Oomycetes are known to secrete a vast arsenal of effectors that modulate the host defense system ... more Oomycetes are known to secrete a vast arsenal of effectors that modulate the host defense system as well as facilitate establishing a parasitic infection in plants. In recent years, tremendous progress has been made in the field of effectromics based on studies of oomycetes, especially the cytoplasmic family of RXLR effectors. Yet, the biology of the RXLR effector family is still poorly understood. There has been a consensus regarding the structure of the RXLR motif in the mycologist community. However, the function of the RXLR motif is still unclear. First, different models have suggested that the role of the RXLR motif is either in translocation to a target destination inside a host cell or in the cleavage of itself followed by secretion. Second, recent studies have suggested different functional models for the RXLR motif. According to a widely accepted model, the RXLR motif is directly involved in the translocation of effectors to target sites. In contrast, a new study has propos...
Targeted genome editing using the CRISPR/Cas9 system is a groundbreaking technology that is being... more Targeted genome editing using the CRISPR/Cas9 system is a groundbreaking technology that is being widely used to produce plants with useful traits. However, for woody plants, only a few successful attempts have been reported. These successes have used Agrobacterium-mediated transformation, which has been reported to be very efficient at producing genetically modified trees. Nonetheless, there are unresolved problems with plasmid sequences that remain in the plant genome. In this study, we demonstrated a DNA-free genome editing technique in which purified CRISPR/Cas9 ribonucleoproteins (RNPs) are delivered directly to the protoplasts of a hybrid poplar (Populus alba × Populus glandulosa). We designed three single-guide RNAs (sgRNAs) to target the stress-associated protein 1 gene (PagSAP1) in the hybrid poplar. Deep sequencing results showed that pre-assembled RNPs had a more efficient target mutagenesis insertion and deletion (indel) frequency than did non-assembled RNPs. Moreover, the RNP of sgRNA3 had a significantly higher editing efficacy than those of sgRNA1 and sgRNA2. Our results suggest that the CRISPR/Cas9 ribonucleoproteinmediated transfection approach is useful for the production of transgene-free genome-edited tree plants.
Caffeoyl shikimate esterase (CSE) has been shown to play an important role in lignin biosynthesis... more Caffeoyl shikimate esterase (CSE) has been shown to play an important role in lignin biosynthesis in plants and is, therefore, a promising target for generating improved lignocellulosic biomass crops for sustainable biofuel production. Populus spp. has two CSE genes (CSE1 and CSE2) and, thus, the hybrid poplar (Populus alba × P. glandulosa) investigated in this study has four CSE genes. Here, we present transgenic hybrid poplars with knockouts of each CSE gene achieved by CRISPR/Cas9. To knockout the CSE genes of the hybrid poplar, we designed three single guide RNAs (sg1–sg3), and produced three different transgenic poplars with either CSE1 (CSE1-sg2), CSE2 (CSE2-sg3), or both genes (CSE1/2-sg1) mutated. CSE1-sg2 and CSE2-sg3 poplars showed up to 29.1% reduction in lignin deposition with irregularly shaped xylem vessels. However, CSE1-sg2 and CSE2-sg3 poplars were morphologically indistinguishable from WT and showed no significant differences in growth in a long-term living modifie...
