ABSTRACT The ERECTA family of receptors (ERfs), encoding leucine-rich repeat receptor-like kinases (LRR-RLKs), can regulate multiple aspects of plant development and abiotic stress responses. The rice (Oryza sativa) genome contains three ERfs, namely ERECTA 1 (OsER1), ERECTA 2 (OsER2), and ERECTA-LIKE (OsERL). However, there is limited information currently available on their expression pattern during the plant developmental stages. The expression pattern of OsERfs genes at reproductive stages from the booting (R2) to milk (R6) stage in Nipponbare cultivar and the transgenic plants expressing a truncated Arabidopsis ERECTA (ER) lacking the kinase domain (∆Kinase) were studied. The ∆Kinase functions as a dominant-negative receptor and presumably disrupts ER signaling in rice. The highest level of rice ERfs (OsERfs) expression was observed at the heading (R3) and flowering (R4) stages. ∆Kinase expression induced the compensation effect and increased the expression of endogenous OsERfs genes, possibly due to the suppression of ER signaling. The response to heat stress was evaluated at the reproductive stages to test the functional effect of the upregulated OsERfs on transgenic plants. Although transgenic plants exhibited enhanced growth during the vegetative stages, they succumbed to heat stress during the grain filling stages. Overall, the ∆Kinase plants generated in this research serve as essential resources to study the role of OsERfs in the response of plants to developmental and environmental stress.

Genome targeting with CRISPR/Cas9 is a popular method for introducing mutations and creating knock-out effects. However, limited information is currently available on the mutagenesis of essential genes. This study investigated the efficiency of CRISPR/Cas9 in targeting rice essential genes: the singleton TARGET OF RAPAMYCIN (OsTOR) and the three paralogs of the Sucrose non-fermenting-1 (SNF1)-related kinase 1 (OsSnRK1α), OsSnRK1αA, OsSnRK1αB and OsSnRK1αC. Strong activity of constitutively expressed CRISPR/Cas9 was effective in creating mutations in OsTOR and OsSnRK1α genes, but inducible CRISPR/Cas9 failed to generate detectable mutations. The rate of OsTOR mutagenesis was relatively lower and only the kinase domain of OsTOR could be targeted, while mutations in the HEAT region were unrecoverable. OsSnRK1α paralogs could be targeted at higher rates; however, sterility or early senescence was observed in >50% of the primary mutants. Additionally, OsSnRK1αB and OsSnRK1αC, which bear high sequence homologies, could be targeted simultaneously to generate double-mutants. Further, although limited types of mutations were found in the surviving mutants, the recovered lines displayed loss-of-function or knockdown tor or snrk1 phenotypes. Overall, our data show that mutations in these essential genes can be created by CRISPR/Cas9 to facilitate investigations on their roles in plant development and environmental response in rice.

Overexpression of Arabidopsis dehydration response element binding 1a (DREB1a) is a well-known approach for developing salinity, cold and/or drought stress tolerance. However, understanding of the genetic mechanisms associated with DREB1a expression in rice is generally limited. In this study, DREB1a-associated early responses were investigated in a transgenic rice line harboring cold-inducible DREB1a at a gene stacked locus. Although the function of other genes in the stacked locus was not relevant to stress tolerance, this study demonstrates DREB1a can be co-localized with other genes for multigenic trait enhancement. As expected, the transgenic lines displayed improved tolerance to salinity stress and water withholding as compared with non-transgenic controls. RNA sequencing and transcriptome analysis showed upregulation of complex transcriptional networks and metabolic reprogramming as DREB1a expression led to the upregulation of multiple transcription factor gene families, suppression of photosynthesis, and induction of secondary metabolism. In addition to the detection of previously described mechanisms such as production of protective molecules, potentially novel pathways were also revealed. These include jasmonate, auxin, and ethylene signaling, induction of JAZ and WRKY regulons, trehalose synthesis, and polyamine catabolism. These genes regulate various stress responses and ensure timely attenuation of the stress signal. Furthermore, genes associated with heat stress response were downregulated in DREB1a expressing lines, suggesting antagonism between heat and dehydration stress response pathways. In summary, through a complex transcriptional network, multiple stress signaling pathways are induced by DREB1a that presumably lead to early perception and prompt response toward stress tolerance as well as attenuation of the stress signal to prevent deleterious effects of the runoff response.

