35S Enhancer Research Articles

Small DNA elements that act as both insulators and silencers in plants.

Insulators are cis -regulatory elements that separate transcriptional units, whereas silencers are elements that repress transcription regardless of their position. In plants, these elements remain largely uncharacterized. Here, we use the massively parallel reporter assay Plant STARR-seq with short fragments of eight large insulators to identify more than 100 fragments that block enhancer activity. The short fragments can be combined to generate more powerful insulators that abolish the capacity of the strong viral 35S enhancer to activate the 35S minimal promoter. Unexpectedly, when tested upstream of weak enhancers, these fragments act as silencers and repress transcription. Thus, these elements are capable of both insulating or repressing transcription dependent upon regulatory context. We validate our findings in stable transgenic Arabidopsis , maize, and rice plants. The short elements identified here should be useful building blocks for plant biotechnology efforts.

Using Machine Learning Approaches to Predict Target Gene Expression in Rice T-DNA Insertional Mutants.

To change the expression of the flanking genes by inserting T-DNA into the genome is commonly used in rice functional gene research. However, whether the expression of a gene of interest is enhanced must be validated experimentally. Consequently, to improve the efficiency of screening activated genes, we established a model to predict gene expression in T-DNA mutants through machine learning methods. We gathered experimental datasets consisting of gene expression data in T-DNA mutants and captured the PROMOTER and MIDDLE sequences for encoding. In first-layer models, support vector machine (SVM) models were constructed with nine features consisting of information about biological function and local and global sequences. Feature encoding based on the PROMOTER sequence was weighted by logistic regression. The second-layer models integrated 16 first-layer models with minimum redundancy maximum relevance (mRMR) feature selection and the LADTree algorithm, which were selected from nine feature selection methods and 65 classified methods, respectively. The accuracy of the final two-layer machine learning model, referred to as TIMgo, was 99.3% based on fivefold cross-validation, and 85.6% based on independent testing. We discovered that the information within the local sequence had a greater contribution than the global sequence with respect to classification. TIMgo had a good predictive ability for target genes within 20 kb from the 35S enhancer. Based on the analysis of significant sequences, the G-box regulatory sequence may also play an important role in the activation mechanism of the 35S enhancer.

Development of a Virus-Based Reporter System for Functional Analysis of Plant rRNA Gene Promoter.

Reporter gene-based expression systems have been intensively used in plants for monitoring the activity of gene promoters. However, rRNA transcripts are unable to efficiently express a reporter gene due to a lack of a 5' cap. Because of this obstacle, plant rRNA gene promoters are less well characterized to this day. We developed a virus-based reporter system to characterize the Nicotiana benthamiana rRNA (NbrRNA) gene promoter. The system utilizes the cap-independent translation strategy of viral genomic mRNA and uses the virus-expressed green fluorescent protein (GFP) as an indicator of the rRNA gene promoter activity in virus-infected plants. Based on the reporter system, some characteristics of the N. benthamiana rRNA gene promoter were revealed. The results showed that the strength of the NbrRNA gene promoter was lower than that of the cauliflower mosaic virus (CaMV) 35S promoter, a well-characterized polymerase II promoter. The sequences between −77 and +42 are sufficient for the NbrRNA gene promoter-mediated transcription and the NbrRNA gene promoter may lack the functional upstream control element (UCE). Interestingly, NbrRNA gene promoter activity was increased when the 35S enhancer was introduced. An intron-excision mediated assay revealed that the NbrRNA gene promoter can be inefficiently used by RNA polymerase II in N. benthamiana cells. This virus-based reporter system is easier to operate and more convenient when compared with the previously Pol I promoter assays. And it offers a promising solution to analyzing the functional architecture of plant rRNA gene promoter.

