Transient RNA Interference Research Articles

PuERF008-PuFAD2 module regulates aroma formation via the fatty acid pathway in response to calcium signaling in ‘Nanguo’ pear

Calcium acts as a secondary messenger in plants and is essential for plant growth and development. However, studies on the pathway of aroma synthesis in ‘Nanguo’ pear (Pyrus ussriensis Maxim.) are scarce. In this study, a bioinformatics analysis of transcriptomic data from calcium-treated ‘Nanguo’ pear was performed, which identified two fatty acid desaturases, PuFAD2 and PuFAD3, and eight AP2/ERF transcription factors, all exhibiting the same expression patterns. Transient expression experiments showed overexpression of PuFAD2 and PuFAD3 significantly increased the levels of aromatic substrates linoleic acid, hexanal, linolenic acid, and (E)-2-hexenal, but RNAi (RNA interference) had the opposite expression. Promoter sequences analysis revealed that PuFAD2 and PuFAD3 have ERE (estrogen response element) motifs on their promoters. The strongest activation of PuFAD2 by PuERF008 was verified using a dual-luciferase reporting system. Additionally, yeast one-hybrid and electrophoretic mobility shift assays revealed PuERF008 could active PuFAD2. Transient overexpression and RNAi analyses of PuERF008 showed a strong correlation with the expression of PuFAD2. This study provides insights into the process of aroma biosynthesis in ‘Nanguo’ pear and offers a theoretical basis for elucidating the role of calcium signaling in aroma synthesis.

DNA methylation controlling abscisic acid catabolism responds to light to mediate strawberry fruit ripening.

Phytohormones, epigenetic regulation and environmental factors regulate fruit ripening but their interplay during strawberry fruit ripening remains to be determined. In this study, bagged strawberry fruit exhibited delayed ripening compared with fruit grown in normal light, correlating with reduced abscisic acid (ABA) accumulation. Transcription of the key ABA catabolism gene, ABA 8'-hydroxylase FaCYP707A4, was induced in bagged fruit. With light exclusion whole genome DNA methylation levels were up-regulated, corresponding to a delayed ripening process, while DNA methylation levels in the promoter of FaCYP707A4 were suppressed, correlating with increases in transcript and decreased ABA content. Experiments indicated FaCRY1, a blue light receptor repressed in bagged fruit and FaAGO4, a key protein involved in RNA-directed DNA methylation, could bind to the promoter of FaCYP707A4. The interaction between FaCRY1 and FaAGO4, and an increased enrichment of FaAGO4 directed to the FaCYP707A4 promoter in fruit grown under light suggests FaCRY1 may influence FaAGO4 to modulate the DNA methylation status of the FaCYP707A4 promoter. Furthermore, transient overexpression of FaCRY1, or an increase in FaCRY1 transcription by blue light treatment, increases the methylation level of the FaCYP707A4 promoter, while transient RNA interference of FaCRY1 displayed opposite phenotypes. These findings reveal a mechanism by which DNA methylation influences ABA catabolism, and participates in light-mediated strawberry ripening.

Development and application of an RNA nanostructure to induce transient RNAi in difficult transgenic plants.

The development of RNA interference (RNAi) is crucial for studying plant gene function. Its use, is limited to a few plants with well-established transgenic techniques. Spray-induced gene silencing (SIGS) introduces exogenous double-stranded RNA (dsRNA) into plants by spraying, injection, or irrigation, triggering the RNAi pathway to instantly silence target genes. As is a transient RNAi technology that does not rely on transgenic methods, SIGS has significant potential for studying gene function in plants lacking advanced transgenic technology. In this study, to enhance their stability and delivery efficiency, siRNAs were used as structural motifs to construct RNA nanoparticles (NPs) of four shapes: triangle, square, pentagon, and hexagon. These NPs, when synthesized by Escherichia coli, showed that triangular and square shapes accumulated more efficiently than pentagon and hexagon shapes. Bioassays revealed that RNA squares had the highest RNAi efficiency, followed by RNA triangles, with GFP-dsRNA showing the lowest efficiency at 4 and 7 days post-spray. We further explored the use of RNA squares in inducing transient RNAi in plants that are difficult to transform genetically. The results indicated that Panax notoginseng-derived MYB2 (PnMYB2) and Camellia oleifera-derived GUT (CoGUT) were significantly suppressed in P. notoginseng and C. oleifera, respectively, following the application of PnMYB2- and CoGUT-specific RNA squares. These findings suggest that RNA squares are highly effective in SIGS and can be utilized for gene function research in plants.

