Deformed wing virus infection affects the neurological function of Apis mellifera by altering extracellular adenosine signaling.

Deformed wing virus (DWV) infection is believed to be closely associated with colony losses of honeybee (Apis mellifera) due to reduced learning and memory of infected bees. The adenosine (Ado) pathway is important for maintaining immunity and memory function in animals, and it enhances antivirus responses by regulating carbohydrate metabolism in insects. Nevertheless, its effect on the memory of invertebrates is not yet clear. This study investigated how the Ado pathway regulates energy metabolism and memory in honeybees following DWV infection. Decreased Ado receptor (Ado-R) expression in the brain of infected bees resulted in a carbohydrate imbalance as well as impairments of glutamate-glutamine (Glu-Gln) cycle and long-term memory. Dietary supplementation with Ado not only increased the brain energy metabolism but also rescued long-term memory loss by upregulating the expression of memory-related genes. The present study demonstrated the regulation of the Ado pathway upon DWV infection and provides insights into the mechanisms underlying energy regulation and the neurological function of honeybees.

Real-time monitoring of deformed wing virus-infected bee foraging behavior following histone deacetylase inhibitor treatment.

Impairment in the learning/memory behavior of bees is responsible for the massive disappearance of bee populations and its consequent agricultural economic losses. Such impairment might be because of o both pesticide exposure and pathogen infection, with a key contributor deformed wing virus (DWV). The present study found that sodium butyrate (NaB) significantly increased survival and reversed the learning/memory impairment of DWV-infected bees. A next-generation sequencing analysis showed that NaB affected the expression of genes involved in glycolytic processes and memory formation, which were suppressed by DWV infection. In addition, we performed a large-scale movement tracking experiment by using a wireless sensor network-based automatic real-time monitoring system and confirmed that NaB could improve the homing ability of DWV-infected bees. In short, we demonstrated the mechanism of how epigenetic regulation can resume the memory function of honeybees and suggest strategies for applying NaB to reduce the incidence of colony losses.

MicroRNAs from Snellenius manilae bracovirus regulate innate and cellular immune responses of its host Spodoptera litura.

To avoid inducing immune and physiological responses in insect hosts, parasitoid wasps have developed several mechanisms to inhibit them during parasitism, including the production of venom, specialized wasp cells, and symbioses with polydnaviruses (PDVs). These mechanisms alter the host physiology to give the wasp offspring a greater chance of survival. However, the molecular mechanisms for most of these alterations remain unclear. In the present study, we applied next-generation sequencing analysis and identified several miRNAs that were encoded in the genome of Snellenius manilae bracovirus (SmBV), and expressed in the host larvae, Spodoptera litura, during parasitism. Among these miRNAs, SmBV-miR-199b-5p and SmBV-miR-2989 were found to target domeless and toll-7 in the host, which are involved in the host innate immune responses. Microinjecting the inhibitors of these two miRNAs into parasitized S. litura larvae not only severely decreased the pupation rate of Snellenius manilae, but also restored the phagocytosis and encapsulation activity of the hemocytes. The results demonstrate that these two SmBV-encoded miRNAs play an important role in suppressing the immune responses of parasitized hosts. Overall, our study uncovers the functions of two SmBV-encoded miRNAs in regulating the host innate immune responses upon wasp parasitism.

Identification of Immune Regulatory Genes in Apis mellifera through Caffeine Treatment.

Plants and pollinators are mutually beneficial: plants provide nectar as a food source and in return their pollen is disseminated by pollinators such as honeybees. Some plants secrete chemicals to deter herbivores as a protective measure, among which is caffeine, a naturally occurring, bitter tasting, and pharmacologically active secondary compound. It can be found in low concentrations in the nectars of some plants and as such, when pollinators consume nectar, they also take in small amounts of caffeine. Whilst caffeine has been indicated as an antioxidant in both mammals and insects, the effect on insect immunity is unclear. In the present study, honeybees were treated with caffeine and the expression profiles of genes involved in immune responses were measured to evaluate the influence of caffeine on immunity. In addition, honeybees were infected with deformed wing virus (DWV) to study how caffeine affects their response against pathogens. Our results showed that caffeine can increase the expression of genes involved in immunity and reduce virus copy numbers, indicating that it has the potential to help honeybees fight against viral infection. The present study provides a valuable insight into the mechanism by which honeybees react to biotic stress and how caffeine can serve as a positive contributor, thus having a potential application in beekeeping.