Analysis of gmo contents as a component in risk-oriented surveillance over food products safety

Control over use of genetically modified products is a vital task within a risk-oriented model for surveillance over food safety products all over the world including the Russian Federation. Our research goal was to examine domestically manufactured food products in order to determine whether they contained certain regulatory sequences typical for genetically modified organisms. We applied polymerase chain reaction with hybridization-fluorescent detection in real time mode to examine 77 food products samples; the task was to determine whether they contained DNA enhancer (E-35S) and promoter (P-35S) of S35 sequence belonging to cauliflower mosaic virus, terminator of nopaline synthase gene from Agrobacterium tumefaciens (T-NOS), 35S enhancer (E-FMV) and promoter (P-FMV) of Figwort mosaic virus, as well as vegetable DNA inducing soya DNA. When analyzing the extracted DNA, we didn’t detect transgenic elements in any samples; however, there were vegetable components reveled in them including 68.8% samples with soya DNA. We established that some sausages were falsified as they contained vegetable elements. In 15.6% cases data on a product structure turned out to be false because soya DNA was not listed on consumer package. Our research on determining soya DNA and transgenic elements in food products indicates that soya ingredients have been added into food products in spite of their absence in relevant documents as recipe components. All the obtained results taken into account, we assume it is necessary to improve control procedures for detecting genetically modified and vegetable components used as ingredients in food products as their falsification can make for changes not only in their consumer properties but also damage consumers’ health.

Анализ содержания компонентов генетически модифицированных организмов как элемент риск-ориентированного надзора за безопасностью пищевых продуктов

Control over use of genetically modified products is a vital task within a risk-oriented model for surveillance over food safety products all over the world including the Russian Federation. Our research goal was to examine domestically manufactured food products in order to determine whether they contained certain regulatory sequences typical for genetically modified organisms. We applied polymerase chain reaction with hybridization-fluorescent detection in real time mode to examine 77 food products samples; the task was to determine whether they contained DNA enhancer (E-35S) and promoter (P-35S) of S35 sequence belonging to cauliflower mosaic virus, terminator of nopaline synthase gene from Agrobacterium tumefaciens (T-NOS), 35S enhancer (E-FMV) and promoter (P-FMV) of Figwort mosaic virus, as well as vegetable DNA inducing soya DNA. When analyzing the extracted DNA, we didn’t detect transgenic elements in any samples; however, there were vegetable components reveled in them including 68.8% samples with soya DNA. We established that some sausages were falsified as they contained vegetable elements. In 15.6% cases data on a product structure turned out to be false because soya DNA was not listed on consumer package. Our research on determining soya DNA and transgenic elements in food products indicates that soya ingredients have been added into food products in spite of their absence in relevant documents as recipe components. All the obtained results taken into account, we assume it is necessary to improve control procedures for detecting genetically modified and vegetable components used as ingredients in food products as their falsification can make for changes not only in their consumer properties but also damage consumers’ health.

Activation tagging identifies Arabidopsis transcription factor AtMYB68 for heat and drought tolerance at yield determining reproductive stages.

Heat stress occurring at reproductive stages can result in significant and permanent damage to crop yields. However, previous genetic studies in understanding heat stress response and signaling were performed mostly on seedling and plants at early vegetative stages. Here we identify, using a developmentally defined, gain-of-function genetic screen with approximately 18000 Arabidopsis thaliana activation-tagged lines, a mutant that maintained productive seed set post-severe heat stress during flowering. Genome walking indicated this phenotype was caused by the insertion of 35S enhancers adjacent to a nuclear localized transcription factor AtMYB68. Subsequent overexpression analysis confirmed that AtMYB68 was responsible for the reproductive heat tolerance of the mutant. Furthermore, these transgenic Arabidopsis plants exhibited enhanced abscisic acid sensitivity at and post-germination, reduced transpirational water loss during a drought treatment, and enhanced seed yield under combined heat and drought stress during flowering. Ectopic expression of AtMYB68 in Brassica napus driven either by 35S or by heat-inducible promoter recapitulated the enhanced reproductive heat stress and drought tolerance phenotypes observed in the transgenic Arabidopsis. The improvement to heat stress is likely due to enhanced pollen viability observed in the transgenic plants. More importantly, the transgenic canola showed significant yield advantages over the non-transgenic controls in multiple locations, multiple season field trials under various drought and heat stress conditions. Together these results suggest that AtMYB68 regulate plant stress tolerance at the most important yield determining stage of plant development, and is an effective target for crop yield protection under current global climate volatility.

