Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is the most commonly-used tool for measurement of gene expression, but its accuracy and reliability depend on appropriate data normalization with the use of one or more stable reference genes. Adelphocoris suturalis is one of the most destructive pests of cotton, but until recently knowledge of its underlying molecular physiology had been hindered by a lack of molecular resources. To facilitate research on this pest, we evaluated 12 common housekeeping genes studied in insects (GAPDH, ACT, βACT, TBP, SDH, βTUB, EF1γ, EF1α, EF1δ, RPL32, RPS15, and RPL27) for their expression stability in A. suturalis when subjected to various experimental treatments, including three biotic (developmental stage and sex, tissue type, and metathoracic scent gland for varying developmental stages and sexes) and one abiotic (RNA interference injection) conditions. Four dedicated algorithms (ΔCt method, geNorm, BestKeeper and NormFinder) were used to analyze gene expression stability. In addition, RefFinder provided an overall ranking of the stability/suitability of these candidates. This study is the first to provide a comprehensive list of suitable reference genes for gene expression analyses in A. suturalis, which can serve to facilitate transcript expression study of related biological processes in this and related species.
Abstract Insect sex pheromones (SPs) are central to mate‐finding behaviour, and play an essential role in the survival and reproduction of organisms. Understanding the roles, biosynthetic pathways and evolution of insect chemical communication systems has been an exciting challenge for biologists. Compared with Lepidoptera, little is known about the mechanisms underlying pheromone biosynthesis in Hemiptera. In this study, we isolated and characterized two new desaturase‐like genes, termed Asutdes1 and Asutdes2 , from Adelphocoris suturalis , an important agricultural pest in China. Although the two genes encode an identical protein, Southern blot analysis revealed that they are duplicated genes. The Asutdes2 transcript is more abundant than Asutdes1 in the tissues tested, in particular the metathoracic scent gland and fat body. Silencing Asutdes expression in females by injecting double‐stranded RNA ( dsAsutdes ) against a portion of the coding sequence shared by the two genes enhanced the production of (E)‐4‐oxo‐2‐hexenal, a component of the A. suturalis SP blend, and dramatically suppressed the sexual attractiveness of A. suturalis females. We conclude that dsAsutdes is associated with the SP biosynthetic pathway in A. suturalis .
Cotton (Gossypium hirsutum) is an allotetraploid species and a typical thermophilic crop that can survive and grow well under temperatures up to 45 °C. CRISPR/LbCpf1 (LbCas12a) is a temperature-sensitive system for plant genome editing (Malzahn et al., 2019) and has been successfully applied in species such as rice, soya bean, tobacco, maize and cotton (Lee et al., 2019; Li et al., 2018; Tang et al., 2017; Xu et al., 2019). However, the temperature sensitivity of LbCpf1 has not been tested in cotton yet, a high temperature-resistant crop. In order to improve LbCpf1 efficiency and determine the optimum temperature for cotton genome editing, we investigated the effects of different temperatures on LbCpf1 activity and genome editing efficiency. Moreover, we created nontransgenic and glandless cotton plants with seeds free of gossypol, representing a valuable germplasm resource for cotton breeding. In a development from our previous study (Li et al., 2019), we found that the observed albino phenotype of transgenic cotton plants containing LbCpf1-GhCLA1 became enhanced and with an increase of temperature in plants grown in the field in Wuhan (Figure 1a). In order to determine the editing efficacy of LbCpf1 under elevated temperatures, 20 T1 seeds from a LbCpf1-GhCLA1 transgenic line with whitish dappled leaves were sown in a growth chamber at either 24 °C, 29 °C, 34 °C or 37 °C for one week. We found that the cotyledons exhibited different degrees of albinism (Figure 1b, upper panel). All 20 plants showed dappled cotyledons at 24 °C. At 29 °C, 16 plants had some white spots and 4 plants had fully white cotyledons. At 34 °C, 13 plants were fully white, 3 plants were dappled and 4 plants had partially