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Engineering Introns to Express RNA Guides for Cas9- and Cpfl-Mediated Multiplex Genome Editing

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Molecular Plant 2018年第4期11卷 542-552页

作者： Dan Ding Kaiyuan Chen Yuedan Chen Hong Li Kabin Xie National Key Laboratory of Crop Genetic Improvement and National Centre of Plant Gene Research (Wuhan) Huazhong Agricultural University Wuhan 430070 China Hubei Key Laboratory of Plant Pathology Huazhong Agricultural University Wuhan 430070 China these authors contributed equally to this article.

The clustered regularly interspaced short palindromic repeat （CRISPR） system has emerged as the revolu- tionary platform for DNA targeting. This system uses a site-specific RNA guide to direct a CRISPR effector （e.g., Cas9 and Cpfl） to a DNA target. Here, we elaborate a general strategy to simultaneously express multiple guide RNAs （gRNA） and CRISPR RNAs （crRNA） from introns of Cas9 and Cpfl. This method utilizes the endogenous tRNA processing system or crRNA processing activity of Cpfl to cleave the spliced intron that contains tRNA-gRNA polycistron or crRNA-crRNA array. We demonstrated that the tRNA-gRNA intron is able to fuse with Cas9 as one gene. Such a hybrid gene could be expressed using one polymerase II promoter, and exhibited high efficiency and robustness in simultaneously targeting multiple sites. We also implemented this strategy in Cpfl-mediated genome editing using intronic tRNA-crRNA and crRNA-crRNA arrays. Interestingly, hybrid genes containing Cpfl and intronic crRNA array exhibited remarkably increased efficiency compared with the conventional Cpfl vectors. Taken together, this study presents a method to express CRISPR reagents from one hybrid gene to increase genome-editing efficiency and capacity. Owing to its simplicity and versatility, this method could be broadly used to develop sophisticated CRISPR tools in eukaryotes.

关键词： CRISPR intron Cas9 Cpfl multiplex

Bi-directional Selection in Upland Rice Leads to Its Adaptive Differentiation from Lowland Rice in Drought Resistance and Productivity

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Molecular Plant 2019年第2期12卷 170-184页

作者： Hui Xia Zhi Luo Jie Xiong Xiaosong Ma Qiaojun Lou Haibin Wei Jie Qiu Hua Yang Guolan Liu Longjiang Fan Liang Chen Lijun Luo Shanghai Agrobiological Gene Center Shanghai China College of Plant Sciences & Tech no logy Huazho ng Agricultural University Wuhan China lnstitute of Crop Science & Institute of Bioinformatics College of Agriculture and Biotechnology Zhejiang University Hangzhou China these authors contributed equally to this article.

Drought resistance is required in rice breeding to address the challenge of frequent droughts . However, the evolutionary mechanism of rice drought resistance is not fully understood. We investigated the genetic differentiation between upland and lowland rice domesticated in agro-ecosystems with contrasting water-soil conditions using genome-wide SNPs. We estimated morphological differences among upland and lowland rice in drought resistance and productivity through common garden experiments. Upland rice had better drought resistance but poorer productivity. The negative correlations between traits of drought resistance and productivity are attributed to the underlying genetic trade-offs through tight linkages (e.g., DCA1 and OsCesA7) or pleiotropic effects (e.g., LAX1). The genetic trade-offs are comm on and greatly shape the evolutio n of drought resista nee in upland rice . In genomic regions associated with both productivity and drought resistance, signs of balancing selection were detected in upland rice, while signs of directional selection were detected in lowland rice, potentially contributing to their adaptive differentiation. Signs of balancing selection in upland rice resulted from bi-directional selection during its domestication in drought-prone upland *** genome-wide association analysis, we ide ntified several valuable quantitative trait loci associated with drought resista nee, for which highly differentiated genes should be considered candidates. Bi-directional selection breaking tight linkages by accumulating recombination events would be applicable in breeding water-saving and droughtresistance rice.

