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1.  Optimization and comparison of knockdown efficacy between polymerase II expressed shRNA and artificial miRNA targeting luciferase and Apolipoprotein B100 
BMC Biotechnology  2012;12:42.
Controlling and limiting the expression of short hairpin RNA (shRNA) by using constitutive or tissue-specific polymerase II (pol II) expression can be a promising strategy to avoid RNAi toxicity. However, to date detailed studies on requirements for effective pol II shRNA expression and processing are not available. We investigated the optimal structural configuration of shRNA molecules, namely: hairpin location, stem length and termination signal required for effective pol II expression and compared it with an alternative strategy of avoiding toxicity by using artificial microRNA (miRNA) scaffolds.
Highly effective shRNAs targeting luciferase (shLuc) or Apolipoprotein B100 (shApoB1 and shApoB2) were placed under the control of the pol II CMV promoter and expressed at +5 or +6 nucleotides (nt) with reference to the transcription start site (TSS). Different transcription termination signals (TTS), namely minimal polyadenylation (pA), poly T (T5) and U1 were also used. All pol II- expressed shRNA variants induced mild inhibition of Luciferase reporters carrying specific targets and none of them showed comparable efficacy to their polymerase III-expressed H1-shRNA controls, regardless of hairpin position and termination signal used. Extending hairpin stem length from 20 basepairs (bp) to 21, 25 or 29 bp yielded only slight improvement in the overall efficacy. When shLuc, shApoB1 and shApoB2 were placed in an artificial miRNA scaffold, two out of three were as potent as the H1-shRNA controls. Quantification of small interfering RNA (siRNA) molecules showed that the artificial miRNA constructs expressed less molecules than H1-shRNAs and that CMV-shRNA expressed the lowest amount of siRNA molecules suggesting that RNAi processing in this case is least effective. Furthermore, CMV-miApoB1 and CMV-miApoB2 were as effective as the corresponding H1-shApoB1 and H1-shApoB2 in inhibiting endogenous ApoB mRNA.
Our results demonstrate that artificial miRNA have a better efficacy profile than shRNA expressed either from H1 or CMV promoter and will be used in the future for RNAi therapeutic development.
PMCID: PMC3424168  PMID: 22827812
2.  Modulation of ethylene- and heat-controlled hyponastic leaf movement in Arabidopsis thaliana by the plant defence hormones jasmonate and salicylate 
Planta  2011;235(4):677-685.
Upward leaf movement (hyponastic growth) is adopted by several plant species including Arabidopsis thaliana, as a mechanism to escape adverse growth conditions. Among the signals that trigger hyponastic growth are, the gaseous hormone ethylene, low light intensities, and supra-optimal temperatures (heat). Recent studies indicated that the defence-related phytohormones jasmonic acid (JA) and salicylic acid (SA) synthesized by the plant upon biotic infestation repress low light-induced hyponastic growth. The hyponastic growth response induced by high temperature (heat) treatment and upon application of the gaseous hormone ethylene is highly similar to the response induced by low light. To test if these environmental signals induce hyponastic growth via parallel pathways or converge downstream, we studied here the roles of Methyl-JA (MeJA) and SA on ethylene- and heat-induced hyponastic growth. For this, we used a time-lapse camera setup. Our study includes pharmacological application of MeJA and SA and biological infestation using the JA-inducing caterpillar Pieris rapae as well as mutants lacking JA or SA signalling components. The data demonstrate that MeJA is a positive, and SA, a negative regulator of ethylene-induced hyponastic growth and that both hormones repress the response to heat. Taking previous studies into account, we conclude that SA is the first among many tested components which is repressing hyponastic growth under all tested inductive environmental stimuli. However, since MeJA is a positive regulator of ethylene-induced hyponastic growth and is inhibiting low light- and heat-induced leaf movement, we conclude that defence hormones control hyponastic growth by affecting stimulus-specific signalling pathways.
PMCID: PMC3313027  PMID: 22009062
Arabidopsis; Ethylene; Heat; Hyponastic growth; Jasmonate; Leaf movement; Salicylate
3.  In vivo knock-down of multidrug resistance transporters ABCC1 and ABCC2 by AAV-delivered shRNAs and by artificial miRNAs 
ABC transporters export clinically-relevant drugs and their over-expression causes multidrug resistance. In order to knock-down ABC transporters, ABCC1 and ABCC2, 13 shRNAs were developed. Four shRNA candidates were tested in vivo using self-complementary adeno-associated virus serotype 8. A strong, specific knock-down of Abbc2 was observed in mice liver, but at the cost of toxicity caused by oversaturation of the RNAi machinery due to high shRNA expression. Subsequent generation of artificial miRNAs showed better efficacy profile. These results demonstrate the feasibility of knocking down Abbc2 via AAV-delivered shRNAs to the liver, and encourage the use of miRNA in further therapeutics development.
