The nuclear lamina is required for proper development and nuclear shape distortion in tomato.

The nuclear lamina in plant cells is composed of plant-specific proteins, including nuclear matrix constituent proteins (NMCPs), which have been postulated to be functional analogs of lamin proteins that provide structural integrity to the organelle and help stabilize the three-dimensional organization of the genome. Using genomic editing, we generated alleles for the three genes encoding NMCPs in cultivated tomato (Solanum lycopersicum) to determine if the consequences of perturbing the nuclear lamina in this crop species were similar to or distinct from those observed in the model Arabidopsis thaliana. Loss of the sole NMCP2-class protein was lethal in tomato but is tolerated in Arabidopsis. Moreover, depletion of NMCP1-type nuclear lamina proteins leads to distinct developmental phenotypes in tomato, including leaf morphology defects and reduced root growth rate (in nmcp1b mutants), compared with cognate mutants in Arabidopsis. These findings suggest that the nuclear lamina interfaces with different developmental and signaling pathways in tomato compared with Arabidopsis. At the subcellular level, however, tomato nmcp mutants resembled their Arabidopsis counterparts in displaying smaller and more spherical nuclei in differentiated cells. This result argues that the plant nuclear lamina facilitates nuclear shape distortion in response to forces exerted on the organelle within the cell.

Expression of Pinellia ternata leaf agglutinin under rolC promoter confers resistance against a phytophagous sap sucking aphid, Myzus persicae

BACKGROUND: Piercing/sucking insect pests in the order Hemiptera causes substantial crop losses by removing photoassimilates and transmitting viruses to their host plants. Cloning and heterologous expression of plantderived insect resistance genes is a promising approach to control aphids and other sap-sucking insect pests. While expression from the constitutive 35S promoter provides broad protection, the phloem-specific rolC promoter provides better defense against sap sucking insects. The selection of plant-derived insect resistance genes for expression in crop species will minimize bio-safety concerns. RESULTS: Pinellia ternata leaf agglutinin gene (pta), encodes an insecticidal lectin, was isolated and cloned under the 35S and rolC promoters in the pGA482 plant transformation vector for Agrobacterium-mediated tobacco transformation. Integration and expression of the transgene was validated by Southern blotting and qRT-PCR, respectively. Insect bioassays data of transgenic tobacco plants showed that expression of pta under rolC promoter caused 100% aphid mortality and reduced aphid fecundity up to 70% in transgenic tobacco line LRP9. These results highlight the better effectivity of pta under rolC promoter to control phloem feeders, aphids. CONCLUSIONS: These findings suggested the potential of PTA against aphids and other sap sucking insect pests. Evaluation of gene in tobacco under two different promoters; 35S constitutive promoter and rolC phloemspecific promoter could be successfully use for other crop plants particularly in cotton. Development of transgenic cotton plants using plant-derived insecticidal, PTA, would be key step towards commercialization of environmentally safe insect-resistant crops.

Agrobacterium tumefaciens-Mediated Transformation of Three Milkweed Species (Asclepias hallii, A. syriaca, and A. tuberosa: Apocynaceae).

Milkweeds have ecological significance for insect herbivores that rely on them as hosts for either part of or the entirety of their life cycles. Interesting interactions, some of which are not completely understood, have evolved over time. To develop these species as models to elucidate the interplay with insect herbivores, we established Agrobacterium tumefaciens-mediated transformation approaches for Asclepias hallii (Hall's milkweed), A. syriaca (common milkweed), and A. tuberosa (butterflyweed). The method is based on infection of stem internodal explants, which were more amenable to transformation than leaf explants. We found that addition of freshly prepared dithiothreitol was critical to prevent browning of stem explants. Depending on the species, the time from infection to the regeneration of transgenic lines ranges from 2 to 4 months. Transformation efficiency for A. hallii was 9%, whereas efficiencies for A. syriaca and A. tuberosa were 6% and 13%, respectively. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Agrobacterium tumefaciens-mediated transformation of Asclepias internodal stem explants Basic Protocol 2: Preparation of Agrobacterium glycerol stocks containing gene constructs.

