Elucidation of genomic organizations of transgenic soybean plants through de novo genome assembly with short paired-end reads.

Elucidation of the genomic organizations of transgene insertion sites is essential for the genetic studies of transgenic plants. Herein, we establish an analysis pipeline that identifies the transgene insertion sites as well as the presence of vector backbones, through de novo genome assembly with high-throughput sequencing data in two transgenic soybean lines, AtYUCCA6-#5 and 35S-UGT72E3/2-#7. Sequencing data of approximately 28× and 29× genome coverages for each line generated by high-throughput sequencing were de novo assembled. The databases generated from the de novo assembled sequences were used to search contigs that contained putative insertion sites and their flanking sequences (integration sites) of transgene fragments using transgenic vector sequences as queries. The predicted integration site sequences, which are located at three annotated genes that might regulate plant development or confer disease resistance, were then confirmed by local alignment against the soybean reference genome and PCR amplification. As results, we revealed the precise transgene-flanking sequences and sequence rearrangements at insertion sites in both the transgenic lines, as well as the aberrant insertion of a transgene fragment. Consequently, relative to experimental or enrichment technologies, our approach is straightforward and time-effective, providing an alternative method for the identification of insertion sites in transgenic plants.

Erratum to: Confirmation of Drought Tolerance of Ectopically Expressed AtABF3 Gene in Soybean.

[This corrects the article on p. 413 in vol. 41.].

Confirmation of Drought Tolerance of Ectopically Expressed AtABF3 Gene in Soybean.

Soybean transgenic plants with ectopically expressed AtABF3 were produced by Agrobacterium-mediated transformation and investigated the effects of AtABF3 expression on drought and salt tolerance. Stable Agrobacterium-mediated soybean transformation was carried based on the half-seed method (Paz et al. 2006). The integration of the transgene was confirmed from the genomic DNA of transformed soybean plants using PCR and the copy number of transgene was determined by Southern blotting using leaf samples from T2 seedlings. In addition to genomic integration, the expression of the transgenes was analyzed by RT-PCR and most of the transgenic lines expressed the transgenes introduced. The chosen two transgenic lines (line #2 and #9) for further experiment showed the substantial drought stress tolerance by surviving even at the end of the 20-day of drought treatment. And the positive relationship between the levels of AtABF3 gene expression and drought-tolerance was confirmed by qRT-PCR and drought tolerance test. The stronger drought tolerance of transgenic lines seemed to be resulted from physiological changes. Transgenic lines #2 and #9 showed ion leakage at a significantly lower level (P < 0.01) than non-transgenic (NT) control. In addition, the chlorophyll contents of the leaves of transgenic lines were significantly higher (P < 0.01). The results indicated that their enhanced drought tolerance was due to the prevention of cell membrane damage and maintenance of chlorophyll content. Water loss by transpiration also slowly proceeded in transgenic plants. In microscopic observation, higher stomata closure was confirmed in transgenic lines. Especially, line #9 had 56% of completely closed stomata whereas only 16% were completely open. In subsequent salt tolerance test, the apparently enhanced salt tolerance of transgenic lines was measured in ion leakage rate and chlorophyll contents. Finally, the agronomic characteristics of ectopically expressed AtABF3 transgenic plants (T2) compared to NT plants under regular watering (every 4 days) or low rate of watering condition (every 10 days) was investigated. When watered regularly, the plant height of drought-tolerant line (#9) was shorter than NT plants. However, under the drought condition, total seed weight of line #9 was significantly higher than in NT plants (P < 0.01). Moreover, the pods of NT plants showed severe withering, and most of the pods failed to set normal seeds. All the evidences in the study clearly suggested that overexpression of the AtABF3 gene conferred drought and salt tolerance in major crop soybean, especially under the growth condition of low watering.

Mitochondrial Porin Isoform AtVDAC1 Regulates the Competence of Arabidopsis thaliana to Agrobacterium-Mediated Genetic Transformation.

