Plant Science & Landscape Architecture Research Works

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    The State of Soil Degradation in Sub-Saharan Africa: Baselines, Trajectories, and Solutions
    (MDPI, 2015-05-26) Tully, Katherine; Sullivan, Clare; Weil, Ray; Sanchez, Pedro
    The primary cause of soil degradation in sub-Saharan Africa (SSA) is expansion and intensification of agriculture in efforts to feed its growing population. Effective solutions will support resilient systems, and must cut across agricultural, environmental, and socioeconomic objectives. While many studies compare and contrast the effects of different management practices on soil properties, soil degradation can only be evaluated within a specific temporal and spatial context using multiple indicators. The extent and rate of soil degradation in SSA is still under debate as there are no reliable data, just gross estimates. Nevertheless, certain soils are losing their ability to provide food and essential ecosystem services, and we know that soil fertility depletion is the primary cause. We synthesize data from studies that examined degradation in SSA at broad spatial and temporal scales and quantified multiple soil degradation indicators, and we found clear indications of degradation across multiple indicators. However, different indicators have different trajectories—pH and cation exchange capacity tend to decline linearly, and soil organic carbon and yields non-linearly. Future research should focus on how soil degradation in SSA leads to changes in ecosystem services, and how to manage these soils now and in the future.
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    A Comparison of Irrigation-Water Containment Methods and Management Strategies Between Two Ornamental Production Systems to Minimize Water Security Threats
    (MDPI, 2019-12-03) Ristvey, Andrew G.; Belayneh, Bruk E.; Lea-Cox, John D.
    Water security in ornamental plant production systems is vital for maintaining profitability. Expensive, complicated, or potentially dangerous treatment systems, together with skilled labor, is often necessary to ensure water quality and plant health. Two contrasting commercial ornamental crop production systems in a mesic region are compared, providing insight into the various strategies employed using irrigation-water containment and treatment systems. The first is a greenhouse/outdoor container operation which grows annual ornamental plants throughout the year using irrigation booms, drip emitters, and/or ebb and flow systems depending on the crop, container size, and/or stage of growth. The operation contains and recycles 50–75% of applied water through a system of underground cisterns, using a recycling reservoir and a newly constructed 0.25 ha slow-sand filtration (SSF) unit. Groundwater provides additional water when needed. Water quantity is not a problem in this operation, but disease and water quality issues, including agrochemicals, are of potential concern. The second is a perennial-plant nursery which propagates cuttings and produces field-grown trees and containerized plants. It has a series of containment/recycling reservoirs that capture rainwater and irrigation return water, together with wells of limited output. Water quantity is a more important issue for this nursery, but poor water quality has had some negative economic effects. Irrigation return water is filtered and sanitized with chlorine gas before being applied to plants via overhead and micro-irrigation systems. The agrochemical paclobutrazol was monitored for one year in the first operation and plant pathogens were qualified and quantified over two seasons for both production systems. The two operations employ very different water treatment systems based on their access to water, growing methods, land topography, and capital investment. Each operation has experienced different water quantity and quality vulnerabilities, and has addressed these threats using a variety of technologies and management techniques to reduce their impacts.
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    Fruit Morphology Measurements of Jujube Cultivar ‘Lingwu Changzao’ (Ziziphus jujuba Mill. cv. Lingwuchangzao) during Fruit Development
    (MDPI, 2021-02-06) Ma, Yaping; Zhang, Dapeng; Wang, Zhuangji; Song, Lihua; Cao, Bing
    ‘Lingwu Changzao’ (Ziziphus jujuba Mill. cv. Lingwuchangzao), a cultivar of Ziziphus in the Rhamnaceae family, is a traditional jujube cultivar in Ningxia, China. For ‘Lingwu Changzao’, morphological traits are prominent in characterizing fruit yield, quality, and consumer acceptance. However, morphological measurements for ‘Lingwu Changzao’ cultivation are limited. Therefore, the objective of this study is to measure the growing patterns of selected morphological traits during ‘Lingwu Changzao’ fruit development. Eight morphological traits, including four fruit traits (fruit length, diameter, weight, and flesh (mesocarp) thickness), three stone traits (stone length, diameter, and weight), and fruit firmness (also known as fruit hardness), were measured over a 3-mo (months) period, covering a completed fruit development period. Results indicate that the growing patterns of fruit traits coincide with double ‘S’ growth curves, which mainly present the growth of ‘Lingwu Changzao’ fruit. Increases of stone traits terminated in the early fruit growth period, while fruit traits continuously increased till the end of the 3-mo period. That implies a high fruit-stone ratio, i.e., a desirable quality attribute for ‘Lingwu Changzao’ as fresh-eating fruits. The results presented in this study can serve as one part of the standard dataset for jujube fruit cultivation in China, and it can also support decisions in plant breeding and field managements for ‘Lingwu Changzao’.