Agrobacterium tumefaciens-Mediated Transformation of Tomato.

Tomato is both an important food crop and serves as a model plant species that is used for various research investigations including understanding gene function. Transformation is commonly utilized to facilitate these investigations in combination with all the extensive genetic and genomic resources available for tomato. The transformation protocol routinely used in our laboratory has been applied to many different tomato genotypes and relies on Agrobacterium tumefaciens infection of young cotyledon sections. We have used vector systems for overexpression, RNA interference for gene silencing, and CRISPR/Cas9 for genome editing. Vectors used to design gene constructs contained selectable marker genes that conferred resistance to kanamycin, hygromycin, and the herbicide component, bialaphos. The protocol we follow for Agrobacterium-mediated transformation of both cultivated and wild species of tomato is detailed in this chapter.

Seasonal dynamics of tetracycline resistance gene transport in the Sumas River agricultural watershed of British Columbia, Canada.

Environmental transport of contaminants that can influence the development of antibiotic resistance in bacteria is an important concern in the management of ecological and human health risks. Agricultural regions are locales where practices linked to food crop and livestock production can introduce contaminants that could alter the selective pressures for the development of antibiotic resistance in microbiota. This is important in regions where the use of animal manure or municipal biosolids as waste and/or fertilizer could influence selection for antibiotic resistance in pathogenic bacterial species. To investigate the environmental transport of contaminants that could lead to the development of antibiotic resistance in bacteria, a watershed with one of the highest levels of intensity of agricultural activity in Canada was studied; the Sumas River located 60 km east of Vancouver, British Columbia. This two-year assessment monitored four selected tetracycline resistance genes (tet(O), tet(M), tet(Q), tet(W)) and water quality parameters (temperature, specific conductivity, turbidity, suspended solids, nitrate, phosphate and chloride) at eight locations across the watershed. The tetracycline resistance genes (Tcr) abundances in the Sumas River network ranged between 1.47 × 102 and 3.49 × 104 copies/mL and ranged between 2.3 and 6.9 copies/mL in a control stream (located far from agricultural activities) for the duration of the study. Further, Tcr abundances that were detected in the wet season months ranged between 1.3 × 103 and 2.29 × 104 copies/mL compared with dry season months (ranging between 0.6 and 31.2 copies/mL). Highest transport rates between 1.67 × 1011 and 1.16 × 1012 copies/s were observed in November 2005 during periods of high rainfall. The study showed that elevated concentrations of antibiotic resistance genes in the order of 102-104 copies/mL can move through stream networks in an agricultural watershed but seasonal variations strongly influenced specific transport patterns of these genes.

How Should We Be Determining Background and Baseline Antibiotic Resistance Levels in Agroecosystem Research?

Although historically, antibiotic resistance has occurred naturally in environmental bacteria, many questions remain regarding the specifics of how humans and animals contribute to the development and spread of antibiotic resistance in agroecosystems. Additional research is necessary to completely understand the potential risks to human, animal, and ecological health in systems altered by antibiotic-resistance-related contamination. At present, analyzing and interpreting the effects of human and animal inputs on antibiotic resistance in agroecosystems is difficult, since standard research terminology and protocols do not exist for studying background and baseline levels of resistance in the environment. To improve the state of science in antibiotic-resistance-related research in agroecosystems, researchers are encouraged to incorporate baseline data within the study system and background data from outside the study system to normalize the study data and determine the potential impact of antibiotic-resistance-related determinants on a specific agroecosystem. Therefore, the aims of this review were to (i) present standard definitions for commonly used terms in environmental antibiotic resistance research and (ii) illustrate the need for research standards (normalization) within and between studies of antibiotic resistance in agroecosystems. To foster synergy among antibiotic resistance researchers, a new surveillance and decision-making tool is proposed to assist researchers in determining the most relevant and important antibiotic-resistance-related targets to focus on in their given agroecosystems. Incorporation of these components within antibiotic-resistance-related studies should allow for a more comprehensive and accurate picture of the current and future states of antibiotic resistance in the environment.