PMCID: PMC3131674  PMID: 21769296
shRNA; miRNA; AAV; Abbc1; Abbc2; multidrug resistance; hepatocellular carcinoma
4.  Kinome Profiling Reveals an Interaction Between Jasmonate, Salicylate and Light Control of Hyponastic Petiole Growth in Arabidopsis thaliana 
PLoS ONE  2010;5(12):e14255.
Plants defend themselves against infection by biotic attackers by producing distinct phytohormones. Especially jasmonic acid (JA) and salicylic acid (SA) are well known defense-inducing hormones. Here, the effects of MeJA and SA on the Arabidopsis thaliana kinome were monitored using PepChip arrays containing kinase substrate peptides to analyze posttranslational interactions in MeJA and SA signaling pathways and to test if kinome profiling can provide leads to predict posttranslational events in plant signaling. MeJA and SA mediate differential phosphorylation of substrates for many kinase families. Also some plant specific substrates were differentially phosphorylated, including peptides derived from Phytochrome A, and Photosystem II D protein. This indicates that MeJA and SA mediate cross-talk between defense signaling and light responses. We tested the predicted effects of MeJA and SA using light-mediated upward leaf movement (differential petiole growth also called hyponastic growth). We found that MeJA, infestation by the JA-inducing insect herbivore Pieris rapae, and SA suppressed low light-induced hyponastic growth. MeJA and SA acted in a synergistic fashion via two (partially) divergent signaling routes. This work demonstrates that kinome profiling using PepChip arrays can be a valuable complementary ∼omics tool to give directions towards predicting behavior of organisms after a given stimulus and can be used to obtain leads for physiological relevant phenomena in planta.
PMCID: PMC2999534  PMID: 21170386
5.  Salicylate-mediated suppression of jasmonate-responsive gene expression in Arabidopsis is targeted downstream of the jasmonate biosynthesis pathway 
Planta  2010;232(6):1423-1432.
Jasmonates (JAs) and salicylic acid (SA) are plant hormones that play pivotal roles in the regulation of induced defenses against microbial pathogens and insect herbivores. Their signaling pathways cross-communicate providing the plant with a regulatory potential to finely tune its defense response to the attacker(s) encountered. In Arabidopsis thaliana, SA strongly antagonizes the jasmonic acid (JA) signaling pathway, resulting in the downregulation of a large set of JA-responsive genes, including the marker genes PDF1.2 and VSP2. Induction of JA-responsive marker gene expression by different JA derivatives was equally sensitive to SA-mediated suppression. Activation of genes encoding key enzymes in the JA biosynthesis pathway, such as LOX2, AOS, AOC2, and OPR3 was also repressed by SA, suggesting that the JA biosynthesis pathway may be a target for SA-mediated antagonism. To test this, we made use of the mutant aos/dde2, which is completely blocked in its ability to produce JAs because of a mutation in the ALLENE OXIDE SYNTHASE gene. Mutant aos/dde2 plants did not express the JA-responsive marker genes PDF1.2 or VSP2 in response to infection with the necrotrophic fungus Alternaria brassicicola or the herbivorous insect Pieris rapae. Bypassing JA biosynthesis by exogenous application of methyl jasmonate (MeJA) rescued this JA-responsive phenotype in aos/dde2. Application of SA suppressed MeJA-induced PDF1.2 expression to the same level in the aos/dde2 mutant as in wild-type Col-0 plants, indicating that SA-mediated suppression of JA-responsive gene expression is targeted at a position downstream of the JA biosynthesis pathway.
PMCID: PMC2957573  PMID: 20839007
Hormone crosstalk; Jasmonic acid; Plant defense; Salicylic acid
6.  Kinome profiling of sugar signaling in plants using multiple platforms 
Plant Signaling & Behavior  2009;4(12):1169-1173.
Although the primary sequence of kinases shows substantial divergence between unrelated eukaryotes, variation in the motifs that are actually phosphorylated by eukaryotic kinases is much smaller. Hence arrays developed for kinome profiling of mammalian cells are useful for kinome profiling of plant tissues as well, facilitating the study of plant signal transduction. We recently employed the Pepscan kinomics chip to reveal the small GTPases in plant sucrose signaling. Here we show that employing a different peptide library (the Pepscan kinase chip) largely similar results are obtained, confirming these earlier data, but such a different library also contributes new insights into the molecular details mediating plant cell responses to a sugar stimulus. Thus when studying plant signal transduction employing peptide arrays, using multiple platforms both increases the confidence of results and provides additional information.
PMCID: PMC2819448  PMID: 20514238
sucrose; arabidopsis; kinome profiling; kinase; MAPK; SnRK; casein kinase; tyrosine phosphorylation; CDC2; AGC kinase
7.  Are Small GTPases Signal Hubs in Sugar-Mediated Induction of Fructan Biosynthesis? 
PLoS ONE  2009;4(8):e6605.