white cotyledons. In a parallel experiment, 20 T1 seeds were sown at 24 °C under the same conditions described above. When the plants developed the first true leaf, they were transferred to 24 °C, 29 °C, 34 °C or 37 °C for 2 weeks, with similar results as described for the previous experiment (Figure 1b, lower panel). However, long and continuous treatment at 37 °C significantly inhibited cotton growth, and some plants died. We determined the editing profiles of T1 seedlings using Sanger sequencing and High-throughput sequencing (Hi-Tom) (Liu et al., 2019). The results showed that each individual plant contained different editing frequencies, and the editing efficiency of GhCLA1 in both the At and Dt subgenomes was 68.4% at 24 °C, 85.3% at 29 °C and 89.7% at 34 °C (Figure 1c). The elevated editing efficiency (genotype) under the higher temperature was in accordance with the observed phenotypic changes under the various temperature regimes. Cotton seeds are a rich source of oil and protein, but are not edible due to the toxicity of the metabolite gossypol, which is stored in dark pigmented glands and is only produced in Gossypium species. Silencing the PIGMENT GLAND FORMATION (PGF) gene by RNAi leads to a glandless phenotype (Ma et al., 2016). To further test the efficiency of CRISPR/LbCpf1 system under different temperatures in cotton, we conducted a targeted editing in the coding sequence of GhPGF by using the tRNA-crRNA1-tRNA-crRNA2 transcription unit developed in our recent report (Wang et al., 2018; Figure 1d). LbCpf1-GhPGF-transformed somatic embyrogenic calli were grown in light incubators at 24 °C, 29 °C, 34 °C or 37 °C for one week. Thirty independent T0 plants were obtained from the treated calli. We found that glands were reduced in number in regenerated plants grown at 24 °C and were completely lacking at 29 °C and 34 °C (Figure 1e). Some calli grown at 37 °C showed abnormal development or death, and no plant data were obtained. Sanger sequencing results showed that the editing profiles at the first target were similar between plants grown under the different temperature treatments. The second target showed very low editing frequencies under 24 °C and 29 °C treatments, while at 34 °C, some editings were detected (Figure 1f). Hi-tom sequencing results also showed that all 10 plants at 24 °C were edited at one GhPGF locus in both the At and Dt subgenomes. At 29 °C and 34 °C, 6/10 and 10/10 plants were mutated at four loci of GhPGF, with editing efficiencies at the GhPGF-crRNA1 site of 15.2% (24 °C), 86.5% (29 °C) and 91.5% (34 °C), and at the GhPGF-crRNA2 site, 13.6% (24 °C), 12.1% (29 °C) and 67.6% (34 °C) (Figure 1c). Correspondingly, both T0 and T1 plants produced glandless bolls, leaves and seeds (Figure 1g). Compared with the expression level at normal temperature (24 °C), transcription of LbCpf1 in transgenic plants was found to be highest at 34 °C (Figure 1h). In order to obtain both glandless and transgene-free plants, T1 plants were obtained by selfing T0 homozygous plants. The LbCpf1 gene was detected by PCR, and the target editing types of GhPGF gene were detected by Sanger sequencing. Two transgene-free T1 plants with the target mutations (Cpgf-1, Cpgf-2) were obtained (Figure 1i), and both plants are glandless. Sequencing demonstrated that no off-target mutations were detected in any predicted off-target sites (Figure 1j). The results presented demonstrate that 34 °C is the optimum temperature for active CRISPR/LbCpf1 in cotton, and the bleached, glandless phenotype facilitates analysis. More importantly, homozygous, nontransgenic and gossypol-free plants provide valuable new germplasm for molecular breeding programs. The opportunity is demonstrated for improved editing efficiency in cotton by simple heat treatment during seed sowing or plant tissue culture. This work was supported by grant from National R&D Project of Transgenic Crops of Ministry of Science and Technology (2019ZX08010-003, 2016ZX08010001-006) and National Key Research and Development Plan (2016YFD0100203-9) of China. SX.J. and XL.Z. designed the project. B. L., S.J.L., F.Q.W., G.Y.W., QQ.W., ZP.X., L.S., L. Y., M.N.Z., H.S., DJ.Y., WF.G. and YQ.W. performed experiments and wrote the manuscript. SX.J., M.A. K.L. revised the manuscript. The authors declare no competing financial interests.
