Method: low-cost delivery of the cotton leaf crumple virus-induced gene silencing system

Background

We previously developed a virus-induced gene silencing (VIGS) vector for cotton from the bipartite geminivirusCotton leaf crumple virus (CLCrV). The original CLCrV VIGS vector was designed for biolistic delivery by a gene gun. This prerequisite limited the use of the system to labs with access to biolistic equipment. Here we describe the adaptation of this system for delivery by Agrobacterium (Agrobacterium tumefaciens). We also describe the construction of two low-cost particle inflow guns.

Results

The biolistic CLCrV vector was transferred into two Agrobacterium binary plasmids. Agroinoculation of the binary plasmids into cotton resulted in silencing and GFP expression comparable to the biolistic vector. Two homemade low-cost gene guns were used to successfully inoculate cotton (G. hirsutum) and N. benthamiana with either the CLCrV VIGS vector or the Tomato golden mosaic virus (TGMV) VIGS vector respectively.

Conclusions

These innovations extend the versatility of CLCrV-based VIGS for analyzing gene function in cotton. The two low-cost gene guns make VIGS experiments affordable for both research and teaching labs by providing a working alternative to expensive commercial gene guns.     

Efficient virus-induced gene silencing in apple, pear and Japanese pear using Apple latent spherical virus vectors

Background

Virus-induced gene silencing (VIGS) is an effective technology for the analysis of gene functions in plants. Though there are many reports on virus vectors for VIGS in plants, no VIGS vectors available for Rosaceae fruit trees were reported so far. We present an effective VIGS system in apple, pear, and Japanese pear using Apple latent spherical virus (ALSV) vectors.

Results

Inoculation of ALSV vectors carrying a partial sequence of endogenous genes from apple [ribulose-1, 5-bisphosphate carboxylase small subunit (rbcS), alpha subunit of chloroplast chaperonin (CPN60a), elongation factor 1 alpha (EF-1a), or actin] to the cotyledons of seeds by a particle bombardment induced highly uniform knock-down phenotypes of each gene on the true leaves of seedlings from 2~3 weeks after inoculation. These silencing phenotypes continued for several months. Northern blot and RT-PCR analyses of leaves infected with ALSV containing a fragment of rbcS gene showed that the levels of rbcS-mRNA drastically decreased in the infected apple and pear leaves, and, in reverse, rbcS-siRNAs were generated in the infected leaves. In addition, some of apple seedlings inoculated with ALSV vector carrying a partial sequence of a TERMINAL FLOWER 1 gene of apple (MdTFL1) showed precocious flowering which is expected as a knock-down phenotype of the silencing of MdTFL1 gene.

Conclusions

The ALSV-based VIGS system developed have provides a valuable new addition to the tool box for functional genomics in apple, pear, and Japanese pear.     

Virus-induced PpCHLH gene silencing in peach leaves (Prunus persica)

Summary

Compared with other model plants or crops, studies on the molecular biology of fruit trees have lagged behind due to technical difficulties in gene transformation and manipulation. Therefore, developing an efficient system for gene manipulation is of particular significance in fruit trees. Here, we report on a method for virus-induced gene silencing (VIGS) by syringe-infiltrating a tobacco rattle virus (TRV) vector containing a specific target gene sequence into peach (Prunus persica) leaves to analyse gene function. The target gene (PpCHLH) was a 4,445 bp sequence encoding the H subunit of magnesium chelatase and was first cloned as a cDNA. This gene (PpCHLH) is reported to be related to chlorophyll biosynthesis, and any loss of function leads to a decrease in chlorophyll content, with concomitant yellow or white colour changes in the leaves. To silence the PpCHLH gene, a 1:1 mixture of Agrobacterium tumefaciens strain GV3101 cultures containing pTRV1 or a pTRV2 vector construct with a 650 bp cDNA fragment of the PpCHLH gene was infiltrated into leaves of 4 – 5 week-old peach seedlings. After 15 d, the inoculated areas of the green leaves faded and finally turned yellow or white. Loss of PpCHLH gene function was confirmed by semi-quantitative RT-PCR, real-time qRT-PCR, and siRNA northern blot analysis. The virus-induced gene silencing (VIGS) technique developed here could be used for further molecular studies on fruit trees.     

