Transgenic Arabidopsis thaliana plants expressing bacterial γ-hexachlorocyclohexane dehydrochlorinase LinA.

BACKGROUND: γ-Hexachlorocyclohexane (γ-HCH), an organochlorine insecticide of anthropogenic origin, is a persistent organic pollutant (POP) that causes environmental pollution concerns worldwide. Although many γ-HCH-degrading bacterial strains are available, inoculating them directly into γ-HCH-contaminated soil is ineffective because of the low survival rate of the exogenous bacteria. Another strategy for the bioremediation of γ-HCH involves the use of transgenic plants expressing bacterial enzyme for γ-HCH degradation through phytoremediation. RESULTS: We generated transgenic Arabidopsis thaliana expressing γ-HCH dehydrochlroninase LinA from bacterium Sphingobium japonicum strain UT26. Among the transgenic Arabidopsis T2 lines, we obtained one line (A5) that expressed and accumulated LinA well. The A5-derived T3 plants showed higher tolerance to γ-HCH than the non-transformant control plants, indicating that γ-HCH is toxic for Arabidopsis thaliana and that this effect is relieved by LinA expression. The crude extract of the A5 plants showed γ-HCH degradation activity, and metabolites of γ-HCH produced by the LinA reaction were detected in the assay solution, indicating that the A5 plants accumulated the active LinA protein. In some A5 lines, the whole plant absorbed and degraded more than 99% of γ-HCH (10 ppm) in the liquid medium within 36 h. CONCLUSION: The transgenic Arabidopsis expressing active LinA absorbed and degraded γ-HCH in the liquid medium, indicating the high potential of LinA-expressing transgenic plants for the phytoremediation of environmental γ-HCH. This study marks a crucial step toward the practical use of transgenic plants for the phytoremediation of POPs.

Plasmodiophora brassicae affects host gene expression by secreting the transcription factor-type effector PbZFE1.

The protist pathogen Plasmodiophora brassicae hijacks the metabolism and development of host cruciferous plants and induces clubroot formation, but little is known about its regulatory mechanisms. Previously, the Pnit2int2 sequence, a sequence around the second intron of the nitrilase gene (BrNIT2) involved in auxin biosynthesis in Brassica rapa ssp. pekinensis, was identified as a specific promoter activated during clubroot formation. In this study, we hypothesized that analysis of the transcriptional regulation of Pnit2int2 could reveal how P. brassicae affects the host gene regulatory system during clubroot development. By yeast one-hybrid screening, the pathogen zinc finger protein PbZFE1 was identified to specifically bind to Pnit2int2. Specific binding of PbZFE1 to Pnit2int2 was also confirmed by electrophoretic mobility shift assay. The binding site of PbZFE1 is essential for promoter activity of Pnit2int2 in clubbed roots of transgenic Arabidopsis thaliana (Pnit2int2-2::GUS), indicating that PbZFE1 is secreted from P. brassicae and functions within plant cells. Ectopic expression of PbZEF1 in A. thaliana delayed growth and flowering time, suggesting that PbZFE1 has significant impacts on host development and metabolic systems. Thus, P. brassicae appears to secrete PbZFE1 into host cells as a transcription factor-type effector during pathogenesis.

Rapid analysis of GBSS1 and Vinv genes expressed in potato tubers using microtubers produced in liquid culture medium.

KEY MESSAGE: This study established a rapid method for the gene expression analysis in potato tubers. The use of microtubers would be useful for primary evaluation of tuber-expressed genes. In the development of transgenic potato or of potato with other genome modifications (e.g., genome editing or RNA-directed DNA methylation (RdDM) and so on) to improve tuber traits, analysis of the target gene is often difficult because of the long cultivation cycle (3-4 months), large areas required, numerous materials for plant cultivation, and considerable efforts needed to obtain transgenic tubers. We demonstrate here rapid and convenient analysis of gene expression in potato microtubers. Enough microtubers for expression analysis can be induced over about 4 weeks in a simple liquid medium in an Erlenmeyer flask. High-quality RNA and protein can be easily prepared from microtubers and used for northern blot, qRT-PCR, and western blot analyses without further purification. We investigated the expression of two tuber-expressed genes (GBSS1 and Vinv) in microtubers derived from the wild-type and from lines derived from RdDM-mediated transcriptional gene silencing. As expected, the expression of both genes was similar between microtubers and normal tubers. Furthermore, we demonstrated that microtubers can be used in western blot and confocal immunofluorescent microscopy analyses. These results suggest that expression analysis using microtubers is a convenient tool for the analysis of tuber-expressed genes such as GBSS1 and Vinv in potato.