External sugar initiates biosynthesis of the reserve carbohydrate fructan, but the molecular processes mediating this response remain obscure. Previously it was shown that a phosphatase and a general kinase inhibitor hamper fructan accumulation. We use various phosphorylation inhibitors both in barley and in Arabidopsis and show that the expression of fructan biosynthetic genes is dependent on PP2A and different kinases such as Tyr-kinases and PI3-kinases. To further characterize the phosphorylation events involved, comprehensive analysis of kinase activities in the cell was performed using a PepChip, an array of >1000 kinase consensus substrate peptide substrates spotted on a chip. Comparison of kinase activities in sugar-stimulated and mock(sorbitol)-treated Arabidopsis demonstrates the altered phosphorylation of many consensus substrates and documents the differences in plant kinase activity upon sucrose feeding. The different phosphorylation profiles obtained are consistent with sugar-mediated alterations in Tyr phosphorylation, cell cycling, and phosphoinositide signaling, and indicate cytoskeletal rearrangements. The results lead us to infer a central role for small GTPases in sugar signaling.
PMCID: PMC2720452  PMID: 19672308
8.  An acceptor-substrate binding site determining glycosyl transfer emerges from mutant analysis of a plant vacuolar invertase and a fructosyltransferase 
Plant Molecular Biology  2008;69(1-2):47-56.
Glycoside hydrolase family 32 (GH32) harbors hydrolyzing and transglycosylating enzymes that are highly homologous in their primary structure. Eight amino acids dispersed along the sequence correlated with either hydrolase or glycosyltransferase activity. These were mutated in onion vacuolar invertase (acINV) according to the residue in festuca sucrose:sucrose 1-fructosyltransferase (saSST) and vice versa. acINV(W440Y) doubles transferase capacity. Reciprocally, saSST(C223N) and saSST(F362Y) double hydrolysis. SaSST(N425S) shows a hydrolyzing activity three to four times its transferase activity. Interestingly, modeling acINV and saSST according to the 3D structure of crystallized GH32 enzymes indicates that mutations saSST(N425S), acINV(W440Y), and the previously reported acINV(W161Y) reside very close together at the surface in the entrance of the active-site pocket. Residues in- and outside the sucrose-binding box determine hydrolase and transferase capabilities of GH32 enzymes. Modeling suggests that residues dispersed along the sequence identify a location for acceptor-substrate binding in the 3D structure of fructosyltransferases.
Electronic supplementary material
The online version of this article (doi:10.1007/s11103-008-9404-7) contains supplementary material, which is available to authorized users.
PMCID: PMC2709226  PMID: 18821058
Invertase; Fructosyltransferase; Sucrose; Transglycosylation; Site-directed mutagenesis; Molecular modeling
9.  Evidence for a Minimal Eukaryotic Phosphoproteome? 
PLoS ONE  2007;2(8):e777.
Reversible phosphorylation catalysed by kinases is probably the most important regulatory mechanism in eukaryotes.
Methodology/Principal Findings
We studied the in vitro phosphorylation of peptide arrays exhibiting the majority of PhosphoBase-deposited protein sequences, by factors in cell lysates from representatives of various branches of the eukaryotic species. We derived a set of substrates from the PhosphoBase whose phosphorylation by cellular extracts is common to the divergent members of different kingdoms and thus may be considered a minimal eukaryotic phosphoproteome. The protein kinases (or kinome) responsible for phosphorylation of these substrates are involved in a variety of processes such as transcription, translation, and cytoskeletal reorganisation.
These results indicate that the divergence in eukaryotic kinases is not reflected at the level of substrate phosphorylation, revealing the presence of a limited common substrate space for kinases in eukaryotes and suggests the presence of a set of kinase substrates and regulatory mechanisms in an ancestral eukaryote that has since remained constant in eukaryotic life.
PMCID: PMC1945084  PMID: 17712425
10.  Kinome profiling of Arabidopsis using arrays of kinase consensus substrates 
Plant Methods  2007;3:3.
Kinome profiling aims at the parallel analysis of kinase activities in a cell. Novel developed arrays containing consensus substrates for kinases are used to assess those kinase activities. The arrays described in this paper were already used to determine kinase activities in mammalian systems, but since substrates from many organisms are present we decided to test these arrays for the determination of kinase activities in the model plant species Arabidopsis thaliana.
Kinome profiling using Arabidopsis cell extracts resulted in the labelling of many consensus peptides by kinases from the plant, indicating the usefulness of this kinome profiling tool for plants. Method development showed that fresh and frozen plant material could be used to make cell lysates containing active kinases. Dilution of the plant extract increased the signal to noise ratio and non-radioactive ATP enhances full development of spot intensities.
Upon infection of Arabidopsis with an avirulent strain of the bacterial pathogen Pseudomonas syringae pv. tomato, we could detect differential kinase activities by measuring phosphorylation of consensus peptides.
We show that kinome profiling on arrays with consensus substrates can be used to monitor kinase activities in plants. In a case study we show that upon infection with avirulent P. syringae differential kinase activities can be found. The PepChip can for example be used to purify (unknown) kinases that play a role in P. syringae infection.
This paper shows that kinome profiling using arrays of consensus peptides is a valuable new tool to study signal-transduction in plants. It complements the available methods for genomics and proteomics research.
PMCID: PMC1803769  PMID: 17295910

Results 1-10 (10)