Oxicam-type nonsteroidal anti-inflammatory drugs enhance Agrobacterium-mediated transient transformation in plants.

Agrobacterium-mediated transformation is a key innovation for plant breeding, and routinely used in basic researches and applied biology. However, the transformation efficiency is often the limiting factor of this technique. In this study, we discovered that oxicam-type nonsteroidal anti-inflammatory drugs, including tenoxicam (TNX), increase the efficiency of Agrobacterium-mediated transient transformation. TNX treatment increased the transformation efficiency of Agrobacterium-mediated transformation of Arabidopsis thaliana mature leaves by agroinfiltration. The increase of efficiency by TNX treatment was not observed in dde2/ein2/pad4/sid2 quadruple mutant, indicating that TNX inhibits the immune system mediated by jasmonic acid, ethylene, and salicylic acid against to Agrobacterium. We also found that TNX-treatment is applicable for the transient expression and subcellular localization analysis of fluorescent-tagged proteins in Arabidopsis leaf cells. In addition, we found that TNX increases the efficiency of Agrobacterium-mediated transient transformation of Jatropha. Given that treatment with oxicam compounds is a simple and cost effective method, our findings will provide a new option to overcome limitations associated with Agrobacterium-mediated transformation of various plant species.

Protocol for Agrobacterium-mediated transformation of tall fescue and future perspective on the application of genome editing.

Tall fescue (Festuca arundinacea Schreb.) is a major cool-season perennial grass grown for forage and turf. We have obtained transgenic tall fescue by Agrobacterium-mediated transformation to improve agronomically important traits. In our protocol, we use embryogenic calli derived from not only mature seeds but also shoot tips. Although tall fescue cultivars consist of various genotypes with different genetic variation, we can produce transgenic plants at any time with calli induced from shoot tips of in vitro-maintained responsive genotypes. When the hygromycin phosphotransferase gene is used as a selectable marker, transformants are selected by incubation with 100 mg l-1 hygromycin in both selection and regeneration media. Since tall fescue is an anemophilous species, the cultivation of transgenic plants poses the risk of transgenic pollen flow. Recently, it has been reported that genome-edited plants without the integration of foreign DNA fragments can be produced by an Agrobacterium-mediated transient gene expression system. We hope that our protocol will contribute to production of transgene-free genome-edited tall fescue.

Identification of a novel major locus for gray leaf spot resistance in Italian ryegrass (Lolium multiflorum Lam.).

BACKGROUND: Gray leaf spot (GLS), caused by Magnaporthe oryzae (anamorph Pyricularia oryzae), in ryegrasses is a very serious problem. Heavily infected small seedlings die within a matter of days, and stands of the grasses are seriously damaged by the disease. Thus, the development of GLS-resistant cultivars has become a concern in ryegrass breeding. RESULTS: Phenotypic segregations in a single cross-derived F1 population of Italian ryegrass (Lolium multiflorum Lam.) indicated that the GLS resistance in the population was possibly controlled by one or two dominant genes with 66.5-77.9% of broad-sense heritability. In bulked segregant analyses, two simple sequence repeat (SSR) markers, which have so far been reported to locate on linkage group (LG) 3 of Italian ryegrass, showed specific signals in the resistant parent and resistant bulk, indicating that the resistance gene locus was possibly in the LG 3. We thus constructed a genetic linkage map of the LG 3 covering 133.6 centimorgan with other SSR markers of the LG 3 of Italian ryegrass and grass anchor probes that have previously been assigned to LG 3 of ryegrasses, and with rice expressed sequence tag (EST)-derived markers selected from a rice EST map of chromosome (Chr) 1 since LG 3 of ryegrasses are syntenic to rice Chr 1. Quantitative trait locus (QTL) analysis with the genetic linkage map and phenotypic data of the F1 population detected a major locus for GLS resistance. Proportions of phenotypic variance explained by the QTL at the highest logarithm of odds scores were 61.0-69.5%. CONCLUSIONS: A resistance locus was confirmed as novel for GLS resistance, because its genetic position was different from other known loci for GLS resistance. Broad-sense heritability and the proportion of phenotypic variance explained by the QTL were similar, suggesting that most of the genetic factors for the resistance phenotype against GLS in the F1 population can be explained by a function of the single resistance locus. We designated the putative gene for the novel resistance locus as LmPi2. LmPi2 will be useful for future development of GLS-resistant cultivars in combination with other resistance genes.

Engineering the Oryza sativa cell wall with rice NAC transcription factors regulating secondary wall formation.