Development of a visible marker trait based on leaf sheath-specific anthocyanin pigmentation applicable to various genotypes in rice.

To overcome a limitation to the breeding of autogamous crops, recurrent selection using transgenic male sterility (RSUTMS) has been proposed. In this system, negatively or positively selectable marker traits are required along with dominant transgenic male sterility. Anthocyanin pigmentation is an excellent marker trait. Two regulatory genes for MYB and bHLH and a structural gene for DFR are required for anthocyanin pigmentation in rice. Therefore, to apply anthocyanin pigmentation as a marker trait in various rice genotypes, coordinated expression of the three genes is required. In this study, we developed a leaf sheath-specific promoter and introduced three genes-DFR and C1/Myb, driven by the 35S promoter, and OsB2/bHLH, driven by the leaf sheath-specific promoter-into the rice genome. Leaf sheath-specific pigmentation was confirmed in all seven genotypes tested, which included japonica and indica cultivars. Analysis of genome sequence data from 25 cultivars showed that the strategy of conferring leaf sheath-specific anthocyanin pigmentation by introduction of these three genes would be effective for a wide range of genotypes and will be applicable to RSUTMS.

The Non-Mendelian Green Cotyledon Gene in Soybean Encodes a Small Subunit of Photosystem II.

Chlorophyll degradation plays important roles in leaf senescence including regulation of degradation of chlorophyll-binding proteins. Although most genes encoding enzymes of the chlorophyll degradation pathway have been identified, the regulation of their activity has not been fully understood. Green cotyledon mutants in legume are stay-green mutants, in which chlorophyll degradation is impaired during leaf senescence and seed maturation. Among them, the soybean (Glycine max) green cotyledon gene cytG is unique because it is maternally inherited. To isolate cytG, we extensively sequenced the soybean chloroplast genome, and detected a 5-bp insertion causing a frame-shift in psbM, which encodes one of the small subunits of photosystem II. Mutant tobacco plants (Nicotiana tabacum) with a disrupted psbM generated using a chloroplast transformation technique had green senescent leaves, confirming that cytG encodes PsbM. The phenotype of cytG was very similar to that of mutant of chlorophyll b reductase catalyzing the first step of chlorophyll b degradation. In fact, chlorophyll b-degrading activity in dark-grown cytG and psbM-knockout seedlings was significantly lower than that of wild-type plants. Our results suggest that PsbM is a unique protein linking photosynthesis in presenescent leaves with chlorophyll degradation during leaf senescence and seed maturation. Additionally, we discuss the origin of cytG, which may have been selected during domestication of soybean.

Development of transgenic male-sterile rice by using anther-specific promoters identified by comprehensive screening of the gene expression profile database 'RiceXPro'.

Because genomic selection is designed for the population breeding of allogamous species, a successive outcrossing system is required for efficient use of genomic selection in autogamous crops, such as Oryza sativa L. (rice). Transgenic and dominant male-sterility is a suitable tool for efficient outcrossing of autogamous crops. Though there have been some reports of dominant male-sterile rice developed using transgenic technology, the flowering habit was substandard. Here, to isolate promoters that, when linked to a lethal gene, induce dominant male-sterility while retaining a good flowering habit, we identified 38 candidate genes with anther-specific expression by using the 'RiceXPro' database. We then evaluated the abilities of the near-upstream regions of these genes to induce male-sterility when linked to the lethal gene barnase and introduced into the rice cultivar 'Nipponbare'. Seven of the 38 promoters induced clear dominant male-sterility; promoters expressed in the later stage of anther development induced male-sterility while retaining better flowering habits when compared to ones expressed in the early stage. These seven promoters could potentially be used to facilitate development of an efficient outcross-based breeding system in rice.

