Tackmin Kwon

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.

Large-scale screening of rice accessions to evaluate resistance to bakanae disease

Bakanae disease is an important fungal disease in the world. No rice (Oryza sativa L.) varieties have been found to be completely resistant to this disease. To facilitate accurate, uniform and simultaneous screening of many rice accessions, we developed an inoculation method for microconidia of Fusarium fujikuroi using a tissue embedding cassette and seedling tray. Standards for evaluating the inoculated rice seedlings as healthy or unhealthy were also established. The method was fast and reproducible for accurately evaluating resistance to bakanae disease in rice.

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.

A double-stranded RNA binding protein, HYL1, regulates plant immunity via the jasmonic acid pathway

MicroRNA (miRNA) and small interference RNA (siRNA) regulate not only plant growth and development, but also various responses to stress, including biotic stress, through post-transcriptional gene regulation. Hyponastic leaves 1 (HYL1), a double-stranded RNA binding protein, plays an important role in miRNA and siRNA processing, along with Dicer-like 1 (DCL1) and Hua Enhancer 1 (HEN1). Of five double-stranded RNA binding protein (DRB) deficient mutants in Arabidopsis, only the hyl1 mutant compromised innate immune responses to Pseudomonas syringae pv. tomato (Pst) DC3000 hrcC− infection when compared to wild-type plants. This was similar to the response of the hen1 mutant. Furthermore, the hyl1 mutant was susceptible to Pst (avrRpt2) infections, and showed a delay in the hypersensitive response that should result from gene-for-gene interactions between RPS2 and AvrRpt2. These results suggest that HYL1 is a major contributor in the DRB family, influencing miRNA and siRNA biogenesis and therefore playing essential roles in PAMP- and effect-triggered immune responses in plants. DNA microarray analysis revealed that the expression of genes involved in jasmonic acid (JA) biosynthesis, signal transduction, and downstream responses was significantly upregulated in the hyl1 mutant compared with wild-type plants. Consistent with this, transgenic plants overexpressing HYL1 are hypersensitive to the necrotrophic pathogen Botrytis cinerea, of which resistance is mainly regulated by JA. These results elucidate the importance of small RNA-mediated JA regulation in plant immunity.

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.

Generation of Transgenic Rice without Antibiotic Selection Marker through Agrobacterium-mediated Co-transformation System

Development of transgenic plant increasing crop yield or disease resistance is good way to solve the world food shortage. However, the persistence of marker genes in crops leads to serious public concerns about the safety of transgenic crops. In the present paper, we developed marker-free transgenic rice inserted high molecular-weight glutenin subunit (HMW-GS) gene (Dx5) from the Korean wheat cultivar ‘Jokyeong’ using Agrobacterium-mediated co-transformation method. Two expression cassettes comprised of separate DNA fragments containing only the Dx5 and hygromycin resistance (HPTII) genes were introduced separately into Agrobacterium tumefaciens EHA105 strain for co-infection. Each EHA105 strain harboring Dx5 or HPTII was infected into rice calli at a 3: 1 ratio of EHA105 with Dx5 gene and EHA105 with HPTII gene expressing cassette. Then, among 66 hygromycin-resistant transformants, we obtained two transgenic lines inserted with both the Dx5 and HPTII genes into the rice genome. We reconfirmed integration of the Dx5 and HPTII genes into the rice genome by Southern blot analysis. Wheat Dx5 transcripts in T1 rice seeds were examined with semi-quantitative RT-PCR. Finally, the marker-free plants containing only the Dx5 gene were successfully screened at the T1 generation. These results show that a co-infection system with two expression cassettes could be an efficient strategy to generate marker-free transgenic rice plants.

