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.     

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.     

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.     

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.     

土蝽——善于土栖生活的半翅目昆虫

土蝽是半翅目异翅亚目蝽总科中多数适应于地栖生活的蝽类。文章简要描述该类昆虫的形态特征、分类历史以及生物学,同时对该类昆虫目前的生物学和系统学研究进展也给予简要介绍。文中还提供形态特征图6幅,臭腺特征扫描电镜照片6幅。     

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。     

丙型病毒性肝炎母婴传播的现状、进展及未来

丙型肝炎病毒(HCV)可引起急性和慢性病毒性肝炎,可发展成肝纤维化、肝硬化,甚至肝细胞癌。HCV经典的传播途径为经血液或血液制品传播,但1992年后献血员HCV的筛检已使输血后肝炎大为减少。在发达国家,HCV传播途径正在发生改变,儿童非血液制品的丙肝日渐增多。母婴间宫内、分娩时及产后感染已成为当前及今后的重要研究课题。研究证实,HCV可经胎盘引起胎儿感染,宫内感染是HCV传播的一条重要途径。尽管人们对HCV母婴传播中所涉及的风险因素逐渐明确,但到目前为止对具体的传播机制和传播时机仍知之甚少。我们就丙型病毒性肝炎母婴传播的现状、进展及未来做简要综述。     

AgroLux: bioluminescent Agrobacterium to improve molecular pharming and study plant immunity

Agroinfiltration in Nicotiana benthamiana is widely used to transiently express heterologous proteins in plants. However, the state of Agrobacterium itself is not well studied in agroinfiltrated tissues, despite frequent studies of immunity genes conducted through agroinfiltration. Here, we generated a bioluminescent strain of Agrobacterium tumefaciens GV3101 to monitor the luminescence of Agrobacterium during agroinfiltration. By integrating a single copy of the lux operon into the genome, we generated a stable ‘AgroLux’ strain, which is bioluminescent without affecting Agrobacterium growth in vitro and in planta. To illustrate its versatility, we used AgroLux to demonstrate that high light intensity post infiltration suppresses both Agrobacterium luminescence and protein expression. We also discovered that AgroLux can detect Avr/Cf-induced immune responses before tissue collapse, establishing a robust and rapid quantitative assay for the hypersensitive response (HR). Thus, AgroLux provides a non-destructive, versatile and easy-to-use imaging tool to monitor both Agrobacterium and plant responses.     

In vivo homopropargylglycine incorporation enables sampling,isolation and characterization of nascent proteins from Arabidopsis thaliana

Determining which proteins are actively synthesized at a given point in time and extracting a representative sample for analysis is important to understand plant responses. Here we show that the methionine (Met) analogue homopropargylglycine (HPG) enables Bio-Orthogonal Non-Canonical Amino acid Tagging (BONCAT) of a small sample of the proteins being synthesized in Arabidopsis plants or cell cultures, facilitating their click-chemistry enrichment for analysis. The sites of HPG incorporation could be confirmed by peptide mass spectrometry at Met sites throughout protein amino acid sequences and correlation with independent studies of protein labelling with ¹⁵N verified the data. We provide evidence that HPG-based BONCAT tags a better sample of nascent plant proteins than azidohomoalanine (AHA)-based BONCAT in Arabidopsis and show that the AHA induction of Met metabolism and greater inhibition of cell growth rate than HPG probably limits AHA incorporation at Met sites in Arabidopsis. We show HPG-based BONCAT provides a verifiable method for sampling, which plant proteins are being synthesized at a given time point and enriches a small portion of new protein molecules from the bulk protein pool for identification, quantitation and subsequent biochemical analysis. Enriched nascent polypeptides samples were found to contain significantly fewer common post-translationally modified residues than the same proteins from whole plant extracts, providing evidence for age-related accumulation of post-translational modifications in plants.