Development and characterization of transgenic dominant male sterile rice toward an outcross-based breeding system.

Genomic selection is attracting attention in the field of crop breeding. To apply genomic selection effectively for autogamous (self-pollinating) crops, an efficient outcross system is desired. Since dominant male sterility is a powerful tool for easy and successive outcross of autogamous crops, we developed transgenic dominant male sterile rice (Oryza sativa L.) using the barnase gene that is expressed by the tapetum-specific promoter BoA9. Barnase-induced male sterile rice No. 10 (BMS10) was selected for its stable male sterility and normal growth characteristics. The BMS10 flowering habits, including heading date, flowering date, and daily flowering time of BMS10 tended to be delayed compared to wild type. When BMS10 and wild type were placed side-by-side and crossed under an open-pollinating condition, the seed-setting rate was <1.5%. When the clipping method was used to avoid the influence of late flowering habits, the seed-setting rate of BMS10 increased to a maximum of 86.4%. Although flowering synchronicity should be improved to increase the seed-setting rate, our results showed that this system can produce stable transgenic male sterility with normal female fertility in rice. The transgenic male sterile rice would promote a genomic selection-based breeding system in rice.

Biodegradation of γ-hexachlorocyclohexane by transgenic hairy root cultures of Cucurbita moschata that accumulate recombinant bacterial LinA.

KEY MESSAGE: γ-HCH was successfully degraded using LinA-expressed transgenic hairy root cultures of Cucurbita moschata . Fusing an endoplasmic reticulum-targeting signal peptide to LinA was essential for stable accumulation in the hairy roots. The pesticide γ-hexachlorocyclohexane (γ-HCH) is a persistent organic pollutant (POP) that raises public health and environmental pollution concerns worldwide. Although several isolates of γ-HCH-degrading bacteria are available, inoculating them directly into γ-HCH-contaminated soil is ineffective because of the bacterial survival rate. Cucurbita species incorporate significant amounts of POPs from soils compared with other plant species. Here, we describe a novel bioremediation strategy that combines the bacterial degradation of γ-HCH and the efficient uptake of γ-HCH by Cucurbita species. We produced transgenic hairy root cultures of Cucurbita moschata that expressed recombinant bacterial linA, isolated from the bacterium Sphingobium japonicum UT26. The LinA protein was accumulated stably in the hairy root cultures by fusing an endoplasmic reticulum (ER)-targeting signal peptide to LinA. Then, we demonstrated that the cultures degraded more than 90 % of γ-HCH (1 ppm) overnight and produced the γ-HCH metabolite 1,2,4-trichlorobenzene, indicating that LinA degraded γ-HCH. These results indicate that the gene linA has high potential for phytoremediation of environmental γ-HCH.

Possibilities of direct introgression from Brassica napus to B. juncea and indirect introgression from B. napus to related Brassicaceae through B. juncea.

The impact of genetically modified canola (Brassica napus) on biodiversity has been examined since its initial stage of commercialization. Various research groups have extensively investigated crossability and introgression among species of Brassicaceae. B. rapa and B. juncea are ranked first and second as the recipients of cross-pollination and introgression from B. napus, respectively. Crossability between B. napus and B. rapa has been examined, specifically in terms of introgression from B. napus to B. rapa, which is mainly considered a weed in America and European countries. On the other hand, knowledge on introgression from B. napus to B. juncea is insufficient, although B. juncea is recognized as the main Brassicaceae weed species in Asia. It is therefore essential to gather information regarding the direct introgression of B. napus into B. juncea and indirect introgression of B. napus into other species of Brassicaceae through B. juncea to evaluate the influence of genetically modified canola on biodiversity. We review information on crossability and introgression between B. juncea and other related Brassicaseae in this report.

Epistatic interactions among multiple copies of FLC genes with naturally occurring insertions correlate with flowering time variation in radish.

