CRISPR/Cas9-mediated disruption of CjACOS5 confers no-pollen formation on sugi trees (Cryptomeria japonica D. Don).

Sugi (Cryptomeria japonica D. Don) is an economically important coniferous tree in Japan. However, abundant sugi pollen grains are dispersed and transported by the wind each spring and cause a severe pollen allergy syndrome (Japanese cedar pollinosis). The use of pollen-free sugi that cannot produce pollen has been thought as a countermeasure to Japanese cedar pollinosis. The sugi CjACOS5 gene is an ortholog of Arabidopsis ACOS5 and rice OsACOS12, which encode an acyl-CoA synthetase that is involved in the synthesis of sporopollenin in pollen walls. To generate pollen-free sugi, we mutated CjACOS5 using the CRISPR/Cas9 system. As a result of sugi transformation mediated by Agrobacterium tumefaciens harboring the CjACOS5-targeted CRISPR/Cas9 vector, 1 bp-deleted homo biallelic mutant lines were obtained. Chimeric mutant lines harboring both mutant and wild-type CjACOS5 genes were also generated. The homo biallelic mutant lines had no-pollen in male strobili, whereas chimeric mutant lines had male strobili with or without pollen grains. Our results suggest that CjACOS5 is essential for the production of pollen in sugi and that its disruption is useful for the generation of pollen-free sugi. In addition to conventional transgenic technology, genome editing technology, including CRISPR/Cas9, can confer new traits on sugi.

Novel Functional Analysis for Pathogenic Proteins of Bursaphelenchus xylophilus in Pine Seed Embryos Using a Virus Vector.

Pine wilt disease (PWD), which is caused by the pine wood nematode Bursaphelenchus xylophilus, is among the most serious tree diseases worldwide. PWD is thought to be initiated by sequential excessive hypersensitive responses to B. xylophilus. Previous studies have reported candidate pathogenic molecules inducing hypersensitive responses in pine trees susceptible to B. xylophilus. The functions of some of these molecules have been analyzed in model plants using transient overexpression; however, whether they can induce hypersensitive responses in natural host pines remains unclear due to the lack of a suitable functional analysis method. In this study, we established a novel functional analysis method for susceptible black pine (Pinus thunbergii) seed embryos using transient overexpression by the Apple latent spherical virus vector and investigated five secreted proteins of B. xylophilus causing cell death in tobacco to determine whether they induce hypersensitive responses in pine. We found that three of five molecules induced significantly higher expression in pathogenesis-related genes ( p < 0.05), indicating hypersensitive response in pine seed embryos compared with mock and green fluorescence protein controls. This result suggests that tobacco-based screening may detect false positives. This study is the first to analyze the function of pathogenic candidate molecules of B. xylophilus in natural host pines using exogenous gene expression, which is anticipated to be a powerful tool for investigating the PWD mechanism.

CRISPR/Cas9-mediated targeted mutagenesis in Japanese cedar (Cryptomeria japonica D. Don).

Cryptomeria japonica (Japanese cedar or sugi) is one of the most important coniferous tree species in Japan and breeding programs for this species have been launched since 1950s. Genome editing technology can be used to shorten the breeding period. In this study, we performed targeted mutagenesis using the CRISPR/Cas9 system in C. japonica. First, the CRISPR/Cas9 system was tested using green fluorescent protein (GFP)-expressing transgenic embryogenic tissue lines. Knock-out efficiency of GFP ranged from 3.1 to 41.4% depending on U6 promoters and target sequences. The GFP knock-out region was mottled in many lines, indicating genome editing in individual cells. However, in 101 of 102 mutated individuals (> 99%) from 6 GFP knock-out lines, embryos had a single mutation pattern. Next, we knocked out the endogenous C. japonica magnesium chelatase subunit I (CjChlI) gene using two guide RNA targets. Green, pale green, and albino phenotypes were obtained in the gene-edited cell lines. Sequence analysis revealed random deletions, insertions, and replacements in the target region. Thus, targeted mutagenesis using the CRISPR/Cas9 system can be used to modify the C. japonica genome.

A protocol for Agrobacterium-mediated transformation of Japanese cedar, Sugi (Cryptomeria japonica D. Don) using embryogenic tissue explants.

Sugi (Cryptomeria japonica D. Don) is the most important afforestation coniferous tree in Japan. Coniferous trees normally have a long juvenile period and require a long cultivation time for breeding. Through a traditional breeding project that began in the 1950s, first generation plus trees with excellent traits were selected primarily from artificial forests and used as seedlings. Recently, the second generation plus trees obtained by crossing between plus trees have been selected. In light of this situation, the improvement of Sugi by a transgenic approach is effective in terms of shortening the breeding period compared with conventional crossing-dependent approaches. There are three key points to an efficient Agrobacterium-mediated transformation system: (1) establishment of explants with high regeneration ability, (2) optimal co-cultivation conditions for explants and Agrobacterium, and (3) efficient elimination of Agrobacterium. Here we describe a protocol for Agrobacterium-mediated transformation of Sugi that meets the above criteria using embryogenic tissues as explants isolated from immature seeds obtained by crossing.