Mechanisms of antimicrobial resistance in finfish aquaculture environments.

Consumer demand for affordable fish drives the ever-growing global aquaculture industry. The intensification and expansion of culture conditions in the production of several finfish species has been coupled with an increase in bacterial fish disease and the need for treatment with antimicrobials. Understanding the molecular mechanisms of antimicrobial resistance prevalent in aquaculture environments is important to design effective disease treatment strategies, to prioritize the use and registration of antimicrobials for aquaculture use, and to assess and minimize potential risks to public health. In this brief article we provide an overview of the molecular mechanisms of antimicrobial resistance in genes found in finfish aquaculture environments and highlight specific research that should provide the basis of sound, science-based policies for the use of antimicrobials in aquaculture.

Tracking Change: A Look at the Ecological Footprint of Antibiotics and Antimicrobial Resistance.

Among the class of pollutants considered as 'emerging contaminants', antibiotic compounds including drugs used in medical therapy, biocides and disinfectants merit special consideration because their bioactivity in the environment is the result of their functional design. Antibiotics can alter the structure and function of microbial communities in the receiving environment and facilitate the development and spread of resistance in critical species of bacteria including pathogens. Methanogenesis, nitrogen transformation and sulphate reduction are among the key ecosystem processes performed by bacteria in nature that can also be affected by the impacts of environmental contamination by antibiotics. Together, the effects of the development of resistance in bacteria involved in maintaining overall ecosystem health and the development of resistance in human, animal and fish pathogens, make serious contributions to the risks associated with environmental pollution by antibiotics. In this brief review, we discuss the multiple impacts on human and ecosystem health of environmental contamination by antibiotic compounds.

Fate of tetracycline resistance genes in aquatic systems: migration from the water column to peripheral biofilms.

Antibiotic resistance genes (ARGs) are emerging contaminants that are being found at elevated levels in sediments and other aquatic compartments in areas of intensive agricultural and urban activity. However, little quantitative data exist on the migration and attenuation of ARGs in natural ecosystems, which is central to predicting their fate after release into receiving waters. Here we examined the fate of tetracycline-resistance genes in bacterial hosts released in cattle feedlot wastewater using field-scale mesocosms to quantify ARG attenuation rate in the water column and also the migration of ARGs into peripheral biofilms. Feedlot wastewater was added to fifteen cylindrical 11.3-m3 mesocosms (some of which had artificial substrates) simulating five different receiving water conditions (in triplicate), and the abundance of six resistance genes (tet(O), tet(W), tet(M), tet(Q), tet(B), and tet(L)) and 16S-rRNA genes was monitored for 14 days. Mesocosm treatments were varied according to light supply, microbial supplements (via river water additions), and oxytetracycline (OTC) level. First-order water column disappearance coefficients (kd) for the sum of the six genes (tetR) were always higher in sunlight than in the dark (-0.72 d(-1) and -0.51 d(-1), respectively). However, water column kd varied among genes (tet(O) < tet(W) < tet(M) < tet(Q); tet(B) and tet(L) were below detection) and some genes, particularly tet(W), readily migrated into biofilms, suggesting that different genes be considered separately and peripheral compartments be included in future fate models. This work provides the first quantitative field data for modeling ARG fate in aquatic systems.

Indirect evidence of transposon-mediated selection of antibiotic resistance genes in aquatic systems at low-level oxytetracycline exposures.

Subinhibitory levels of antibiotics can promote the development of antibiotic resistance in bacteria. However, it is unclear whether antibiotic concentrations released into aquatic systems exert adequate pressure to select populations with resistance traits. To examine this issue, 15 mesocosms containing pristine surface water were treated with oxytetracycline (OTC) for 56 days at five levels (0, 5, 20, 50, and 250 microg L(-1)), and six tetracycline-resistance genes (tet(B), tet(L), tet(M), ted(O), tet(Q), and tet(W)), the sum of those genes (tet(R)), "total" 16S-rRNA genes, and transposons (Tn916 and Tn 1545) were monitored using real-time PCR. Absolute water-column resistance-gene abundances did not change at any OTC exposure. However, an increase was observed in the ratio of tet(R) to 16S-rRNA genes in the 250 microg L(-1) OTC units, and an increase in the selection rate of Tc(r) genes (relative to 16S-rRNA genes) was seen when OTC levels were at 20 microg L(-1). Furthermore, tet(M) and Tn916/1545 gene abundances correlated among all treatments (r2 = 0.701, p = 0.05), and there were similar selection patterns of tetR and Tn916/1545 genes relative to the OTC level, suggesting a possible mechanism for retention of specific resistance genes within the systems.