Attenuation of Chronic Inflammation in Intestinal Organoids with Graphene Oxide-Mediated Tumor Necrosis Factor-α_Small Interfering RNA Delivery.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract with a complex and multifactorial etiology, making it challenging to treat. While recent advances in immunomodulatory biologics, such as antitumor necrosis factor-α (TNF-α) antibodies, have shown moderate success, systemic administration of antibody therapeutics may lead to several adverse effects, including the risk of autoimmune disorders due to systemic cytokine depletion. Transient RNA interference using exogenous short interfering RNA (siRNA) to regulate target gene expression at the transcript level offers an alternative to systemic immunomodulation. However, siRNAs are susceptible to premature degradation and have poor cellular uptake. Graphene oxide (GO) nanoparticles have been shown to be effective nanocarriers for biologics due to their reduced cytotoxicity and enhanced bioavailability. In this study, we evaluate the therapeutic efficacy of GO mediated TNF-α_siRNA using in vitro models of chronic inflammation generated by treating murine small intestines (enteroids) and large intestines (colonoids) with inflammatory agents IL-1β, TNF-α, and LPS. The organotypic mouse enteroids and colonoids developed an inflammatory phenotype similar to that of IBD, characterized by impaired epithelial homeostasis and an increased production of inflammatory cytokines such as TNF-α, IL-1β, and IL-6. We assessed siRNA delivery to these inflamed organoids using three different GO formulations. Out of the three, small-sized GO with polymer and dendrimer modifications (smGO) demonstrated the highest transfection efficiency, which led to the downregulation of inflammatory cytokines, indicating an attenuation of the inflammatory phenotype. Moreover, the transfection efficiency and inflammation-ameliorating effects could be further enhanced by increasing the TNF-α_siRNA/smGO ratio from 1:1 to 3:1. Overall, the results of this study demonstrate that ex vivo organoids with disease-specific phenotypes are invaluable models for assessing the therapeutic potential of nanocarrier-mediated drug and biologic delivery systems.

DNA methylation mediated by RdDM pathway and demethylation affects furanone accumulation through regulation of QUINONE OXIDOREDUCTASE in strawberry.

Recently, increasing evidence suggests that DNA methylation plays a crucial role in fruit ripening. However, the role of DNA methylation in regulating specific traits, such as flavor, remains unclear. Here, we report a role of DNA methylation in affecting furanone biosynthesis in strawberry. Strawberry quinone oxidoreductase (FaQR) is a key enzyme in furanone biosynthesis. There are four FaQR homologs in strawberry cultivar 'Yuexin', and one of them, FaQR3, contributes ~50% of FaQR transcripts, indicating a major role of FaQR3 in furanone biosynthesis. Through characterization of levels of DNA methylation and FaQR3 transcript and furanone contents during fruit ripening and after the application of DNA methylation inhibitor, we found that the DNA methylation level of the FaQR3 promoter was negatively correlated with FaQR3 expression and furanone accumulation, suggesting that DNA methylation may be involved in furanone biosynthesis through adjusting FaQR3 expression, and responded to different temperatures consistently. In addition, transient expression of a gene in the RNA-directed DNA methylation (RdDM) pathway, FaAGO4, and enrichment analysis of the 24-nucleotide siRNAs suggested that DNA methylation in the FaQR3 promoter is mediated by the RdDM pathway. Transient RNA interference (RNAi) of FaDML indicated that the demethylation pathway may be involved in regulating furanone accumulation. These findings provide new insights into the role of DNA methylation and demethylation in affecting flavor quality in strawberry during fruit ripening.

Identification of AP2/ERF transcription factors in Tetrastigma hemsleyanum revealed the specific roles of ERF46 under cold stress.