Plant transformation with multiple genes is a major challenge, rendering multi-trait engineering extremely difficult in crop plants. One of the hurdles in multigene transformation is the uncontrolled integration process that leads to low quality transgenic lines that are unsuitable for practical application. Recombinase-mediated site-specific integration has been tested and validated for developing high quality transgenic lines expressing one, two, or multiple genes. Of the numerous recombinase systems tested, Cre-lox and FLP-FRT show high efficiency in plants. Recently, Cre-lox system was successfully used to stack a set of 3 constitutive, 1 heat-induced, and 1 cold-induced gene. A number of transgenic lines were obtained through a relatively small effort, and the resulting transgenic lines all expressed the genes properly as determined by their promoter-specificity. Here, a method of Cre-lox mediated stacking of a multigene construct is described using rice as a model crop.

Efficient methods for multigene transformation are important for developing novel crop varieties. Methods based on random integrations of multiple genes have been successfully used for metabolic engineering in plants. However, efficiency of co-integration and co-expression of the genes could present a bottleneck. Recombinase-mediated integration into the engineered target sites is arguably a more efficient method of targeted integration that leads to the generation of stable transgenic lines at a high rate. This method has the potential to streamline multigene transformation for metabolic engineering and trait stacking in plants. Therefore, empirical testing of transgene(s) stability from the multigene site-specific integration locus is needed. Here, the recombinase technology based on Cre-lox recombination was evaluated for developing multigenic lines harboring constitutively-expressed and inducible genes. Targeted integration of a five genes cassette in the rice genome generated a precise full-length integration of the cassette at a high rate, and the resulting multigenic lines expressed each gene reliably as defined by their promoter activity. The stable constitutive or inducible expression was faithfully transmitted to the progeny, indicating inheritance-stability of the multigene locus. Co-localization of two distinctly inducible genes by heat or cold with the strongly constitutive genes did not appear to interfere with each other's expression pattern. In summary, high rate of co-integration and co-expression of the multigene cassette installed by the recombinase technology in rice shows that this approach is appropriate for multigene transformation and introduction of co-segregating traits.SIGNIFICANCE STATEMENT:Recombinase-mediated site-specific integration approach was found to be highly efficacious in multigene transformation of rice showing proper regulation of each gene driven by constitutive or inducible promoter. This approach holds promise for streamlining gene stacking in crops and expressing complex multigenic traits.

A selectable marker free, highly expressed single copy locus flanked by insulators was created as landing pad for transgene stacking in sugarcane. These events displayed superior transgene expression compared to single-copy transgenic lines lacking insulators. Excision of the selectable marker gene from transgenic sugarcane lines was supported by FLPe/FRT site-specific recombination. Sugarcane, a tropical C4 grass in the genus Saccharum (Poaceae), accounts for nearly 80% of sugar produced worldwide and is also an important feedstock for biofuel production. Generating transgenic sugarcane with predictable and stable transgene expression is critical for crop improvement. In this study, we generated a highly expressed single copy locus as landing pad for transgene stacking. Transgenic sugarcane lines with stable integration of a single copy nptII expression cassette flanked by insulators supported higher transgene expression along with reduced line to line variation when compared to single copy events without insulators by NPTII ELISA analysis. Subsequently, the nptII selectable marker gene was efficiently excised from the sugarcane genome by the FLPe/FRT site-specific recombination system to create selectable marker free plants. This study provides valuable resources for future gene stacking using site-specific recombination or genome editing tools.