A modified in-planta transformation technique to generate stable gain-in function transformants in a recalcitrant indica rice genotype

A tissue culture independent in-planta transformation technique is an alternate option to generate transgenics in crop cultivars recalcitrant for regeneration. The T0 plants generated by in-planta technique are chimeric hence very large number of T1 seeds needs to be screened to identify the putative transformants. Besides, low transformation efficiency and stable integration is another lacuna, because the T0 seedling explants are not subjected for selection. To overcome these constraints, a modified in-planta technique was developed in this study to generate gain-in-function activation tagged mutants in the background of a recalcitrant rice genotype, KMP-153. A binary vector was developed with three glyphosate selectable marker genes and 4X CaMV 35S enhancer elements for developing rice plants. Simultaneous expression of Glyphosate insensitive CP4-EPSPS with two other glyphosate degrading enzymes, PsAKR1 (Aldo–Keto reductases) and GO (Glycine Oxidase) substantially improved tolerance up to 100 ppm of glyphosate in the seedling bioassay system. Selecting the seedling explants and T1 generation seeds at higher concentration (100 ppm) of glyphosate resulted in recovering fewer transformants but with stable integration and expression of all the transgenes compared to those screened at lower levels of glyphosate. Further, T2 generation plants from these transgenic lines grown in contained nethouse conditions were tolerant when sprayed up to 1500 ppm concentrations of glyphosate. These transgenics showed substantial lower levels of shikimic acid compared to wild type plants treated with glyphosate signifies the expression of the transgenes and less inhibition of shikimic acid pathway by glyphosate. The proteins extracted from the transgenic plants showed significant degradation of glyphosate. The stable integration was confirmed through flanking sequence information and event specific PCR information. Our results clearly demonstrate that the modified in-planta technique is a potential transformation protocol to generate stable transformants in rice cultivars.

A 100 bp GAGA motif-containing sequence in AGAMOUS second intron is able to suppress the activity of CaMV35S enhancer in vegetative tissues

Flower-specific promoters enable genetic manipulation of floral organs to improve crop yield and quality without affecting vegetative growth. However, the identification of strong tissue-specific promoters is a challenge. In addition, information on cis elements that is able to repress gene expression in vegetative tissues remains limited. Here, we report that fusing a 35S enhancer to the stamen- and carpel-specific NtAGIP1 promoter derived from the tobacco AGAMOUS second intron (AGI) can significantly increase the promoter activity. Interestingly, although the activity of the new promoter extends to sepals and pedicles, it does not cross the boundary of the reproductive organs. Serial deletion of the AGI and chromatin immunoprecipitation (ChIP) assay reveal a 100-bp fragment that contains a conserved GAGA factor binding motif contributes to the flower specificity by mediating histone H3 lysine 27 trimethylation (H3K27me3) modification of the promoter. Furthermore, this fragment shows significant suppressive effect on the activity of the 35S enhancer in vegetative tissues, consequently, resulting in a significant increase of the activity of 35S enhancer:AGI chimeric promoter without sacrifice of its specificity in inflorescence.

SHOOT MERISTEMLESS is Required for the Proper Internode Patterning and the Sepal Separation in Arabidopsis

SHOOT MERISTMLESS (STM) encoding a homeodomain protein plays a vital role in the specification of shoot meristem, the source of undifferentiated cells for the above-ground organ development in Arabidopsis thaliana. The roles of STM still remain to be elucidated as stm is deficient in the late organ development. Here, we have isolated a recessive filamentous gynoecium (fig)-1 displaying slightly reduced floral meristem and weak sepal fusion defect by introducing the 35S enhancer tag into the poltergeist-6. A truncated tag was localized at the 4628 base pairs (bps) upstream from the STM start codon in fig-1 led to the decrease in the STM transcripts. Two additional knocked-down mutant alleles, stm-p1 and stm-p3 harboring T-DNA insertions at 468 and 2225 bps upstream from the start codon, respectively displayed the pennywise (pny)-like clustered internode together with the sepal fusion defects. In stm-p1, the expression of KNOTTED-1 LIKE FROM A. THALIANA6 was enhanced and stm-p1 brevipedicelus-1 developed extremely compact internodes. The sepal fusion phenotypes of stm-p1 and stm-p3 were comparable to that of cup-shaped cotyledon1 (cuc1) and CUC1 expression was reduced in stm-p1. Together, these results suggest that STM functions genetically in association with PNY and CUC1 for the proper inflorescence development and the sepal separation, respectively.

EAT-Rice: A predictive model for flanking gene expression of T-DNA insertion activation-tagged rice mutants by machine learning approaches.