Plant tissues that require structural rigidity synthesize a thick, strong secondary cell wall of lignin, cellulose and hemicelluloses in a complicated bridged structure. Master regulators of secondary wall synthesis were identified in dicots, and orthologs of these regulators have been identified in monocots, but regulation of secondary cell wall formation in monocots has not been extensively studied. Here we demonstrate that the rice transcription factors SECONDARY WALL NAC DOMAIN PROTEINs (SWNs) can regulate secondary wall formation in rice (Oryza sativa) and are potentially useful for engineering the monocot cell wall. The OsSWN1 promoter is highly active in sclerenchymatous cells of the leaf blade and less active in xylem cells. By contrast, the OsSWN2 promoter is highly active in xylem cells and less active in sclerenchymatous cells. OsSWN2 splicing variants encode two proteins; the shorter protein (OsSWN2S) has very low transcriptional activation ability, but the longer protein (OsSWN2L) and OsSWN1 have strong transcriptional activation ability. In rice, expression of an OsSWN2S chimeric repressor, driven by the OsSWN2 promoter, resulted in stunted growth and para-wilting (leaf rolling and browning under normal water conditions) due to impaired vascular vessels. The same OsSWN2S chimeric repressor, driven by the OsSWN1 promoter, caused a reduction of cell wall thickening in sclerenchymatous cells, a drooping leaf phenotype, reduced lignin and xylose contents and increased digestibility as forage. These data suggest that OsSWNs regulate secondary wall formation in rice and manipulation of OsSWNs may enable improvements in monocotyledonous crops for forage or biofuel applications.

Male-sterile and cleistogamous phenotypes in tall fescue induced by chimeric repressors of SUPERWOMAN1 and OsMADS58.

Since tall fescue (Festuca arundinacea Schreb.) is an anemophilous (wind-pollinated) grass species, male sterility is strongly desired for transgenic tall fescue to prevent pollen dispersal. To create male-sterile tall fescue, we applied Chimeric REpressor gene-Silencing Technology (CRES-T) based on rice APETALA3 (AP3) and AGAMOUS (AG) orthologues that specify the formation of stamens. We fused the coding regions of rice AP3 orthologue SUPERWOMAN1 (SPW1), and rice AG orthologues, Os12g0207000, Os01g0886200 and OsMADS58, respectively with the artificial sequence encoding the modified EAR-like motif repression domain (SRDX). We first introduced Os12g0207000SRDX, Os01g0886200SRDX and OsMADS58SRDX into rice for evaluation of their abilities to induce male sterility. The transgenic rice expressing OsMADS58SRDX had reiterated formation of lodicule-like organs instead of stamens and carpel, a typical phenotype of ag mutant. Thus, we found that OsMADS58SRDX was most suitable for our purpose. Next, we introduced SPW1SRDX and OsMADS58SRDX into tall fescue. Although the transgenic tall fescue did not have the stamen alterations seen in SPW1SRDX and OsMADS58SRDX rice, they either produced no pollen or produced immature pollen; thus, the anthers were not dehiscent and the plants were male-sterile. In addition to the male sterility, SPW1SRDX tall fescue showed a cleistogamous (closed) phenotype in which anthers were not observed outside the glumes, with thin, abnormally elongated lodicules. Some lines of OsMADS58SRDX tall fescue showed a cleistogamous phenotype in which the lodicules were homeotically transformed into lemma-like organs. In both cases, cleistogamous phenotype was associated with morphological changes to the lodicules. We also obtained a mild phenotype of OsMADS58SRDX tall fescue, which exhibited only the male sterility. In this study, we produced novel male-sterile phenotypes using chimeric repressors and thus suggest CRES-T as a tool for transgenic improvement of forage and turf grasses.

Assessing the effects of cultivating genetically modified glyphosate-tolerant varieties of soybeans (Glycine max (L.) Merr.) on populations of field arthropods.

We assessed the effects of cultivating two genetically modified (GM) glyphosate-tolerant soybean varieties (Glycine max (L.) Merr.) derived from Event 40-3-2 and a Japanese conventional variety on arthropods under field conditions, with weed control using glyphosate and conventional weed control for two years. Plant height and dry weight of the conventional variety were significantly larger than those of the GM varieties, but the GM varieties bore more pods than the conventional variety. We found arthropods of nine taxonomic orders (Araneae, Acari, Thysanoptera, Homoptera, Heteroptera, Coleoptera, Diptera, Lepidoptera, and Hymenoptera) on the plants. The arthropod incidence (number per plant unit weight pooled for each taxonomic order) on the soybean stems and leaves generally did not differ significantly between the GM and conventional varieties. However, the incidence of Thysanoptera and total incidence (all orders combined) were greater on the GM variety in the second year. The weed control regimes had no significant influence on the arthropod incidence on the soybean stems and leaves. The number of flower-inhabiting Thysanoptera (the dominant arthropod in the flowers) was not significantly different between the GM and conventional varieties. Asphondylia yushimai (Diptera, Cecidomyiidae) was more numerous on the pods of the GM variety in both years. Neither the soybean variety nor the weed control regime significantly affected the density of soil macro-organisms. However, the glyphosate weed control affected arthropods between the rows of plants by decreasing the abundances of Homoptera, Heteroptera, Coleoptera and Lepidoptera, and diversity of arthropods.

