Achieving High Expression of Cry in Green Tissues and Negligible Expression in Endosperm Simultaneously via rbcS Gene Fusion Strategy in Rice.

To allay excessive public concern about the safety of transgenic foods, and to optimize insect-resistant genes expression to delay the evolution of resistance in pests, we developed a promising strategy to fuse the GOI (gene of interest) with OsrbcS (rice small subunit of ribulose bisphosphate carboxylase/oxygenase) in transgenic rice, which acted as a carrier, driven by the OsrbcS native promoter to sequester its expression in green tissues. Using eYFP as a trial, we reported a high-level accumulation of eYFP in green tissue and almost none in the seed and root of the fused construct compared to the non-fused construct. After applying this fusion strategy in insect-resistant rice breeding, recombinant OsrbcS-Cry1Ab/Cry1Ac expressed rice plants conferred high resistance to leaffolders and striped stem borers, among which two single-copy lines possessed normal agronomic performance in the field. Specifically, Cry1Ab/Cry1Ac protein levels in single-copy construct transgenic lines ranged from 1.8 to 11.5 µg g-1 in the leaf, higher than the Actin I promoter-driven control, T51-1, about 1.78 µg g-1 in the leaf, but negligible (only 0.00012-0.00117 µg g-1) in endosperm by ELISA analysis. Our study provided a novel approach to creating Cry1Ab/Cry1Ac-free endosperm rice with a high level of insect-resistant protein in green tissues through the simultaneous usage of the OsrbcS promoter and OsrbcS as a fusion partner.

Moringa oleifera smoke induced positive changes in biochemical, metabolic, and antioxidant profile of rice seedling under cadmium stress.

Cadmium as a heavy metal contaminates the agricultural soil and effect plant growth due to rapid increases in industrialization and anthropogenic activities. Smoke water of Moringa oleifera was used in the current study to alleviate the effect of cadmium on the physiological, biochemical, metabolic, and antioxidant profile of Basmati 385 and Shaheen Basmati seedling. Cadmium stress of 100, 200, and 400 µM were given to 28 days-old seedlings along with smoke water (1:1,000) for one week in hydroponic culture. As a result, Cd+2 toxicity negatively affects the seedling length, fresh and dry weight, photosynthetic pigment, and electrolytes leakage, while the application of smoke water alleviated those effects. Furthermore, Cd+2 content, cell injury, metabolic parameters (proline, total soluble sugar), and antioxidants (peroxidase, catalase) were increased with increasing Cd+2 concentration while smoke water-treated seedlings showed reduction at high concentration. From present study, it can be concluded that smoke water had some regulatory compound which could reduce the Cd+2 stress level in rice seedlings and improve plant growth.

Moringa (Moringa oleifera) is a famous medicinal plant. Its fruits, roots, leaves, and flowers are used as vegetables in different part of the world. Moringa leaves are rich source of vitamin A, C riboflavin, beta carotenoid, iron, and phenolic acid and also reported for antioxidant properties. The unique aspect of current study is use to M. oliferia leaves for the preparation of smoke water, because of its nutritional and antioxidant properties and further its effects was observed on rice seedling under cadmium stress, which has not been evaluated or reported earlier.

Osj10gBTF3-Mediated Import of Chloroplast Protein Is Essential for Pollen Development in Rice.

Chloroplasts are crucial organelles for the generation of fatty acids and starch required for plant development. Nascent polypeptide-associated complex (NAC) proteins have been implicated in development as transcription factors. However, their chaperone roles in chloroplasts and their relationship with pollen development in plants remain to be elucidated. Here, we demonstrated that Osj10gBTF3, a NAC protein, regulates pollen and chloroplast development in rice by coordinating with a Hsp90 family chaperone OsHSP82 to mediate chloroplast import. Knockout of Osj10gBTF3 affects pollen and chloroplast development and significantly reduces the accumulation of fertility-related chloroplast protein OsPPR676. Both Osj10gBTF3 and OsHSP82 interact with OsPPR676. Interestingly, the interaction between OsHSP82 and OsPPR676 is only found in the cytoplasm, while the interaction between Osj10gBTF3 and OsPPR676 also occurs inside the chloroplast. The chloroplast stroma chaperone OsCpn60 can also be co-precipitated with Osj10gBTF3, but not with OsHSP82. Further investigation indicates that Osj10gBTF3 enters the chloroplast stroma possibly through the inner chloroplast membrane channel protein Tic110 and then recruits OsCpn60 for the folding or assembly of OsPPR676. Our results reveal a chaperone role of Osj10gBTF3 in chloroplast import different from Hsp90 and provide a link between chloroplast transport and pollen development in rice.