Phosphate uptake and growth characteristics of transgenic rice with phosphate transporter 1 (OsPT1 ) gene overexpression under high phosphate soils

Farmers have used phosphate fertilizer to provide sufficient yields. However, overuse of phosphorus accumulate in soil and causes soil and water pollution. We evaluated the phosphate acquisition and growth characteristics of OsPT1 transgenic rice (OsPT1-OX, over-expressing the high affinity phosphate transporter 1) in high phosphate soils with different level of nitrogen fertilizer treatment to investigate its removal ability of excessive phosphate from soil. OsPT1-OX had shorter culm length but more tillers than those of wild-type plants in each soil conditions. Phosphate content per dry weight of OsPT1-OX was 1.8 times higher than that of wild-type under control fertilizer treated conditions. Although the dry weight of OsPT1-OX was not different from that of wild-type plants, whole plant phosphate content was 1.7 times higher than that of wild-type plants under control fertilizer conditions. Tiller number and phosphate content per dry weight of wild-type plants increased following high levels of phosphate application, but did not change following additional nitrogen application. Tiller number and phosphate content per dry weight of OsPT1-OX did not also change under the high phosphate condition, but increased following nitrogen application under similar conditions. Whole plant phosphate content was also highest under high nitrogen and high phosphate application conditions. These results suggest that OsPT1-OX may reduce phosphate content in soils containing excess phosphate and may be further effective under high nitrogen condition. Key words : Phosphate content, fertilizer treatment, phosphate transporter, rice, soil.

Functional Analysis of the High Affinity Phosphate Transporter Genes Derived from Oryza sativa in Arabidopsis thaliana.

Phosphate, a favorable phosphorous form for plant, is one of major nutrient elements for growth and development in plants. Plants exhibit various physiological and biochemical responses in reaction to phosphate starvation in order to maintain phosphate homeostasis. Of them, expression of high affinity phosphate transporter gene family and efficient uptake of phosphate via them is a major physiological process for adaption to phosphate deficient environment. Although the various genetic resources of high affinity phosphate transporter are identified recently, little is known about their functions in plant that is prerequisite information before applying to crop plants to generate valuable transgenic plants. We demonstrated that Arabidopsis transgenic plants over-expressing two different high affinity phosphate transporter gens, OsPT1 and OsPT7, derived from rice, exhibit better growth responses compared with wild-type under phosphate starvation condition. Specially, OsPT7 gene has proven to be more effective to generate Arabidopsis transgenic plant tolerant to phosphate deficiency than OsPT1. Furthermore, the expression level of AtPT1 gene that is one of reporter genes specifically induced by phosphate starvation was significantly low compared with wild-type during phosphate starvation. Taken together, these results collectively suggest that over expression of OsPTl and OsPT7 genes derived from monocotyledonous plant function efficiently in the dicotyledonous plant, relieving stress response caused by phosphate starvation and leading to better growth rate.

AtERF11 is a positive regulator for disease resistance against a bacterial pathogen, Pseudomonas syringae, in Arabidopsis thaliana

AvrRpt2 protein triggers hypersensitive response (HR) and strong disease resistance when it is translocated from a bacterial pathogen Pseudomonas sp. to host plant cells containing a cognate RPS2 resistance protein through Type III Secretion System (TTSS). However, AvrRpt2 protein can function as the effector that suppresses a basal defense and enhances the disease symptom when functional RPS2 resistance protein is absent in the infected plant cells. Using Affymetrix Arabidopsis DNA chip, we found that many genes were specifically regulated by AvrRpt2 protein in the rps2 Arabidopsis mutant. Here, we showed that expression of AtERF11 that is known as a member of B1a subcluster of AP2/ERF transcription factor family was down regulated specifically by AvrRpt2. To determine its function in plant resistance, we also generated the Arabidopsis thaliana transgenic plants constitutively overexpressing AtERF11 under CaMV 355 promoter, which conferred an enhanced resistance against a bacterial pathogen, Pseudomonas syringae pv. tomato DC3000. Thus, these results collectively suggest that AtERF11 plays a role as a positive regulator for disease resistance against biotrophic bacterial pathogen in plant.