关键词： drought resistance upland rice ecological adaptation ecotype trade-off domestication

Identification and Validation of a Major Quantitative Trait Locus for Slow-rusting Resistance to Stripe Rust in Wheat

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Journal of Integrative Plant Biology 2012年第5期54卷 330-344页

作者： Xiaohua Cao Jianghong Zhou Xiaoping Gong Guangyao Zhao Jizeng Jia Xiaoquan Qi Key Laboratory of Plant Molecular Physiology Institute of BotanyChinese Academy of SciencesBeijing 100093China Graduate University of Chinese Academy of Sciences Beijing 100049China Institute of Crop Sciences Chinese Academy of Agricultural SciencesBeijing 100081China these authors contributed equally to this article.

Stripe （yellow） rust, caused by Puccinia striiformis Westend. f. sp. tritici Eriks （Pst）, is one of the most important wheat （Triticum aestivum L.） diseases and causes significant yield losses. A recombinant inbred （RI） population derived from a cross between Yanzhan 1 and Xichang 76-9 cultivars was evaluated for resistance to wheat stripe rust strain CYR32 at both the seedling and adult plant stages. Four resistance quantitative trait loci （QTLs） were detected in this population, in which the major one, designated as Yrql, was mapped on chromosome 2DS. The strategy of using the Brachypodium distachyon genome, wheat expressed sequence tags and a draft DNA sequences （scaffolds） of the D-genome （Aegilops tauschfi Coss.） for the development of simple sequence repeat （SSR） markers was successfully used to identify 147 SSRs in hexaploid wheat. Of the 19 polymorphic SSRs in the RI population, 17 SSRs were mapped in the homeologous group 2 chromosomes near Yrql region and eight SSRs were genetically mapped in the 2.7 cM region of Yrql, providing abundant DNA markers for fine-mapping of Yrql and marker-assisted selection in wheat breeding program. The effectiveness of Yrql was validated in an independent population, indicating that this resistance QTL can be successfully transferred into a susceptible cultivar for improvement of stripe rust resistance.

关键词： Quantitative trait locus slow-rusting stripe rust wheat.

Photoactivated CRY1 and phyB Interact Directly with AUX/IAA Proteins to Inhibit Auxin Signaling in Arabidopsis

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Molecular Plant 2018年第4期11卷 523-541页

作者： Feng Xu Shengbo He Jingyi Zhang Zhilei Mao Wenxiu Wang Ting Li Jie Hua Shasha Du Pengbo Xu Ling Li Hongli Lian Hong-Quan Yang State Key Laboratory of Genetic Engineering Collaborative Innovation Center for Genetics and Development School of Life Sciences Fudan University Shanghai 200438 China School of Agriculture and Biology/School of Life Sciences and Biotechnotogy Shanghai Jiaotong University Shanghai 200240 China these authors contributed equally to this article.

Light is a key environmental cue that inhibits hypocotyl cell elongation through the blue and red/far-red light photoreceptors cryptochrome- and phytochrome-mediated pathways in Arabidopsis. In contrast, as a pivotal endogenous phytohormone auxin promotes hypocotyl elongation through the auxin receptors TIR1/AFBs-mediated degradation of AUX/IAA proteins （AUX/IAAs）. However, the molecular mechanisms underlying the antagonistic interaction of light and auxin signaling remain unclear. Here, we report that light inhibits auxin signaling through stabilization of AUX/IAAs byblue and red light-dependent interactions of cryptochrome 1 （CRY1） and phytochrome B with AUX/IAAs, respectively. Blue light-triggered interactions of CRY1 with AUX/IAAs inhibit the associations of TIR1 with AUX/IAAs, leading to the repression of auxin- induced degradation of these proteins. Our results indicate that photoreceptors share AUX/IAAs with auxin receptors as the same direct downstream signaling components. We propose that antagonistic regulation of AUX/IAA protein stability by photoreceptors and auxin receptors allows plants to balance light and auxin signals to optimize their growth.