An improved protocol for efficient transformation and regeneration of diverse indica rice cultivars

Background

Common bean (Phaseolus vulgaris L.) is a crop of economic and nutritious importance in many parts of the world. The lack of genomic resources have impeded the advancement of common bean genomics and thereby crop improvement. Although concerted efforts from the "Phaseomics" consortium have resulted in the development of several genomic resources, functional studies have continued to lag due to the recalcitrance of this crop for genetic transformation.

Results

Here we describe the use of a bean pod mottle virus (BPMV)-based vector for silencing of endogenous genes in common bean as well as for protein expression. This BPMV-based vector was originally developed for use in soybean. It has been successfully employed for both protein expression and gene silencing in this species. We tested this vector for applications in common bean by targeting common bean genes encoding nodulin 22 and stearoyl-acyl carrier protein desaturase for silencing. Our results indicate that the BPMV vector can indeed be employed for reverse genetics studies of diverse biological processes in common bean. We also used the BPMV-based vector for expressing the green fluorescent protein (GFP) in common bean and demonstrate stable GFP expression in all common bean tissues where BPMV was detected.

Conclusions

The availability of this vector is an important advance for the common bean research community not only because it provides a rapid means for functional studies in common bean, but also because it does so without generating genetically modified plants. Here we describe the detailed methodology and provide essential guidelines for the use of this vector for both gene silencing and protein expression in common bean. The entire VIGS procedure can be completed in 4-5 weeks.     

Development of a gene silencing DNA vector derived from a broad host range geminivirus

Background  

Gene silencing is proving to be a powerful tool for genetic, developmental, and physiological analyses. The use of viral induced gene silencing (VIGS) offers advantages to transgenic approaches as it can be potentially applied to non-model systems for which transgenic techniques are not readily available. However, many VIGS vectors are derived from Gemini viruses that have limited host ranges. We present a new, unipartite vector that is derived from a curtovirus that has a broad host range and will be amenable to use in many non-model systems.     

茄子SmMsrA基因VIGS表达载体的构建及表达分析

利用RT-PCR从茄子单性结实品系D-10中获得甲硫氨酸亚砜还原酶A基因(Sm Msr A)的编码区。通过Gateway同源重组技术,构建了3个分别含Sm Msr A基因不同片段的烟草脆裂病毒(Tobacco rattle virus,TRV)介导的基因沉默(VIGS)表达载体p TRV2-Sm Msr Ai。表达载体转入农杆菌GV3101后,用注射压迫法侵染茄子叶片。采用表型观察、TRV病毒分子检测和q RT-PCR技术,评价构建的VIGS体系对Sm Msr A基因的沉默效果。结果表明,报告基因PDS沉默后叶片呈现明显的光漂白现象,Sm Msr A基因沉默后叶片呈花叶状,果实变小,叶片和果实中的Sm Msr A基因的表达量明显降低,表明Sm Msr A基因是正向调控茄子果实发育的相关基因。     

Transient co-expression with three <Emphasis Type="Italic">O</Emphasis>-glycosylation enzymes allows production of GalNAc-<Emphasis Type="Italic">O</Emphasis>-glycosylated Granulocyte-Colony Stimulating Factor in <Emphasis Type="Italic">N. benthamiana</Emphasis>

Background

Expression of economically relevant proteins in alternative expression platforms, especially plant expression platforms, has gained significant interest in recent years. A special interest in working with plants as bioreactors for the production of pharmaceutical proteins is related to low production costs, product safety and quality. Among the different properties that plants can also offer for the production of recombinant proteins, protein glycosylation is crucial since it may have an impact on pharmaceutical functionality and/or stability.

Results

The pharmaceutical glycoprotein human Granulocyte-Colony Stimulating Factor was transiently expressed in Nicotiana benthamiana plants and subjected to mammalian-specific mucin-type O-glycosylation by co-expressing the pharmaceutical protein together with the glycosylation machinery responsible for such post-translational modification.