Effects of dietary exposure of 4-nonylphenol on growth and smoltification of juvenile coho salmon (Oncorhynchus kisutch).

There is considerable concern that endocrine disrupting substances such as 4-nonylphenol (4-NP) in the freshwater environment may have adverse effects on the growth, survival, and osmoregulatory ability of salmonids during and after their transfer to sea water. This study was conducted to examine the effects of dietary exposure of coho salmon (Oncorhynchus kisutch) to 4-NP during the parr-smolt transformation phase of their life cycle. Under laboratory conditions, juvenile fish were fed by hand twice daily to satiation diets dosed with one of several concentrations of 4-NP (doses varied between 0 (control) and 2000 mg/kg) for 4 weeks, then immediately transferred to sea water. Growth was observed for two successive 6-week periods following sea water transfer when all groups were fed the control diet (no supplemental 4-NP) only. In addition to 4-NP measurement in fish tissues, thyroid hormone concentrations in blood plasma were followed and related to diet treatment and sampling time. Dietary treatment of 4-NP did not influence the growth and smoltification of coho salmon, a result that conflicts to some extent with other reports in which deleterious effects of water-borne 4-NP on the smoltification process of salmonids were linked to disruption of the endocrine system. Appreciable concentrations of 4-NP were present in the livers, gall bladders and tissues after the 4-week exposure of coho salmon to the highest dietary dose of 4-NP, but 4-NP appeared to be effectively eliminated from the fish by the biliary-fecal pathway after sea water transfer.

Sources, pathways, and relative risks of contaminants in surface water and groundwater: a perspective prepared for the Walkerton inquiry.