OseIF3h Regulates Plant Growth and Pollen Development at Translational Level Presumably through Interaction with OsMTA2.

The initiation stage of protein biosynthesis is a sophisticated process tightly regulated by numerous initiation factors and their associated components. However, the mechanism underlying translation initiation has not been completely understood in rice. Here, we showed knock-out mutation of the rice eukaryotic translation initiation factor 3 subunit h (OseIF3h) resulted in plant growth retardation and seed-setting rate reduction as compared to the wild type. Further investigation demonstrated an interaction between OseIF3h and OsMTA2 (mRNA adenosine methylase 2), a rice homolog of METTL3 (methyltransferase-like 3) in mammals, which provided new insight into how N6-methyladenosine (m6A) modification of messenger RNA (mRNA) is engaged in the translation initiation process in monocot species. Moreover, the RIP-seq (RNA immunoprecipitation sequencing) data suggested that OseIF3h was involved in multiple biological processes, including photosynthesis, cellular metabolic process, precursor metabolites, and energy generation. Therefore, we infer that OseIF3h interacts with OsMTA2 to target a particular subset of genes at translational level, regulating plant growth and pollen development.

OsPPR939, a nad5 splicing factor, is essential for plant growth and pollen development in rice.

KEY MESSAGE: P-subfamily PPR protein OsPPR939, which can be phosphorylated by OsS6K1, regulates plant growth and pollen development by involving in the splicing of mitochondrial nad5 introns 1, 2, and 3. In land plants, pentatricopeptide repeat (PPR) proteins play key roles in mitochondrial group II intron splicing, but how these nucleus-encoded proteins are imported into mitochondria is unknown. To date, a few PPR proteins have been characterized in rice (Oryza sativa). Here, we demonstrate that the mitochondrion-localized P-subfamily PPR protein OsPPR939 is required for the splicing of nad5 introns 1, 2, and 3 in rice. Complete knockout or partial disruption of OsPPR939 function resulted in different degrees of growth retardation and pollen sterility. The dramatically reduced splicing efficiency of these introns in osppr939-4 and osppr939-5 led to reduced mitochondrial complex I abundance and activity and enhanced expression of alternative respiratory pathway genes. Complementation with OsPPR939 rescued the defective plant morphology of osppr939-4 and restored its decreased splicing efficiency of nad5 introns 1, 2, and 3. Therefore, OsPPR939 plays crucial roles in plant growth and pollen development by splicing mitochondrial nad5 introns 1, 2, and 3. More importantly, the 12th amino acid Ser in the N-terminal targeting sequence of OsPPR939 is phosphorylated by OsS6K1, and truncated OsPPR939 with a non-phosphorylatable S12A mutation in its presequence could not be imported into mitochondria, suggesting that phosphorylation of this amino acid plays an important role in the mitochondrial import of OsPPR939. To our knowledge, the 12th residue Ser on OsPPR939 is the first experimentally proven phosphorylation site in PPR proteins. Our results provide a basis for investigating the regulatory mechanism of PPR proteins at the post-translational level.

Detection and Analysis of C-to-U RNA Editing in Rice Mitochondria-Encoded ORFs.

Cytidine to uridine (C-to-U) RNA editing is an important type of substitutional RNA modification and is almost omnipresent in plant chloroplasts and mitochondria. In rice mitochondria, 491 C-to-U editing sites have been identified previously, and case studies have elucidated the function of several C-to-U editing sites in rice, but the functional consequence of most C-to-U alterations needs to be investigated further. Here, by means of Sanger sequencing and publicly available RNA-seq data, we identified a total of 569 C-to-U editing sites in rice mitochondria-encoded open reading frames (ORFs), 85.41% of these editing sites were observed on the first or the second base of a codon, resulting in the alteration of encoded amino acid. Moreover, we found some novel editing sites and several inaccurately annotated sites which may be functionally important, based on the highly conserved amino acids encoded by these edited codons. Finally, we annotated all 569 C-to-U RNA editing sites in their biological context. More precise information about C-to-U editing sites in rice mitochondria-encoded ORFs will facilitate our investigation on the function of C-to-U editing events in rice and also provide a valid benchmark from rice for the analysis of mitochondria C-to-U editing in other plant species.

