Gene silencing in Escherichia coli using antisense RNAs expressed from doxycycline‐inducible vectors

Here, we report on the construction of doxycycline (tetracycline analogue)‐inducible vectors that express antisense RNAs in Escherichia coli. Using these vectors, the expression of genes of interest can be silenced conditionally. The expression of antisense RNAs from the vectors was more tightly regulated than the previously constructed isopropyl‐β‐D‐galactopyranoside‐inducible vectors. Furthermore, expression levels of antisense RNAs were enhanced by combining the doxycycline‐inducible promoter with the T7 promoter‐T7 RNA polymerase system; the T7 RNA polymerase gene, under control of the doxycycline‐inducible promoter, was integrated into the lacZ locus of the genome without leaving any antibiotic marker. These vectors are useful for investigating gene functions or altering cell phenotypes for biotechnological and industrial applications.

Significance and Impact of the Study

A gene silencing method using antisense RNAs in Escherichia coli is described, which facilitates the investigation of bacterial gene function. In particular, the method is suitable for comprehensive analyses or phenotypic analyses of genes essential for growth. Here, we describe expansion of vector variations for expressing antisense RNAs, allowing choice of a vector appropriate for the target genes or experimental purpose.     

A double‐mutant collection targeting MAP kinase related genes in Arabidopsis for studying genetic interactions

Mitogen‐activated protein kinase cascades are conserved in all eukaryotes. In Arabidopsis thaliana there are approximately 80 genes encoding MAP kinase kinase kinases (MAP3K), 10 genes encoding MAP kinase kinases (MAP2K), and 20 genes encoding MAP kinases (MAPK). Reverse genetic analysis has failed to reveal abnormal phenotypes for a majority of these genes. One strategy for uncovering gene function when single‐mutant lines do not produce an informative phenotype is to perform a systematic genetic interaction screen whereby double‐mutants are created from a large library of single‐mutant lines. Here we describe a new collection of 275 double‐mutant lines derived from a library of single‐mutants targeting genes related to MAP kinase signaling. To facilitate this study, we developed a high‐throughput double‐mutant generating pipeline using a system for growing Arabidopsis seedlings in 96‐well plates. A quantitative root growth assay was used to screen for evidence of genetic interactions in this double‐mutant collection. Our screen revealed four genetic interactions, all of which caused synthetic enhancement of the root growth defects observed in a MAP kinase 4 (MPK4) single‐mutant line. Seeds for this double‐mutant collection are publicly available through the Arabidopsis Biological Resource Center. Scientists interested in diverse biological processes can now screen this double‐mutant collection under a wide range of growth conditions in order to search for additional genetic interactions that may provide new insights into MAP kinase signaling.     

A dual gene‐silencing vector system for monocot and dicot plants

Plant virus‐based gene‐silencing vectors have been extensively and successfully used to elucidate functional genomics in plants. However, only limited virus‐induced gene‐silencing (VIGS) vectors can be used in both monocot and dicot plants. Here, we established a dual gene‐silencing vector system based on Bamboo mosaic virus (BaMV) and its satellite RNA (satBaMV). Both BaMV and satBaMV vectors could effectively silence endogenous genes in Nicotiana benthamiana and Brachypodium distachyon. The satBaMV vector could also silence the green fluorescent protein (GFP) transgene in GFP transgenic N. benthamiana. GFP transgenic plants co‐agro‐inoculated with BaMV and satBaMV vectors carrying sulphur and GFP genes, respectively, could simultaneously silence both genes. Moreover, the silenced plants could still survive with the silencing of genes essential for plant development such as heat‐shock protein 90 (Hsp90) and Hsp70. In addition, the satBaMV‐ but not BaMV‐based vector could enhance gene‐silencing efficiency in newly emerging leaves of N. benthamiana deficient in RNA‐dependant RNA polymerase 6. The dual gene‐silencing vector system of BaMV and satBaMV provides a novel tool for comparative functional studies in monocot and dicot plants.     

Precision genome editing in plants via gene targeting and piggyBac‐mediated marker excision

Precise genome engineering via homologous recombination (HR)‐mediated gene targeting (GT) has become an essential tool in molecular breeding as well as in basic plant science. As HR‐mediated GT is an extremely rare event, positive–negative selection has been used extensively in flowering plants to isolate cells in which GT has occurred. In order to utilize GT as a methodology for precision mutagenesis, the positive selectable marker gene should be completely eliminated from the GT locus. Here, we introduce targeted point mutations conferring resistance to herbicide into the rice acetolactate synthase (ALS) gene via GT with subsequent marker excision by piggyBac transposition. Almost all regenerated plants expressing piggyBac transposase contained exclusively targeted point mutations without concomitant re‐integration of the transposon, resulting in these progeny showing a herbicide bispyribac sodium (BS)‐tolerant phenotype. This approach was also applied successfully to the editing of a microRNA targeting site in the rice cleistogamy 1 gene. Therefore, our approach provides a general strategy for the targeted modification of endogenous genes in plants.     