OBJECTIVES:Removal of selection marker genes from transgenic plants is highly desirable for their regulatory approval and public acceptance. This study evaluated the use of two nucleases, the yeast homing endonuclease, I-SceI, and the designed zinc finger nuclease, CCR5-ZFN, in excising marker genes from plants using rice and Arabidopsis as the models.RESULTS:In an in vitro culture assay, both nucleases were effective in precisely excising the DNA fragments marked by the nuclease target sites. However, rice cultures were found to be refractory to transformation with the I-SceI and CCR5-ZFN overexpressing constructs. The inducible I-SceI expression was also problematic in rice as the progeny of the transgenic lines expressing the heat-inducible I-SceI did not inherit the functional gene. On the other hand, heat-inducible I-SceI expression in Arabidopsis was effective in creating somatic excisions in transgenic plants but ineffective in generating heritable excisions. The inducible expression of CCR5-ZFN in rice, although transmitted stably to the progeny, appeared ineffective in creating detectable excisions. Therefore, toxicity of these nucleases in plant cells poses major bottleneck in their application in plant biotechnology, which could be avoided by expressing them transiently in cultures in vitro.

SummaryTransient expression of CRISPR/Cas9 is an effective approach for limiting its activities and improving its precision in genome editing. Here, we describe the heat‐shock‐inducible CRISPR/Cas9 for controlled genome editing, and demonstrate its efficiency in the model crop, rice. Using the soybean heat‐shock protein gene promoter and the rice U3 promoter to express Cas9 and sgRNA, respectively, we developed the heat‐shock (HS)‐inducible CRISPR/Cas9 system, and tested its efficacy in targeted mutagenesis. Two loci were targeted in rice, and the presence of targeted mutations was determined before and after the HS treatment. Only a low rate of targeted mutagenesis was detected before HS (~16%), but an increased rate of mutagenesis was observed after the HS treatment among the transgenic lines (50–63%). Analysis of regenerated plants harboring HS‐CRISPR/Cas9 revealed that targeted mutagenesis was suppressed in the plants but induced by HS, which was detectable by Sanger sequencing after a few weeks of HS treatments. Most importantly, the HS‐induced mutations were transmitted to the progeny at a high rate, generating monoallelic and biallelic mutations that independently segregated from the Cas9 gene. Additionally, off‐target mutations were either undetectable or found at a lower rate in HS‐CRISPR/Cas9 lines as compared to the constitutive‐overexpression CRISPR/Cas9 lines. Taken together, this work shows that HS‐CRISPR/Cas9 is a controlled and reasonably efficient platform for genome editing, and therefore, a promising tool for limiting genome‐wide off‐target effects and improving the precision of genome editing.

The present study investigated the efficiency of CRISPR/Cas9 in creating genomic deletions as the basis of its application in removing selection marker genes or the intergenic regions. Three loci, representing a transgene and two rice genes, were targeted at two sites each, in separate experiments, and the deletion of the defined fragments was investigated by PCR and sequencing. Genomic deletions were found at a low rate among the transformed callus lines that could be isolated, cultured, and regenerated into plants harboring the deletion. However, randomly regenerated plants showed mixed genomic effects, and generally did not harbor heritable genomic deletions. To determine whether point mutations occurred at each targeted site, a total of 114 plants consisting of primary transgenic lines and their progeny were analyzed. Ninety-three plants showed targeting, 60 of which were targeted at both sites. The presence of point mutations at both sites was correlated with the guide RNA efficiency. In summary, genomic deletions through dual-targeting by the paired-guide RNAs were generally observed in callus, while de novo point mutations at one or both sites occurred at high rates in transgenic plants and their progeny, generating a variety of insertion-deletions or single-nucleotide variations. In this study, point mutations were exceedingly favored over genomic deletions; therefore, for the recovery of plant lines harboring targeted deletions, identifying early transformed clones harboring the deletions, and isolating them for plant regeneration is recommended.

