Gene Silencing Technology Research Articles

Cloning and functional analysis of mitogen‐activated protein kinases 6 gene in lettuce

AbstractLettuce (Lactuca sativa L.) being a cool‐season crop can bolt if the temperatures increase above 20 °C. When lettuce bolts, the plants start the reproductive growth cycle. The improvement in resistance to high temperature bolting has been an important goal of lettuce breeding. This paper verified that Lactuca sativa mitogen‐activated protein kinases 6 (LsMAPK6) played an important role in high temperature bolting. The full‐length CDS of LsMAPK6 gene was amplified by real‐time polymerase chain reaction (RT‐PCR) and analyzed by bioinformatics software. The expression of LsMAPK6 was determined by quantitative real‐time polymerase chain reaction (qRT‐PCR). The results showed that the length of LsMAPK6 was 1,182 bp, encoding 393 amino acids. The protein domain was STKc_TEY_MAPK and closely relevant to sunflower (Helianthus annuus L.), mouse‐ear cress [Arabidopsis thaliana (L.) Heynh.], and chrysanthemum (Chrysanthemum morifolium Ramat.). The qRT‐PCR showed that Lsmapk6 was mainly expressed in stem, stem apex, and taproot, and high temperature significantly promoted its expression. Virus‐induced gene silencing (VIGS) technology successfully silenced LsMAPK6 in lettuce, and the bolting of silenced lines was delayed under the action of high temperature and exogenous gibberellin. These suggest that LsMAPK6 plays an important role in promoting bolting. This study laid a foundation for further study on the function and regulation mechanism of MAPK6.

Genome-wide expression analysis of carboxylesterase (CXE) gene family implies GBCXE49 functional responding to alkaline stress in cotton

BackgroundCarboxylesterase (CXE) is a type of hydrolase with α/β sheet hydrolase activity widely found in animals, plants and microorganisms, which plays an important role in plant growth, development and resistance to stress.ResultsA total of 72, 74, 39, 38 CXE genes were identified in Gossypium barbadense, Gossypium hirsutum, Gossypium raimondii and Gossypium arboreum, respectively. The gene structure and expression pattern were analyzed. The GBCXE genes were divided into 6 subgroups, and the chromosome distribution of members of the family were mapped. Analysis of promoter cis-acting elements showed that most GBCXE genes contain cis-elements related to plant hormones (GA, IAA) or abiotic stress. These 6 genes we screened out were expressed in the root, stem and leaf tissues. Combined with the heat map, GBCXE49 gene was selected for subcellular locate and confirmed that the protein was expressed in the cytoplasm.ConclusionsThe collinearity analysis of the CXE genes of the four cotton species in this family indicated that tandem replication played an indispensable role in the evolution of the CXE gene family. The expression patterns of GBCXE gene under different stress treatments indicated that GBCXE gene may significantly participate in the response to salt and alkaline stress through different mechanisms. Through the virus-induced gene silencing technology (VIGS), it was speculated that GBCXE49 gene was involved in the response to alkaline stress in G. barbadense.

PpePL1 and PpePL15 Are the Core Members of the Pectate Lyase Gene Family Involved in Peach Fruit Ripening and Softening.

Pectin is the major component in the primary cell wall and middle lamella, maintaining the physical stability and mechanical strength of the cell wall. Pectate lyase (PL), a cell wall modification enzyme, has a major influence on the structure of pectin. However, little information and no comprehensive analysis is available on the PL gene family in peach (Prunus persica L. Batsch). In this study, 20 PpePL genes were identified in peach. We characterized their physicochemical characteristics, sequence alignments, chromosomal locations, and gene structures. The PpePL family members were classified into five groups based on their phylogenetic relationships. Among those, PpePL1, 9, 10, 15, and 18 had the higher expression abundance in ripe fruit, and PpePL1, 15, and 18 were upregulated during storage. Detailed RT-qPCR analysis revealed that PpePL1 and PpePL15 were responsive to ETH treatment (1 g L−1 ethephon) with an abundant transcript accumulation, which suggested these genes were involved in peach ripening and softening. In addition, virus-induced gene silencing (VIGS) technology was used to identify the roles of PpePL1 and PpePL15. Compared to controls, the RNAi fruit maintained greater firmness in the early storage stage, increased acid-soluble pectin (ASP), and reduced water-soluble pectin (WSP). Moreover, transmission electron microscopy (TEM) showed that cell wall degradation was reduced in the fruit of RNAi-1 and RNAi-15, which indicated that softening of the RNAi fruit has been delayed. Our results indicated that PpePL1 and PpePL15 play an important role in peach softening by depolymerizing pectin and degrading cell wall.