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    Non-Target Site Mechanisms of Fungicide Resistance in Crop Pathogens: A Review
    (MDPI, 2021-02-27) Hu, Mengjun; Chen, Shuning
    The rapid emergence of resistance in plant pathogens to the limited number of chemical classes of fungicides challenges sustainability and profitability of crop production worldwide. Understanding mechanisms underlying fungicide resistance facilitates monitoring of resistant populations at large-scale, and can guide and accelerate the development of novel fungicides. A majority of modern fungicides act to disrupt a biochemical function via binding a specific target protein in the pathway. While target-site based mechanisms such as alternation and overexpression of target genes have been commonly found to confer resistance across many fungal species, it is not uncommon to encounter resistant phenotypes without altered or overexpressed target sites. However, such non-target site mechanisms are relatively understudied, due in part to the complexity of the fungal genome network. This type of resistance can oftentimes be transient and noninheritable, further hindering research efforts. In this review, we focused on crop pathogens and summarized reported mechanisms of resistance that are otherwise related to target-sites, including increased activity of efflux pumps, metabolic circumvention, detoxification, standing genetic variations, regulation of stress response pathways, and single nucleotide polymorphisms (SNPs) or mutations. In addition, novel mechanisms of drug resistance recently characterized in human pathogens are reviewed in the context of nontarget-directed resistance.
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    The Effects of Pedestrian Environments on Walking Behaviors and Perception of Pedestrian Safety
    (MDPI, 2021-08-05) Kweon, Byoung-Suk; Rosenblatt-Naderi, Jody; Ellis, Christopher D.; Shin, Woo-Hwa; Danies, Blair H.
    We investigated the effects of pedestrian environments on parents’ walking behavior, their perception of pedestrian safety, and their willingness to let their children walk to school. This study was a simulated walking environment experiment that created six different pedestrian conditions using sidewalks, landscape buffers, and street trees. We used within subjects design where participants were exposed to all six simulated conditions. Participants were 26 parents with elementary school children. Sidewalks, buffer strips, and street trees affected parents’ decisions to: walk themselves; let their children walk to school; evaluate their perception whether the simulated environment was safe for walking. We found that the design of pedestrian environments does affect people’s perceptions of pedestrian safety and their willingness to walk. The presence of a sidewalk, buffer strip, and street trees affected parents’ decision to walk, their willingness to let their children walk to school and perceived the pedestrian environment as safer for walking. The effects of trees on parents’ walking and perception of pedestrian safety are greater when there is a wide buffer rather than a narrow buffer. It was found that parents are more cautious about their children’s walking environments and safety than their own.
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    Two Triacylglycerol Lipases Are Negative Regulators of Chilling Stress Tolerance in Arabidopsis
    (MDPI, 2022-03-21) Wang, Lang; Qian, Bilian; Zhao, Lei; Liang, Ming-Hua; Zhan, Xiangqiang; Zhu, Jianhua
    Cold stress is one of the abiotic stress conditions that severely limit plant growth and development and productivity. Triacylglycerol lipases are important metabolic enzymes for the catabolism of triacylglycerols and, therefore, play important roles in cellular activities including seed germination and early seedling establishment. However, whether they play a role in cold stress responses remains unknown. In this study, we characterized two Arabidopsis triacylglycerol lipases, MPL1 and LIP1 and defined their role in cold stress. The expression of MPL1 and LIP1 is reduced by cold stress, suggesting that they may be negative factors related to cold stress. Indeed, we found that loss-of-function of MPL1 and LIP1 resulted in increased cold tolerance and that the mpl1lip1 double mutant displayed an additive effect on cold tolerance. We performed RNA-seq analysis to reveal the global effect of the mpl1 and lip1 mutations on gene expression under cold stress. The mpl1 mutation had a small effect on gene expression under both under control and cold stress conditions whereas the lip1 mutation caused a much stronger effect on gene expression under control and cold stress conditions. The mpl1lip1 double mutant had a moderate effect on gene expression under control and cold stress conditions. Together, our results indicate that MPL1 and LIP1 triacylglycerol lipases are negative regulators of cold tolerance without any side effects on growth in Arabidopsis and that they might be ideal candidates for breeding cold-tolerant crops through genome editing technology.