Tetrastigma hemsleyanum (T. hemsleyanum) is a traditional medicinal plant that is widely used in China. Cultivated T. hemsleyanum usually encounters cold stress, limiting its growth and quality at key developmental stages. APETALA2 (AP2)/ethylene-responsive factor (ERF) transcription factors (TFs) comprise one of the largest gene superfamilies in plants and are widely involved in biotic and abiotic stresses. To reveal the roles of AP2/ERF TFs during T. hemsleyanum development, 70 AP2/ERF TFs were identified in T. hemsleyanum. Among them, 18 and 2 TFs were classified into the AP2 and RAV families, respectively. The other 50 TFs belonged to the ERF family and were further divided into the ERF and (dehydration reaction element binding factor) DREB subfamilies. The ERF subfamily contained 46 TFs, while the DREB subfamily contained 4 TFs. Phylogenetic analysis indicated that AP2/ERF TFs could be classified into five groups, in which 10 conserved motifs were confirmed. Several motifs were group- or subgroup-specific, implying that they were significant for the functions of the AP2/ERF TFs of these clades. In addition, 70 AP2/ERF TFs from the five groups were used for an expression pattern analysis under three low-temperature levels, namely, –4, 0, and 4°C. The majority of these AP2/ERF TFs exhibited a positive response to cold stress conditions. Specifically, ThERF5, ThERF31, ThERF46, and ThERF55 demonstrated a more sensitive response to cold stress. Moreover, AP2/ERF TFs exhibited specific expression patterns under cold stress. Transient overexpression and RNA interference indicated that ThERF46 has a specific tolerance to cold stress. These new insights provide the basis for further studies on the roles of AP2/ERF TFs in cold stress tolerance in T. hemsleyanum.

A non-photosynthetic green alga illuminates the reductive evolution of plastid electron transport systems

BackgroundPlastid electron transport systems are essential not only for photosynthesis but also for dissipating excess reducing power and sinking excess electrons generated by various redox reactions. Although numerous organisms with plastids have lost their photoautotrophic lifestyles, there is a spectrum of known functions of remnant plastids in non-photosynthetic algal/plant lineages; some of non-photosynthetic plastids still retain diverse metabolic pathways involving redox reactions while others, such as apicoplasts of apicomplexan parasites, possess highly reduced sets of functions. However, little is known about underlying mechanisms for redox homeostasis in functionally versatile non-photosynthetic plastids and thus about the reductive evolution of plastid electron transport systems.ResultsHere we demonstrated that the central component for plastid electron transport systems, plastoquinone/plastoquinol pool, is still retained in a novel strain of an obligate heterotrophic green alga lacking the photosynthesis-related thylakoid membrane complexes. Microscopic and genome analyses revealed that the Volvocales green alga, chlamydomonad sp. strain NrCl902, has non-photosynthetic plastids and a plastid DNA that carries no genes for the photosynthetic electron transport system. Transcriptome-based in silico prediction of the metabolic map followed by liquid chromatography analyses demonstrated carotenoid and plastoquinol synthesis, but no trace of chlorophyll pigments in the non-photosynthetic green alga. Transient RNA interference knockdown leads to suppression of plastoquinone/plastoquinol synthesis. The alga appears to possess genes for an electron sink system mediated by plastid terminal oxidase, plastoquinone/plastoquinol, and type II NADH dehydrogenase. Other non-photosynthetic algae/land plants also possess key genes for this system, suggesting a broad distribution of an electron sink system in non-photosynthetic plastids.ConclusionThe plastoquinone/plastoquinol pool and thus the involved electron transport systems reported herein might be retained for redox homeostasis and might represent an intermediate step towards a more reduced set of the electron transport system in many non-photosynthetic plastids. Our findings illuminate a broadly distributed but previously hidden step of reductive evolution of plastid electron transport systems after the loss of photosynthesis.

RNA Interference-Based Screen Reveals Concerted Functions of MEKK2 and CRCK3 in Plant Cell Death Regulation.