As the basic building blocks of all living species, cells play a crucial role in sustaining life activities. Traditional bulk methods only give us molecular insight into tissue, organ, or/and individual. With the rapid development of sequencing technology, especially the 10× Genomics based on microfluidic omics, biological research has entered the era of single-cell level, which enables us to gain insight into life activities from more microscopic perspective. With this cutting-edge technology, it is now possible to mine heterogeneity between tissue types and within cells like never before. However, the preparation of single-cell suspension (namely, protoplasm suspension) and the annotation of cell clusters are still two main obstacles faced by single-cell research. A few marker gene databases are currently available, including CellMarker, PanglaoDB, and SignatureDB (https://lymphochip.nih.gov/signaturedb/). Single-cell research in mouse and human is at the rapid development stage. However, in plant science, the research for the single-cell profiling is still in its infancy and quite limited resource were available, such as in Arabidopsis thaliana (Gala et al., 2021; Zhang and Chen, 2021b; Zhang et al., 2019), Zea mays (Marand et al., 2021; Xu et al., 2021), Oryza sativa (Liu, Liang, et al., 2021; Wang et al., 2021; Zhang et al., 2021a), Solanum lycopersicum (Tian et al., 2020), and Arachis hypogaea (Liu, Hu, et al., 2021). The existence of cell wall makes the preparation of plant single-cell suspension more difficult than that of animals. On the other hand, due to the lack of effective marker gene database, single-cell research in plant was usually time-consuming for performing large amount of in situ RNA hybridization or genetic transformation with reporter gene to provide reliable experimental evidence for the identification of tissue type of cell cluster. It is in urgent demands to establish a comprehensive database and exploit efficient tools to analyze such scattered over thousands marker genes of specific cell types from different plant species. Therefore, based on the Shiny and bootstrap frameworks, we developed the Plant Single Cell Transcriptome Hub (PsctH) (http://jinlab.hzau.edu.cn/PsctH/), aiming to provide a comprehensive and accurate resource of cell markers and web tool for various cell types in tissues of plant species (Figure 1a). All marker genes included in the PsctH must have been evidenced via RNA in situ hybridization or expression of GFP reporter. Based on this standard, we presented over 20 published plant single-cell reports including 98 cell markers from 51 cell types in 9 plant tissues/sub-tissues of five plant species (Arabidopsis thaliana, Zea mays, Oryza sativa, Arachis hypogaea, and Solanum lycopersicum), and the data were collected and deposited in PsctH (Figure 1b–d). There have been more than 2 marker genes for phloem parenchyma cells and veins from leaf, cortex, lateral root primordia, and stele from root; inflorescence meristem; and spikelet meristem from staminate primordia. Notably, the majority of the cell marker entries are derived from roots, involving 26 cell types, and the most abundant root cell type is lateral root primordia cell. All marker genes were displayed in page of 'MarkerGeneDB' page and can be searched via keywords (Figure 1e). Meanwhile, all experimental evidence derived from RNA in situ hybridization or GFP reporter were accompanied for each marker gene. In addition, all marker genes' information can be download as a tab-delimited file on the 'Download' page and can be easily used in the command line. Another challenge of applying single-cell analysis in plants is to prepare integrity and living single-cell suspension. The first step of preparing plant single-cell suspension usually starts with digestion of cell wall via cellulase and hemicellulase. In PsctH, we compared and evaluated different protocols used to prepare protoplasts and offer a feasible and efficient experimental pipeline for reference, which included preparation and isolation of tissue samples, enzyme digestion, purification, and the detection of the integrity of single cells (Figure 1f). To standardize and link the process of plant single-cell data mining, a flexible pipeline of plant single-cell transcriptome (scRNA-seq) analysis was also provided, including the process of quality control, normalization and scale, clustering, and marker genes identification (Figure 1g). Users can configure key parameters to obtain a dedicated R analysis script. In addition, we also provide configuration files (SingleCellCondaEnvironment.yml) that can easily reproduce the analysis environment of single-cell transcriptome through conda and run the R script obtained previously. The scRNA-Seq data are particularly powerful in resolving progressions in gene expression. Single-cell sequencing data from different tissues of different species will help us to construct a complete plant single-cell landscape, dissecting cellular heterogeneity, identify the key regulatory genes involved in life activities, and insight into specific biological mechanisms. In this case, we collected all published high throughput sequencing data of plant single-cell and accessible in 'SingleCellDB' page, which were categorized by species and tissues. To best serve the community, we build a user interface that allows intuitive searching of plant single-cell literature and performed text mining using natural language processing (NLP) to highlight the most important keywords and explore their associations. The results are compiled by searching for keywords in abstracts and titles using data from PubMed. In summary, PsctH will be a comprehensive and valuable resource for researchers applying single-cell experiments to plants. The isolation of protoplast, pipeline of data processing, and manually curated resource of cell markers collected from experimental researches will be expected to promote plant science research into a single-cell resolution. Indeed, the rapidly growing field of single-cell biology has given us an opportunity to constantly improve and enrich the database. Therefore, we will continue to track the single-cell sequencing studies and update the database by frequent additions of new cell markers across all plant species. Meanwhile, we hope researchers can contribute PsctH by submitting data to us by 'Submit' page. This research was supported by the National Natural Science Foundation of China (31872077) and Science and Technology Major Project of Guangxi (Gui Ke AA18118046) to Dr. Fang Ding and Fundamental Research Funds for the Central Universities (2021ZKPY003) to Dr. Shuangxia Jin. The authors declare no conflict of interest. S.J. and F.D. conceptualized and supervised the research. Z.X. designed and coded this database. Z.X. and G.W. wrote the manuscript. Q.W., X.Z., and G.W. help collected literature and manually curated data. Q.Y., L.T., X.Z., and H.D. provided constructive comments and suggestions on this research. All authors contributed to the writing of the final manuscript.