Analysis of expressed sequence tags in apomictic guineagrass (Panicum maximum).

Apomixis is an intriguing asexual mode of reproduction, because it produces maternal clones that permit vegetative reproduction through seeds. Guineagrass (Panicum maximum) has both facultative aposporous apomixis and obligate sexual modes of reproduction. Despite the importance of apomixis in guineagrass, expressed sequence tags (ESTs) for this condition have not been studied in this species. We constructed a guineagrass cDNA library from two aposporous strains, Ku5954 and GM64-3A, and utilized them as microarray probes. To find genes uniquely expressed in the immature pistils of apomicts, we performed a microarray analysis using target RNA from another apomict, OKI64. Of the 4608 probes in the microarray, only 394 showed clear gene expression in the immature pistils. Of the 394 expressed probes, 196 were successfully sequenced. Of these, 181 had significant homology with other species, including 10 ESTs with matches in a pistil cDNA library from another aposporous species, Cenchrus ciliaris. Of the remaining ESTs, three showed significant homology only with animal database sequences and the other 12 ESTs showed no homology with any previously registered sequence. In reverse-transcriptase PCR and real-time quantitative PCR, nine ESTs reliably detected ovary-specific gene expression. Of these, three revealed aposporous ovary-specific genes expressed in the early developmental stage, suggesting that these could be apomixis-related genes.

Development of simple sequence repeat (SSR) markers and construction of an SSR-based linkage map in Italian ryegrass (Lolium multiflorum Lam.).

In order to develop simple sequence repeat (SSR) markers in Italian ryegrass, we constructed a genomic library enriched for (CA)n-containing SSR repeats. A total of 1,544 clones were sequenced, of which 1,044 (67.6%) contained SSR motifs, and 395 unique clones were chosen for primer design. Three hundred and fifty-seven of these clones amplified products of the expected size in both parents of a two-way pseudo-testcross F(1) mapping population, and 260 primer pairs detected genetic polymorphism in the F(1) population. Genetic loci detected by a total of 218 primer pairs were assigned to locations on seven linkage groups, representing the seven chromosomes of the haploid Italian ryegrass karyotype. The SSR markers covered 887.8 cM of the female map and 795.8 cM of the male map. The average distance between two flanking SSR markers was 3.2 cM. The SSR markers developed in this study will be useful in cultivar discrimination, linkage analysis, and marker-assisted selection of Italian ryegrass and closely related species.

Molecular cloning and expression analysis of the replacement histone H3 gene of Italian ryegrass (Lolium multiflorum).

The replacement histone H3 gene and its 5'-flanking sequence were isolated from Italian ryegrass by polymerase chain reaction and inverse polymerase chain reaction, respectively. Expression analysis showed that this gene is constitutively expressed in the entire plant. The expression level in leaves was found to be significantly low when compared with that in other tissues. However, the gene expression level in leaves was increased by the treatment with abscisic acid and abiotic stresses such as cold, heat and high-salinity (NaCl). The motif search of the 5'-flanking sequence of the replacement histone H3 gene revealed the presence of several potential cis-acting elements that could respond to the above-mentioned abiotic stresses. In addition to defence-related elements, we also found type I and II-/III-like elements, which are highly conserved motifs in the 5'-regulatory sequence of plant histone genes that are expressed specifically during the S-phase. Experiments using transgenic Italian ryegrass plants proved that the isolated 5'-flanking sequence of the replacement histone H3 gene, which was fused to a beta-glucuronidase reporter gene, was fully functional for inducing gene expression under various abiotic stress conditions.

Increased resistance to crown rust disease in transgenic Italian ryegrass (Lolium multiflorum Lam.) expressing the rice chitinase gene.

We introduced the rice chitinase (Cht-2; RCC2) gene into calli of Italian ryegrass (Lolium multiflorum Lam.), with a hygromycin phosphotransferase (HPT) gene as a selectable marker, by particle bombardment. Hygromycin-resistant calli were selected and transferred to regeneration medium for shoot formation. Polymerase chain reaction (PCR) analysis revealed regenerants containing the HPT gene. The RCC2 gene was detected in 65.5% of those regenerants. Southern hybridization detected both HPT and RCC2 genes and indicated that the transgenic plants were independently transformed. Expression of the RCC2 gene in the transgenic plants was confirmed by Northern hybridization, reverse transcription-PCR and Western blotting. Bioassay of detached leaves indicated increased resistance to crown rust (Puccinia coronata) in transgenic plants, which exhibited higher chitinase activity than a nontransgenic plant.