A wide variety of intrinsic and extrinsic cues lead to cell death with unclear mechanisms. The infertility of some death mutants often hurdles the classical suppressor screens for death regulators. We have developed a transient RNA interference (RNAi)-based screen using a virus-induced gene silencing approach to understand diverse cell death pathways in Arabidopsis (Arabidopsis thaliana). One death pathway is due to the depletion of a MAP kinase (MAPK) cascade, consisting of MAPK kinase kinase 1 (MEKK1), MKK1/2, and MPK4, which depends on a nucleotide-binding site Leu-rich repeat (NLR) protein SUMM2. Silencing of MEKK1 by virus-induced gene silencing resembles the mekk1 mutant with autoimmunity and defense activation. The RNAi-based screen toward Arabidopsis T-DNA insertion lines identified SUMM2, MEKK2, and Calmodulin-binding receptor-like cytoplasmic kinase 3 (CRCK3) to be vital regulators of RNAi MEKK1-induced cell death, consistent with the reports of their requirement in the mekk1-mkk1/2-mpk4 death pathway. Similar with MEKK2, overexpression of CRCK3 caused dosage- and SUMM2-dependent cell death, and the transcripts of CRCK3 were up-regulated in mekk1, mkk1/2, and mpk4 MEKK2-induced cell death depends on CRCK3. Interestingly, CRCK3-induced cell death also depends on MEKK2, consistent with the biochemical data that MEKK2 complexes with CRCK3. Furthermore, the kinase activity of CRCK3 is essential, whereas the kinase activity of MEKK2 is dispensable, for triggering cell death. Our studies suggest that MEKK2 and CRCK3 exert concerted functions in the control of NLR SUMM2 activation and MEKK2 may play a structural role, rather than function as a kinase, in regulating CRCK3 protein stability.

Discovery and characterization of tannase genes in plants: roles in hydrolysis of tannins.

Plant tannins, including condensed tannins (CTs) and hydrolyzable tannins (HTs), are widely distributed in the plant kingdom. To date, tannase (TA) - is a type of tannin acyl-hydrolase hydrolyzing HTs, CT monomer gallates and depsides - has been reported in microbes only. Whether plants express TA remains unknown. Herein, we report plant TA genes. A native Camellia sinensis TA (CsTA) is identified from leaves. Six TAs are cloned from tea, strawberry (Fragaria×ananassa, Fa) and four other crops. Biochemical analysis shows that the native CsTA and six recombinant TAs hydrolyze tannin compounds, depsides and phenolic glycosides. Transcriptional and metabolic analyses reveal that the expression of CsTA is oppositely associated with the accumulation of galloylated catechins. Moreover, the transient overexpression and RNA interference of FaTA are positively associated with the accumulation of ellagitannins in strawberry fruit. Phylogenetic analysis across different kingdoms shows that 29 plant TA homologs are clustered as a plant-specific TA clade in class I carboxylesterases. Further analysis across the angiosperms reveals that these TA genes are dispersed in tannin-rich plants, which share a single phylogenetic origin c. 120 million yr ago. Plant TA is discovered for the first time in the plant kingdom and is shown to be valuable to improve tannin compositions in plants.

Tandem 13-Lipoxygenase Genes in a Cluster Confers Yellow-Green Leaf in Cucumber.

Some lipoxygenase (LOX) isoenzymes can co-oxidize carotenoids. Carotenoids are collectors of light energy for photosynthesis and can protect plants from reactive oxygen species and coloration. This study isolated the cucumber (Cucumis sativus L.) yellow-green leaf mutant (ygl1), which had yellow-green leaves with decreased chlorophyll synthesis, increased relative carotenoid content, and delayed chloroplast development. Genetic analysis demonstrated that the phenotype of ygl1 was caused by a recessive mutation in a nuclear gene. The bulked segregants were resequenced, and the candidate ygl1 locus identified was mapped to the 9.2 kb region of the chromosome 4. Sequence analysis revealed that ygl1 encodes the tandem 13-LOX genes in a cluster. Four missense mutations were found in four tandem 13-LOX genes (Csa4M286960, Csa4M287550, Csa4M288070, and Csa4M288080) in the ygl1 mutant, and the four 13-LOX genes showed high similarity with one another. The transient RNA interference and virus-induced gene silencing of these genes simultaneously resulted in yellow-green leaves with a reduced amount of chloroplasts and increased relative carotenoid content, which were observed in the ygl1 mutant. This evidence supported the non-synonymous SNPs (Single Nucleotide Polymorphism) in the four tandem 13-LOX genes as being the causative mutation for the yellow-green leaves. Furthermore, this study provides a new allele for breeding cucumbers with yellow-green leaves and serves as an additional resource for studying carotenoid biosynthesis.