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.

Somatic embryogenesis in artificially pollinated seed families of 2nd generation plus trees and cryopreservation of embryogenic tissue in Cryptomeria japonica D. Don (Sugi).

Cryptomeria japonica D. Don (common name is Sugi or Japanese cedar) is the most important forestation tree species in Japan, and 2nd generation plus trees with superior traits have been selected by breeding projects. Biotechnological approaches such as genetic transformation and genome editing are expected to accelerate to add useful traits (e.g., no-pollen traits) to superior trees in short time. To develop a platform for genetic transformation and genome editing of C. japonica superior trees, this study investigated the embryogenic potential of 2nd generation plus trees and obtained good cell lines with high embryogenic potential, which could be useful material for adding new and useful traits to superior trees by genetic transformation. However, the maintenance of embryogenic cell lines is laborious, and prolonged subculture leads to a loss of embryogenesis potential. Therefore, cell lines need to be cryopreserved for long without subculture. Therefore, in this study we made a simple cryopreservation protocol suitable for most C. japonica cell lines. We showed that cryopreserved cells using this protocol formed somatic embryos, which were then converted to plantlets. Transgenic cells produced from cryopreserved cells expressed transgene, gfp. These results indicated that our cryopreservation protocol can be used for prolonged storage of genetic transformation target materials in C. japonica.

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.

Improvement of Agrobacterium-mediated transformation of cucumber (Cucumis sativus L.) by combination of vacuum infiltration and co-cultivation on filter paper wicks.

An improved method for genetic transformation of cucumber (Cucumis sativus L. cv. Shinhokusei No. 1) was developed. Vacuum infiltration of cotyledonary explants with Agrobacterium suspension enhanced the efficiency of Agrobacterium infection in the proximal regions of explants. Co-cultivation on filter paper wicks suppressed necrosis of explants, leading to increased regeneration efficiency. Putative transgenic plants were screened by kanamycin resistance and green fluorescent protein (GFP) fluorescence, and integration of the transgene into the cucumber genome was confirmed by genomic polymerase chain reaction (PCR) and Southern blotting. These transgenic plants grew normally and T1 seeds were obtained from 7 lines. Finally, stable integration and transmission of the transgene in T1 generations were confirmed by GFP fluorescence and genomic PCR. The average transgenic efficiency for producing cucumbers with our method was 11.9 ± 3.5 %, which is among the highest values reported until date using kanamycin as a selective agent.

The promoter of an A9 homolog from the conifer Cryptomeria japonica imparts male strobilus-dominant expression in transgenic trees.

KEY MESSAGE : GUS analysis in Cryptomeria japonica revealed that the CjMALE1 promoter is activated in the male strobilus of C. japonica. Toward the development of male sterile technology for Cryptomeria japonica, a male strobilus-dominant promoter of C. japonica was isolated. The CjMALE1 gene was isolated from a male strobilus-specific suppression subtractive hybridization (SSH) library, and the promoter was isolated by the TAIL-PCR method. To characterize the CjMALE1 promoter, ß-glucuronidase (GUS)-fused genes were constructed and introduced into C. japonica using Agrobacterium tumefaciens. GUS expression from CjMALE1-2.5 K (2,718 bp fragment)::GUS C. japonica and CjMALE1-1 K (1,029 bp fragment)::GUS C. japonica was detected in the tapetum and microspore mother cells. These promoter fragments were comparably active in the pre-meiotic stage of the male strobilus of C. japonica. Our analysis showed that the 1,029 bp promoter had all the cis-elements necessary for male strobilus-dominant expression of CjMALE1. When CjMALE1-1 K::GUS was introduced into Arabidopsis, GUS expression was detected in the same spatiotemporal pattern as in C. japonica. These results suggest that the CjMALE1 promoter is subject to transcriptional regulatory systems consisting of cis- and trans-elements that have been highly conserved during evolution.

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.

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.

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.

Efficient production of genetically engineered, male-sterile Arabidopsis thaliana using anther-specific promoters and genes derived from Brassica oleracea and B. rapa.

Prevention of transgene flow from genetically modified crops to food crops and wild relatives is of concern in agricultural biotechnology. We used genes derived from food crops to produce complete male sterility as a strategy for gene confinement as well as to reduce the food purity concerns of consumers. Anther-specific promoters (A3, A6, A9, MS2, and MS5) were isolated from Brassica oleracea and B. rapa and fused to the beta-glucuronidase (GUS) reporter gene and candidate genes for male sterility, including the cysteine proteases BoCysP1 and BoCP3, and negative regulatory components of phytohormonal responses involved in male development. These constructs were then introduced into Arabidopsis thaliana. GUS analyses revealed that A3, A6, and A9 had tapetum-specific promoter activity from the anther meiocyte stage. Male sterility was confirmed in tested constructs with protease or gibberellin insensitive (gai) genes. In particular, constructs with BoCysP1 driven by the A3 or A9 promoter most efficiently produced plants with complete male sterility. The tapetum and middle layer cells of anthers expressing BoCysP1 were swollen and excessively vacuolated when observed in transverse section. This suggests that the ectopic expression of cysteine protease in the meiocyte stage may inhibit programmed cell death. The gai gene also induced male sterility, although at a low frequency. This is the first report to show that plant cysteine proteases and gai from food crops are available as a novel tool for the development of genetically engineered male-sterile plants.