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    Acclimation and Compensating Metabolite Responses to UV-B Radiation in Natural and Transgenic Populus spp. Defective in Lignin Biosynthesis
    (MDPI, 2022-08-20) Wong, Tiffany M.; Sullivan, Joe H.; Eisenstein, Edward
    Plants have evolved to protect leaf mesophyll tissue from damage caused by UV-B radiation by producing an array of UV-absorbing secondary metabolites. Flavonoids (phenolic glycosides) and sinapate esters (hydroxycinnamates) have been implicated as UV-B protective compounds because of the accumulation in the leaf epidermis and the strong absorption in the wavelengths corresponding to UV. Environmental adaptations by plants also generate a suite of responses for protection against damage caused by UV-B radiation, with plants from high elevations or low latitudes generally displaying greater adaptation or tolerance to UV-B radiation. In an effort to explore the relationships between plant lignin levels and composition, the origin of growth elevation, and the hierarchical synthesis of UV-screening compounds, a collection of natural variants as well as transgenic Populus spp. were examined for sensitivity or acclimation to UV-B radiation under greenhouse and laboratory conditions. Noninvasive, ecophysiological measurements using epidermal transmittance and chlorophyll fluorescence as well as metabolite measurements using UPLC-MS generally revealed that the synthesis of anthocyanins, flavonoids, and lignin precursors are increased in Populus upon moderate to high UV-B treatment. However, poplar plants with genetic modifications that affect lignin biosynthesis, or natural variants with altered lignin levels and compositions, displayed complex changes in phenylpropanoid metabolites. A balance between elevated metabolic precursors to protective phenylpropanoids and increased biosynthesis of these anthocyanins, flavonoids, and lignin is proposed to play a role in the acclimation of Populus to UV-B radiation and may provide a useful tool in engineering plants as improved bioenergy feedstocks.
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    School Walk Zone: Identifying Environments That Foster Walking and Biking to School
    (MDPI, 2023-02-06) Kweon, Byoung-Suk; Shin, Woo-Hwa; Ellis, Christopher D.
    Today, few children walk or bike to school. According to the National Household Travel Survey, only 11% of children walk or bike to school. In 1969, almost 50% of children walked or biked to school in the US. Although our understanding is limited, previous research has shown that physical environments can influence non-automobile mode choices for travel to school. For example, landscape buffers and trees affect parents’ perceptions of their children’s safety and increase their willingness to let their children walk to school. We investigated how a number of physical attributes in the pedestrian environment influence children’s commutes to school. A total of 186 parents from four school walk zones in College Station, TX, participated in this study. We found that children walked more in neighborhoods with mature trees. Moreover, the mean walking and biking distances differed from each other, and both were influenced by the location of the school within the walk zones. Concerns about traffic safety and convenience were negatively related to walking and biking. The findings here suggest ways to shape better school walk zone guidelines that include neighborhood design, planning, and engagement in support of active and healthy children.
