Live‐cell CRISPR imaging in plants reveals dynamic telomere movements

Elucidating the spatiotemporal organization of the genome inside the nucleus is imperative to our understanding of the regulation of genes and non‐coding sequences during development and environmental changes. Emerging techniques of chromatin imaging promise to bridge the long‐standing gap between sequencing studies, which reveal genomic information, and imaging studies that provide spatial and temporal information of defined genomic regions. Here, we demonstrate such an imaging technique based on two orthologues of the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR associated protein 9 (Cas9). By fusing eGFP/mRuby2 to catalytically inactive versions of Streptococcus pyogenes and Staphylococcus aureus Cas9, we show robust visualization of telomere repeats in live leaf cells of Nicotiana benthamiana. By tracking the dynamics of telomeres visualized by CRISPR–dCas9, we reveal dynamic telomere movements of up to 2 μm over 30 min during interphase. Furthermore, we show that CRISPR–dCas9 can be combined with fluorescence‐labelled proteins to visualize DNA–protein interactions in vivo. By simultaneously using two dCas9 orthologues, we pave the way for the imaging of multiple genomic loci in live plants cells. CRISPR imaging bears the potential to significantly improve our understanding of the dynamics of chromosomes in live plant cells.     

Reconstitution of the plant ubiquitination cascade in bacteria using a synthetic biology approach

Ubiquitination modulates nearly all aspects of plant life. Here, we reconstituted the Arabidopsis thaliana ubiquitination cascade in Escherichia coli using a synthetic biology approach. In this system, plant proteins are expressed and then immediately participate in ubiquitination reactions within E. coli cells. Additionally, the purification of individual ubiquitination components prior to setting up the ubiquitination reactions is omitted. To establish the reconstituted system, we co‐expressed Arabidopsis ubiquitin (Ub) and ubiquitination substrates with E1, E2 and E3 enzymes in E. coli using the Duet expression vectors. The functionality of the system was evaluated by examining the auto‐ubiquitination of a RING (really interesting new gene)‐type E3 ligase AIP2 and the ubiquitination of its substrate ABI3. Our results demonstrated the fidelity and specificity of this system. In addition, we applied this system to assess a subset of Arabidopsis E2s in Ub chain formation using E2 conjugation assays. Affinity‐tagged Ub allowed efficient purification of Ub conjugates in milligram quantities. Consistent with previous reports, distinct roles of various E2s in Ub chain assembly were also observed in this bacterial system. Therefore, this reconstituted system has multiple advantages, and it can be used to screen for targets of E3 ligases or to study plant ubiquitination in detail.     

High‐efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR/Cas9

The ability to edit plant genomes through gene targeting (GT) requires efficient methods to deliver both sequence‐specific nucleases (SSNs) and repair templates to plant cells. This is typically achieved using Agrobacterium T‐DNA, biolistics or by stably integrating nuclease‐encoding cassettes and repair templates into the plant genome. In dicotyledonous plants, such as Nicotinana tabacum (tobacco) and Solanum lycopersicum (tomato), greater than 10‐fold enhancements in GT frequencies have been achieved using DNA virus‐based replicons. These replicons transiently amplify to high copy numbers in plant cells to deliver abundant SSNs and repair templates to achieve targeted gene modification. In the present work, we developed a replicon‐based system for genome engineering of cereal crops using a deconstructed version of the wheat dwarf virus (WDV). In wheat cells, the replicons achieve a 110‐fold increase in expression of a reporter gene relative to non‐replicating controls. Furthermore, replicons carrying CRISPR/Cas9 nucleases and repair templates achieved GT at an endogenous ubiquitin locus at frequencies 12‐fold greater than non‐viral delivery methods. The use of a strong promoter to express Cas9 was critical to attain these high GT frequencies. We also demonstrate gene‐targeted integration by homologous recombination (HR) in all three of the homoeoalleles (A, B and D) of the hexaploid wheat genome, and we show that with the WDV replicons, multiplexed GT within the same wheat cell can be achieved at frequencies of ~1%. In conclusion, high frequencies of GT using WDV‐based DNA replicons will make it possible to edit complex cereal genomes without the need to integrate GT reagents into the genome.     

A removable virus vector suitable for plant genome editing

Plant genome editing is achieved by the expression of sequence‐specific nucleases (SSNs). RNA virus vector‐mediated expression of SSNs is a promising approach for transgene integration‐free targeted mutagenesis in plants. However, the removal of virus vectors from infected plants is challenging because no antiviral drugs are available against plant viruses. Here, we developed a removable RNA virus vector that carries the target site of tobacco microRNA398 (miR398) whose expression is induced during shoot regeneration. In the inoculated leaves in which expression of miR398 is not induced, insertion of the miR398 target site did not affect the practicability of the virus vector. When shoots were regenerated from the infected leaves, miR398 was expressed and viral RNA was eliminated. The virus vector successfully expressed SSNs in inoculated leaves, from which virus‐free genome‐edited plants were regenerated via tissue culture.     