Conclusions

The pharmaceutical glycoprotein human Granulocyte-Colony Stimulating Factor can be expressed in N. benthamiana plants via agroinfiltration with its native mammalian-specific mucin-type O-glycosylation.

     

茄子TRV-VIGS体系的优化及SmIAA19基因功能初步分析

以经过改造的烟草脆裂病毒（Tobacco rattle virus,TRV）载体PYL156为工具,采用叶片注射法侵染茄子（Solanum melongena L.）叶片,通过表型比较、TRV病毒分子检测和荧光定量PCR技术分析,探究环境温度、植株大小、目的基因插入片段大小对茄子TRV-VIGS沉默体系的影响。结果表明,昼夜温度为（25 ± 3）℃和（20 ± 2）℃时,接种茄子幼苗子叶的植株沉默效果明显,侵染后植株叶片中目的基因表达量与阴性对照相比明显降低;早春日光温室和秋季日光温室条件下,侵染植株表型性状和基因表达量差异不显著。沉默片段大小为200 bp左右时目的基因的沉默效果最好;侵染茄子幼苗子叶期植株出现明显病毒斑,而接种6周龄真叶则无明显表型差异。用茄子生长素诱导基因（SmIAA19）为靶基因对其进行验证,结果表明显著抑制了SmIAA19的表达,其在叶片中的表达量和生长素含量均显著降低,表明SmIAA19是1个与生长素代谢途径相关的基因。     

High-level diterpene production by transient expression in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis>

Background

Characterization of plant terpene synthases is typically done by production of recombinant enzymes in Escherichia coli. This is often difficult due to solubility and codon usage issues. Furthermore, plant terpene synthases which are targeted to the plastids, such as diterpene synthases, have to be shortened in a more or less empirical approach to improve expression. We report here an optimized Agrobacterium-mediated transient expression assay in Nicotiana benthamiana for plant diterpene synthase expression and product analysis.

Results

Agrobacterium-mediated transient expression of plant diterpene synthases in N. benthamiana led to the accumulation of diterpenes within 3 days of infiltration and with a maximum at 5 days. Over 50% of the products were exported onto the leaf surface, thus considerably facilitating the analysis by reducing the complexity of the extracts. The robustness of the method was tested by expressing three different plant enzymes, cembratrien-ol synthase from Nicotiana sylvestris, casbene synthase from Ricinus communis and levopimaradiene synthase from Gingko biloba. Furthermore, co-expression of a 1-deoxy-D-xylulose-5-phosphate synthase from tomato and a geranylgeranyl diphosphate synthase from tobacco led to a 3.5-fold increase in the amount of cembratrien-ol produced, with maximum yields reaching 2500 ng/cm².

Conclusion

With this optimized method for diterpene synthase expression and product analysis, a single infiltrated leaf of N. benthamiana would be sufficient to produce quantities required for the structure elucidation of unknown diterpenes. The method will also be of general use for gene function discovery, pathway reconstitution and metabolic engineering of diterpenoid biosynthesis in plants.

     

Evaluation of alternative RNA labeling protocols for transcript profiling with Arabidopsis AGRONOMICS1 tiling arrays

Background

The genus Populus is accepted as a model system for molecular tree biology. To investigate gene functions in Populus spp. trees, generating stable transgenic lines is the common technique for functional genetic studies. However, a limited number of genes have been targeted due to the lengthy transgenic process. Transient transformation assays complementing stable transformation have significant advantages for rapid in vivo assessment of gene function. The aim of this study is to develop a simple and efficient transient transformation for hybrid aspen and to provide its potential applications for functional genomic approaches.

Results

We developed an in planta transient transformation assay for young hybrid aspen cuttings using Agrobacterium-mediated vacuum infiltration. The transformation conditions such as the infiltration medium, the presence of a surfactant, the phase of bacterial growth and bacterial density were optimized to achieve a higher transformation efficiency in young aspen leaves. The Agrobacterium infiltration assay successfully transformed various cell types in leaf tissues. Intracellular localization of four aspen genes was confirmed in homologous Populus spp. using fusion constructs with the green fluorescent protein. Protein-protein interaction was detected in transiently co-transformed cells with bimolecular fluorescence complementation technique. In vivo promoter activity was monitored over a few days in aspen cuttings that were transformed with luciferase reporter gene driven by a circadian clock promoter.