Edonerpic maleate regulates glutamate receptors through CRMP2- and Arc-mediated mechanisms in response to brain trauma

Dysfunction of ionotropic glutamate receptors (iGluRs) is a key molecular mechanism of excitotoxic neuronal injury following traumatic brain injury (TBI). Edonerpic maleate is a low molecular-weight compound that was screened as a candidate neuroprotective agent. In this study, we investigated its effects on TBI and GluRs signaling. Traumatic neuronal injury (TNI) induced by scratch followed by glutamate treatment was performed to mimic TBI in vitro. Edonerpic maleate at 1 and 10 μM exerted protective activity when it was added within 2 h following injury. The protective activities were also confirmed by the reduction of lipid peroxidation and oxidative stress. In addition, edonerpic maleate inhibited the expression of surface NR2B, total GluR1, and surface GluR1, and mitigated the intracellular Ca2+ responses following injury in vitro. Western blot analysis showed that edonerpic maleate reduced the cleavage of collapsing response mediator protein 2 (CRMP2), but increased the expression of postsynaptic protein Arc. By using gene overexpression and silencing technologies, CRMP2 was overexpressed and Arc was knockdown in cortical neurons. The results showed that the effect of edonerpic maleate on NMDA receptor expression was mediated by CRMP2, whereas the edonerpic maleate-induced AMPA receptor regulation was dependent on Arc activation. In in vivo TBI model, 30 mg/kg edonerpic maleate alleviated the TBI-induced brain edema, neuronal loss, and microglial activation, with no effect on locomotor function at 24 h. However, edonerpic maleate improves long-term neurological function after TBI. Furthermore, edonerpic maleate inhibited CRMP2 cleavage but increased Arc activation in vivo. In summary, our results identify edonerpic maleate as a clinically potent small compound with which to attenuate TBI-related brain damage through regulating GluRs signaling.

Phytophthora sojae Transformation Based on the CRISPR/Cas9 System.

Phytophthora sojae is a model species for the study of plant pathogenic oomycetes. The initial research on gene function using Phytophthora was mainly based on gene silencing technology. Recently, the CRISPR/Cas9-mediated genome editing technology was successfully established in P. sojae and widely used in oomycetes. In this protocol, we describe the operating procedures for the use of CRISPR/Cas9-based genome editing technology and PEG-mediated stable transformation of P. sojae protoplasts. Two plasmids were co-transformed into P. sojae: pYF515 expressing Cas9 and the single guide RNA, and the homologous replacement vector of the candidate gene. Finally, the ORF of candidate gene were replaced with the ORF of the entire hygromycin B phosphotransferase gene (HPH), to achieve precise knockout.