Adenosine Receptor Modulates Permissiveness of Baculovirus (Budded Virus) Infection via Regulation of Energy Metabolism in Bombyx mori.

Although the modulation of host physiology has been interpreted as an essential process supporting baculovirus propagation, the requirement of energy supply for host antivirus reactions could not be ruled out. Our present study showed that metabolic induction upon AcMNPV (budded virus) infection of Bombyx mori stimulated virus clearance and production of the antivirus protein, gloverin. In addition, we demonstrated that adenosine receptor signaling (AdoR) played an important role in regulating such metabolic reprogramming upon baculovirus infection. By using a second lepidopteran model, Spodoptera frugiperda Sf-21 cells, we demonstrated that the glycolytic induction regulated by adenosine signaling was a conservative mechanism modulating the permissiveness of baculovirus infection. Another interesting finding in our present study is that both BmNPV and AcMNPV infection cause metabolic activation, but it appears that BmNPV infection moderates the level of ATP production, which is in contrast to a dramatic increase upon AcMNPV infection. We identified potential AdoR miRNAs induced by BmNPV infection and concluded that BmNPV may attempt to minimize metabolic activation by suppressing adenosine signaling and further decreasing the host's anti-baculovirus response. Our present study shows that activation of energy synthesis by adenosine signaling upon baculovirus infection is a host physiological response that is essential for supporting the innate immune response against infection.

Identification of Regulatory Host Genes Involved in Sigma Virus Replication Using RNAi Knockdown in Drosophila.

The Drosophila melanogaster sigma virus, a member of the Rhabdoviridae family, specifically propagates itself in D. melanogaster. It contains six genes in the order of 3'-N-P-X-M-G-L-5'. The sigma virus is the only arthropod-specific virus of the Rhabdoviridae family. Sigma-virus-infected Drosophila may suffer from irreversible paralysis when exposed to a high CO2 concentration, but generally, no other symptoms are reported. A recent study reported that host gene expression in immune pathways was not changed in sigma-virus-infected Drosophila, which does not necessarily suggest that they are not involved in virus-host interactions. The present study aimed to identify host genes associated with sigma virus replication. Immune pathways JAK-STAT and IMD were selected for detailed study. The results showed that the genome copy number of the sigma virus increased after knocking down the immune pathway genes domeless and PGRP-LC in Drosophila S2 cells. The knocking down of domeless and PGRP-LC significantly up-regulated the expression of the L gene compared to the other viral genes. We propose that the immune pathways respond to sigma virus infection by altering L expression, hence suppressing viral replication. This effect was further tested in vivo, when D. melanogaster individuals injected with dsdome and dsPGRP-LC showed not only an increase in sigma virus copy number, but also a reduced survival rate when treated with CO2. Our study proved that host immunity influences viral replication, even in persistent infection. Knocking down the key components of the immune process deactivates immune controls, thus facilitating viral expression and replication. We propose that the immunity system of D. melanogaster regulates the replication of the sigma virus by affecting the L gene expression. Studies have shown minimal host-virus interaction in persistent infection. However, our study demonstrated that the immunity continued to affect viral replication even in persistent infection because knocking down the key components of the immune process disabled the relevant immune controls and facilitated viral expression and replication.

MicroRNAs derived from the insect virus HzNV-1 promote lytic infection by suppressing histone methylation.

Heliothis zea nudivirus-1 (HzNV-1) is an insect virus that can induce both lytic and latent infections in various insect cell lines. During latent infection, several microRNAs (miRNAs) are produced from persistency-associated gene 1 (pag1) as the only detectable HzNV-1 transcript. Previous studies have shown that the pag1 gene suppresses the immediate-early gene hhi1 and promotes host switching into a latent infection via miRNAs derived from pag1. Although other functions of the miRNAs derived from pag1 have not yet been elucidated, several studies have suggested that miRNAs encoded from latency-associated genes can regulate histone-associated enzymes. Because pag1 is a noncoding transcript, it potentially regulates host chromatin structure through miRNAs upon infection. Nevertheless, the exact mechanism by which pag1 alters viral infections remains unknown. In this study, we found that the pag1-encoded miRNA miR-420 suppresses expression of the histone modification-associated enzyme su(var)3-9. Therefore, this miRNA causes histone modification to promote HzNV-1 infection. These results suggest that HzNV-1 may directly influence epigenetic regulation in host cells through interactions with pag1 miRNAs to promote lytic infection. This study provides us with a better understanding of both the HzNV-1 infection pathway and the relationship between viral miRNAs and epigenetic regulation.

