPR/Cas9 single guide RNA (sgRNA) for lncRNA is not easy with an unwarrantable effectiveness. Thus, it is worthy of collecting validated sgRNAs, to assist in efficiently choosing sgRNA with an expected activity. CRISPRlnc ( or is a manually curated database of validated CRISPR/Cas9 sgRNAs for lncRNAs from all species...
October 4, 2018: Nucleic Acids Research
Thomas W Kirchner, Markus Niehaus, Kim L Rössig, Timo Lauterbach, Marco Herde, Helge Küster, Manfred K Schenk
Formation of longer root hairs under limiting phosphate (P) conditions can increase the inorganic P (Pi) uptake. Here, regulatory candidate genes for Pi deficiency-induced root hair growth were identified by comparison of massive analysis of cDNA ends (MACE) provided expression profiles of two Brassica carinata cultivars (cv.) differing in their root hair response to Pi deficiency: cv. Bale develops longer root hairs under Pi deficiency, but not cv. Bacho. A split-root experiment was conducted for the differentiation between locally and systemically regulated genes...
2018: Frontiers in Plant Science
Rukmini Mishra, Raj Kumar Joshi, Kaijun Zhao
Rice ( Oryza sativa L.) is the major food source for more than three billion people of the world. In the last few decades, the classical, mutational, and molecular breeding approaches have brought about tremendous increase in rice productivity with the development of novel rice varieties. However, stagnation in rice yield has been reported in recent decade owing to several factors including the emergence of pests and phyto pathogens, climate change, and other environmental issues posing great threat to global food security...
2018: Frontiers in Plant Science
Shouhong Zhu, Xiuli Yu, Yanjun Li, Yuqiang Sun, Qianhao Zhu, Jie Sun
The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) gene editing system has been shown to be able to induce highly efficient mutagenesis in the targeted DNA of many plants, including cotton, and has become an important tool for investigation of gene function and crop improvement. Here, we developed a simple and easy to operate CRISPR/Cas9 system and demonstrated its high editing efficiency in cotton by targeting-ALARP, a gene encoding alanine-rich protein that is preferentially expressed in cotton fibers...
October 1, 2018: International Journal of Molecular Sciences
Agustin Zsögön, Tomáš Čermák, Emmanuel Rezende Naves, Marcela Morato Notini, Kai H Edel, Stefan Weinl, Luciano Freschi, Daniel F Voytas, Jörg Kudla, Lázaro Eustáquio Pereira Peres
Breeding of crops over millennia for yield and productivity has led to reduced genetic diversity. As a result, beneficial traits of wild species, such as disease resistance and stress tolerance, have been lost. We devised a CRISPR-Cas9 genome engineering strategy to combine agronomically desirable traits with useful traits present in wild lines. We report that editing of six loci that are important for yield and productivity in present-day tomato crop lines enabled de novo domestication of wild Solanum pimpinellifolium...
October 1, 2018: Nature Biotechnology
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