关键词： Arabidopsis light auxin cryptochrome phytochrome AUX/IAA proteins

A simple and efficient in planta transformation method based on the active regeneration capacity of plants

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Plant Communications 2024年第4期5卷 166-176页

作者： Guoguo Mei Ao Chen Yaru Wang Shuquan Li Minyi Wu Yilong Hu Xu Liu Xingliang Hou Guangdong Provincial Key Laboratory of Applied Botany&State Key Laboratory of Plant Diversity and Specialty Crops South China Botanical GardenChinese Academy of SciencesGuangzhou 510650China University of Chinese Academy of Sciences Beijing 100049China these authors contributed equally to this article.

Plant genetic transformation strategies serve as essential tools for the genetic engineering and advanced molecular breeding of ***,the complicated operational protocols and low efficiency of cur-rent transformation strategies restrict the genetic modification of most plant *** paper de-scribes the development of the regenerative activity–dependent in planta injection delivery(RAPID)method based on the active regeneration capacity of *** this method,Agrobacterium tumefaciens is delivered to plant meristems via injection to induce transfected nascent *** transgenic plants can be obtained by subsequent vegetative propagation of the positive nascent *** method was successfully used for transformation of plants with strong regeneration capacity,including different genotypes of sweet potato(Ipomoea batatas),potato(Solanum tuberosum),and bayhops(Ipo-moea pes-caprae).Compared with traditional transformation methods,RAPID has a much higher trans-formation efficiency and shorter duration,and it does not require tissue culture *** RAPID method therefore overcomes the limitations of traditional methods to enable rapid in planta transformation and can be potentially applied to a wide range of plant species that are capable of active regeneration.

关键词： plant genetic transformation RAPID active regeneration sweet potato potato bayhops

CRISPR-Mediated Engineering across the Central Dogma in Plant Biology for Basic Research and Crop Improvement

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Molecular Plant 2021年第1期14卷 127-150页

作者： Dibyajyoti Pramanik Rahul Mahadev Shelake Mi Jung Kim Jae-Yean Kim Division of Applied Life Science(BK21 Four Program) Plant Molecular Biology and Biotechnology Research CenterGyeongsang National UniversityJinju 52828Korea These authors contributed equally to this article

The central dogma(CD)of molecular biology is the transfer of genetic information from DNA to RNA to *** CD processes governing genetic flow include the cell cycle,DNA replication,chromosome packaging,epigenetic changes,transcription,posttranscriptional alterations,translation,and posttranslational *** CD processes are tightly regulated in plants to maintain genetic integrity throughout the life cycle and to pass genetic materials to next *** of various CD processes involved in gene regulation will accelerate crop improvement to feed the growing world *** technology enables programmable editing of CD processes to alter DNA,RNA,or protein,which would have been impossible in the ***,an overview of recent advancements in CRISPR tool development and CRISPR-based CD modulations that expedite basic and applied plant research is ***,CRISPR applications in major thriving areas of research,such as gene discovery(allele mining and cryptic gene activation),introgression(de novo domestication and haploid induction),and application of desired traits beneficial to farmers or consumers(biotic/abiotic stress-resilient crops,plant cell factories,and delayed senescence),are ***,the global regulatory policies,challenges,and prospects for CRISPR-mediated crop improvement are discussed.

关键词： central dogma CRISPR crop improvement gene regulation genome editing

Cellular basis of legume-rhizobium symbiosis

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Plant Communications 2024年第11期5卷 41-53页

作者： Xiaxia Zhang Jingxia Wu Zhaosheng Kong State Key Laboratory of Plant Genomics Institute of MicrobiologyChinese Academy of SciencesBeijing 100101China Houji Laboratory in Shanxi Province Academy of AgronomyShanxiChina These authors contributed equally to this article