Identification of a high-efficiency baculovirus DNA replication origin that functions in insect and mammalian cells.

UNLABELLED: The p143 gene from Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) has been found to increase the expression of luciferase, which is driven by the polyhedrin gene promoter, in a plasmid with virus coinfection. Further study indicated that this is due to the presence of a replication origin (ori) in the coding region of this gene. Transient DNA replication assays showed that a specific fragment of the p143 coding sequence, p143-3, underwent virus-dependent DNA replication in Spodoptera frugiperda IPLB-Sf-21 (Sf-21) cells. Deletion analysis of the p143-3 fragment showed that subfragment p143-3.2a contained the essential sequence of this putative ori. Sequence analysis of this region revealed a unique distribution of imperfect palindromes with high AT contents. No sequence homology or similarity between p143-3.2a and any other known ori was detected, suggesting that it is a novel baculovirus ori. Further study showed that the p143-3.2a ori can replicate more efficiently in infected Sf-21 cells than baculovirus homologous regions (hrs), the major baculovirus ori, or non-hr oris during virus replication. Previously, hr on its own was unable to replicate in mammalian cells, and for mammalian viral oris, viral proteins are generally required for their proper replication in host cells. However, the p143-3.2a ori was, surprisingly, found to function as an efficient ori in mammalian cells without the need for any viral proteins. We conclude that p143 contains a unique sequence that can function as an ori to enhance gene expression in not only insect cells but also mammalian cells. IMPORTANCE: Baculovirus DNA replication relies on both hr and non-hr oris; however, so far very little is known about the latter oris. Here we have identified a new non-hr ori, the p143 ori, which resides in the coding region of p143. By developing a novel DNA replication-enhanced reporter system, we have identified and located the core region required for the p143 ori. This ori contains a large number of imperfect inverted repeats and is the most active ori in the viral genome during virus infection in insect cells. We also found that it is a unique ori that can replicate in mammalian cells without the assistance of baculovirus gene products. The identification of this ori should contribute to a better understanding of baculovirus DNA replication. Also, this ori is very useful in assisting with gene expression in mammalian cells.

A novel exo-cellulase from white spotted longhorn beetle (Anoplophora malasiaca).

Wood feeding insects depends heavily on the secretion of a combination of cellulases, mainly endoglucanases and other glucanases such as exoglucanases and xylanases, for efficient digestion of the cellulosic materials. To date, although a high number of endoglucanases have been found in xytophagous insects, little is known about exoglucanases encoded in the genome of these insects. Here we report the identification and isolation of an exoglucanase, designated as AmCel-5B, from the white spotted longhorn beetle, Anoplophora malasiaca. The optimal condition of enzymatic activity was found to be 50 °C and pH 4.0. Interestingly, this enzyme is not only exhibited exo-ß-glucanase activity, but also with obvious endo-ß-glucanase activity. Furthermore, this enzyme is unique in that, although it recognizes Avicel, evidenced as an exo-ß-glucanase, it cannot recognize oligosaccharides smaller than cellohexaose. This may explain why longhorn beetle can well digest hard "living" wood, which contains primarily rigid long fibers. Although it is known that metal ions can enhance the activity of some cellulases, we further demonstrated that reducing agent could work synergistically with metal ions for significant activity enhancement of AmCel-5B. The discovery and investigation of an insect exoglucanase should lead to a greater understanding of the mechanism for efficient digestion of cellulosic materials by wood feeding insects, as well as facilitate their potential applications in the production of bioenergy and biomaterials from lignocellulosic biomass in the future.

Heliothis zea nudivirus 1 gene hhi1 induces apoptosis which is blocked by the Hz-iap2 gene and a noncoding gene, pag1.