Conclusions

The Agrobacterium infiltration assay developed here is a simple and enhanced throughput method that requires minimum handling and short transgenic process. This method will facilitate functional analyses of Populus genes in a homologous plant system.     

Molecular cloning and characterization of nitrate reductase gene from non-heading Chinese cabbage

Nitrate reductase (NR) is a key enzyme that catalyzes the first step in plants nitrogen metabolization. A cDNA of a NR gene from non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) cultivar ‘Suzhouqing’ was isolated by RT-PCR and (5′/3′)-RACE techniques. The full-length cDNA sequence of 3049 bp contained an open reading frame of 2733 bp encoding 910 amino acids, a 5′-untranslated region of 113 bp and a 3′-untranslated sequence of 203 bp with a poly (A) tail. This protein shares common structural features with NRs from other higher plants and eukaryotes. It was classified as NR by sequence alignment, motif search and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, and consequently nominated Brassica campestris ssp. chinensis Makino NR (BcNR). Southern blot analysis indicated that BcNR gene represented as 3 copies in the non-heading Chinese cabbage genome. Accumulation of BcNR mRNA was transiently induced by nitrate supply and correlated with nitrate concentration, the highest mRNA levels of BcNR was induced by 30 mM NO₃⁻ and reached a maximum at 4 h at this optimal concentration treatment, in contrast, NH₄⁺ cannot induce the accumulation of BcNR mRNA. The BcNR was heterologously expressed in Escherichia coli BL21 (DE3) as a fusion protein. There was a specific band at about 110 kDa in size by SDS-PAGE analysis which was according with the expected molecular weight of the recombinant protein. To further explore its biological function, BcNR under regulation of the cauliflower mosaic virus (CaMV) 35S promoter was transferred into Arabidopsis thaliana. The transgenic plants exhibited an enhanced level of NR and nitrate reductase activity (NRA) in leaves under NO₃⁻ inducement.     

A high-resolution method for the localization of proanthocyanidins in plant tissues

Background

Histochemical staining of plant tissues with 4-dimethylaminocinnamaldehyde (DMACA) or vanillin-HCl is widely used to characterize spatial patterns of proanthocyanidin accumulation in plant tissues. These methods are limited in their ability to allow high-resolution imaging of proanthocyanidin deposits.

Results

Tissue embedding techniques were used in combination with DMACA staining to analyze the accumulation of proanthocyanidins in Lotus corniculatus (L.) and Trifolium repens (L.) tissues. Embedding of plant tissues in LR White or paraffin matrices, with or without DMACA staining, preserved the physical integrity of the plant tissues, allowing high-resolution imaging that facilitated cell-specific localization of proanthocyanidins. A brown coloration was seen in proanthocyanidin-producing cells when plant tissues were embedded without DMACA staining and this was likely to have been due to non-enzymatic oxidation of proanthocyanidins and the formation of colored semiquinones and quinones.

Conclusions

This paper presents a simple, high-resolution method for analysis of proanthocyanidin accumulation in organs, tissues and cells of two plant species with different patterns of proanthocyanidin accumulation, namely Lotus corniculatus (birdsfoot trefoil) and Trifolium repens (white clover). This technique was used to characterize cell type-specific patterns of proanthocyanidin accumulation in white clover flowers at different stages of development.     

Isolation of plant transcription factors using a modified yeast one-hybrid system

Background  

The preparation of expressional cDNA libraries for use in the yeast two-hybrid system is quick and efficient when using the dedicated Clontech™ product, the MATCHMAKER Library Construction and Screening Kit 3. This kit employs SMART technology for the amplification of full-length cDNAs, in combination with cloning using homologous recombination.     