Brassicaceae crops, which underwent whole-genome triplication during their evolution, have multiple copies of flowering-related genes. Interactions among multiple gene copies may be involved in flowering time regulation; however, this mechanism is poorly understood. In this study, we performed comprehensive, high-throughput RNA sequencing analysis to identify candidate genes involved in the extremely late-bolting (LB) trait in radish. Then, we examined the regulatory roles and interactions of radish FLOWERING LOCUS C (RsFLC) paralogs, the main flowering repressor candidates. Seven flowering integrator genes, five vernalization genes, nine photoperiodic/circadian clock genes and eight genes from other flowering pathways were differentially expressed in the early-bolting (EB) cultivar 'Aokubinagafuto' and LB radish cultivar 'Tokinashi' under different vernalization conditions. In the LB cultivar, RsFLC1 and RsFLC2 expression levels were maintained after 40 days of cold exposure. Bolting time was significantly correlated with the expression rates of RsFLC1 and RsFLC2. Using the EB × LB F2 population, we performed association analyses of genotypes with or without 1910- and 1627-bp insertions in the first introns of RsFLC1 and RsFLC2, respectively. The insertion alleles prevented the repression of their respective FLC genes under cold conditions. Interestingly, genotypes homozygous for RsFLC2 insertion alleles maintained high RsFLC1 and RsFLC3 expression levels under cold conditions, and two-way analysis of variance revealed that RsFLC1 and RsFLC3 expression was influenced by the RsFLC2 genotype. Our results indicate that insertions in the first introns of RsFLC1 and RsFLC2 contribute to the late-flowering trait in radish via different mechanisms. The RsFLC2 insertion allele conferred a strong delay in bolting by inhibiting the repression of all three RsFLC genes, suggesting that radish flowering time is determined by epistatic interactions among multiple FLC gene copies.

Molecular analysis of transgenic melon plants showing virus resistance conferred by direct repeat of movement gene of Cucumber green mottle mosaic virus.

Cucumber green mottle mosaic virus (CGMMV) is a major limiting factor in the production of melon plants worldwide. For effective control of this virus using the transgenic approach, the direct repeat of the movement protein gene of CGMMV was used for transforming melon plants by Agrobacterium tumefaciens. PCR and Southern blot analyses of T3 confirmed that they carried the transgene. Northern blot analysis with total RNA showed that transgene transcript RNA as well as siRNA was observed in all plants tested. Separate leaves or individual plants were inoculated with CGMMV and subjected to ELISA and RNA blot analysis using the coat protein gene probe of the virus. Compared to nontransgenic control, these plants were shown to have high virus resistance. Furthermore, cytosine of the transgene DNA in the plants was methylated. Thus, these results reveal that the transgenic lines were highly resistant to the virus through RNA silencing. Key message High virus resistance was obtained in transgenic melon plants with direct repeat of movement protein gene of Cucumber green mottle mosaic tobamovirus through RNA silencing.

Persistent C genome chromosome regions identified by SSR analysis in backcross progenies between Brassica juncea and B. napus.

Given that feral transgenic canola (Brassica napus) from spilled seeds has been found outside of farmer's fields and that B. juncea is distributed worldwide, it is possible that introgression to B. juncea from B. napus has occurred. To investigate such introgression, we characterized the persistence of B. napus C genome chromosome (C-chromosome) regions in backcross progenies by B. napus C-chromosome specific simple sequence repeat (SSR) markers. We produced backcross progenies from B. juncea and F(1) hybrid of B. juncea × B. napus to evaluate persistence of C-chromosome region, and screened 83 markers from a set of reported C-chromosome specific SSR markers. Eighty-five percent of the SSR markers were deleted in the BC(1) obtained from B. juncea × F(1) hybrid, and this BC(1) exhibited a plant type like that of B. juncea. Most markers were deleted in BC(2) and BC(3) plants, with only two markers persisting in the BC(3). These results indicate a small possibility of persistence of C-chromosome regions in our backcross progenies. Knowledge about the persistence of B. napus C-chromosome regions in backcross progenies may contribute to shed light on gene introgression.

Relationship between hybridization frequency of Brassica juncea × B. napus and distance from pollen source (B. napus) to recipient (B. juncea) under field conditions in Japan.