Horticulture, Environment, and Biotechnology, 2020
Grape ripe rot is a fungal disease, which is almost inextinguishable particularly before and afte... more Grape ripe rot is a fungal disease, which is almost inextinguishable particularly before and after harvest in most vineyards. The management of this disease largely depends on the breeding of resistant varieties. Thus, the search for resistant varieties should parallel the molecular analysis of the markers associated with disease resistance. Here, we tested the leaf inoculation assay of Colletotrichum acutatum and C. gloeosporioides by inoculating them into 350 grapevine varieties. As a result of the phenotypes common to both species, 119 varieties (34%) were classified as resistant. Consequently, we discovered the locus with nucleotide-binding sites and carboxyterminal leucine-rich repeat regions (NBS-LRR) associated with grape ripe rot resistance through genotyping-by-sequencing and genome-wide association study. Ultimately, we identified the Vitis vinifera Colletotrichum resistance protein (VvCRP) markers from chromosome 3, which belongs to the CC-NBS-LRR type, as disease-resistant proteins. These markers were verified as the melting peak, and their chromatograms of nucleotide sequences were visualized in each single nucleotide polymorphism (SNP) loci. All the SNP markers differentiated successfully the resistant from the susceptible cultivars. Interestingly, we also found that a resistant cultivar named Bailey Alicante A has a heterozygous genotype, substantiating its potential usefulness for crops such as grapevine with many hybrids. Altogether, our results indicate that VvCRP markers can be utilized to distinguish grape ripe rot-resistant cultivars, particularly in crossbreeding populations.
We previously isolated Nicotiana benthamiana matrix metalloprotease 1 (NMMP1) from tobacco leaves... more We previously isolated Nicotiana benthamiana matrix metalloprotease 1 (NMMP1) from tobacco leaves. The NMMP1 gene encodes a highly conserved, Zn-containing catalytic protease domain that functions as a factor in the plant's defense against bacterial pathogens. Expression of NMMP1 was strongly induced during interactions between tobacco and one of its pathogens, Phytophthora infestans. To elucidate the role of the NMMP1 in defense of N. benthamiana against fungal pathogens, we performed gain-of-function and loss-of-function studies. NMMP1-overexpressing plants had stronger resistance responses against P. infestans infections than control plants, while silencing of NMMP1 resulted in greater susceptibility of the plants to the pathogen. This greater susceptibility correlated with fewer NMMP1 transcripts than the non-silenced control. We also examined cell death as a measure of disease. The amount of cell death induced by the necrosis-inducing P. infestans protein 1, PiNPP1, was dependent on NMMP1 in N. benthamiana. Potato plants overexpressing NMMP1 also had enhanced disease resistance against P. infestans. RT-PCR analysis of these transgenic potato plants revealed constitutive upregulation of the potato defense gene NbPR5. NMMP1-overexpressing potato plants were taller and produced heavier tubers than control plants. We suggest a role for NMMP1in pathogen defense and development.
The objectives of this study were to 1) evaluate the efficiency of the protocol of Agrobacterium-... more The objectives of this study were to 1) evaluate the efficiency of the protocol of Agrobacterium-mediated transformation of cucumber to introduce phytoene synthase-2a carotene desaturase (PAC genes); 2) demonstrate the integration of PAC genes into the genome of putative transgenic cucumber based on growth on selection medium, PCR and Southern analysis; 3) evaluate the expression of PAC genes in transgenic cucumber based on the analysis of RT-PCR and Northern blot hybridization. Out of 5,945 cotyledonary-node explants inoculated with Agrobacterium, 65 (1.1%) explants produced 238 shoots. Integration of PAC genes into the genome of the cucumber was demonstrated based on the analysis of gDNA-PCR, 21 out of the 238 plants regenerated; while 6 plants proved positive for Southern blot hybridization. Transgene expression was demonstrated based on analysis of RT-PCR, 6 plants proved positive out of the 6 plants analyzed; while 4 plants out of 6 proved positive during Northern blot hy-bridization. This study successfully demonstrated the production of transgenic cucumber, integration, and expression of the PAC gene in cucumber.
Agrobacterium tumefaciens-mediated cotyledonary-node explants transformation was used to produce ... more Agrobacterium tumefaciens-mediated cotyledonary-node explants transformation was used to produce transgenic cucumber. Cotyledonary-node explants of cucumber (Cucumis sativus L. cv., Eunsung) were co-cultivated with Agrobacterium strains (EHA101) containing the binary vector (pPZP211) carrying with CaMV 35S promoter-nptII gene as selectable marker gene and 35S promoter-DQ gene (unpublished data) as target gene. The average of transformation efficiency (4.01%) was obtained from three times experiments and the maximum efficiency was shown at 5.97%. A total of 9 putative transgenic plants resistant to paromomycin were produced from the cultures of cotyledonary-node explants on selection medium. Among them, 6 transgenic plants showed that the nptII gene integrated into each genome of cucumber by Southern blot analysis.