T-DNA activation-tagging technology is widely used to study rice gene functions. When T-DNA inserts into genome, the flanking gene expression may be altered using CaMV 35S enhancer, but the affected genes still need to be validated by biological experiment. We have developed the EAT-Rice platform to predict the flanking gene expression of T-DNA insertion site in rice mutants. The three kinds of DNA sequences including UPS1K, DISTANCE, and MIDDLE were retrieved to encode and build a forecast model of two-layer machine learning. In the first-layer models, the features nucleotide context (N-gram), cis-regulatory elements (Motif), nucleotide physicochemical properties (NPC), and CG-island (CGI) were used to build SVM models by analysing the concealed information embedded within the three kinds of sequences. Logistic regression was used to estimate the probability of gene activation which as feature-encoding weighting within first-layer model. In the second-layer models, the NaiveBayesUpdateable algorithm was used to integrate these first layer-models, and the system performance was 88.33% on 5-fold cross-validation, and 79.17% on independent-testing finally. In the three kinds of sequences, the model constructed by Middle had the best contribution to the system for identifying the activated genes. The EAT-Rice system provided better performance and gene expression prediction at further distances when compared to the TRIM database. An online server based on EAT-rice is available at http://predictor.nchu.edu.tw/EAT-Rice.

A 64-bp sequence containing the GAAGA motif is essential for CaMV-35S promoter methylation in gentian

This study investigated sequence specificity and perenniality of DNA methylation in the cauliflower mosaic virus (CaMV) 35S promoter of transgenic gentian (Gentiana triflora×G. scabra) plants. Unlike conventional transgene silencing models, 35S promoter hypermethylation in gentian is species-specific and occurs irrespective of the T-DNA copy number and genomic location. Modified 35S promoters were introduced into gentian, and single-copy transgenic lines were selected for methylation analysis. Modified 35S promoter lacking a core (−90) region [35S(Δcore)] in gentian conferred hypermethylation and high levels of de novo methylation of the CpHpH/CpCpG sites in the 35S enhancer regions (−298 to −241 and −148 to −85). Therefore, promoter transcription may not be an absolute requirement for the methylation machinery. In vitro, de novo methylation persisted for more than eight years. In another modified 35S promoter, two “GAAGA” motifs (−268 to −264 and −135 to −131) were replaced by “GTTCA” in the two highly de novo methylated regions. It did not support hypermethylation and showed transgene expression. A 64-bp fragment of the 35S enhancer region (−148 to −85) was introduced into gentian and the resultant transgenic lines analyzed. The 64-bp region exhibited hypermethylation at the CpG/CpWpG sites, but the CpHpH/CpCpG methylation frequency was lower than those of the unmodified 35S- and 35S(Δcore) promoters. Nevertheless, a distinct CpHpH/CpCpG methylation peak was found in the 64-bp region of all single-copy transgenic lines. These results suggest that the 64-bp region may contain an element required for 35S methylation but insufficient for high de novo methylation compared with those in the unmodified 35S and 35S(Δcore) promoters.

Novel rice mutants overexpressing the brassinosteroid catabolic gene CYP734A4.

Moderate overexpression of CYP734A4 improves grain number per main panicle and seed setting rate. Brassinosteroid (BR) homeostasis and signaling are crucial for plant growth and development. CYP734A genes encode cytochrome P450 monooxygenases that control the level of bioactive BRs by degrading BRs. However, fertile plants overexpressing CYP734As have not been reported in rice. Here, we isolated a novel semi-dominant mutant brd3-D, in which T-DNA was inserted approximately 4kb upstream of the CYP734A4 gene (GenBank Accession AB488667), causing its overexpression. The mutant is characterized by dwarfism, small grains, and erect leaves and is less sensitive to brassinolide-induced lamina joint inclination and primary root elongation. However, increased grain number per main panicle and improved seed setting rate were also found in heterozygous brd3-D. To our knowledge, these traits have not been reported in other BR deficient mutants. Quantitative real-time PCR analysis indicated that phenotypic severity of the brd3-D mutant is positively correlated with the CYP734A4 transcription level. In accordance with the increased expression of CYP734A4, a lower castasterone (a rice BR) content was detected in the brd3-D mutants. Knockout of brd3-D by using the CRISPR/Cas9 system rescued the mutation. In addition, transgenic plants overexpressing CYP734A4 with the 35S enhancer mimicked the brd3-D phenotypes, confirming that moderate overexpression of the CYP734A4 gene can improve grain number per main panicle and the seed setting rate in rice. Further studies showed that overexpression of CYP734A4 influences the expressions of multiple genes involved in the BR pathway, and the expression of CYP734A4 is induced by exogenous brassinolide, confirming the negative regulatory role of CYP734A4 in the BR pathway. CYP734A4 might provide a useful gene resource for developing new high-yielding rice varieties.