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    Reducing the generation time in winter wheat cultivars using speed breeding
    (Wiley, 2023-05-02) Schoen, Adam; Wallace, Sydney; Holbert, Meghan Fisher; Brown-Guidera, Gina; Harrison, Stephen; Murphy, Paul; Sanantonio, Nicholas; Van Sanford, David; Boyles, Richard; Mergoum, Mohamed; Rawat, Nidhi; Tiwari, Vijay
    Reducing generation time is critical to achieving the goals of genetic gain in important crops like wheat (Triticum aestivum). Speed breeding (SB) has been shown to considerably reduce generation times in crop plants. Unlike spring wheat cultivars, winter wheat varieties require typically 6–9 weeks of cold treatment, called vernalization, for flowering which extends the generation time for the development of improved winter wheat cultivars. Here, we optimized the SB method using a set of 48 diverse soft red winter wheat (SRWW) cultivars by testing vernalization duration, light and temperature requirements, and the viability of seeds harvested after different durations post-anthesis under extended daylight conditions. We have found that using a 22-h setting (22 h day/2 h night, 25°C/22°C) in high-density 50-cell trays results in rapid generation advancement. We used genotypic data for a panel of soft red winter wheat varieties from the regional programs to determine the impact of photoperiod and vernalization alleles on the efficiency of the SB approach. Using a set of 48 SRWW cultivars and germplasm from Maryland and four other public breeding programs, we establish that this protocol can allow for the advancement of four generations per year in controlled conditions for winter wheat varieties, experimental lines, or emerging cultivars. Our work shows the potential to reduce generation time by ∼30 days per generation faster than what had been reported in the SB strategies for winter wheat, thus allowing for a quicker turnaround time from original cross to genetically stable experimental genotypes that can be tested in field settings.
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    Expanding the targeting scope of FokI-dCas nuclease systems with SpRY and Mb2Cas12a
    (Wiley, 2022-04-04) Cheng, Yanhao; Sretenovic, Simon; Zhang, Yingxiao; Pan, Changtian; Huang, Ji; Qi, Yiping
    CRISPR-Cas9 and Cas12a are widely used sequence-specific nucleases (SSNs) for genome editing. The nuclease domains of Cas proteins can induce DNA double strand breaks upon RNA guided DNA targeting. Zinc finger nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs) have been popular SSNs prior to CRISPR. Both ZFNs and TALENs are based on reconstitution of two monomers with each consisting of a DNA binding domain and a FokI nuclease domain. Inspired by the configuration of ZFNs and TALENs, dimeric FokI-dCas9 systems were previously demonstrated in human cells. Such configuration, based on a pair of guide RNAs (gRNAs), offers great improvement on targeting specificity. To expand the targeting scope of dimeric FokI-dCas systems, the PAM (protospacer adjacent motif)-less SpRY Cas9 variant and the PAM-relaxed Mb2Cas12a system were explored. Rice cells showed that FokI-dSpRY had more robust editing efficiency than a paired SpRY nickase system. Furthermore, a dimeric FokI-dMb2Cas12a system was developed that displayed comparable editing activity to Mb2Cas12a nuclease in rice cells. Finally, a single-chain FokI-FokI-dMb2Cas12a system was developed that cuts DNA outside its targeting sequence, which could be useful for many versatile applications. Together, this work greatly expanded the FokI based CRISPR-Cas systems for genome editing.
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    High throughput sequencing reveals novel and abiotic stress-regulated microRNAs in the inflorescences of rice
    (Springer Nature, 2012-08-03) Barrera-Figueroaroa, Blanca E; Gao, Lei; Wu, Zhigang; Zhou, Xuefeng; Zhu, Jianhua; Jin, Hailing; Liu, Renyi; Zhu, Jian-Kang
    MicroRNAs (miRNAs) are small RNA molecules that play important regulatory roles in plant development and stress responses. Identification of stress-regulated miRNAs is crucial for understanding how plants respond to environmental stimuli. Abiotic stresses are one of the major factors that limit crop growth and yield. Whereas abiotic stress-regulated miRNAs have been identified in vegetative tissues in several plants, they are not well studied in reproductive tissues such as inflorescences. We used Illumina deep sequencing technology to sequence four small RNA libraries that were constructed from the inflorescences of rice plants that were grown under control condition and drought, cold, or salt stress. We identified 227 miRNAs that belong to 127 families, including 70 miRNAs that are not present in the miRBase. We validated 62 miRNAs (including 10 novel miRNAs) using published small RNA expression data in DCL1, DCL3, and RDR2 RNAi lines and confirmed 210 targets from 86 miRNAs using published degradome data. By comparing the expression levels of miRNAs, we identified 18, 15, and 10 miRNAs that were regulated by drought, cold and salt stress conditions, respectively. In addition, we identified 80 candidate miRNAs that originated from transposable elements or repeats, especially miniature inverted-repeat elements (MITEs). We discovered novel miRNAs and stress-regulated miRNAs that may play critical roles in stress response in rice inflorescences. Transposable elements or repeats, especially MITEs, are rich sources for miRNA origination.