Heliothis zea nudivirus 1 (HzNV-1 or Hz-1 virus), previously regarded as a nonoccluded baculovirus, recently has been placed in the Nudivirus genus. This virus generates HzNV-1 HindIII-I 1 (hhi1) and many other transcripts during productive viral infection; during latent viral infection, however, persistency-associated gene 1 (pag1) is the only gene expressed. In this report, we used transient expression assays to show that hhi1 can trigger strong apoptosis in transfected cells, which can be blocked, at least partially, by the inhibitor of apoptosis genes Autographa californica iap2 (Ac-iap2) and H. zea iap2 (Hz-iap2). In addition to these two genes, unexpectedly, pag1, which encodes a noncoding RNA with no detectable protein product, was found to efficiently suppress hhi1-induced apoptosis. The assay of pro-Sf-caspase-1 processing by hhi1 transfection did not detect the small P12 subunit at any of the time intervals tested, suggesting that hhi1 of HzNV-1 induces apoptosis through alternative caspase pathways.

Membrane penetrating peptides greatly enhance baculovirus transduction efficiency into mammalian cells.

The baculovirus group of insect viruses is widely used for foreign gene introduction into mammalian cells for gene expression and protein production; however, the efficiency of baculovirus entry into mammalian cells is in general still low. In this study, two recombinant baculoviruses were engineered and their ability to improve viral entry was examined: (1) cytoplasmic transduction peptide (CTP) was fused with baculovirus envelope protein, GP64, to produce a cytoplasmic membrane penetrating baculovirus (vE-CTP); and (2) the protein transduction domain (PTD) of HIV TAT protein was fused with the baculovirus capsid protein VP39 to form a nuclear membrane penetrating baculovirus (vE-PTD). Transduction experiments showed that both viruses had better transduction efficiency than vE, a control virus that only expresses EGFP in mammalian cells. Interestingly, vE-CTP and vE-PTD were also able to improve the transduction efficiency of a co-transduced baculovirus, resulting in higher levels of gene expression. Our results have described new routes to further enhance the development of baculovirus as a tool for gene delivery into mammalian cells.

A non-coding RNA of insect HzNV-1 virus establishes latent viral infection through microRNA.

Heliothis zea nudivirus-1 (HzNV-1) is an insect virus previously known as Hz-1 baculovirus. One of its major early genes, hhi1, is responsible for the establishment of productive viral infection; another gene, pag1, which expresses a non-coding RNA, is the only viral transcript detectable during viral latency. Here we showed that this non-coding RNA was further processed into at least two distinct miRNAs, which targeted and degraded hhi1 transcript. This is a result strikingly similar to a recent report that herpes simplex virus produces tightly-regulated latent specific miRNAs to silence its own key early transcripts. Nevertheless, proof for the establishment of viral latency by miRNA is still lacking. We further showed that HzNV-1 latency could be directly induced by pag1-derived miRNAs in cells infected with a pag1-deleted, latency-deficient virus. This result suggests the existence of a novel mechanism, where miRNAs can be functional for the establishment of viral latency.

Autographa californica multiple nucleopolyhedrovirus LEF-2 is a capsid protein required for amplification but not initiation of viral DNA replication.

The late expression factor 2 gene (lef-2) of baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) has been identified as one of the factors essential for origin-dependent DNA replication in transient expression assays and has been shown to be involved in late/very late gene expression. To study the function of lef-2 in the life cycle of AcMNPV, lef-2 knockout and repair bacmids were generated by homologous recombination in Escherichia coli. Growth curve analysis showed that lef-2 was essential for virus production. Interestingly, a DNA replication assay indicated that lef-2 is not required for the initiation of viral DNA replication and that, rather, it is required for the amplification of DNA replication. lef-2 is also required for the expression of late and very late genes, as the expression of these genes was abolished by lef-2 deletion. Temporal and spatial distributions of LEF-2 protein in infected cells were also analyzed, and the data showed that LEF-2 protein was localized to the virogenic stroma in the nuclei of the infected cells. Analysis of purified virus particles revealed that LEF-2 is a viral protein component of both budded and occlusion-derived virions, predominantly in the nucleocapsids of the virus particles. This observation suggests that LEF-2 may be required immediately after virus entry into host cells for efficient viral DNA replication.

The early gene hhi1 reactivates Heliothis zea nudivirus 1 in latently infected cells.