Application of Cre-lox gene switch to limit the Cry expression in rice green tissues.

The presence of genetically modified (GM) protein in the endosperm is important information for the public when considering the biological safety of transgenic rice. To limit the expression of GM proteins to rice green tissues, we developed a modified Cre-lox gene switch using two cassettes named KEY and LOCK. KEY contains a nuclear-localized Cre recombinase driven by the green-tissue-specific promoter rbcS. LOCK contains a Nos terminator (NosT), which is used to block the expression of the gene of interest (GOI), bounded by two loxP sites. When KEY and LOCK are pyramided into hybrid rice, a complete gene switch system is formed. The Cre recombinase from KEY excises loxP-NosT in LOCK and unlocks the GOI in green tissues but keeps it locked in the endosperm. This regulatory effect was demonstrated by eYFP and Bt expression assays. The presence of eYFP and Cre were confirmed in the leaf, sheath, stem, and glume but not in the root, anther or seed of the gene-switch-controlled eYFP hybrids. Meanwhile, gene switch-controlled Bt hybrid rice not only confined the expression of Bt protein to the green tissues but also showed high resistance to striped stem borers and leaffolders.

A Plastid-Localized Pentatricopeptide Repeat Protein is Required for Both Pollen Development and Plant Growth in Rice.

Several mitochondrial-targeted pentatricopeptide repeat (PPR) proteins involved in pollen development have been reported to be fertility restorer (Rf) proteins. However, the roles of plastid-localized PPR proteins in plant male reproduction are poorly defined. Here, we described a plastid-localized PPR-SMR protein, OsPPR676, which is required for plant growth and pollen development in rice. In this study, OsPPR676 was confirmed to be an interacted protein with Osj10gBTF3, ß-subunit of nascent polypeptide-associated complex (ß-NAC), by bimolecular fluorescence complementation assays, indicating that both proteins are probably involved in the same regulatory pathway of pollen development. Compared with other chloroplast-rich tissues, OsPPR676 was only weakly expressed in anther, but in the Mei and YM stages of pollen development, its expression was relatively strong in the tapetum. Disruption of OsPPR676 resulted in growth retardation of plants and partial sterility of pollens. Phenotypic analysis of different osppr676 mutant lines implied that the SMR domain was not essential for the function of OsPPR676. We further demonstrated that OsPPR676 is essential for production of plastid atpB subunit, and then plays crucial roles in biosynthesis of fatty acids, carbohydrates, and other organic matters via affecting activity of ATP synthase.

A rapid and efficient method to study the function of crop plant transporters in Arabidopsis.

Iron (Fe) is an essential micronutrient for humans. Fe deficiency disease is widespread and has led to extensive studies on the mechanisms of Fe uptake and storage, especially in staple food crops such as rice. However, studies of functionally related genes in rice and other crops are often time and space demanding. Here, we demonstrate that transgenic Arabidopsis suspension culture cells and Arabidopsis plants can be used as an efficient expression system for gain-of-function study of selected transporters, using Fe transporters as a proof-of-principle. The vacuolar membrane transporters OsVIT1 and OsVIT2 have been described to be important for iron sequestration, and disruption of these two genes leads to Fe accumulation in rice seeds. In this study, we have taken advantage of the fluorescent-tagged protein GFP-OsVIT1, which functionally complements the Fe hypersensitivity of ccc1 yeast mutant, to generate transgenic Arabidopsis suspension cell lines and plants. GFP-OsVIT1 was shown to localize on the vacuolar membrane using confocal microscopy and immunogold EM. More importantly, the Fe concentration, as well as the concentration of Zn, in the transgenic cell lines and plants were significantly increased compared to that in the WT. Taken together, our study shows that the heterologous expression of rice vacuolar membrane transporter OsVIT1 in Arabidopsis system is functional and effectively enhances iron accumulation, indicating an useful approach for studying other putative transporters of crop plants in this system.

Pyramiding and evaluation of both a foreign Bacillus thuringiensis and a Lysine-rich protein gene in the elite indica rice 9311.