Rice ONAC106 Inhibits Leaf Senescence and Increases Salt Tolerance and Tiller Angle.

NAM/ATAF1/ATAF2/CUC2 (NAC) is a plant-specific transcription factor (TF) family, and NACs participate in many diverse processes during the plant life cycle. Several Arabidopsis thaliana NACs have important roles in positively or negatively regulating leaf senescence, but in other plant species, including rice, the senescence-associated NACs (senNACs) remain largely unknown. Here we show that the rice senNAC TF ONAC106 negatively regulates leaf senescence. Leaves of onac106-1D (insertion of the 35S enhancer in the promoter region of the ONAC106 gene) mutants retained their green color under natural senescence and dark-induced senescence conditions. Genome-wide transcriptome analysis revealed that key senescence-associated genes (SGR, NYC1, OsNAC5, OsNAP, OsEIN3 and OsS3H) were differentially expressed in onac106-1D during dark-induced senescence. In addition to delayed senescence, onac106-1D also showed a salt stress-tolerant phenotype; key genes that down-regulate salt response signaling (OsNAC5, OsDREB2A, OsLEA3 and OsbZIP23) were rapidly up-regulated in onac106-1D under salt stress. Interestingly, onac106-1D also exhibited a wide tiller angle phenotype throughout development, and the tiller angle-related gene LPA1 was down-regulated in onac106-1D. Using yeast one-hybrid assays, we found that ONAC106 binds to the promoter regions of SGR, NYC1, OsNAC5 and LPA1. Taking these results together, we propose that ONAC106 functions in leaf senescence, salt stress tolerance and plant architecture by modulating the expression of its target genes that function in each signaling pathway.

Characterization of a potato activation-tagged mutant, nikku, and its partial revertant.

A potato mutant with a strong stress-response phenotype, and a partial mutant revertant, were characterized. Gene expression patterns and DNA cytosine methylation varied between these and wild-type, indicating a role for DNA cytosine methylation changes in the gene expression and visible phenotypes. Morphological and molecular studies were conducted to compare potato cv. Bintje, a Bintje activation-tagged mutant (nikku), and nikku revertant phenotype plants. Morphological studies revealed that nikku plants exhibited an extremely dwarf phenotype, had small hyponastic leaves, were rootless, and infrequently produced small tubers compared to wild-type Bintje. The overall phenotype was suggestive of a constitutive stress response, which was further supported by the greater expression level of several stress-responsive genes in nikku. Unlike the nikku mutant, the revertant exhibited near normal shoot elongation, larger leaves and consistent rooting. The reversion appeared partial, and was not the result of a loss of 35S enhancer copies from the original nikku mutant. Southern blot analyses indicated the presence of a single T-DNA insertion on chromosome 12 in the mutant. Gene expression studies comparing Bintje, nikku and revertant phenotype plants indicated transcriptional activation/repression of several genes flanking both sides of the insertion in the mutant, suggesting that activation tagging had pleiotropic effects in nikku. In contrast, gene expression levels for many, but not all, of the same genes in the revertant were similar to Bintje, indicating some reversion at the gene expression level as well. DNA methylation studies indicated differences in cytosine methylation status of the 35S enhancers between the nikku mutant and its revertant. In addition, global DNA cytosine methylation varied between Bintje, the nikku mutant and the revertant, suggesting involvement in gene expression changes, as well as mutant phenotype.

A detailed analysis of the leaf rolling mutant sll2 reveals complex nature in regulation of bulliform cell development in rice (Oryza sativa L.).