Heliothis zea nudivirus 1 (HzNV-1), previously known as Hz-1 virus, is an insect virus able to establish both productive and latent infections in several lepidopteran insect cells. Here, we have cloned and characterized one of the HzNV-1 early genes, hhi1, which maps to the HindIII-I fragment of the viral genome. During the productive viral infection, a 6.2-kb hhi1 transcript was detectable as early as 0.5 h postinfection (hpi). The level of transcript reached a maximum at 2 hpi and gradually decreased after 4 hpi. The transcript was not detectable during the latent phase of viral infection. Upon cycloheximide treatment, much higher levels of hhi1 transcript were detected throughout the productive viral infection cycle, suggesting that newly synthesized proteins are not needed for the expression of hhi1. Nevertheless, viral coinfection can further stimulate the expression of transfected hhi1 promoter in a plasmid. Transient hhi1 expression in latently infected cells resulted in a significant increase in virus titer and viral DNA propagation, suggesting that hhi1 plays a critical role in viral reactivation. Additional experiments showed that six early genes, which possibly function in transcription or DNA replication, were activated in the latent cells upon hhi1 transfection. Among these six genes, orf90 and orf121 expression could be induced by hhi1 alone without the need for other viral genes. Our discovery should be useful for future mechanistic study of the switches of latent/productive HzNV-1 viral infections.

RING and coiled-coil domains of baculovirus IE2 are critical in strong activation of the cytomegalovirus major immediate-early promoter in mammalian cells.

In recent years, baculovirus has emerged as a tool for high-efficiency gene transfer into mammalian cells. However, the level of gene expression is often limited by the strength of the mammalian promoter used. Here, we show that the baculovirus RING protein IE2 is a strong, promiscuous trans-activator in mammalian cells, dramatically upregulating the cytomegalovirus (CMV) promoter in both Vero E6 and U-2OS cells. Further study of the cellular mechanism for the activation led to the discovery of a novel IE2 nuclear body structure which contains a high concentration of G-actin and closely associates with RNA polymerase II, PML, and SUMO1. IE2 mutagenesis studies indicated that the RING and coiled-coil domains of IE2 were necessary for nuclear body formation, as well as for strong activation of the CMV promoter in mammalian cells. Overall, this study shows that the IE2 trans-activator could significantly advance the use of baculovirus in mammalian gene transfer and protein production.

Identification of baculoviral factors required for the activation of enhancer-like polyhedrin upstream (pu) sequence.

Previously, we identified a novel enhancer-like element, the polyhedrin upstream (pu) sequence, in the genome of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), which activates several early promoters. The activation requires co-infection of AcMNPV, suggesting that viral gene products are needed for pu-mediated promoter activation. DNA replication assay showed that the pu sequence did not assist in DNA replication and suggested its involvement in activated transcription from target promoters. In order to identify the viral genes responsible for pu-dependent activation of early promoters, a set of overlapping cosmid clones covering the entire 134-kb AcMNPV genome were constructed and screened. Our results identified three viral genes ie1, ie2, and pe38, which function in concert with pu to activate target promoters. In addition, these three viral factors can substitute for the entire virus for the synergistic promoter activation mediated by pu and the known baculovirus enhancer, the homologous region.

Cooperation of ie1 and p35 genes in the activation of baculovirus AcMNPV and HzNV-1 promoters.

HzNV-1 is a non-occluded virus belongs to the family of the baculovirus. One of the first detectable transcripts expressed by HzNV-1 virus infection is a 6.2 kb gene, hhi1, located in the HindIII-I fragment of the viral genome. Here we show that infection of baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) could activate the expression of the hhi1 promoter. By using constructs containing progressive deletions of the upstream regulatory regions of the hhi1 gene, we demonstrated that the most highly activated area was located between nucleotides -62 to +277 of the hhi1 promoter. We subsequently searched the entire 130 kb AcMNPV genome and identified two baculovirus genes, ie1 and p35, that their cooperation is required for the activation of the hhi1 promoter. Further, by taking advantages of a baculovirus DNA chip and low background baculovirus gene expressions in the mammalian cells, we went on to identify a specific set of baculoviral genes, including orf21 and orf25, that could be specifically activated by the combination of ie1 and p35 genes. We conclude that a unique cooperative mechanism of ie1 and p35 exists in the genome of AcMNPV, which can activate the expression of a specific set of AcMNPV and HzNV-1 promoters.