Several imported transgenic canola (Brassica napus) seeds have been spilled and have grown along roadsides around import ports. B. juncea, a relative of B. napus with which it has high interspecific crossability, is widely distributed throughout Japan. There is public concern about the harmful impacts of feral B. napus plants on biodiversity, but spontaneous hybridization between spilled B. napus and weedy B. juncea populations is hardly revealed. We evaluated the relationship between the hybridization frequency of B. juncea × B. napus and their planting distance in field experiments using the mutagenic herbicide-tolerant B. napus cv. Bn0861 as a pollen source for hybrid screening. The recipient B. juncea cv. Kikarashina was planted in an experimental field with Bn0861 planted in the center. No hybrids were detected under natural flowering conditions in 2009. However, the flowering period was artificially kept overlapping in 2010, leading to a hybridization frequency of 1.62% in the mixed planting area. The hybridization frequency decreased drastically with distance from the pollen source, and was lower under field conditions than estimated from the high crossability, implying that spontaneous hybridization between spilled B. napus and weedy B. juncea is unlikely in the natural environment.

Agrobacterium-mediated transformation of kabocha squash (Cucurbita moschata Duch) induced by wounding with aluminum borate whiskers.

An efficient genetic transformation method for kabocha squash (Cucurbita moschata Duch cv. Heiankogiku) was established by wounding cotyledonary node explants with aluminum borate whiskers prior to inoculation with Agrobacterium. Adventitious shoots were induced from only the proximal regions of the cotyledonary nodes and were most efficiently induced on Murashige-Skoog agar medium with 1 mg/L benzyladenine. Vortexing with 1% (w/v) aluminum borate whiskers significantly increased Agrobacterium infection efficiency in the proximal region of the explants. Transgenic plants were screened at the T(0) generation by sGFP fluorescence, genomic PCR, and Southern blot analyses. These transgenic plants grew normally and T(1) seeds were obtained. We confirmed stable integration of the transgene and its inheritance in T(1) generation plants by sGFP fluorescence and genomic PCR analyses. The average transgenic efficiency for producing kabocha squashes with our method was about 2.7%, a value sufficient for practical use.

Estrogen-inducible GFP expression patterns in rice (Oryza sativa L.).

We investigated estrogen-inducible green fluorescent protein (GFP) expression patterns using an estrogen receptor fused chimeric transcription activator, XVE, in the monocotyledonous model plant rice (Oryza sativa L.). This system has been shown to be an effective chemical-inducible gene expression system in Arabidopsis and has been applied to other plants in order to investigate gene functions or produce marker-free transgenic plants. However, limited information is available on the correlation between inducer concentration and the expression level of the gene induced in monocots. Here, we produced a transgenic rice integrated estrogen-inducible GFP expression vector, pLex:GFP, and investigated dose-response and time-course patterns of GFP induction in rice calli and seedlings for the first time. With 17-ß-estradiol treatment at >5 µM, GFP signals were detected in the entire surface of calli within 2 days of culture. Highest GFP signals were extended for 8 days with estradiol treatment at 25 µM. In three-leaf-stage seedlings, GFP signals in the leaves of pLex:GFP-integrated transgenic lines were weaker than those in the leaves of p35S:GFP-integrated transgenic lines. However, GFP signals in the roots of pLex:GFP- and p35S:GFP-integrated transgenic lines were similar with estradiol treatment at >10 µM. With regard to controlling appropriate gene expression, these results might provide helpful indications on estradiol treatment conditions to be used for the XVE system in rice and other monocots.

Occurrence of metaxenia and false hybrids in Brassica juncea L. cv. Kikarashina × B. napus.

Imported genetically modified (GM) canola (Brassica napus) is approved by Japanese law. Some GM canola varieties have been found around importation sites, and there is public concern that these may have any harmful effects on related species such as reduction of wild relatives. Because B. juncea is distributed throughout Japan and is known to be high crossability with B. napus, it is assumed to be a recipient of B. napus. However, there are few reports for introgression of cross-combination in B. juncea × B. napus. To assess crossability, we artificially pollinated B. juncea with B. napus. After harvesting a large number of progeny seeds, we observed false hybrids and metaxenia of seed coats. Seed coat color was classified into four categories and false hybrids were confirmed by morphological characteristics and random amplified polymorphic DNA (RAPD) markers. Furthermore, the occurrence of false hybrids was affected by varietal differences in B. napus, whereas that of metaxenia was related to hybridity. Therefore, we suggest that metaxenia can be used as a marker for hybrid identification in B. juncea L. cv. Kikarashina × B. napus. Our results suggest that hybrid productivity in B. juncea × B. napus should not be evaluated by only seed productivity, crossability ought to be assessed the detection of true hybrids.