Gene pyramiding is an efficient approach for the genetic improvement of multiple agronomic traits simultaneously. In this study, we pyramided two foreign genes, cry1Ac driven by the rice Actin I promoter, and lysine-rich protein (LRP), driven by the endosperm-specific GLUTELIN1 (GT1) promoter, into the elite indica cultivar 9311. These two genes were chosen in an attempt to enhance insect-resistance and Lysine (Lys) content. In the pyramided line, the foreign gene cry1Ac was efficiently expressed in the leaves and stems, and exhibited highly efficient resistance to striped stem borer (SSB, Chilo suppressalis Walker) in the laboratory and rice leaf folder (RLF, Cnaphalocrocis medinalis Guenee) in the field. Furthermore, the LRP gene was highly expressed in the endosperm and produced a remarkable increase of Lys content in the seeds of the pyramided line. The data from field trials demonstrated that most of the agronomic traits including yield were well maintained in the pyramided line compared to the parental control. These results strongly suggest that the foreign cry1Ac and LRP genes have remarkable application potential in rice, and the resultant pyramided line serves as an ideal bridge material for the improvement of insect-resistance and high Lys rice in the future.

The Rice Eukaryotic Translation Initiation Factor 3 Subunit e (OseIF3e) Influences Organ Size and Pollen Maturation.

Eukaryotic translation initiation factor 3 (eIF3) is a large protein complex that participates in most translation initiation processes. While eIF3 has been well characterized, less is known about the roles of individual eIF3 subunits, particularly in plants. Here, we identified and characterized OseIF3e in rice (Oryza sativa L.). OseIF3e was constitutively expressed in various tissues, but most strongly in vigorously growing organs. Transgenic OseIF3e-silenced rice plants showed inhibited growth in seedling and vegetative stages. Repression of OseIF3e led to defects in pollen maturation but did not affect pollen mitosis. In rice, eIF3e interacted with eIF3 subunits b, d, e, f, h, and k, and with eIF6, forming homo- and heterodimers to initiate translation. Furthermore, OseIF3e was shown by yeast two-hybrid assay to specifically bind to inhibitors of cyclin-dependent kinases 1, 5, and 6. This interaction was mediated by the sequence of amino acid residues at positions 118-138, which included a conserved motif (IGPEQIETLYQFAKF). These results suggested although OseIF3e is not a "functional core" subunit of eIF3, it still plays crucial roles in rice growth and development, in combination with other factors. We proposed a pathway by which OseIF3e influence organ size and pollen maturation in rice, providing an opportunity to optimize plant architecture for crop breeding.

Development of high-lysine rice via endosperm-specific expression of a foreign LYSINE RICH PROTEIN gene.

BACKGROUND: Lysine (Lys) is considered to be the first limiting essential amino acid in rice. Although there have been extensive efforts to improve the Lys content of rice through traditional breeding and genetic engineering, no satisfactory products have been achieved to date. RESULTS: We expressed a LYSINE-RICH PROTEIN gene (LRP) from Psophocarpus tetragonolobus (L.) DC using an endosperm-specific GLUTELIN1 promoter (GT1) in Peiai64S (PA64S), an elite photoperiod-thermo sensitive male sterility (PTSMS) line. The expression of the foreign LRP protein was confirmed by Western blot analysis. The Lys level in the transgenic rice seeds increased more than 30 %, the total amount of other amino acids also increased compared to wild-type. Persistent investigation of amino acids in 3 generations showed that the Lys content was significantly increased in seeds of transgenic rice. Furthermore, Lys content in the hybrid of the transgenic plants also had an approximate 20 % increase compared to hybrid control. At the grain-filling stage, we monitored the transcript abundance of many genes encoding key enzymes involved in amino acid metabolism, and the results suggested that reduced amino acid catabolism led to the accumulation of amino acids in the transgenic plants. The genetically engineered rice showed unfavorable grain phenotypes compared to wild-type, however, its hybrid displayed little negative effects on grain. CONCLUSIONS: Endosperm-specific expression of foreign LRP significantly increased the Lys content in the seeds of transgenic plant, and the the Lys increase was stably heritable with 3 generation investigation. The hybrid of the transgenic plants also showed significant increases of Lys content in the seeds. These results indicated that expression of LRP in rice seeds may have promising applications in improving Lys levels in rice.