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    Elucidating the evolutionary history and expression patterns of nucleoside phosphorylase paralogs (vegetative storage proteins) in Populusand the plant kingdom
    (Springer Nature, 2013-08-19) Pettengill, Emily A; Pettengill, James B; Coleman, Gary D
    Nucleoside phosphorylases (NPs) have been extensively investigated in human and bacterial systems for their role in metabolic nucleotide salvaging and links to oncogenesis. In plants, NP-like proteins have not been comprehensively studied, likely because there is no evidence of a metabolic function in nucleoside salvage. However, in the forest trees genus Populus a family of NP-like proteins function as an important ecophysiological adaptation for inter- and intra-seasonal nitrogen storage and cycling. We conducted phylogenetic analyses to determine the distribution and evolution of NP-like proteins in plants. These analyses revealed two major clusters of NP-like proteins in plants. Group I proteins were encoded by genes across a wide range of plant taxa while proteins encoded by Group II genes were dominated by species belonging to the order Malpighiales and included the Populus Bark Storage Protein (BSP) and WIN4-like proteins. Additionally, we evaluated the NP-like genes in Populus by examining the transcript abundance of the 13 NP-like genes found in the Populus genome in various tissues of plants exposed to long-day (LD) and short-day (SD) photoperiods. We found that all 13 of the Populus NP-like genes belonging to either Group I or II are expressed in various tissues in both LD and SD conditions. Tests of natural selection and expression evolution analysis of the Populus genes suggests that divergence in gene expression may have occurred recently during the evolution of Populus, which supports the adaptive maintenance models. Lastly, in silico analysis of cis-regulatory elements in the promoters of the 13 NP-like genes in Populus revealed common regulatory elements known to be involved in light regulation, stress/pathogenesis and phytohormone responses. In Populus, the evolution of the NP-like protein and gene family has been shaped by duplication events and natural selection. Expression data suggest that previously uncharacterized NP-like proteins may function in nutrient sensing and/or signaling. These proteins are members of Group I NP-like proteins, which are widely distributed in many plant taxa. We conclude that NP-like proteins may function in plants, although this function is undefined.
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    Proteomic analysis of Staphylococcus aureus biofilm cells grown under physiologically relevant fluid shear stress conditions
    (Springer Nature, 2014-04-30) Islam, Nazrul; Kim, Yonghyun; Ross, Julia M; Marten, Mark R
    The biofilm forming bacterium Staphylococcus aureus is responsible for maladies ranging from severe skin infection to major diseases such as bacteremia, endocarditis and osteomyelitis. A flow displacement system was used to grow S. aureus biofilms in four physiologically relevant fluid shear rates (50, 100, 500 and 1000 s-1) to identify proteins that are associated with biofilm. Global protein expressions from the membrane and cytosolic fractions of S. aureus biofilm cells grown under the above shear rate conditions are reported. Sixteen proteins in the membrane-enriched fraction and eight proteins in the cytosolic fraction showed significantly altered expression (p < 0.05) under increasing fluid shear. These 24 proteins were identified using nano-LC-ESI-MS/MS. They were found to be associated with various metabolic functions such as glycolysis / TCA pathways, protein synthesis and stress tolerance. Increased fluid shear stress did not influence the expression of two important surface binding proteins: fibronectin-binding and collagen-binding proteins. The reported data suggest that while the general metabolic function of the sessile bacteria is minimal under high fluid shear stress conditions, they seem to retain the binding capacity to initiate new infections.