CURRENT STATUS, CHALLENGES, MANAGEMENT AND FUTURE PERSPECTIVES OF THE RED PALM WEEVIL Rhynchophorus ferrugineus OLIVIER (COLEOPTERA, CURCULIONIDAE) ERADICATION - A REVIEW

The date palm is a cultural and economic heritage of many nations. The red palm weevil, Rhynchophorus ferrugineus Olivier (Coleoptera, Curculionidae) is among the world’s most serious insect pests of palms that have rapidly expanded its geographical distribution and host plant range during the last decades. Integrated pest management (IPM) is followed to suppress the pest using the most economical and least hazardous methods to humans and the environment. Since R. ferrugineus is a serious pest of date palm production worldwide, farmers, researchers, and scientists have developed many tactics to eradicate this pest. However, there was no published article covering and integrating the current status, biology, ecology, and future control tactics of R. ferrugineus and proposes an IPM program. Therefore, in this review, R. ferrugineus geographical distribution, host plant range, economic importance, infestation symptoms, morphology, biology, and its management tactics were thoroughly discussed. These tactics include early detection of R. ferrugineus infestation, trapping, chemical applications, use of bio-agents, bioinsecticides (plant extracts), resistance cultivars, cultural practices, sterile insect technique, gene silencing technology, quarantine, and geographical information system. In conclusion, all available control tactics suggest that R. ferrugineus could be successfully managed by developing IPM programs comprising several means of control. It is hoped that this review will highlight some aspects of date palm management and raise research gaps and directions deserving further investigations to develop a better understanding of R. ferrugineus management and therefore contributes to the sustainability of date palm cultivation worldwide.

Spatio-Temporal Profiling of Metarhizium anisopliae-Responsive microRNAs Involved in Modulation of Plutella xylostella Immunity and Development.

Metarhizium anisopliae, a ubiquitous pathogenic fungus, regulates a wide array of the insect pest population. The fungus has been employed to control Plutella xylostella, an insecticide-resistant destructive lepidopteran pest, which causes substantial economic losses in crops worldwide. Integration of modern gene-silencing technologies in pest control strategies has become more crucial to counter pesticide-resistant insects. MicroRNAs (miRNA) play essential roles in the various biological process via post-transcriptional gene regulation. In the present study, RNA-seq analysis of control (CK36h, CK72h) and fungal-infected (T36h, T72h) midguts was performed to reveal underlying molecular mechanisms occurring in larval midgut at different time courses. We aimed at exploring M. anisopliae-responsive miRNAs and their target genes involved in development and immunity. After data filtration, a combined set of 170 miRNAs were identified from all libraries. Interestingly, miR-281, miR-263, miR-1, miR-6094 and miR-8 were listed among the most abundantly expressed conserved miRNAs. Furthermore, we experimentally studied the role of differentially expressed miR-11912-5p in regulating corresponding target trypsin-like serine proteinase (Px_TLSP). The luciferase assay (in vitro) revealed that miRNA-11912-5p significantly downregulated its target gene, suggesting it might play a crucial role in defense mechanism of P. xylostella against M.+ anisopliae infection. We used synthetic miRNA mimic/inhibitor (in vivo), to overexpress/silence miRNA, which showed harmful effects on larval duration, survival and adult fecundity. Additionally, fungal application in the presence of mimics revealed enhanced sensitivity of P. xylostella to infection. Our finding provides an insight into the relatively obscure molecular mechanisms involved in insect midgut during the fungal infection.

VvANR silencing promotes expression of VvANS and accumulation of anthocyanin in grape berries.

Virus-induced gene silencing (VIGS) technology was applied to silence VvANR in cv. Zaoheibao grape berries, and the effects of VvANR silencing on berries phenotype; gene expression level of ANS, LAR1, LAR2, and UFGT; enzyme activity of ANS; and accumulations of anthocyanin and flavan-3-ol were investigated. At the third day after treatment, the VvANR silenced grape berries began to turn red slightly, which was 2days earlier than that of the control group. And the flavan-3-ol content in VvANR-silenced grape berries had been remarkable within 1 to 5days, the ANR enzyme activity in VvANR-silenced grapes extremely significantly decreased in 3days, and LAR enzyme activity also decreased, but the difference was not striking. The ANS enzyme activity of the transformed berries was significantly higher than that of the control after 3days of infection, and it was exceedingly significantly higher than that of the control after 5 to 10days. The content of anthocyanin in transformed berries increased of a very marked difference within 3 to 15days. pTRV2-ANR infection resulted in an extremely significant decrease in the expression of VvANR gene, and the expression of VvLAR1, VvLAR2, VvMYBPA1, VvMYBPA2, and VvDFR were also down-regulated. However, the expression of VvANS and VvUFGT was up-regulated significantly. After VvANR silencing via VIGS, VvANR expression in grape berries was extremely significantly decreased, resulting in decreased ANR enzyme activity and flavan-3-ol content; berries turned red and deeper in advance. In addition, VvANR silencing can induce up-regulation of VvANS and VvUFGT expression, significantly increase ANS enzyme activity, and increase of anthocyanin accumulation.