Ubiquitin Extension Protein UEP1 Modulates Cell Death and Resistance to Various Pathogens in Tobacco.

Ubiquitin (Ub) extension proteins (UEPs) are fusion proteins of a Ub at the N terminus to a ribosomal protein. They are the main source of Ub and the only source of extension ribosomal protein. Although important roles of the Ub-26S proteasome system in various biological processes have been well established, direct evidence for the role of UEP genes in plant defense is rarely reported. In this study, we cloned a Ub-S27a-type UEP gene (NbUEP1) from Nicotiana benthamiana and demonstrated its function in cell death and disease resistance. Virus-induced gene silencing of NbUEP1 led to intensive cell death, culminating in whole-seedling withering. Transient RNA interference (RNAi) of NbUEP1 caused strong cell death in infiltrated areas, while stable NbUEP1-RNAi tobacco plants constitutively formed necrotic lesions in leaves. NbUEP1-RNAi plants exhibited increased resistance to the oomycete Pythium aphanidermatum and viruses Tobacco mosaic virus and Cucumber mosaic virus while displaying decreased resistance to the nematode Meloidogyne incognita compared with non-RNAi control plants. Transcription profiling analysis indicated that jasmonate and ethylene pathways, lipid metabolism, copper amine oxidase-mediated active species generation, glycine-rich proteins, vacuolar processing enzyme- and RD21-mediated cell death and defense regulation, and autophagy might be associated with NbUEP1-mediated cell death and resistance. Our results provided evidence for the important roles of plant UEPs in modulating plant cell death and disease resistance.

Automated Image Acquisition and Morphological Analysis of Cell Growth Mutants in Physcomitrella patens.

This protocol describes the automated imaging and a quantitative analysis of the morphology of small plants from the moss Physcomitrella patens. This method can be used for the analysis of growth phenotypes produced by transient RNA interference or for the analysis of stable mutant plants. Furthermore, we describe how to acquire higher resolution images via the acquisition of a collection of multiple overlapping tiles from the same image. Information is presented to guide the investigator in the choice of vectors and basic conditions to perform transient RNA interference in moss. Detailed directions and examples for fluorescence image acquisition of small regenerating moss plants are provided. Instructions for stitching image tiles and for using an ImageJ-based macro for the quantitative morphological analysis of moss plants are also provided.

Pig IFITMs are restriction factors for Japanese encephalitis virus

Objective: Japanese encephalitis virus (JEV) is responsible for one of the most serious epidemics of encephalitis in the world. JEV uses pigs as its main hosts and spreads among vertebrates and humans mediated by Culex mosquitoes. The prevention and control of JEV spread in pigs is one of the most effective measures to protect global public health. Interferon-inducible transmembrane proteins (IFITMs) are small membrane-spanning proteins that were identified as innate antiviral factors against multiple pathogenic viruses, especially enveloped viral pathogens. This study aims to verify whether pig interferon-inducible transmembrane proteins (pIFITMs) inhibit JEV and investigate the related molecular mechanisms of anti-JEV. Methods: Transient expression and RNA interference technology were used to overexpress and silence IFITMs gene. Three different cell lines, PK15, HEK293 and Huh7, were transfected with recombinant pIFITMs-expressing plasmids. The lentiviral vectors harboring RNAi sequences targeting pIFITMs were introduced into PK15 cells. Quantitative real-time PCR was used to determine the antiviral activities of pIFITMs through measuring and analyzing the virus copy number of JEV (SA14-14-2 strain) in the supernant of pIFITMs overexpression or silencing cells 48 hours post transfection. The expression of the related proteins was examined by western blot. The fusion vectors inserted with pig IFITM1-EGFP were constructed and introduced into three different cell lines respectively. Then Laser Co-focus light microscopy was used to observe the subcellular localization. The key active amino acids of pIFITM1 were analyzed by investigating the anti-JEV effect of the cysteine mutants produced with PCR site directed mutagenesistechnology. Results: In three different cell lines, PK15, HEK293 and Huh7, all of three pig IFITM proteins, pIFITM1, pIFITM2, and pIFITM3 could inhibit the replication of JEV whether through transient gene over-expression methods or RNA interference silencing. And that, among three pig IFITMs, pIFITM1 showed the strongest anti-JEV effect. The anti-JEV activity of pIFITM1 manifested at the early entry stage. In PK15, BHK21, and HEK293 cells, before virus infection, pIFITM1 was located in the plasma membrane area, and after infection, transferred to the membranous structures outside the nucleus. The S-palmitoylated cysteines at position 50, 51 and 84 of pIFITM1 had significant effect on virus replication. Conclusions: Pig interferon-inducible transmembrane proteins are restriction factors for JEV infection and have potentials in the prevention of virus spread. Our results provide some new sights into understanding the antiviral activity of pig IFITMs.