ABSTRACT Plant receptor-like kinases (RLKs) are proteins that are involved in the regulation of d... more ABSTRACT Plant receptor-like kinases (RLKs) are proteins that are involved in the regulation of development, hormone signaling, abiotic, and biotic stress responses. It has been suggested that cysteine-rich receptor-like kinases (CRKs), which are one of the largest RLK groups, is significant in pathogen defense and programmed cell death. The CRK1 gene is isolated and characterized from tomato (Solanum lycopersicum L.). The SlCRK1 has two C-X8-C-X2-C motifs: a trans-membrane region and a kinase domain similar to other CRKs. The semi-quantitative RT-PCR exhibits the specific expression of SlCRK1 in the flower, but not in the root, leaf, seed, and fruit of the tomato. In addition, SlCRK1 exhibits pollen-specific expression in the floral organ. SlCRK1 has pollen-specific cis-acting elements in the promoter region, and its promoter has pollen-specific activity in the homozygous transgenic plants of tomato and Arabidopsis as confirmed through histochemical GUS assays. Moreover, the expression of SlCRK1 is not detected via stress treatment or hormone treatment. In this study, SlCRK1 from tomato is characterized and its promoter can be useful in developing transgenic plants with foreign genes that should be expressed in pollens.
Oh, S.-K., Jang, H. A., Kim, J., Choi, D., Park, Y.-I. and Kwon, S.-Y. 2014. Expression of cucumb... more Oh, S.-K., Jang, H. A., Kim, J., Choi, D., Park, Y.-I. and Kwon, S.-Y. 2014. Expression of cucumber LOX genes in response to powdery mildew and defense-related signal molecules. Can. J. Plant Sci. 94: 845–850. The cucumber genome contains 23 lipoxygenase (LOX) genes. The expression of seven type-I and six type-II LOX genes was induced when cucumber leaves were challenged with Sphaerotheca fuliginea and treated with salicylic acid, methyl jasmonate, and abscisic acid. These 13 CsLOX genes were differentially regulated during biotic and abiotic stresses.
Kim, W. B., Lim, C. J., Jang, H. A., Yi, S. Y., Oh, S.-K., Lee, H. Y., Kim, H. A., Park, Y.-I. an... more Kim, W. B., Lim, C. J., Jang, H. A., Yi, S. Y., Oh, S.-K., Lee, H. Y., Kim, H. A., Park, Y.-I. and Kwon, S.-Y. 2014. SlPMEI, a pollen-specific gene in tomato. Can. J. Plant Sci. 94: 73–83. Pectin is one of the main components of plant cell walls, and its biosynthesis is controlled by pectin methylesterase (PME). Pectin methylesterase inhibitors (PMEIs) are key regulators of PME. We report here the cloning and characterization of a novel Solanum lycopersicum L. PMEI gene, SlPMEI. RT-PCR studies of leaf, seed, fruit, flower, and flower organs confirmed that SlPMEI is expressed specifically in pollen. Promoter analysis of SlPMEI revealed pollen-specific cis-acting elements (pollen lat52 and g10). In addition, SlPMEI is expressed independently of abiotic stress, pathogen exposure, and growth stage in tomato, and a histochemical analysis of promoter activity revealed pollen-specific expression in both Arabidopsis and tomato. Under the microscope, we observed pollen-specific GUS expressio...