Alternative transcription initiation of the nitrilase gene (BrNIT2) caused by infection with Plasmodiophora brassicae Woron. in Chinese cabbage (Brassica rapa L.).

In clubroot disease, gall formation is induced by infection with the obligate biotroph Plasmodiophora brassicae, and cell hypertrophy is dependent on increased auxin levels. The enzyme nitrilase is suggested to play an important role in auxin biosynthesis in plants. Here, we investigated the expression of nitrilase genes in clubroot disease in Chinese cabbage (Brassica rapa L.). We isolated four isogenes of nitrilase (BrNIT1, BrNIT2, BrNIT3, and BrNIT4) from Chinese cabbage. When a BrNIT2-specific probe was used for Northern blot hybridization, enhanced accumulation of a 1.4 kb mRNA and additional shorter transcripts (1.1 kb) were only detected in clubbed roots 25 days postinoculation (dpi) onward. The expression of BrNIT1 was not strongly affected by infection with P. brassicae. BrNIT3 expression was detected in the roots at 10 and 20 dpi, and the expression was less in clubbed roots than in healthy roots at 20 dpi. Analysis of the transcription initiation point of the BrNIT2 gene suggests that 1.1 kb transcripts were generated by alternative transcription initiation between the second intron and the third exon. The sequence from the second intron to half of the third exon (+415 to +1037, 623 bp) had promoter activity in Arabidopsis during clubroot formation. Therefore, our results suggest that transcriptional regulation of BrNIT2 might be involved in auxin overproduction during clubroot development.

Increase in BrAO1 gene expression and aldehyde oxidase activity during clubroot development in Chinese cabbage (Brassica rapa L.).

SUMMARY In clubroot disease, gall formation is induced by infection with the obligate biotroph Plasmodiophora brassicae due to increased levels of auxins and cytokinins. Because aldehyde oxidase (AO) may be involved in auxin biosynthesis in plants, we isolated two AO genes (BrAO1 and BrAO2) from Chinese cabbage (Brassica rapa ssp. pekinensis cv. Muso), which are the most similar to AAO1 among Arabidopsis AO genes, and examined their expressions during clubroot development. The expression of BrAO1 was enhanced in inoculated roots from 15 days post-inoculation (dpi) when visible clubroots were still undetectable. Thereafter, BrAO1 expression increased with clubroot development compared with uninoculated roots, although BrAO2 expression was repressed. In situ hybridization revealed that BrAO1 was strongly expressed in tissues that were invaded by immature plasmodia at 35 dpi, suggesting that BrAO1 expression was enhanced by the pathogen in order to establish its pathogenesis. In addition, we detected AO activity, as evidenced by the occurrence of at least six bands (BrAO-a to BrAO-f) in the roots of Chinese cabbage using an active staining method with benzaldehyde and indlole-3-aldehyde as the substrate. Coincidental with BrAO1 expression, the signals of BrAO-a and BrAO-d increased with inoculation by P. brassicae during clubroot development compared with healthy roots, resulting in an increase in total AO activity. By contrast, the band BrAO-b decreased post-inoculation, in parallel with the expression of BrAO2. The other bands of activity were not clearly influenced by the infection. Based on these results, we discuss the involvement of AO in auxin-overproduction during clubroot development in Chinese cabbage.

Interaction of tomato mosaic virus movement protein with tobacco RIO kinase.

Tomato mosaic virus (ToMV) has a regulatory gene encoding a movement protein (MP) that is involved in the cell-to-cell movement of viral RNA through plasmodesmata. To identify the host cell factors interacting with ToMV MP, we used a recombinant MP probe to isolate cDNA clones from a phage expression library of Nicotiana tabacum by a far-Western screening method. One of the cDNA clones encoded an MP-interacting protein, MIP-T7, homologous to the yeast novel protein kinase, Rio1p. We isolated a full-length cDNA by RT-PCR. The putative gene product was designated NtRIO, and shared 33 and 73% amino acid identity with yeast and Arabidopsis RIO kinases, respectively. In vitro analyses using recombinant proteins showed that NtRIO also interacted with a different MP derived from Cucumber mosaic virus. NtRIO had autophosphorylation activity and phosphorylated ToMV MP. Addition of recombinant tobacco casein kinase 2 resulted in a marked increase in the phosphorylation of NtRIO. The interaction between NtRIO and ToMV MP was inhibited by phosphorylation of NtRIO.