Abstract B31: The MUC4 oncomucin mediates resistance of human pancreatic cancer cells to FOLFIRINOX drugs

Abstract Introduction: Pancreatic adenocarcinoma (PDAC) is one of the most deadly cancers in western countries with an extremely poor prognosis because of a lack of efficient therapeutic tools. Gemcitabine, a fluorinated analog of deoxycytidine, is the main chemotherapy in PDAC, but survival remains poor. FOLFIRINOX, a more aggressive protocol combining 5-fluorouracil (5-FU), irinotecan/SN-38, oxaliplatin and leucovorin, emerged as a new option in patients with metastatic pancreatic cancer. MUC4 is a membrane-bound oncomucin involved in pancreatic cancer (PC) pathogenesis and chemoresistance to gemcitabine. MUC4 is not expressed in healthy pancreas whereas its expression increases constantly during carcinogenetic progression. In this work, we aimed to decipher the involvement of MUC4 and associated cellular mechanisms in chemoresistance to FOLFIRINOX drugs. Material and Methods: MUC4 expression was knocked down (KD) by stable ShRNA in CAPAN-1/-2 PC cells. IC50 and cell viability to FOLFIRINOX drugs were determined by tetrazolium salt (MTT) assays. Expression of genes involved in drug metabolism was assessed by quantitative RT-PCR (qRT-PCR) and Western blot. MiRnomes of Mock and MUC4-KD cells were determined using miRNA array (8x15K Agilent). Results: MUC4 depletion in PC cells leads to an increase of sensitivity to 5-FU and to an increased resistance to oxaliplatin. Combined treatments of 5-FU and oxaliplatin (FOX) lead to an intermediate survival rate suggesting additional effects. This altered chemosensitivity is associated with an altered expression of drug transporters/channels and drug metabolism actors. Notably, an increase of CTR1 and ATP7B transporters and DPYD enzyme was observed in MUC4-KD cells. Furthermore, we have observed downregulation of miR-96-5p, miR-132-3p and miR-210 suggesting a possible link between MUC4 expression, drug resistance and regulation by miRNAs. The involvement of these molecules is currently validated by transient RNA interference and miRNA transfections. Conclusion: this work led to the identification of proteins and miRNAs specifically involved in priming PC chemoresistance. These factors may represent critical therapeutic targets and also robust prognostic/predictive markers, and thus provide better healthcare and management of PC patients. Citation Format: Nicolas Skrypek, Nihad Boukrout, Fatima Lahdaoui, Céline Schulz, Romain Vasseur, Belinda Duchêne, Isabelle Van Seuningen, Nicolas Jonckheere.{Authors}. The MUC4 oncomucin mediates resistance of human pancreatic cancer cells to FOLFIRINOX drugs. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B31.

The role of ZmWRKY4 in regulating maize antioxidant defense under cadmium stress

WRKY transcription factors act as positive regulators in abiotic stress responses by activation of the cellular antioxidant systems. However, there are few reports on the response of WRKY genes to cadmium (Cd) stress. In this study, the role of maize ZmWRKY4 in regulating antioxidant enzymes in Cd stress was investigated. The results indicated that Cd induced up-regulation of the expression and the activities of ZmWRKY4 and superoxide dismutase (SOD) and ascorbate peroxidase (APX).Transient expression and RNA interference (RNAi) silencing of ZmWRKY4 in maize mesophyll protoplasts further revealed that ZmWRKY4 was required for the abscisic acid (ABA)-induced increase in expression and activity of SOD and APX. Overexpression of ZmWRKY4 in protoplasts upregulated the expression and the activities of antioxidant enzymes, whereas ABA induced increases in the expression and the activities of antioxidant enzymes were blocked by the RNAi silencing of ZmWRKY4.Bioinformatic analysis indicated that ZmSOD4 and ZmcAPX both harbored two W-boxes, binding motif for WRKY transcription factors, in their promoter region. Intriguingly, ZmWRKY4 belongs to group I WRKYs with two WRKY domains. Moreover, the synchronized expression patterns indicate that ZmWRKY4 might play a critical role in either regulating the ZmSOD4 and ZmcAPX expression or cooperating with them in response to stress and phytohormone.