Bulliform cells are large, thin-walled and highly vacuolated cells, and play an important role in controlling leaf rolling in response to drought and high temperature. However, the molecular mechanisms regulating bulliform cell development have not been well documented. Here, we report isolation and characterisation of a rice leaf-rolling mutant, named shallot-like 2 (sll2). The sll2 plants exhibit adaxially rolled leaves, starting from the sixth leaf stage, accompanied by increased photosynthesis and reduced plant height and tiller number. Histological analyses showed shrinkage of bulliform cells, resulting in inward-curved leaves. The mutant is recessive and revertible at a rate of 9%. The leaf rolling is caused by a T-DNA insertion. Cloning of the insertion using TAIL-PCR revealed that the T-DNA was inserted in the promoter region of LOC_Os07g38664. Unexpectedly, the enhanced expression of LOC_Os07g38664 by the 35S enhancer in the T-DNA is not responsible for the leaf rolling phenotype. Further, the enhancer also exerted a long-distance effect, including up-regulation of several bulliform cell-related genes. sll2 suppressed the outward leaf rolling of oul1 in the sll2oul1 double mutant. We conclude that leaf rolling in sll2 could be a result of the combined effect of multi-genes, implying a complex network in regulation of bulliform cell development.

Constitutive overexpression of the pollen specific gene SKS13 in leaves reduces aphid performance on Arabidopsis thaliana.

BackgroundPlants have developed a variety of mechanisms to counteract aphid attacks. They activate their defences by changing the expression of specific genes. Previously we identified an activation tag mutant of Arabidopsis thaliana on which Myzus persicae population development was reduced. Activation tag mutants are gain-of-function in which the expression of a gene is increased by the insertion of the Cauliflower mosaic virus 35S enhancer that acts on the natural promoter. By further characterizing this previously identified mutant we identified a gene that reduces performance of M. persicae and also provided clues about the mechanism involved.ResultsWe show that SKU5 SIMILAR 13 (SKS13), a gene whose expression in wild type plants is restricted to pollen and non-responsive to M. persicae attack, is overexpressed in the A. thaliana mutant showing reduced performance of M. persicae. Monitoring M. persicae feeding behaviour on SKS13 overexpressing plants indicated that M. persicae have difficulties feeding from the phloem. The constitutive expression of SKS13 results in accumulation of reactive oxygen species, which is possibly regulated through the jasmonic acid pathway. The enhanced resistance is not aphid species specific as also the population development of Brevicoryne brassicae was affected.ConclusionsWe demonstrate that constitutive expression in leaves of the pollen-specific gene SKS13 can enhance plant defence, resulting in a reduction of M. persicae population development and also decreases the transmission of persistent viruses. Overexpression of SKS13 in A. thaliana also affects B. brassicae and possibly other phloem feeding insects as well. Identifying genes that can enhance plant defence against insects will be important to open up new avenues for the development of insect resistant crop plants.

Construction of Flower-specific Chimeric Promoters and Analysis of Their Activities in Transgenic Torenia

Flower-specific promoters can enable transgenic enhancement of valuable ornamental traits, including flower shape and color. However, the identification of strong, tissue-specific promoters remains a limiting factor. To obtain enhanced flower-specific promoters, we constructed four chimeric promoters (p35S-PCHS-Ω, p35S-LCHS-Ω, pOCS-PCHS-Ω and pOCS-LCHS-Ω) combining the 35S or OCS enhancer fused to a 302 bp CHSA core promoter fragment from petunia (PCHS) or a 307 bp CHS core promoter fragment from lily (LCHS), and also containing an omega element (Ω). Each promoter was fused to the β-glucuronidase (GUS) reporter gene, and we examined the levels and tissue specificity of GUS expression in transgenic Torenia fournieri. p35S-PCHS-Ω and p35S-LCHS-Ω drove strong, constitutive GUS expression in all tissues, especially in colored corollas (p35S-PCHS-Ω) or in colored corollas and roots (p35S-LCHS-Ω). pOCS-PCHS-Ω drove stronger GUS expression in colored corollas than in other tissues but expression was weaker than that of p35S-PCHS-Ω. pOCS-LCHS-Ω drove GUS in colored corollas but also in roots. Among the four chimeric promoters, pOCS-PCHS-Ω exhibited stronger activity only in colored corollas, making it useful for transgenic enhancement of floral traits, such as expressing ‘blue genes’ in lily to produce new lines with blue flowers.