Both overexpression and suppression of an Oryza sativa NB-LRR-like gene OsLSR result in autoactivation of immune response and thiamine accumulation.

Tight and accurate regulation of immunity and thiamine biosynthesis is critical for proper defence mechanisms and several primary metabolic cycles in plants. Although thiamine is known to enhance plant defence by priming, the mechanism by which thiamine biosynthesis responds to immune signals remains poorly understood. Here we identified a novel rice (Oryza sativa L.) NB-LRR gene via an insertion mutation, this mutant confesses a low seed setting phenotype and the corresponding genetic locus was named OsLSR (Low seed setting related). Comparing with wildtype plant, both overexpression and suppression of OsLSR lead to the autoactivation of the rice immune system and accumulation of thiamine, which result in a great fitness cost and yield penalty. Moreover, when fused with eGFP at their C terminus, two fragments, OsLSR1-178 and OsLSR464-546, localized to chloroplasts where thiamine is produced. Our result suggests that OsLSR differs from traditional NB-LRR genes. Its expression is closely related to the immune status and thiamine level in plant cells and should be maintained within a narrow range for rice growth.

The RING Finger Ubiquitin E3 Ligase OsHTAS Enhances Heat Tolerance by Promoting H2O2-Induced Stomatal Closure in Rice.

Heat stress often results in the generation of reactive oxygen species, such as hydrogen peroxide, which plays a vital role as a secondary messenger in the process of abscisic acid (ABA)-mediated stomatal closure. Here, we characterized the rice (Oryza sativa) HEAT TOLERANCE AT SEEDLING STAGE (OsHTAS) gene, which plays a positive role in heat tolerance at the seedling stage. OsHTAS encodes a ubiquitin ligase localized to the nucleus and cytoplasm. OsHTAS expression was detected in all tissues surveyed and peaked in leaf blade, in which the expression was concentrated in mesophyll cells. OsHTAS was responsive to multiple stresses and was strongly induced by exogenous ABA. In yeast two-hybrid assays, OsHTAS interacted with components of the ubiquitin/26S proteasome system and an isoform of rice ascorbate peroxidase. OsHTAS modulated hydrogen peroxide accumulation in shoots, altered the stomatal aperture status of rice leaves, and promoted ABA biosynthesis. The results suggested that the RING finger ubiquitin E3 ligase OsHTAS functions in leaf blade to enhance heat tolerance through modulation of hydrogen peroxide-induced stomatal closure and is involved in both ABA-dependent and DROUGHT AND SALT TOLERANCE-mediated pathways.

Exploring the roles of basal transcription factor 3 in eukaryotic growth and development.

Basal transcription factor 3 (BTF3) has been reported to play a significant part in the transcriptional regulation linking with eukaryotes growth and development. Alteration in the BTF3 gene expression patterns or variation in their activities adds to the explanation of different signaling pathways and regulatory networks. Moreover, BTF3s often respond to numerous stresses, and subsequently they are involved in regulation of various mechanisms. BTF3 proteins also function through protein-protein contact, which can assist us to identify the multifaceted processes of signaling and transcriptional regulation controlled by BTF3 proteins. In this review, we discuss current advances made in starting to explore the roles of BTF3 transcription factors in eukaryotes especially in plant growth and development.

Development of photoperiod- and thermo-sensitive male sterility rice expressing transgene Bacillus thuringiensis.

Stem borers and leaffolders are the main pests that cause severe damage in rice (Oryza sativa L.) production worldwide. We developed the first photoperiod- and thermo-sensitive male sterility (PTSMS) rice 208S with the cry1Ab/1Ac Bacillus thuringiensis (Bt) gene, through sexual crossing with Huahui 1 (elite line with the cry1Ab/1Ac gene). The novel 208S and its hybrids presented high and stable resistance to stem borers and leaffolders, and the content of Cry1Ab/1Ac protein in chlorophyllous tissues achieved the identical level as donor and showed little accumulation in non-chlorophyllous tissue. No dominant dosage effect in the Bt gene was observed in 208S and its derived hybrids. An analysis of fertility transition traits indicated that 208S was completely sterile under long day length/high temperature, but partially fertile under short day length/low temperature. With fine grain quality and favorable combining ability, 208S had no observed negative effects on fertility and agronomic traits from Bt (cry1Ab/1Ac). Additionally, 208S as a male sterile line showed no fertility decrease caused by Bt transgenic process, as it is the case in Huahui 1. Thus, 208S has great application value in two-line hybrid production for insect resistance, and can also be used as a bridge material in rice Bt transgenic breeding.