Cyclin E1 Inhibition can Overcome Sorafenib Resistance in Hepatocellular Carcinoma Cells Through Mcl-1 Suppression.

To clarify the effects of cyclin E1 suppression on antitumor efficacy of sorafenib in hepatocellular carcinoma cells and to explore the potential of combining sorafenib with cyclin-dependent kinase (CDK) inhibition in therapy. The effects of cyclin E1 suppression on sorafenib-induced apoptosis were tested in both sorafenib-sensitive (Huh-7 and HepG2, IC50 5-6 μmol/L) and sorafenib-resistant (Huh-7R and HepG2R, IC50 14-15 μmol/L) hepatocellular carcinoma cells. The activity of pertinent signaling pathways and the expression of cell cycle and apoptosis-related proteins were measured using Western blotting. Efficacy of sorafenib combined with the pan-CDK inhibitor flavopiridol was tested both in vitro and in xenograft experiments. The pertinent downstream mediators of antitumor efficacy were tested in transient transfection and RNA interference experiments. Cyclin E1 mRNA and protein expressions were suppressed after sorafenib treatment in sorafenib-sensitive but not in sorafenib-resistant hepatocellular carcinoma cells. Changes in cyclin E2 or D1 were not correlated with sorafenib sensitivity. The knockdown of cyclin E1 expression reversed the resistance of hepatocellular carcinoma cells to sorafenib in terms of cell growth and apoptosis induction, whereas the overexpression of cyclin E1 increased the resistance to sorafenib. The growth-inhibitory and apoptosis-inducing effects of sorafenib were enhanced by flavopiridol, and Mcl-1 suppression was determined to play a critical role in mediating this enhancing effect. The cyclin E1 suppression in hepatocellular carcinoma cells may serve as a pharmacodynamic biomarker for predicting sorafenib efficacy. The combination of sorafenib and CDK inhibitors may improve the efficacy of sorafenib in hepatocellular carcinoma. Clin Cancer Res; 22(10); 2555-64. ©2015 AACR.

Role of TFEB-driven autophagy regulation in pancreatic cancer treatment.

Autophagy pathways promote the growth of pancreatic ductal adenocarcinoma (PDAC), but the critical role is yet to be determined. Transcription factor EB (TFEB) centrally controls lysosomal and autophagy biogenesis. This study aimed to explore the role of TFEB for autophagy regulation in PDAC. We found that TFEB expression was significantly elevated in human PDAC samples (n=45), and localized to the cytoplasm and nucleus in 11 of 15 cases. In primary PDAC cell lines, TFEB nuclear expression was evident even under basal conditions, and further nuclear enrichment was achieved by starvation. Transient RNA interference reduced TFEB expression to 11-23%, but starvation-induced accumulation of the lipidated, autophagosome-associated LC3-II and the autophago-to-lysosome route was maintained after TFEB silencing. Likewise, gemcitabine treatment of the cancer cells augmented apoptosis and LC3-II as an indicator of autophagy, regardless of the TFEB expression levels. Moreover, the interplay of oncogenic KRAS with TFEB and autophagy was investigated. KRAS silencing caused PDAC cell apoptosis and a reciprocal increase in TFEB expression. This inverse correlation could be confirmed in published data sets of genetically engineered mouse models and human PDAC samples using the the Pubmed GEO and BioPortal databases, and was independent of KRAS mutation status. In conclusion, the central autophagy regulator TFEB is expressed and active in PDAC, but autophagy is sustained after TFEB knockdown, suggesting alternative bypass signaling. TFEB is dispensable for gemcitabine-induced cell death, but inversely correlated with KRAS expression.