The cotton 70-kDa heat shock protein GhHSP70-26 plays a positive role in the drought stress response

The 70-kDa heat shock protein (HSP70) is a molecular chaperone that plays an important role in the response of plants to abiotic stress, but the function and molecular mechanism of HSP70 in the cotton drought stress response are unclear. In this study, the HSP70-encoding gene GhHSP70-26, which belongs to the cytoplasmic HSP70 subgroup, was cloned from upland cotton (Gossypium hirsutum L.). The transcript level of GhHSP70-26 in cotton leaves was higher than that in cotton stems and roots. GhHSP70-26 was found to respond at the transcriptional level to polyethylene glycol (PEG) and abscisic acid (ABA) treatment and to cold, salt, and heat stress, and its transcript level was positively correlated with the drought resistance of different cotton varieties. Heterologous overexpression of the GhHSP70-26 gene improved the drought resistance of transgenic tobacco compared with that of wild-type (WT) tobacco, and the transgenic tobacco plants were characterized by low leaf wilting, high survival, low leaf water loss, increased root length and high chlorophyll content. Moreover, the transgenic tobacco plants overexpressing GhHSP70-26 had higher proline contents, higher superoxide dismutase (SOD) and peroxidase (POD) enzyme activities, and lower malondialdehyde (MDA) and reactive oxygen species (ROS) contents than the WT tobacco plants. The transcript levels of the stress response-related genes NtLEA, NtERD10D, NtPOD, NtSOD, and NtNCED3-1 in transgenic tobacco overexpressing GhHSP70-26 were significantly higher than those in WT tobacco. In contrast, under drought stress, unlike the results obtained with the pTRV2 empty vector (EV)-transformed and WT cotton plants, the use of virus-induced gene silencing (VIGS) technology to silence the GhHSP70-26 gene in cotton resulted in plants with severely wilted leaves, an increased water loss rate, a higher MDA content, and higher relative electrical conductivity (REC). Furthermore, yeast one-hybrid assays showed that GhbZIP43 and GhHSF8 bind to ABA-responsive elements (ABREs) and heat shock elements (HSEs) in the GhHSP70-26 promoter, respectively. Moreover, yeast two-hybrid, bimolecular fluorescence complementation and luciferase complementation assays showed that the GhHSP70-26 protein interacts with the ascorbate peroxidase (APX) GhAPX2 protein. These results indicate that by reducing the degrees of cell membrane damage and cellular damage caused by ROS stress, the GhHSP70-26 protein plays a positive role in the response of plants to drought stress.

Two loss‐of‐function alleles of the glutathione S‐transferase (GST) gene cause anthocyanin deficiency in flower and fruit skin of peach (Prunus persica)