Gene editing by co-transformation of TALEN and chimeric RNA/DNA oligonucleotides on the rice OsEPSPS gene and the inheritance of mutations.

Although several site-specific nucleases (SSNs), such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas, have emerged as powerful tools for targeted gene editing in many organisms, to date, gene targeting (GT) in plants remains a formidable challenge. In the present study, we attempted to substitute a single base in situ on the rice OsEPSPS gene by co-transformation of TALEN with chimeric RNA/DNA oligonucleotides (COs), including different strand composition such as RNA/DNA (C1) or DNA/RNA (C2) but contained the same target base to be substituted. In contrast to zero GT event obtained by the co-transformation of TALEN with homologous recombination plasmid (HRP), we obtained one mutant showing target base substitution although accompanied by undesired deletion of 12 bases downstream the target site from the co-transformation of TALEN and C1. In addition to this typical event, we also obtained 16 mutants with different length of base deletions around the target site among 105 calli lines derived from transformation of TALEN alone (4/19) as well as co-transformation of TELAN with either HRP (5/30) or C1 (2/25) or C2 (5/31). Further analysis demonstrated that the homozygous gene-edited mutants without foreign gene insertion could be obtained in one generation. The induced mutations in transgenic generation were also capable to pass to the next generation stably. However, the genotypes of mutants did not segregate normally in T1 population, probably due to lethal mutations. Phenotypic assessments in T1 generation showed that the heterozygous plants with either one or three bases deletion on target sequence, called d1 and d3, were more sensitive to glyphosate and the heterozygous d1 plants had significantly lower seed-setting rate than wild-type.

Assembling long heteroduplexes by asymmetric polymerase chain reaction and annealing the resulting single-stranded DNAs.

We developed an effective protocol for generating high-purity heteroduplexes via annealing single-stranded DNAs (ssDNAs) derived from plasmid DNA by asymmetric polymerase chain reaction (A-PCR). With the addition of dimethyl sulfoxide, a one-step A-PCR procedure can generate ssDNAs stably at a range of reaction temperatures. Several annealing buffers can anneal two ssDNAs into heteroduplexes effectively. We further developed a simple strategy to create d(GATC) hemimethylated heteroduplexes by annealing fully methylated homoduplexes in the presence of excessive unmethylated ssDNAs. The constructed heteroduplexes have been well tested as substrates for mismatch repair in Escherichia coli and, thus, can be used in various biotechnology applications.

Efficient generation of marker-free transgenic rice plants using an improved transposon-mediated transgene reintegration strategy.

Marker-free transgenic plants can be developed through transposon-mediated transgene reintegration, which allows intact transgene insertion with defined boundaries and requires only a few primary transformants. In this study, we improved the selection strategy and validated that the maize (Zea mays) Activator/Dissociation (Ds) transposable element can be routinely used to generate marker-free transgenic plants. A Ds-based gene of interest was linked to green fluorescent protein in transfer DNA (T-DNA), and a green fluorescent protein-aided counterselection against T-DNA was used together with polymerase chain reaction (PCR)-based positive selection for the gene of interest to screen marker-free progeny. To test the efficacy of this strategy, we cloned the Bacillus thuringiensis (Bt) δ-endotoxin gene into the Ds elements and transformed transposon vectors into rice (Oryza sativa) cultivars via Agrobacterium tumefaciens. PCR assays of the transposon empty donor site exhibited transposition in somatic cells in 60.5% to 100% of the rice transformants. Marker-free (T-DNA-free) transgenic rice plants derived from unlinked germinal transposition were obtained from the T1 generation of 26.1% of the primary transformants. Individual marker-free transgenic rice lines were subjected to thermal asymmetric interlaced-PCR to determine Ds(Bt) reintegration positions, reverse transcription-PCR and enzyme-linked immunosorbent assay to detect Bt expression levels, and bioassays to confirm resistance against the striped stem borer Chilo suppressalis. Overall, we efficiently generated marker-free transgenic plants with optimized transgene insertion and expression. The transposon-mediated marker-free platform established in this study can be used in rice and possibly in other important crops.