The efficiency of Agrobacterium-mediated transformation in plants depends on the virulence of Agrobacterium strains, the plant tissue culture conditions, and the susceptibility of host plants. Understanding the molecular interactions between Agrobacterium and host plant cells is crucial when manipulating the susceptibility of recalcitrant crop plants and protecting orchard trees from crown gall disease. It was discovered that Arabidopsis voltage-dependent anion channel 1 (atvdac1) mutant has drastic effects on Agrobacterium-mediated tumorigenesis and growth developmental phenotypes, and that these effects are dependent on a Ws-0 genetic background. Genetic complementation of Arabidopsis vdac1 mutants and yeast porin1-deficient strain with members of the AtVDAC gene family revealed that AtVDAC1 is required for Agrobacterium-mediated transformation, and there is weak functional redundancy between AtVDAC1 and AtVDAC3, which is independent of porin activity. Furthermore, atvdac1 mutants were deficient in transient and stable transformation by Agrobacterium, suggesting that AtVDAC1 is involved in the early stages of Agrobacterium infection prior to transferred-DNA (T-DNA) integration. Transgenic plants overexpressing AtVDAC1 not only complemented the phenotypes of the atvdac1 mutant, but also showed high efficiency of transient T-DNA gene expression; however, the efficiency of stable transformation was not affected. Moreover, the effect of phytohormone treatment on competence to Agrobacterium was compromised in atvdac1 mutants. These data indicate that AtVDAC1 regulates the competence of Arabidopsis to Agrobacterium infection.

Enzymatic and metabolic engineering for efficient production of syringin, sinapyl alcohol 4-O-glucoside, in Arabidopsis thaliana.

To promote efficient production of syringin, a plant-derived bioactive monolignol glucoside, synergistic effects of enzymatic and metabolic engineering were combined. Recombinant UGT72E3/E2 chimeras, generated by exchanging parts of the C-terminal domain including the Putative Secondary Plant Glycosyltransferase (PSPG) motif of UGT72E3 and UGT72E2, were expressed in leaves of transgenic Arabidopsis plants; syringin production was measured in vivo and by enzymatic assays in vitro. In both tests, UGT72E3/2 displayed substrate specificity for sinapyl alcohol like the parental enzyme UGT72E3, and the syringin production was significantly increased compared to UGT72E3. In particular, in the in vitro assay, which was performed in the presence of a high concentration of sinapyl alcohol, the production of syringin by UGT72E3/2 was 4-fold higher than by UGT72E3. Furthermore, to enhance metabolic flow through the phenylpropanoid pathway and maintain a high basal concentration of sinapyl alcohol in the leaves, UGT72E3/2 was combined with the sinapyl alcohol synthesis pathway gene F5H encoding ferulate 5-hydroxylase and the lignin biosynthesis transcriptional activator MYB58. The resulting UGT72E3/2+F5H+MYB58 OE plants, which simultaneously overexpress these three genes, accumulated a 56-fold higher level of syringin in their leaves than wild-type plants.

Fine mapping and identification of candidate rice genes associated with qSTV11(SG), a major QTL for rice stripe disease resistance.

Rice stripe disease, caused by rice stripe virus (RSV) is a serious constraint to rice production in subtropical regions of East Asia. We performed fine mapping of a RSV resistance QTL on chromosome 11, qSTV11 ( SG ), using near-isogenic lines (NILs, BC(6)F(4)) derived from a cross between the highly resistant variety, Shingwang, and the highly susceptible variety, Ilpum, using 11 insertion and deletion (InDel) markers. qSTV11 ( SG ) was localized to a 150-kb region between InDel 11 (17.86 Mbp) and InDel 5 (18.01 Mbp). Among the two markers in this region, InDel 7 is diagnostic of RSV resistance in 55 Korean japonica and indica rice varieties. InDel 7 could also distinguish the allele type of Nagdong, Shingwang, Mudgo, and Pe-bi-hun from Zenith harboring the Stv-b ( i ) allele. As a result, qSTV11 ( SG ) is likely to be the Stv-b ( i ) allele. There were 21 genes in the 150-kb region harboring the qSTV11 ( SG ) locus. Three of these genes, LOC_Os11g31430, LOC_Os11g31450, and LOC_Os11g31470, were exclusively expressed in the susceptible variety. These expression profiles were consistent with the quantitative nature along with incomplete dominance of RSV resistance. Sequencing of these genes showed that there were several amino acid substitutions between susceptible and resistant varieties. Putative functions of these candidate genes for qSTV11 (SG) are discussed.

Increased expression of OsPT1, a high-affinity phosphate transporter, enhances phosphate acquisition in rice.

Most high-affinity phosphate transporter genes (OsPTs) in rice were highly induced in roots when phosphate was depleted. OsPT1, however, was highly expressed in primary roots and leaves regardless of external phosphate concentrations. This finding was confirmed histochemically using transgenic rice plants that express the GUS reporter gene under the control of the OsPT1 promoter, which exhibited high GUS activity even in the phosphate sufficient condition. Furthermore, transgenic rice plants overexpressing the OsPT1 gene accumulated almost twice as much phosphate in the shoots as did wild-type plants. As a result, transgenic plants had more tillers than did wild-type plants, which is a typical physiological indicator for phosphate status in rice.