SummaryFlower and fruit colors are important agronomic traits. To date, there is no forward genetic evidence that the glutathione S‐transferase (GST) gene is responsible for the white flower color in peach (Prunus persica). In this study, genetic analysis indicated that the white‐flower trait is monogenetic, is recessive to the non‐white allele, and shows pleiotropic effects with non‐white‐flowered types. The genetic locus underpinning this trait was mapped onto chromosome 3 between 0.421951 and 3.227115 Mb by using bulked segregant analysis in conjunction with whole‐genome sequencing, and was further mapped between 0 and 1.178149 Mb by using the backcross 1 (BC1) population. Finally, the locus was fine‐mapped within 535.974‐ and 552.027‐kb intervals by using 151 F2 individuals and 75 individuals from a BC1 self‐pollinated (BC1S1) population, respectively. Pp3G013600, encoding a GST that is known to transport anthocyanin, was identified within the mapping interval. The analysis of genome sequence data showed Pp3G013600 in white flowers has a 2‐bp insertion or a 5‐bp deletion in the third exon. These variants likely render the GST non‐functional because of early stop codons that reduce the protein length from 215 amino acids to 167 and 175 amino acids, respectively. Genetic markers based on these variants validated a complete correlation between the GST loss‐of‐function alleles and white flower in 128 peach accessions. This correlation was further confirmed by silencing of Pp3G013600 using virus‐induced gene silencing technology, which reduced anthocyanin accumulation in peach fruit. The new knowledge from this study is useful for designing peach breeding programs to generate cultivars with white flower and fruit skin.

A naked antisense oligonucleotide with phosphorothioate linkages is taken up intracellularly more efficiently but functions less effectively

We have been developing a gene silencing technology by harnessing a tRNA 3′ processing endoribonuclease, tRNase ZL, with antisense oligonucleotides. Here, to further improve this technology, we investigated how the length and the modifications of naked oligonucleotides affect the efficiency of their uptake by HeLa, HEK293, and HL60 cells by flow cytometry and fluorescence microscopy. 7–30-nt Alexa-Fluor-568-labeled DNAs with phosphorothioate linkages and 7–30-nt Alexa-Fluor-568-labeled, 2′-O-methylated RNAs without phosphorothioate linkages were examined, and, on the whole, longer oligonucleotides were shown to be intracellularly taken up more efficiently. In addition, a 2′-O-methoxyethylated RNA without phosphorothioate linkages, a 2′-fluoriated RNA without phosphorothioate linkages, a 2′-O-methylated RNA with phosphorothioate linkages, and a 2′-O-methylated RNA with phosphorothioate linkages and LNA modifications of 5′-/3′-terminal nucleotides were examined. The oligonucleotides with phosphorothioate linkages were taken up by the cells more efficiently than those without the linkages. Furthermore, we examined how the phosphorothioate linkages of oligonucleotides affect their antisense effects using 22-nt anti-miR16 oligonucleotides with and without phosphorothioate linkages. The latter oligonucleotide decreased the miR16 level much more intensively than the former, although the latter was intracellularly taken up much less efficiently. These observations may be not generalized and differ depending on features of oligonucleotides and cell types. Taken together these results suggest that the productive uptake efficiency for an antisense oligonucleotide needs to be considered to select its length and modifications.

Enhancing preclinical predictivity: what is going wrong between preclinical in vitro/in vivo and clinical in vivo settings?

<div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://cdn.insights.bio/uploads/Christian Schmees copy.jpg" data-image="1"></div><div class="author-det"> <b>Christian Schmees</b> is heading the Tumor Biology group at the Natural and Medical Sciences Institute (NMI) at the University of Tuebingen, Germany. He received his undergraduate degree in biochemistry from Tuebingen University in 2002. Since 2006 he holds a PhD (with highest honors) in cancer immunology from the Technical University of Munich, Germany. His thesis resulted in the identification of gamma-glutamyl-transpeptidase as the major factor for T cell specific immune evasion of the tumorigenic bacterium Helicobacter pylori. As a post-doc he joined the laboratories of Carl-Henrik Heldin at the Ludwig Institute for Cancer Research (LICR) in Uppsala, Sweden (2006-2010) and Philippe Bastiaens at the Max-Planck Institute of Molecular Physiology in Dortmund, Germany (2010-2011). He received fellowships from the German Research Foundation and the LICR to support his research on differential regulation of intracellular PDGF α- and β-receptor trafficking in cancer cells. In 2011 he joined the Natural and Medical Sciences Institute at the University of Tuebingen (NMI) as a senior scientist in tumor biology and was appointed Head of Tumor Biology in 2014. Ongoing projects in his group are focusing on the development of clinically relevant cellular model systems for drug development and target validation in translational oncology. These approaches are combined with gene editing and silencing technologies as well as protein profiling and immunophenotyping platforms for mode-of-action analyses of different compound classes. Dr. Schmees is member of the Association for Cancer Immunotherapy (CIMT). </div></div></div>