Overexpression of IRM1 Enhances Resistance to Aphids in Arabidopsis thaliana

Aphids are insects that cause direct damage to crops by the removal of phloem sap, but more importantly they spread devastating viruses. Aphids use their sophisticated mouthpart (i.e. stylet) to feed from the phloem sieve elements of the host plant. To identify genes that affect host plant resistance to aphids, we previously screened an Arabidopsis thaliana activation tag mutant collection. In such mutants, tagged genes are overexpressed by a strong 35S enhancer adjacent to the natural promoter, resulting in a dominant gain-of-function phenotype. We previously identified several of these mutants on which the aphid Myzus persicae showed a reduced population development compared with wild type. In the present study we show that the gene responsible for the phenotype of one of the mutants is At5g65040 and named this gene Increased Resistance to Myzus persicae 1 (IRM1). Overexpression of the cloned IRM1 gene conferred a phenotype identical to that of the original mutant. Conversely, an IRM1 knockout mutant promoted aphid population development compared to the wild type. We performed Electrical Penetration Graph analysis to investigate how probing and feeding behaviour of aphids was affected on plants that either overexpressed IRM1 or contained a knockout mutation in this gene. The EPG results indicated that the aphids encounter resistance factors while reaching for the phloem on the overexpressing line. This resistance mechanism also affected other aphid species and is suggested to be of mechanical nature. Interestingly, genetic variation for IRM1 expression in response to aphid attack was observed. Upon aphid attack the expression of IRM1 was initially (after 6 hours) induced in ecotype Wassilewskija followed by suppression. In Columbia-0, IRM1 expression was already suppressed six hours after the start of the infestation. The resistance conferred by the overexpression of IRM1 in A. thaliana trades off with plant growth.

The CaMV 35S enhancer has a function to change the histone modification state at insertion loci in Arabidopsis thaliana

Chromatin regions with different states usually harbor distinct epigenetic information, through which gene expression is regulated. Recent studies using mammalian cells showed that a chromatin state signature is associated with active developmental enhancers, defined by high levels of histone H3 lysine 27 acetylation (H3K27ac) and strong depletion of H3K27 trimethylation (H3K27me3). These findings also imply that active enhancers may play a role in creating a chromatin state by changing histone modification markers, which in turn affects gene expression. To explore whether an active enhancer in plants affect histone modifications, we investigated the cauliflower mosaic virus 35S enhancer (35Senh) for understanding its action model in Arabidopsis. We report that the 35Senh has a function to change the histone modification pattern at its presenting loci, by characterization of the 35Senh activated BREVIPEDICELLUS (BP) silencing lines and the randomly selected 35Senh activation tagging lines. By analyzing histone modification markers reflecting the plant chromatin state, we show that the 35Senh is generally correlated with the reduced level of H3K27me3 and the increased level of H3K4me3 at the insertion loci. Our data are consistent with those in mammals and suggest that the enhancer sequence correlating with the active chromatin state signature may be generally present in the eukaryotic kingdom.

An ActiveAc/DsTransposon System for Activation Tagging in Tomato Cultivar M82 Using Clonal Propagation

Tomato (Solanum lycopersicum) is a model organism for Solanaceae in both molecular and agronomic research. This project utilized Agrobacterium tumefaciens transformation and the transposon-tagging construct Activator (Ac)/Dissociator (Ds)-ATag-Bar_gosGFP to produce activation-tagged and knockout mutants in the processing tomato cultivar M82. The construct carried hygromycin resistance (hyg), green fluorescent protein (GFP), and the transposase (TPase) of maize (Zea mays) Activator major transcript X054214.1 on the stable Ac element, along with a 35S enhancer tetramer and glufosinate herbicide resistance (BAR) on the mobile Ds-ATag element. An in vitro propagation strategy was used to produce a population of 25 T0 plants from a single transformed plant regenerated in tissue culture. A T1 population of 11,000 selfed and cv M82 backcrossed progeny was produced from the functional T0 line. This population was screened using glufosinate herbicide, hygromycin leaf painting, and multiplex polymerase chain reaction (PCR). Insertion sites of transposed Ds-ATag elements were identified through thermal asymmetric interlaced PCR, and resulting product sequences were aligned to the recently published tomato genome. A population of 509 independent, Ds-only transposant lines spanning all 12 tomato chromosomes has been developed. Insertion site analysis demonstrated that more than 80% of these lines harbored Ds insertions conducive to activation tagging. The capacity of the Ds-ATag element to alter transcription was verified by quantitative real-time reverse transcription-PCR in two mutant lines. The transposon-tagged lines have been immortalized in seed stocks and can be accessed through an online database, providing a unique resource for tomato breeding and analysis of gene function in the background of a commercial tomato cultivar.