Gossypium hirsutum is an allotetraploid with a complex genome. Most genes have multiple copies that belong to At and Dt subgenomes. Sequence similarity is also very high between gene homologues. To efficiently achieve site/gene-specific mutation is quite needed. Due to its high efficiency and robustness, the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has exerted broad site-specific genome editing from prokaryotes to eukaryotes. In this study, we utilized a CRISPR/Cas9 system to generate two sgRNAs in a single vector to conduct multiple sites genome editing in allotetraploid cotton. An exogenously transformed gene Discosoma red fluorescent protein2(DsRed2) and an endogenous gene GhCLA1 were chosen as targets. The DsRed2-edited plants in T0 generation reverted its traits to wild type, with vanished red fluorescence the whole plants. Besides, the mutated phenotype and genotype were inherited to their T1 progenies. For the endogenous gene GhCLA1, 75% of regenerated plants exhibited albino phenotype with obvious nucleotides and DNA fragments deletion. The efficiency of gene editing at each target site is 66.7-100%. The mutation genotype was checked for both genes with Sanger sequencing. Barcode-based high-throughput sequencing, which could be highly efficient for genotyping to a population of mutants, was conducted in GhCLA1-edited T0 plants and it matched well with Sanger sequencing results. No off-target editing was detected at the potential off-target sites. These results prove that the CRISPR/Cas9 system is highly efficient and reliable for allotetraploid cotton genome editing.
It is widely recognized that biofuel production from lignocellulosic materials is limited by inadequate technology to efficiently and economically release fermentable sugars from the complex multi-polymeric raw materials. Therefore, endoglucanases, exoglucanase, pectate lyases, cutinase, swollenin, xylanase, acetyl xylan esterase, beta glucosidase and lipase genes from bacteria or fungi were expressed in Escherichia coli or tobacco chloroplasts. A PCR-based method was used to clone genes without introns from Trichoderma reesei genomic DNA. Homoplasmic transplastomic lines showed normal phenotype and were fertile. Based on observed expression levels, up to 49, 64 and 10, 751 million units of pectate lyases or endoglucanase can be produced annually, per acre of tobacco. Plant production cost of endoglucanase is 3100-fold, and pectate lyase is 1057 or 1480-fold lower than the same recombinant enzymes sold commercially, produced via fermentation. Chloroplast-derived enzymes had higher temperature stability and wider pH optima than enzymes expressed in E. coli. Plant crude-extracts showed higher enzyme activity than E. coli with increasing protein concentration, demonstrating their direct utility without purification. Addition of E. coli extracts to the chloroplast-derived enzymes significantly decreased their activity. Chloroplast-derived crude-extract enzyme cocktails yielded more (up to 3625%) glucose from filter paper, pine wood or citrus peel than commercial cocktails. Furthermore, pectate lyase transplastomic plants showed enhanced resistance to Erwina soft rot. This is the first report of using plant-derived enzyme cocktails for production of fermentable sugars from lignocellulosic biomass. Limitations of higher cost and lower production capacity of fermentation systems are addressed by chloroplast-derived enzyme cocktails.
Although the regulatory function of miRNAs and their targets have been characterized in model plants, a possible underlying role in the cotton response to herbivore infestation has not been determined. To investigate this, we performed small RNA and degradome sequencing between resistant and susceptible cotton cultivar following infestation with the generalist herbivore whitefly. In total, the 260 miRNA families and 241 targets were identified. Quantitative-PCR analysis revealed that several miRNAs and their corresponding targets exhibited dynamic spatio-temporal expression patterns. Moreover, 17 miRNA precursors were generated from 29 long intergenic non-coding RNA (lincRNA) transcripts. The genome-wide analysis also led to the identification of 85 phased small interfering RNA (phasiRNA) loci. Among these, nine PHAS genes were triggered by miR167, miR390, miR482a, and two novel miRNAs, including those encoding a leucine-rich repeat (LRR) disease resistance protein, an auxin response factor (ARF) and MYB transcription factors. Through combined modeling and experimental data, we explored and expanded the miR390-tasiARF cascade during the cotton response to whitefly. Virus-induced gene silencing (VIGS) of ARF8 from miR390 target in whitefly-resistant cotton plants increased auxin and jasmonic acid (JA) accumulation, resulting in increased tolerance to whitefly infestation. These results highlight the provides a useful transcriptomic resource for plant-herbivore interaction.
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