Trps1 targets Ccnd1 to regulate mouse Leydig cell proliferation.

The tricho-rhino-phalangeal syndrome-1 gene (Trps1) is an atypical GATA family member. Although current studies of Trps1 mainly focus on tumors, whether Trps1 plays a role in the male reproductive system remains unknown. The purpose of this study was to elucidate the function of Trps1 in Leydig cells, indicating its regulatory mechanism on the cell cycle. Gene-silencing technology, RNA-seq, RT-qPCR, and western blotting were used to evaluate the function of Trps1 in mouse primary Leydig cells and MLTC-1 cells. In addition, ChIP-base sets and ChIP-qPCR were employed to further assess the regulatory mechanism of Trps1 in MLTC-1 cells. Knockdown of Trps1 in Leydig cells significantly suppressed phosphorylation of Src and Akt and expression of Ccnd1, which was accompanied by impairment of cell proliferative ability. Trps1 may affect the cell cycle through the Src/Akt/Ccnd1 signaling pathway. In addition, Trps1 may bind to the promoter of Srcin1 to regulate its transcription, thus influencing Src phosphorylation levels and the proliferation of Leydig cells. Src increases in Leydig cells during pubertal development, suggesting its functional involvement in differentiated adult Leydig cells. Inhibition of the Src/Akt pathway would reduce Ccnd1 expression. In the present study, we found that Trps1 may regulate the phosphorylation level of Src and Akt through Srcin1, targeting Ccnd1 to influence mouse Leydig cell proliferation. These findings shed light on the regulation of Trps1 on cell proliferation and differentiation of mouse Leydig cells.

The downregulation of PpPG21 and PpPG22 influences peach fruit texture and softening.

The downregulation of PpPG21 and PpPG22 expression in melting-flesh peach delays fruit softening and hinders texture changes by influencing pectin solubilization and depolymerization. The polygalacturonase (PG)-catalyzed solubilization and depolymerization of pectin plays a central role in the softening and texture formation processes in peach fruit. In this study, the expression characteristics of 15 PpPG members in peach fruits belonging to the melting flesh (MF) and non-melting flesh (NMF) types were analyzed, and virus-induced gene silencing (VIGS) technology was used to identify the roles of PpPG21 (ppa006839m) and PpPG22 (ppa006857m) in peach fruit softening and texture changes. In both MF and NMF peaches, the expression of PpPG1, 10, 12, 23, and 25 was upregulated, whereas that of PpPG14, 24, 35, 38, and 39 was relatively stable or downregulated during shelf life. PpPG1 was highly expressed in NMF fruit, whereas PpPG21 and 22 were highly expressed in MF peaches. Suppressing the expression of PpPG21 and 22 by VIGS in MF peaches significantly reduced PG enzyme activity, maintained the firmness of the fruit during the late shelf life stage, and suppressed the occurrence of the "melting" stage compared with the control fruits. Moreover, the downregulation of PpPG21 and 22 expression also reduced the water-soluble pectin (WSP) content, increased the contents of ionic-soluble pectin (ISP) and covalent-soluble pectin (CSP) and affected the expression levels of ethylene synthesis- and pectin depolymerization-related genes in the late shelf life stage. These results indicate that PpPG21 and 22 play a major role in the development of the melting texture trait of peaches by depolymerizing cell wall pectin. Our results provide direct evidence showing that PG regulates peach fruit softening and texture changes.