A method of transformation and current progress in transgenic research on cucumbers and Cucurbita species.

Cucumber (Cucumis sativus L.) and Cucurbita species (squashes, pumpkins, and gourds), belonging to the Cucurbitaceae family, are among the major vegetable crops in the world. Transgenic approaches could contribute to the accumulation of new knowledge of these species and to the development of elite cultivars. Despite this, research reports using transformants of these species are very limited so far. One of the reasons for this may be that although there are effective transformation methods, these methods are not well known among researchers. In the present review, we describe efficient protocols for the transformation of cucumber and squash plants and mention possible pitfalls in and advice for following these protocols. In addition, we discuss the current progress of genetic transformation research using cucumbers and squash, including genome editing.

A novel strategy for promoting homoplasmic plastid transformant production using the barnase-barstar system.

Plastid transformants form biofactories that are able to produce extra proteins in plastids when they are in a homoplasmic state. To date, plastid transformation has been reported in about twenty plant species; however, the production of homoplasmic plastid transformants is not always successful or easy. Heteroplasmic plants that contain wild-type plastids produce fewer target proteins and do not always successfully transfer transgenes to progeny. In order to promote the generation of homoplasmic plants, we developed a novel system using barnase-barster to eliminate wild-type plastids from heteroplasmic cells systematically. In this system, a chemically inducible cytotoxic barnase under a plastid transit signal was introduced into nuclear DNA and barster, which inhibits barnase, was integrated into plastid DNA with the primary selection markers aminoglycoside 3'-adenylyltransferase (aadA) and green fluorescence protein (GFP) gene. As expected, the expression of the plastid barnase was lethal to cells as seen in leaf segments, but barster expression in plastids rescued them. We then investigated the regeneration frequency of homoplasmic shoots from heteroplasmic leaf segments with or without barnase expression. The regeneration frequency of homoplasmic-like shoots expressing barnase-barster system was higher than that of shoots not expressing this. We expect that the application of this novel strategy for transformation of plastids will be supportive to generate homoplasmic plastid transformants in other plant species.

Analyzing Twitter Conversation on Genome-Edited Foods and Their Labeling in Japan.

In recent years, the research and development of genome editing technology have been progressing rapidly, and the commercial use of genome-edited soybean started in the United States in 2019. A preceding study's results found that there is public concern with regard to the safety of high-tech foods, such as genetically modified foods and genome-edited foods. Twitter, one of the most popular social networks, allows users to post their opinions instantaneously, making it an extremely useful tool to collect what people are actually saying online in a timely manner. Therefore, it was used for collecting data on the users' concerns with and expectations of high-tech foods. This study collected and analyzed Twitter data on genome-edited foods and their labeling from May 25 to October 15 in 2019. Of 14,066 unique user IDs, 94.9% posted 5 or less tweets, whereas 64.8% tweeted only once, indicating that the majority of users who tweeted on this issue are not as intense, as they posted tweets consistently. After a process of refining, there were 28,722 tweets, of which 2,536 tweets (8.8%) were original, 326 (1.1%) were replies, and 25,860 (90%) were retweets. The numbers of tweets increased in response to government announcements and news content in the media. A total of six prominent peaks were detected during the investigation period, proving that Twitter could serve as a tool for monitoring degree of users' interests in real time. The co-occurrence network of original and reply tweets provided different words from various tweets that appeared with a certain frequency. However, the network derived from all tweets seemed to concentrate on words from specific tweets with negative overtones. As a result of sentiment analysis, 54.5% to 62.8% tweets were negative about genome-edited food and the labeling policy of the Consumer Affairs Agency, respectively, indicating a strong demand for mandatory labeling. These findings are expected to contribute to the communication strategy of genome-edited foods toward social implementation by government officers and science communicators.