On a global scale, pathogenic contamination of drinking water poses the most significant health risk to humans, and there have been countless numbers of disease outbreaks and poisonings throughout history resulting from exposure to untreated or poorly treated drinking water. However, significant risks to human health may also result from exposure to nonpathogenic, toxic contaminants that are often globally ubiquitous in waters from which drinking water is derived. With this latter point in mind, the objective of this commission paper is to discuss the primary sources of toxic contaminants in surface waters and groundwater, the pathways through which they move in aquatic environments, factors that affect their concentration and structure along the many transport flow paths, and the relative risks that these contaminants pose to human and environmental health. In assessing the relative risk of toxic contaminants in drinking water to humans, we have organized our discussion to follow the classical risk assessment paradigm, with emphasis placed on risk characterization. In doing so, we have focused predominantly on toxic contaminants that have had a demonstrated or potential effect on human health via exposure through drinking water. In the risk assessment process, understanding the sources and pathways for contaminants in the environment is a crucial step in addressing (and reducing) uncertainty associated with estimating the likelihood of exposure to contaminants in drinking water. More importantly, understanding the sources and pathways of contaminants strengthens our ability to quantify effects through accurate measurement and testing, or to predict the likelihood of effects based on empirical models. Understanding the sources, fate, and concentrations of chemicals in water, in conjunction with assessment of effects, not only forms the basis of risk characterization, but also provides critical information required to render decisions regarding regulatory initiatives, remediation, monitoring, and management. Our discussion is divided into two primary themes. First we discuss the major sources of contaminants from anthropogenic activities to aquatic surface and groundwater and the pathways along which these contaminants move to become incorporated into drinking water supplies. Second, we assess the health significance of the contaminants reported and identify uncertainties associated with exposures and potential effects. Loading of contaminants to surface waters, groundwater, sediments, and drinking water occurs via two primary routes: (1) point-source pollution and (2) non-point-source pollution. Point-source pollution originates from discrete sources whose inputs into aquatic systems can often be defined in a spatially explicit manner. Examples of point-source pollution include industrial effluents (pulp and paper mills, steel plants, food processing plants), municipal sewage treatment plants and combined sewage-storm-water overflows, resource extraction (mining), and land disposal sites (landfill sites, industrial impoundments). Non-point-source pollution, in contrast, originates from poorly defined, diffuse sources that typically occur over broad geographical scales. Examples of non-point-source pollution include agricultural runoff (pesticides, pathogens, and fertilizers), storm-water and urban runoff, and atmospheric deposition (wet and dry deposition of persistent organic pollutants such as polychlorinated biphenyls [PCBs] and mercury). Within each source, we identify the most important contaminants that have either been demonstrated to pose significant risks to human health and/or aquatic ecosystem integrity, or which are suspected of posing such risks. Examples include nutrients, metals, pesticides, persistent organic pollutants (POPs), chlorination by-products, and pharmaceuticals. Due to the significant number of toxic contaminants in the environment, we have necessarily restricted our discussion to those chemicals that pose risks to human health via exposure through drinking water. A comprehensive and judicious consideration of the full range of contaminants that occur in surface waters, sediments, and drinking water would be a large undertaking and clearly beyond the scope of this article. However, where available, we have provided references to relevant literature to assist the reader in undertaking a detailed investigation of their own. The information collected on specific chemicals within major contaminant classes was used to determine their relative risk using the hazard quotient (HQ) approach. Hazard quotients are the most widely used method of assessing risk in which the exposure concentration of a stressor, either measured or estimated, is compared to an effect concentration (e.g., no-observed-effect concentration or NOEC). A key goal of this assessment was to develop a perspective on the relative risks associated with toxic contaminants that occur in drinking water. Data used in this assessment were collected from literature sources and from the Drinking Water Surveillance Program (DWSP) of Ontario. For many common contaminants, there was insufficient environmental exposure (concentration) information in Ontario drinking water and groundwater. Hence, our assessment was limited to specific compounds within major contaminant classes including metals, disinfection by-products, pesticides, and nitrates. For each contaminant, the HQ was estimated by expressing the maximum concentration recorded in drinking water as a function of the water quality guideline for that compound. There are limitations to using the hazard quotient approach of risk characterization. For example, HQs frequently make use of worst-case data and are thus designed to be protective of almost all possible situations that may occur. However, reduction of the probability of a type II error (false negative) through the use of very conservative application factors and assumptions can lead to the implementation of expensive measures of mitigation for stressors that may pose little threat to humans or the environment. It is important to realize that our goal was not to conduct a comprehensive, in-depth assessment of risk for each chemical; more comprehensive assessments of managing risks associated with drinking water are addressed in a separate issue paper by Krewski et al. (2001a). Rather, our goal was to provide the reader with an indication of the relative risk of major contaminant classes as a basis for understanding the risks associated with the myriad forms of toxic pollutants in aquatic systems and drinking water. For most compounds, the estimated HQs were < 1. This indicates that there is little risk associated with exposure from drinking water to the compounds tested. There were some exceptions. For example, nitrates were found to commonly yield HQ values well above 1 in- many rural areas. Further, lead, total trihalomethanes, and trichloroacetic acid yielded HQs > 1 in some treated distribution waters (water distributed to households). These latter compounds were further assessed using a probabilistic approach; these assessments indicated that the maximum allowable concentrations (MAC) or interim MACs for the respective compounds were exceeded <5% of the time. In other words, the probability of finding these compounds in drinking water at levels that pose risk to humans through ingestion of drinking water is low. Our review has been carried out in accordance with the conventional principles of risk assessment. Application of the risk assessment paradigm requires rigorous data on both exposure and toxicity in order to adequately characterize potential risks of contaminants to human health and ecological integrity. Weakness rendered by poor data, or lack of data, in either the exposure or effects stages of the risk assessment process significantly reduces the confidence that can be placed in the overall risk assessment. (ABSTRACT TRUNCATED)