Update on the management and treatment of viral hepatitis.

This review aims to summarize the current evidence on the treatment of viral hepatitis, focusing on its clinical management. Also, future treatment options and areas of potential research interest are detailed. PubMed and Scopus databases were searched for primary studies published within the last ten years. Keywords included hepatitis A virus, hepatitis B virus (HBV), hepatitis C virus, hepatitis D virus (HDV), hepatitis E virus, and treatment. Outcomes reported in the studies were summarized, tabulated, and synthesized. Significant advances in viral hepatitis treatment were accomplished, such as the advent of curative therapies for hepatitis C and the development and improvement of hepatitis A, hepatitis B, and hepatitis E vaccination. Drugs that cure hepatitis B, going beyond viral suppression, are so far unavailable; however, targeted antiviral drugs against HBV (immunomodulatory therapies and gene silencing technologies) are promising approaches to eradicating the virus. Ultimately, high vaccination coverage and large-scale test-and-treat programmes with high screening rates may eliminate viral hepatitis and mitigate their burden on health systems. The development of curative hepatitis C treatment renewed the enthusiasm for curing hepatitis B, albeit further investigation is required. Novel therapeutic options targeting HDV life cycle are currently under clinical investigation.

Climate Mismatch between Introduced Biological Control Agents and Their Invasive Host Plants: Improving Biological Control of Tropical Weeds in Temperate Regions.

Simple SummaryMismatched distributions between biological control agents and their host plants occur for a variety of reasons but are often linked to climate, specifically differences in their low-temperature tolerances. How to measure and use low-temperature tolerances of control agents to inform agent prioritization, selection for redistribution, or predict efficacy is vitally important, but has not been previously synthesized in a single source. We discuss causes of climate mismatches between agents and target weeds, the traditional and non-traditional approaches that could be used to decrease the degree of mismatch and improve control, and regulatory issues to consider when taking such approaches. We also discuss the variety of cold tolerance metrics, their measurement and ecological value, and the types of modeling that can be carried out to improve predictions about potential distributions of agents. We also briefly touch on molecular bases for cold tolerance and opportunities for improving cold tolerance of agents using modern molecular tools.Many weed biological control programs suffer from large-scale spatial variation in success due to restricted distributions or abundances of agents in temperate climates. For some of the world’s worst aquatic weeds, agents are established but overwintering conditions limit their survival in higher latitudes or elevations. The resulting need is for new or improved site- or region-specific biological control tools. Here, we review this challenge with a focus on low-temperature limitations of agents and propose a roadmap for improving success. Investigations across spatial scales, from global (e.g., foreign exploration), to local (selective breeding), to individual organisms (molecular modification), are discussed. A combination of traditional (foreign) and non-traditional (introduced range) exploration may lead to the discovery and development of better-adapted agent genotypes. A multivariate approach using ecologically relevant metrics to quantify and compare cold tolerance among agent populations is likely required. These data can be used to inform environmental niche modeling combined with mechanistic modeling of species’ fundamental climate niches and life histories to predict where, when, and at what abundance agents will occur. Finally, synthetic and systems biology approaches in conjunction with advanced modern genomics, gene silencing and gene editing technologies may be used to identify and alter the expression of genes enhancing cold tolerance, but this technology in the context of weed biological control has not been fully explored.

Characterization of BoaCRTISO Reveals Its Role in Carotenoid Biosynthesis in Chinese Kale.