The legume-rhizobium symbiosis represents the most important system for terrestrial biological nitrogen fixation on *** nitrogen fixation during this symbiosis depends on successful rhizobial infection and complete endosymbiosis,which are achieved by complex cellular events including cell-wall remodeling,cytoskeletal reorganizations,and extensive membrane expansion and *** this review,we explore the dynamic remodeling of the plant-specific cell wall-membrane system-cytoskeleton(WMC)continuum during symbiotic nitrogen *** focus on key processes linked to efficient nitrogen fixation,including rhizobial uptake,infection thread formation and elongation,rhizobial droplet release,cytoplasmic bridge formation,and rhizobial ***,we discuss the advanced techniques for investigating the cellular basis of root-nodule symbiosis and provide insights into the unsolved mysteries of robust symbiotic nitrogen fixation.

关键词： root nodule symbiosis infection threads symbiosomes rhizobia endosymbiosis cell wall-membrane system-cytoskeleton continuum

BZR1 Positively Regulates Freezing Tolerance via CBF-Dependent and CBF-Independent Pathways in Arabidopsis

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Molecular Plant 2017年第4期10卷 545-559页

作者： Hui Li Keyi Ye Yiting Shi Jinkui Cheng Xiaoyan Zhang Shuhua Yang State Key Laboratory of Plant Physiology and Biochemistry College of Biological Sciences China Agricultural University Beijing 100193 China these authors contributed equally to this article.

Cold stress is a major environmental factor that adversely affects plant growth and development. The C-repeat binding factor/DRE binding factor 1 （CBF/DREB1） transcriptional regulatory cascade has been shown to play important roles in plant response to cold. Here we demonstrate that two key components of brassinosteroid （BR） signaling modulate freezing tolerance of Arabidopsis plants. The loss-of-function mutant of the GSK3-1ike kinases involved in BR signaling, bin2-3 bill bil2, showed increased freezing tolerance, whereas overexpression of BIN2 resulted in hypersensitivity to freezing stress under both non-acclimated and acclimated conditions. By contrast, gain-of-function mutants of the transcription factors BZR1 and BES1 displayed enhanced freezing tolerance, and consistently cold treatment could induce the accumulation of dephosphorylated BZR1. Biochemical and genetic analyses showed that BZR1 acts upstream of CBF1 and CBF2 to directly regulate their expression. Moreover, we found that BZR1 also regulated other COR genes uncoupled with CBFs, such as WKRY6, PYL6, SOCl, JMT, and SAG21, to modulate plant response to cold stress. Consistently, wrky6 mutants showed decreased freezing tolerance. Taken together, our results indicate that BZR1 positively modulates plant freezing tolerance through CBF-dependent and CBF-independent pathways.

关键词： BIN2 BZR1 freezing tolerance CBF Arabidopsis

Evolutionary Genomics of Plant Gametophytic Selection

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Plant Communications 2020年第6期1卷 18-33页

作者： Felix E.G.Beaudry Joanna L.Rifkin Spencer C.H.Barrett Stephen I.Wright Department of Ecology and Evolutionary Biology The University of Toronto25 Willcocks StreetTorontoON M5S 3B2Canada These authors contributed equally to this article

It has long been recognized that natural selection during the haploid gametophytic phase of the plant life cycle may have widespread importance for rates of evolution and the maintenance of genetic *** theoretical advances have further highlighted the significance of gametophytic selection for diverse evolutionary *** approaches offer exciting opportunities to address key questions about the extent and effects of gametophytic selection on plant evolution and ***,we review the progress and prospects for integrating functional and evolutionary genomics to test theoretical predictions,and to examine the importance of gametophytic selection on genetic diversity and rates of *** is growing evidence that selection during the gametophyte phase of the plant life cycle has important effects on both gene and genome evolution and is likely to have important pleiotropic effects on the *** discuss the opportunities to integrate comparative population genomics,genome-wide association studies,and experimental approaches to further distinguish how differential selection in the two phases of the plant life cycle contributes to genetic diversity and adaptive evolution.

关键词： gametophyte pollen ovule evolution genomics selection