Nuclear gene targeting in Chlamydomonas using engineered zinc‐finger nucleases

The unicellular green alga Chlamydomonas reinhardtii is a versatile model for fundamental and biotechnological research. A wide range of tools for genetic manipulation have been developed for this alga, but specific modification of nuclear genes is still not routinely possible. Here, we present a nuclear gene targeting strategy for Chlamydomonas that is based on the application of zinc‐finger nucleases (ZFNs). Our approach includes (i) design of gene‐specific ZFNs using available online tools, (ii) evaluation of the designed ZFNs in a Chlamydomonas in situ model system, (iii) optimization of ZFN activity by modification of the nuclease domain, and (iv) application of the most suitable enzymes for mutagenesis of an endogenous gene. Initially, we designed a set of ZFNs to target the COP3 gene that encodes the light‐activated ion channel channelrhodopsin‐1. To evaluate the designed ZFNs, we constructed a model strain by inserting a non‐functional aminoglycoside 3′‐phosphotransferase VIII (aphVIII) selection marker interspaced with a short COP3 target sequence into the nuclear genome. Upon co‐transformation of this recipient strain with the engineered ZFNs and an aphVIII DNA template, we were able to restore marker activity and select paromomycin‐resistant (Pm‐R) clones with expressing nucleases. Of these Pm‐R clones, 1% also contained a modified COP3 locus. In cases where cells were co‐transformed with a modified COP3 template, the COP3 locus was specifically modified by homologous recombination between COP3 and the supplied template DNA. We anticipate that this ZFN technology will be useful for studying the functions of individual genes in Chlamydomonas.     

Flip‐flop organization in the chloroplast genome of Capsosiphon fulvescens (Ulvophyceae,Chlorophyta)

To better understand organelle genome evolution of the ulvophycean green alga Capsosiphon fulvescens, we sequenced and characterized its complete chloroplast genome. The circular chloroplast genome was 111,561 bp in length with 31.3% GC content that contained 108 genes including 77 protein‐coding genes, two copies of rRNA operons, and 27 tRNAs. In this analysis, we found the two types of isoform, called heteroplasmy, were likely caused by a flip‐flop organization. The flip‐flop mechanism may have caused structural variation and gene conversion in the chloroplast genome of C. fulvescens. In a phylogenetic analysis based on all available ulvophycean chloroplast genome data, including a new C. fulvescens genome, we found three major conflicting signals for C. fulvescens and its sister taxon Pseudoneochloris marina within 70 individual genes: (i) monophyly with Ulotrichales, (ii) monophyly with Ulvales, and (iii) monophyly with the clade of Ulotrichales and Ulvales. Although the 70‐gene concatenated phylogeny supported monophyly with Ulvales for both species, these complex phylogenetic signals of individual genes need further investigations using a data‐rich approach (i.e., organelle genome data) from broader taxon sampling.     

Lotus SHAGGY‐like kinase 1 is required to suppress nodulation in Lotus japonicus

Glycogen synthase kinase/SHAGGY‐like kinases (SKs) are a highly conserved family of signaling proteins that participate in many developmental, cell‐differentiation, and metabolic signaling pathways in plants and animals. Here, we investigate the involvement of SKs in legume nodulation, a process requiring the integration of multiple signaling pathways. We describe a group of SKs in the model legume Lotus japonicus (LSKs), two of which respond to inoculation with the symbiotic nitrogen‐fixing bacterium Mesorhizobium loti. RNAi knock‐down plants and an insertion mutant for one of these genes, LSK1, display increased nodulation. Ηairy‐root lines overexpressing LSK1 form only marginally fewer mature nodules compared with controls. The expression levels of genes involved in the autoregulation of nodulation (AON) mechanism are affected in LSK1 knock‐down plants at low nitrate levels, both at early and late stages of nodulation. At higher levels of nitrate, these same plants show the opposite expression pattern of AON‐related genes and lose the hypernodulation phenotype. Our findings reveal an additional role for the versatile SK gene family in integrating the signaling pathways governing legume nodulation, and pave the way for further study of their functions in legumes.