Multiplex single‐cell quantification of rare RNA transcripts from protoplasts in a model plant system

Here we demonstrate multiplex and simultaneous detection of four different rare RNA species from plant, Arabidopsis thaliana, using surface‐enhanced Raman spectroscopy (SERS) and gold nanoprobes at single‐cell resolution. We show the applicability of nanoparticle‐based Raman spectroscopic sensor to study intracellular RNA copies. First, we demonstrate that gold‐nanoparticles decorated with Raman probes and carrying specific nucleic acid probe sequences can be uptaken by the protoplasts. We confirm the internalization of gold nanoprobes by transmission electron microscopy, inductively‐coupled plasma‐mass spectrometry and fluorescence imaging. Second, we show the utility of a SERS platform to monitor individual alternatively spliced (AS) variants and miRNA copies within single cells. Finally, the distinctive spectral features of Raman‐active dyes were exploited for multiplex analysis of AtPTB2, AtDCL2, miR156a and miR172a. Furthermore, single‐cell studies were validated by in vitro quantification and evaluation of nanotoxicity of gold probes. Raman tag functionalized gold nanosensors yielded an approach for the tracking of rare RNAs within the protoplasts. The SERS‐based approach for quantification of RNAs has the capability to be a highly sensitive, accurate and discerning method for single‐cell studies including AS variants quantification and rare miRNA detection in specific plant species.     

Identification of the phosphorylation targets of symbiotic receptor‐like kinases using a high‐throughput multiplexed assay for kinase specificity

Detecting the phosphorylation substrates of multiple kinases in a single experiment is a challenge, and new techniques are being developed to overcome this challenge. Here, we used a multiplexed assay for kinase specificity (MAKS) to identify the substrates directly and to map the phosphorylation site(s) of plant symbiotic receptor‐like kinases. The symbiotic receptor‐like kinases nodulation receptor‐like kinase (NORK) and lysin motif domain‐containing receptor‐like kinase 3 (LYK3) are indispensable for the establishment of root nodule symbiosis. Although some interacting proteins have been identified for these symbiotic receptor‐like kinases, very little is known about their phosphorylation substrates. Using this high‐throughput approach, we identified several other potential phosphorylation targets for both these symbiotic receptor‐like kinases. In particular, we also discovered the phosphorylation of LYK3 by NORK itself, which was also confirmed by pairwise kinase assays. Motif analysis of potential targets for these kinases revealed that the acidic motif xxxsDxxx was common to both of them. In summary, this high‐throughput technique catalogs the potential phosphorylation substrates of multiple kinases in a single efficient experiment, the biological characterization of which should provide a better understanding of phosphorylation signaling cascade in symbiosis.     

日本手取群研究的新进展

介绍日本中生代晚期手取群研究的最新进展,并简要评述与中国的对比。手取群从下到上分为九头龙亚群、石白亚群、赤岩亚群。庄川地区九头龙亚群御手洗组新发现菊石,属Tithonian-Berriasian期,改变了以往Callovian期的传统意见。石白亚群发现菊石,属Late Hauterivian-Early Barremian期;亚群上部发现海相双壳类,属Hauterivian期,半咸水相和淡水双壳类也与外带Hauterivian期的对比;绝对年龄测定为135±7Ma和128±8Ma,分别属Hauterivian早期和Barremian早期,其中135Ma被普遍接受;植物群反映的古气候特征与俄罗斯东部Valanginian-Hauterivian期温湿化一致。石白亚群的时代综合为Valanginian-Hauterivian期。赤岩亚群的Trigonioides(Wakinoa)tetoriensis也发现于外带,时代属latest Hauterivian-Early Barremian期;绝对年龄测定数据不稳定,自127±8Ma(Barremian中期)至106±7Ma(Albian中期)。对亚群的Nippononaia ryosekiana作了修订,认为应属N.linhaiensis,与浙江馆头组的同种,比濑林组(Lower Aptian)的N.ryosekiana原始,故时代早于Aptian期。赤岩亚群的时代综合为Barremian期,但不排除上延的可能。区域对比结果认为,中国东北龙爪沟群及绥滨地区的相当地层可与九头龙亚群和石白亚群对比;馆头组、下城子组可与赤岩亚群对比;热河群上部沙海组-阜新组煤系地层可与石白亚群对比;并对义县组的时代提出了看法。     

岷江干旱河谷-山地森林交错带震后生态恢复的关键科学技术问题

作为典型的生态过渡区,岷江干旱河谷-山地森林交错带不仅是藏羌居民生活的重要区域,而且在抑制干旱河谷上延和延伸亚高山森林生态系统的功能等方面具有十分重要的作用。但这种脆弱生态系统极易受到人类活动干扰和自然灾害的损害,使其成为“5．12”汶川大地震中受损程度较高、灾后生态恢复与重建的重点区域之一。基于岷江干旱河谷-山地森林交错带受汶川大地震破坏的特点以及该区的生态重要性和本身的脆弱性,损毁生态系统的快速评估与生态重建规划、生产与生态双赢共建关键技术、震后残存植被保育、水源涵养地植被保护与恢复、震毁植被恢复与重建、耕地生产恢复与重建、边坡综合治理、低效薪炭林改良以及居民聚居点风景林营造等被认为是震后生态恢复的关键科学技术问题。震毁生态系统的生态恢复过程监测与评估、干旱河谷-山地森林交错带生态系统的脆弱性机制及生态学过程、震后生态系统对变化环境的响应与适应机制等可能是未来的重点研究领域。     

植物挥发性气体(VOCs)研究进展

植物挥发性气体(VOCs)在植物一植食性昆虫-天敌三级营养关系、植物间信息传递及适应性改变上都发挥着重要作用.植物释放VOCs具特异性、系统性、时序性与节律性等特点,VOCs主要在寄主选择行为、产卵行为、求偶行为、引来昆虫夭敌干涉等方面影响植食性昆虫.VOCs-介导的植物间信息传递作用包括4个过程:"释放者"植物合成及释放气体、气体在空气中的运输、气体在植物表面的吸附及"接收者"植株对气体信号的感知.收集VOCs的方法主要有吸附-溶剂洗脱法和吸附-热脱附法.