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    Directed plant cell-wall accumulation of iron: embedding co-catalyst for efficient biomass conversion
    (Springer Nature, 2016-10-21) Lin, Chien-Yuan; Jakes, Joseph E.; Donohoe, Bryon S.; Ciesielski, Peter N.; Yang, Haibing; Gleber, Sophie-Charlotte; Vogt, Stefan; Ding, Shi-You; Peer, Wendy A.; Murphy, Angus S.; McCann, Maureen C.; Himmel, Michael E.; Tucker, Melvin P.; Wei, Hui
    Plant lignocellulosic biomass is an abundant, renewable feedstock for the production of biobased fuels and chemicals. Previously, we showed that iron can act as a co-catalyst to improve the deconstruction of lignocellulosic biomass. However, directly adding iron catalysts into biomass prior to pretreatment is diffusion limited, and increases the cost of biorefinery operations. Recently, we developed a new strategy for expressing iron-storage protein ferritin intracellularly to accumulate iron as a catalyst for the downstream deconstruction of lignocellulosic biomass. In this study, we extend this approach by fusing the heterologous ferritin gene with a signal peptide for secretion into Arabidopsis cell walls (referred to here as FerEX). The transgenic Arabidopsis plants. FerEX. accumulated iron under both normal and iron-fertilized growth conditions; under the latter (iron-fertilized) condition, FerEX transgenic plants showed an increase in plant height and dry weight by 12 and 18 %, respectively, compared with the empty vector control plants. The SDS- and native-PAGE separation of cell-wall protein extracts followed by Western blot analyses confirmed the extracellular expression of ferritin in FerEX plants. Meanwhile, Perls' Prussian blue staining and X-ray fluorescence microscopy (XFM) maps revealed iron depositions in both the secondary and compound middle lamellae cell-wall layers, as well as in some of the corner compound middle lamella in FerEX. Remarkably, their harvested biomasses showed enhanced pretreatability and digestibility, releasing, respectively, 21 % more glucose and 34 % more xylose than the empty vector control plants. These values are significantly higher than those of our recently obtained ferritin intracellularly expressed plants. This study demonstrated that extracellular expression of ferritin in Arabidopsis can produce plants with increased growth and iron accumulation, and reduced thermal and enzymatic recalcitrance. The results are attributed to the intimate colocation of the iron co-catalyst and the cellulose and hemicellulose within the plant cell-wall region, supporting the genetic modification strategy for incorporating conversion catalysts into energy crops prior to harvesting or processing at the biorefinery.
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    Plant genome editing with TALEN and CRISPR
    (Springer Nature, 2017-04-24) Malzahn, Aimee; Lowder, Levi; Qi, Yiping
    Genome editing promises giant leaps forward in advancing biotechnology, agriculture, and basic research. The process relies on the use of sequence specific nucleases (SSNs) to make DNA double stranded breaks at user defined genomic loci, which are subsequently repaired by two main DNA repair pathways: non-homologous end joining (NHEJ) and homology directed repair (HDR). NHEJ can result in frameshift mutations that often create genetic knockouts. These knockout lines are useful for functional and reverse genetic studies but also have applications in agriculture. HDR has a variety of applications as it can be used for gene replacement, gene stacking, and for creating various fusion proteins. In recent years, transcription activator-like effector nucleases and clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR associated protein 9 or CRISPR from Prevotella and Francisella 1 have emerged as the preferred SSNs for research purposes. Here, we review their applications in plant research, discuss current limitations, and predict future research directions in plant genome editing.