Elevating the lipid content in vegetative tissues has emerged as a new strategy for increasing energy density and biofuel yield of crops. Storage lipids in contrast to structural and signaling lipids are mainly composed of glycerol esters of fatty acids, also known as triacylglycerol (TAG). TAGs are one of the most energy-rich and abundant forms of reduced carbon available in nature. Therefore, altering the carbon-partitioning balance in favour of TAG in vegetative tissues of sugarcane, one of the highest yielding biomass crops, is expected to drastically increase energy yields. Here we report metabolic engineering to elevate TAG accumulation in vegetative tissues of sugarcane. Constitutive co-expression of WRINKLED1 (WRI1), diacylglycerol acyltransferase1-2 (DGAT1-2) and oleosin1 (OLE1) and simultaneous cosuppression of ADP-glucose pyrophosphorylase (AGPase) and a subunit of the peroxisomal ABC transporter1 (PXA1) in transgenic sugarcane elevated TAG accumulation in leaves or stems by 95- or 43-fold to 1.9% or 0.9% of dry weight (DW), respectively, while expression or suppression of one to three of the target genes increased TAG levels by 1.5- to 9.5-fold. Accumulation of TAG in vegetative progeny plants was consistent with the results from primary transgenics and contributed to a total fatty acid content of up to 4.7% or 1.7% of DW in mature leaves or stems, respectively. Lipid droplets were visible within mesophyll cells of transgenic leaves by confocal fluorescence microscopy. These results provide the basis for optimizations of TAG accumulation in sugarcane and other high yielding biomass grasses and will open new prospects for biofuel applications.

There is evidence that vaginal delivery confers respiratory benefit in preterm birth. We set out to analyze whether birthweight alters the effect of mode of delivery on preterm neonatal outcomes. We hypothesize that vaginal delivery is associated with benefit, independent of birthweight. We conducted a retrospective cohort study of preterm infants admitted to the neonatal intensive care unit between March 31, 1996 and May 31, 2014. We included neonates born at ≥ 24 and ≤ 30 6/7 weeks' gestation. We excluded the following: presence of fetal anomaly, multiple gestation, intrauterine fetal demise, operative vaginal delivery, delivery outside of labor and delivery, or breech presentation attempting vaginal delivery. Data were collected on maternal characteristics, labor characteristics, neonatal resuscitation, and short-term neonatal outcomes. Comparisons of neonatal outcomes by intended and actual mode of delivery were made using chi-square and t-tests. The cohort was then stratified by birthweight with a cutoff of 1250g. Multiple logistic regression was performed to control for confounding variables. Outcomes were analyzed for 652 births. Among infants weighing <1250g, outcomes were no different when comparing by actual or attempted mode of delivery. Among infants weighing ≥1250g, those born vaginally had shorter NICU stays (34.9± 20.2 vs 44.8± 22.2 days, p=0.03). When comparing by attempted mode of delivery, infants weighing ≥1250g who attempted a vaginal delivery had lower rates of RDS (OR 0.41; 95% CI, 0.18-0.95) and shorter NICU stays (35.1± 21.8 vs 47.2± 22.9 days, p<0.01). Among infants weighing <1250g, there is no difference when comparing outcomes by actual or attempted mode of delivery. Infants weighing ≥1250g who are born vaginally have shorter NICU stays than those born by Cesarean. When comparing by attempted mode of delivery, infants weighing ≥1250g who attempt vaginal delivery have reduced RDS and shorter NICU stays compared with those who undergo planned Cesarean.

Objective We set out to test the hypothesis that infants born vaginally at <= 30 weeks gestation have less respiratory distress syndrome (RDS) than those born by cesarean delivery.Study Design We conducted a retrospective cohort study of 652 infants born between 24 and 30(6/7) weeks gestation from March 31, 1996 to May 31, 2014. Comparisons of neonatal outcomes by intended and actual mode of delivery were made using chi-square and t-tests (alpha = 0.05). Multiple logistic regression was performed to control for confounding variables.Results Neonates born by cesarean delivery were more likely to have RDS (odds ratio [OR], 1.79; 95% confidence interval [CI], 1.10-2.90), require intubation (OR, 1.80; 95% CI, 1.12-2.88), and have longer neonatal intensive care unit stay (70.0 +/- 37.1 vs. 57.3 +/- 40.1 days, p = 0.02).Conclusion Compared with cesarean delivery, vaginal delivery is associated with a significant reduction in RDS among infants born at <= 30 weeks gestation.