FOXM1 is overexpressed in B-acute lymphoblastic leukemia (B-ALL) and its inhibition sensitizes B-ALL cells to chemotherapeutic drugs.

The Forkhead box protein M1 (FOXM1) is a transcription factor that plays a central role in the regulation of cell cycle, proliferation, DNA repair, and apoptosis. FOXM1 is overexpressed in many human tumors and its upregulation has been linked to high proliferation rates and poor prognosis. We therefore studied the role of FOXM1 in B-lymphoblastic leukemia (B-ALL) in order to understand whether FOXM1 could be a key target for leukemia therapy. RT-PCR and western blot analysis were carried out in a small cohort of pediatric B-ALL patients to evaluate FOXM1 levels. To assess its biological relevance, its expression was down-modulated by transient RNA interference in B-ALL cell lines (REH and NALM-6). Our results show that FOXM1 expression is higher in both B-ALL patients and cell lines when compared to PBMC or normal B-cells (CD19+) from healthy donors. Furthermore, blocking FOXM1 activity in two B-ALL cell lines, by either knockdown or treatment with the FOXM1 inhibitor thiostrepton, causes significant decrease in their cell proliferation. This decrease in cell proliferation was coupled with both an induction of the G2/M cell cycle arrest and with a reduction in the S phase population. Finally, we noted how thiostrepton synergises with chemotherapeutic agents commonly used in B-ALL therapy, thus increasing their efficiency. Therefore our results suggest that FOXM1 is highly expressed in both patients and B-ALL cell lines, and that targeting FOXM1 could be an attractive strategy for leukemia therapy and for overcoming drug resistance.

An ACCUMULATION AND REPLICATION OF CHLOROPLASTS 5 gene mutation confers light green peel in cucumber.

The peel color of fruit is an important commercial trait in cucumber, but the underlying molecular basis is largely unknown. A mutant showing light green exocarp was discovered from ethyl methane sulfonate (EMS) mutagenized cucumber line 406 with dark green exocarp. Genetic analysis showed the mutant phenotype is conferred by a single recessive gene, here designated as lgp (light green peel). By re-sequencing of bulked segregants, we identified the candidate gene Csa7G051430 encoding ACCUMULATION AND REPLICATION OF CHLOROPLASTS 5 (ARC5) that plays a vital role in chloroplast division in Arabidopsis. A single nucleotide polymorphism (SNP) causing amino acid alteration in the conserved GTPase domain of Csa7G051430 showed co-segregation with the altered phenotype. Furthermore, the transient RNA interference of this gene resulted in reduced number and enlarged size of chloroplasts, which were also observed in the lgp mutant. This evidence supports that the non-synonymous SNP in Csa7G051430 is the causative mutation for the light green peel. This study provides a new allele for cucumber breeding for light green fruits and additional resource for the study of chloroplast development.

OsHK3 is a crucial regulator of abscisic acid signaling involved in antioxidant defense in rice

In this study, the role of the rice (Oryza sativa L.) histidine kinase OsHK3 in abscisic acid (ABA)-induced antioxidant defense was investigated. Treatments with ABA, H2 O2 , and polyethylene glycol (PEG) induced the expression of OsHK3 in rice leaves, and H2 O2 is required for ABA-induced increase in the expression of OsHK3 under water stress. Subcellular localization analysis showed that OsHK3 is located in the cytoplasm and the plasma membrane. The transient expression analysis and the transient RNA interference test in rice protoplasts showed that OsHK3 is required for ABA-induced upregulation in the expression of antioxidant enzymes genes and the activities of antioxidant enzymes. Further analysis showed that OsHK3 functions upstream of the calcium/calmodulin-dependent protein kinase OsDMI3 and the mitogen-activated protein kinase OsMPK1 to regulate the activities of antioxidant enzymes in ABA signaling. Moreover, OsHK3 was also shown to regulate the expression of nicotinamide adenine dinucleotide phosphate oxidase genes, OsrbohB and OsrbohE, and the production of H2 O2 in ABA signaling. Our data indicate that OsHK3 play an important role in the regulation of ABA-induced antioxidant defense and in the feedback regulation of H2 O2 production in ABA signaling.