Overexpressing both ATP sulfurylase and selenocysteine methyltransferase enhances selenium phytoremediation traits in Indian mustard.

A major goal of our selenium (Se) phytoremediation research is to use genetic engineering to develop fast-growing plants with an increased ability to tolerate, accumulate, and volatilize Se. To this end we incorporated a gene (encoding selenocysteine methyltransferase, SMT) from the Se hyperaccumulator, Astragalus bisulcatus, into Indian mustard (LeDuc, D.L., Tarun, A.S., Montes-Bayón, M., Meija, J., Malit, M.F., Wu, C.P., AbdelSamie, M., Chiang, C.-Y., Tagmount, A., deSouza, M., Neuhierl, B., Böck, A., Caruso, J., Terry, N., 2004. Overexpression of selenocysteine methyltransferase in Arabidopsis and Indian mustard increases selenium tolerance and accumulation Plant Physiol. 135, 377-383.). The resulting transgenic plants successfully enhanced Se phytoremediation in that the plants tolerated and accumulated Se from selenite significantly better than wild type. However, the advantage conferred by the SMT enzyme was much less when Se was supplied as selenate. In order to enhance the phytoremediation of selenate, we developed double transgenic plants that overexpressed the gene encoding ATP sulfurylase (APS) in addition to SMT, i.e., APSxSMT. The results showed that there was a substantial improvement in Se accumulation from selenate (4 to 9 times increase) in transgenic plants overexpressing both APS and SMT.

Enhancement of correct protein folding in vivo by a non-lytic baculovirus.

The BEVS (baculovirus expression vector system) is widely used for the production of proteins. However, engineered proteins frequently experience the problem of degradation, possibly due to the lytic nature of the conventional BEVS (herein referred to as L-BEVS). In the present study, a non-lytic BEVS (N-BEVS) was established by random mutagenesis of viral genomes. At 5 days post-infection, N-BEVS showed only 7% cell lysis, whereas L-BEVS showed 60% lysis of cells. The quality of protein expressed in both N- and L-BEVSs was examined further using a novel FRET (fluorescence resonance energy transfer)-based assay. To achieve this, we constructed a concatenated fusion protein comprising LUC (luciferase) sandwiched between EYFP (enhanced yellow fluorescent protein) and ECFP (enhanced cyan fluorescent protein). The distance separating the two fluorescent proteins in the fusion protein EYFP-LUC-ECFP (designated hereafter as the YLC construct) governs energy transfer between EYFP and ECFP. FRET efficiency thus reflects the compactness of LUC, indicating its folding status. We found more efficient FRET in N-BEVS compared with that obtained in L-BEVS, suggesting that more tightly folded LUC was produced in N-BEVS. YLC expression was also analysed by Western blotting, revealing significantly less protein degradation in N-BEVS than in L-BEVS, in which extensive degradation was observed. This FRET-based in vivo folding technology showed that YLC produced in N-BEVS is more compact, correlating with improved resistance to degradation. N-BEVS is thus a convenient alternative for L-BEVS for the production of proteins vulnerable to degradation using baculoviruses.

Overexpression of selenocysteine methyltransferase in Arabidopsis and Indian mustard increases selenium tolerance and accumulation.

A major goal of phytoremediation is to transform fast-growing plants with genes from plant species that hyperaccumulate toxic trace elements. We overexpressed the gene encoding selenocysteine methyltransferase (SMT) from the selenium (Se) hyperaccumulator Astragalus bisulcatus in Arabidopsis and Indian mustard (Brassica juncea). SMT detoxifies selenocysteine by methylating it to methylselenocysteine, a nonprotein amino acid, thereby diminishing the toxic misincorporation of Se into protein. Our Indian mustard transgenic plants accumulated more Se in the form of methylselenocysteine than the wild type. SMT transgenic seedlings tolerated Se, particularly selenite, significantly better than the wild type, producing 3- to 7-fold greater biomass and 3-fold longer root lengths. Moreover, SMT plants had significantly increased Se accumulation and volatilization. This is the first study, to our knowledge, in which a fast-growing plant was genetically engineered to overexpress a gene from a hyperaccumulator in order to increase phytoremediation potential.