Carotenoids are organic pigments that play an important role in both plant coloration and human health; they are a critical subject in molecular breeding due to growing demand for natural molecules in both food and medicine. In this study, we focus upon characterizing BoaCRTISO, the carotenoid isomerase gene before the branch of the carotenoid biosynthetic pathway, which is expressed in all organs and developmental stages of Chinese kale, and BoaCRTISO, which is located in the chloroplast. The expression of BoaCRTISO is induced by strong light, red and blue combined light, and gibberellic acid treatment, but it is suppressed by darkness and abscisic acid treatment. We obtained BoaCRTISO-silenced plants via virus-induced gene silencing technology, and the silence efficiencies ranged from 52 to 77%. The expressions of most carotenoid and chlorophyll biosynthetic genes in BoaCRTISO-silenced plants were downregulated, and the contents of carotenoids and chlorophyll were reduced. Meanwhile, BoaCRTISO-silenced plants exhibited phenotypes of yellowing leaves and inhibited growth. This functional characterization of BoaCRTISO provides insight for the biosynthesis and regulation of carotenoid in Chinese kale.

HSP17.4 mediates salicylic acid and jasmonic acid pathways in the regulation of resistance to Colletotrichum gloeosporioides in strawberry.

In this study, we used virus‐mediated gene silencing technology and found that the HSP17.4 gene‐silenced cultivar Sweet Charlie plants were more susceptible to Colletotrichum gloeosporioides than the wild‐type Sweet Charlie, and the level of infection was even higher than that of the susceptible cultivar Benihopp. The results of differential quantitative proteomics showed that after infection with the pathogen, the expression of the downstream response genes NPR1, TGA, and PR‐1 of the salicylic acid (SA) signalling pathway was fully up‐regulated in the wild‐type Sweet Charlie, and the expression of the core transcription factor MYC2 of the jasmonic acid (JA) pathway was significantly down‐regulated. The expression of the proteins encoded by these genes did not change significantly in the HSP17.4‐silenced Sweet Charlie, indicating that the expression of HSP17.4 activated the up‐regulation of downstream signals of SA and inhibited the JA signal pathway. The experiments that used SA, methyl jasmonate, and their inhibitors to treat plants provide additional evidence that the antagonism between SA and JA regulates the resistance of strawberry plants to C. gloeosporioides.

CELL CYCLE SEITCH 52 regulates tillering by interacting with LATERAL SUPPRESSOR in non-heading Chinese cabbage

With the discovery of essential genes regulating tillering, such as MONOCULM 1 (MOC1) in rice and LATERAL SUPPRESSOR (LAS in Arabidopsis, LS in tomato), research on tillering mechanisms has made great progress; however, the study of tillering in non-heading Chinese cabbage (NHCC) is rare. Here, we report that BcLAS, as a member of the GRAS family, plays an important role in the tillering of NHCC during its vegetative growth. BcLAS was almost not expressed in other examed parts except leaf axils throughout life. When the expression of BcLAS was silenced utilizing virus-induced gene silencing (VIGS) technology, we found that the tiller number of ‘Maertou’ decreased sharply. In ‘Suzhouqing’, overexpression of BcLAS significantly promoted tillering. BcCCS52, the orthologue to CELL CYCLE SEITCH 52 (CCS52), interacts with BcLAS. Downregulation of the expression of BcCCS52 promoted tillering of ‘Suzhouqing’; therefore, we conclude that BcCCS52 plays a negative role in tillering regulation. Our findings reveal the tillering regulation mechanism of NHCCs at the vegetative stage and report an orthologue of CCS52 regulating tillering in NHCC.

Host-induced gene silencing - mechanisms and applications.

Host-induced gene silencing (HIGS) technology has emerged as a powerful alternative to chemical treatments for protecting plants from pathogens or pests. More than 170 HIGS studies have been published so far, and HIGS products have been launched. First, we discuss the strengths and limitations of this technology in a pathosystem-specific context. Next, we highlight the requirement for fundamental knowledge on the molecular mechanisms (i.e. uptake, processing and translocation of transgene-expressed double-stranded RNAs) that determine the efficacy and specificity of HIGS. Additionally, we speculate on the contribution of host and target RNA interference machineries, which may be incompatible depending on the lifestyle of the pathogen or pest. Finally, we predict that closing these gaps in knowledge will lead to the development of novel integrative concepts, precise risk assessment and tailor-made HIGS therapy for plant diseases.