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    Genome editing is revolutionizing biology
    (Springer Nature, 2017-07-14) Qi, Yiping
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    A large-scale whole-genome sequencing analysis reveals highly specific genome editing by both Cas9 and Cpf1 (Cas12a) nucleases in rice
    (Springer Nature, 2018-07-04) Tang, Xu; Liu, Guanqing; Zhou, Jianping; Ren, Qiurong; You, Qi; Tian, Li; Xin, Xuhui; Zhong, Zhaohui; Liu, Binglin; Zheng, Xuelian; Zhang, Dengwei; Malzahn, Aimee; Gong, Zhiyun; Qi, Yiping; Zhang, Tao; Zhang, Yong
    Targeting specificity has been a barrier to applying genome editing systems in functional genomics, precise medicine and plant breeding. In plants, only limited studies have used whole-genome sequencing (WGS) to test off-target effects of Cas9. The cause of numerous discovered mutations is still controversial. Furthermore, WGS-based off-target analysis of Cpf1 (Cas12a) has not been reported in any higher organism to date. We conduct a WGS analysis of 34 plants edited by Cas9 and 15 plants edited by Cpf1 in T0 and T1 generations along with 20 diverse control plants in rice. The sequencing depths range from 45× to 105× with read mapping rates above 96%. Our results clearly show that most mutations in edited plants are created by the tissue culture process, which causes approximately 102 to 148 single nucleotide variations (SNVs) and approximately 32 to 83 insertions/deletions (indels) per plant. Among 12 Cas9 single guide RNAs (sgRNAs) and three Cpf1 CRISPR RNAs (crRNAs) assessed by WGS, only one Cas9 sgRNA resulted in off-target mutations in T0 lines at sites predicted by computer programs. Moreover, we cannot find evidence for bona fide off-target mutations due to continued expression of Cas9 or Cpf1 with guide RNAs in T1 generation. Our comprehensive and rigorous analysis of WGS data across multiple sample types suggests both Cas9 and Cpf1 nucleases are very specific in generating targeted DNA modifications and off-targeting can be avoided by designing guide RNAs with high specificity.
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    Comparative genome analyses reveal sequence features reflecting distinct modes of host-adaptation between dicot and monocot powdery mildew
    (Springer Nature, 2018-09-25) Wu, Ying; Ma, Xianfeng; Pan, Zhiyong; Kale, Shiv D.; Song, Yi; King, Harlan; Zhang, Qiong; Presley, Christian; Deng, Xiuxin; Wei, Cheng-I; Xiao, Shunyuan
    Powdery mildew (PM) is one of the most important and widespread plant diseases caused by biotrophic fungi. Notably, while monocot (grass) PM fungi exhibit high-level of host-specialization, many dicot PM fungi display a broad host range. To understand such distinct modes of host-adaptation, we sequenced the genomes of four dicot PM biotypes belonging to Golovinomyces cichoracearum or Oidium neolycopersici. We compared genomes of the four dicot PM together with those of Blumeria graminis f.sp. hordei (both DH14 and RACE1 isolates), B. graminis f.sp. tritici, and Erysiphe necator infectious on barley, wheat and grapevine, respectively. We found that despite having a similar gene number (6620–6961), the PM genomes vary from 120 to 222 Mb in size. This high-level of genome size variation is indicative of highly differential transposon activities in the PM genomes. While the total number of genes in any given PM genome is only about half of that in the genomes of closely related ascomycete fungi, most (~ 93%) of the ascomycete core genes (ACGs) can be found in the PM genomes. Yet, 186 ACGs were found absent in at least two of the eight PM genomes, of which 35 are missing in some dicot PM biotypes, but present in the three monocot PM genomes, indicating remarkable, independent and perhaps ongoing gene loss in different PM lineages. Consistent with this, we found that only 4192 (3819 singleton) genes are shared by all the eight PM genomes, the remaining genes are lineage- or biotype-specific. Strikingly, whereas the three monocot PM genomes possess up to 661 genes encoding candidate secreted effector proteins (CSEPs) with families containing up to 38 members, all the five dicot PM fungi have only 116–175 genes encoding CSEPs with limited gene amplification. Compared to monocot (grass) PM fungi, dicot PM fungi have a much smaller effectorome. This is consistent with their contrasting modes of host-adaption: while the monocot PM fungi show a high-level of host specialization, which may reflect an advanced host-pathogen arms race, the dicot PM fungi tend to practice polyphagy, which might have lessened selective pressure for escalating an with a particular host.