Chromatin immunoprecipitation: optimization, quantitative analysis and data normalization

Background

Chromatin remodeling, histone modifications and other chromatin-related processes play a crucial role in gene regulation. A very useful technique to study these processes is chromatin immunoprecipitation (ChIP). ChIP is widely used for a few model systems, including Arabidopsis, but establishment of the technique for other organisms is still remarkably challenging. Furthermore, quantitative analysis of the precipitated material and normalization of the data is often underestimated, negatively affecting data quality.

Results

We developed a robust ChIP protocol, using maize (Zea mays) as a model system, and present a general strategy to systematically optimize this protocol for any type of tissue. We propose endogenous controls for active and for repressed chromatin, and discuss various other controls that are essential for successful ChIP experiments. We experienced that the use of quantitative PCR (QPCR) is crucial for obtaining high quality ChIP data and we explain why. The method of data normalization has a major impact on the quality of ChIP analyses. Therefore, we analyzed different normalization strategies, resulting in a thorough discussion of the advantages and drawbacks of the various approaches.

Conclusion

Here we provide a robust ChIP protocol and strategy to optimize the protocol for any type of tissue; we argue that quantitative real-time PCR (QPCR) is the best method to analyze the precipitates, and present comprehensive insights into data normalization.     

High Resolution Melt (HRM) analysis is an efficient tool to genotype EMS mutants in complex crop genomes

Background

Targeted Induced Loci Lesions IN Genomes (TILLING) is increasingly being used to generate and identify mutations in target genes of crop genomes. TILLING populations of several thousand lines have been generated in a number of crop species including Brassica rapa. Genetic analysis of mutants identified by TILLING requires an efficient, high-throughput and cost effective genotyping method to track the mutations through numerous generations. High resolution melt (HRM) analysis has been used in a number of systems to identify single nucleotide polymorphisms (SNPs) and insertion/deletions (IN/DELs) enabling the genotyping of different types of samples. HRM is ideally suited to high-throughput genotyping of multiple TILLING mutants in complex crop genomes. To date it has been used to identify mutants and genotype single mutations. The aim of this study was to determine if HRM can facilitate downstream analysis of multiple mutant lines identified by TILLING in order to characterise allelic series of EMS induced mutations in target genes across a number of generations in complex crop genomes.

Results

We demonstrate that HRM can be used to genotype allelic series of mutations in two genes, BraA.CAX1a and BraA.MET1.a in Brassica rapa. We analysed 12 mutations in BraA.CAX1.a and five in BraA.MET1.a over two generations including a back-cross to the wild-type. Using a commercially available HRM kit and the Lightscanner? system we were able to detect mutations in heterozygous and homozygous states for both genes.

Conclusions

Using HRM genotyping on TILLING derived mutants, it is possible to generate an allelic series of mutations within multiple target genes rapidly. Lines suitable for phenotypic analysis can be isolated approximately 8-9 months (3 generations) from receiving M₃ seed of Brassica rapa from the RevGenUK TILLING service.     

Molecular cloning and characterization of a cucumber MAP kinase gene in response to excess NO3 and other abiotic stresses

Mitogen-activated protein kinases (MAPKs) play important roles in the transduction of extracellular signals to the intracellular targets in all eukaryotes. Here, a cucumber cDNA designated CsNMAPK, encoding a mitogen-activated protein kinase was isolated using RT-PCR, 3′ and 5′ RACE. The full-length cDNA sequence contains 1636 bp and an open reading frame (ORF) of 1113 bp, which encodes 370 amino acid residues. According to the phylogenetic analysis, CsNMAPK belongs to subgroup I MAPK in plants. Northern blot analysis revealed that CsNMAPK expressed differently in response to excess NO₃⁻. And the CsNMAPK expression kinetics between a salt-resistant cultivar (Xintaimici) and a salt-sensitive cultivar (Shennongchunwu) was slightly different under 182 mmol L⁻¹ NO₃⁻ treatment. The mRNA levels also increased after 24 h treatments with H₂O₂ and salicylic acid (SA), but decreased with abscisic acid (ABA) and low-temperature. However, there was no significant induction of CsNMAPK gene after 24 h drought and high-temperature treatments. Our results suggested that a MAP kinase cascade may function in excess NO₃⁻ and other abiotic stresses in cucumber.