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    Virome analyses of Hevea brasiliensis using small RNA deep sequencing and PCR techniques reveal the presence of a potential new virus
    (Springer Nature, 2018-11-26) Fonseca, Paula L. C.; Badotti, Fernanda; de Oliveira, Tatiana F. P.; Fonseca, Antônio; Vaz, Aline B. M.; Tomé, Luiz M. R.; Abrahão, Jônatas S.; Marques, João T.; Trindade, Giliane S.; Chaverri, Priscila; Aguiar, Eric R. G. R.; Góes-Neto, Aristóteles
    Hevea brasiliensis is an important commercial crop due to the high quality of the latex it produces; however, little is known about viral infections in this plant. The only virus described to infect H. brasiliensis until now is a Carlavirus, which was described more than 30 years ago. Virus-derived small interfering RNA (vsiRNAs) are the product of the plant’s antiviral defense triggered by dsRNA viral intermediates generated, during the replication cycle. These vsiRNAs are complementar to viral genomes and have been widely used to identify and characterize viruses in plants. In the present study, we investigated the virome of leaf and sapwood samples from native H. brasiliensis trees collected in two geographic areas in the Brazilian Amazon. Small RNA (sRNA) deep sequencing and bioinformatic tools were used to assembly, identify and characterize viral contigs. Subsequently, PCR amplification techniques were performed to experimentally verify the presence of the viral sequences. Finally, the phylogenetic relationship of the putative new virus with related viral genomes was analyzed. Our strategy allowed the identification of 32 contigs with high similarity to viral reference genomes, from which 23 exhibited homology to viruses of the Tymoviridae family. The reads showed a predominant size distribution at 21 nt derived from both strands, which was consistent with the vsiRNAs profile. The presence and genome position of the viral contigs were experimentally confirmed using droplet digital PCR amplifications. A 1913 aa long fragment was obtained and used to infer the phylogenetic relationship of the putative new virus, which indicated that it is taxonomically related to the Grapevine fleck virus, genus Maculavirus. The putative new virus was named Hevea brasiliensis virus (HBrV) in reference to its host. The methodological strategy applied here proved to be efficient in detecting and confirming the presence of new viral sequences on a ‘very difficult to manage’ sample. This is the second time that viral sequences, that could be ascribed as a putative novel virus, associated to the rubber tree has been identified.
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    Application of CRISPR-Cas12a temperature sensitivity for improved genome editing in rice, maize, and Arabidopsis
    (Springer Nature, 2019-01-31) Malzahn, Aimee A.; Tang, Xu; Lee, Keunsub; Ren, Qiurong; Sretenovic, Simon; Zhang, Yingxiao; Chen, Hongqiao; Kang, Minjeong; Bao, Yu; Zheng, Xuelian; Deng, Kejun; Zhang, Tao; Salcedo, Valeria; Wang, Kan; Zhang, Yong; Qi, Yiping
    CRISPR-Cas12a (formerly Cpf1) is an RNA-guided endonuclease with distinct features that have expanded genome editing capabilities. Cas12a-mediated genome editing is temperature sensitive in plants, but a lack of a comprehensive understanding on Cas12a temperature sensitivity in plant cells has hampered effective application of Cas12a nucleases in plant genome editing. We compared AsCas12a, FnCas12a, and LbCas12a for their editing efficiencies and non-homologous end joining (NHEJ) repair profiles at four different temperatures in rice. We found that AsCas12a is more sensitive to temperature and that it requires a temperature of over 28 °C for high activity. Each Cas12a nuclease exhibited distinct indel mutation profiles which were not affected by temperatures. For the first time, we successfully applied AsCas12a for generating rice mutants with high frequencies up to 93% among T0 lines. We next pursued editing in the dicot model plant Arabidopsis, for which Cas12a-based genome editing has not been previously demonstrated. While LbCas12a barely showed any editing activity at 22 °C, its editing activity was rescued by growing the transgenic plants at 29 °C. With an early high-temperature treatment regime, we successfully achieved germline editing at the two target genes, GL2 and TT4, in Arabidopsis transgenic lines. We then used high-temperature treatment to improve Cas12a-mediated genome editing in maize. By growing LbCas12a T0 maize lines at 28 °C, we obtained Cas12a-edited mutants at frequencies up to 100% in the T1 generation. Finally, we demonstrated DNA binding of Cas12a was not abolished at lower temperatures by using a dCas12a-SRDX-based transcriptional repression system in Arabidopsis. Our study demonstrates the use of high-temperature regimes to achieve high editing efficiencies with Cas12a systems in rice, Arabidopsis, and maize and sheds light on the mechanism of temperature sensitivity for Cas12a in plants.