Homologs of the cytoplasmic protein kinase Pti1 are found in diverse plant species. A clear role ... more Homologs of the cytoplasmic protein kinase Pti1 are found in diverse plant species. A clear role of Pti1 in plant defense response has not been established. We identified a Pti1 homolog in cucumber (CsPti1-L). CsPti1-L expression was induced when cucumber plants were challenged with the fungal pathogen Sphaerotheca fuliginea or with salt treatment. CsPti1-L expression in cucumber leaves also was induced by methyl jasmonate, salicylic acid, and abscisic acid. CsPti1-L exhibited autophosphorylation activity and was targeted to the cytoplasm. Transgenic Nicotiana benthamiana expressing CsPti1-L exhibited greater cell death and increased ion leakage in response to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000, resistance to Botrytis cinerea infection, and higher tolerance to salt stress. RT-PCR analysis of transgenic N. benthamiana overexpressing CsPti1-L revealed constitutive upregulation of multiple genes involved in plant-defense and osmotic-stress responses. Our results suggest a functional role for CsPti1-L as a positive regulator of pathogen-defense and salt-stress responses.
Oomycetes are known to secrete a vast arsenal of effectors that modulate the host defense system ... more Oomycetes are known to secrete a vast arsenal of effectors that modulate the host defense system as well as facilitate establishing a parasitic infection in plants. In recent years, tremendous progress has been made in the field of effectromics based on studies of oomycetes, especially the cytoplasmic family of RXLR effectors. Yet, the biology of the RXLR effector family is still poorly understood. There has been a consensus regarding the structure of the RXLR motif in the mycologist community. However, the function of the RXLR motif is still unclear. First, different models have suggested that the role of the RXLR motif is either in translocation to a target destination inside a host cell or in the cleavage of itself followed by secretion. Second, recent studies have suggested different functional models for the RXLR motif. According to a widely accepted model, the RXLR motif is directly involved in the translocation of effectors to target sites. In contrast, a new study has propos...
Targeted genome editing using the CRISPR/Cas9 system is a groundbreaking technology that is being... more Targeted genome editing using the CRISPR/Cas9 system is a groundbreaking technology that is being widely used to produce plants with useful traits. However, for woody plants, only a few successful attempts have been reported. These successes have used Agrobacterium-mediated transformation, which has been reported to be very efficient at producing genetically modified trees. Nonetheless, there are unresolved problems with plasmid sequences that remain in the plant genome. In this study, we demonstrated a DNA-free genome editing technique in which purified CRISPR/Cas9 ribonucleoproteins (RNPs) are delivered directly to the protoplasts of a hybrid poplar (Populus alba × Populus glandulosa). We designed three single-guide RNAs (sgRNAs) to target the stress-associated protein 1 gene (PagSAP1) in the hybrid poplar. Deep sequencing results showed that pre-assembled RNPs had a more efficient target mutagenesis insertion and deletion (indel) frequency than did non-assembled RNPs. Moreover, the RNP of sgRNA3 had a significantly higher editing efficacy than those of sgRNA1 and sgRNA2. Our results suggest that the CRISPR/Cas9 ribonucleoproteinmediated transfection approach is useful for the production of transgene-free genome-edited tree plants.
Caffeoyl shikimate esterase (CSE) has been shown to play an important role in lignin biosynthesis... more Caffeoyl shikimate esterase (CSE) has been shown to play an important role in lignin biosynthesis in plants and is, therefore, a promising target for generating improved lignocellulosic biomass crops for sustainable biofuel production. Populus spp. has two CSE genes (CSE1 and CSE2) and, thus, the hybrid poplar (Populus alba × P. glandulosa) investigated in this study has four CSE genes. Here, we present transgenic hybrid poplars with knockouts of each CSE gene achieved by CRISPR/Cas9. To knockout the CSE genes of the hybrid poplar, we designed three single guide RNAs (sg1–sg3), and produced three different transgenic poplars with either CSE1 (CSE1-sg2), CSE2 (CSE2-sg3), or both genes (CSE1/2-sg1) mutated. CSE1-sg2 and CSE2-sg3 poplars showed up to 29.1% reduction in lignin deposition with irregularly shaped xylem vessels. However, CSE1-sg2 and CSE2-sg3 poplars were morphologically indistinguishable from WT and showed no significant differences in growth in a long-term living modifie...
Horticulture, Environment, and Biotechnology, 2020
Grape ripe rot is a fungal disease, which is almost inextinguishable particularly before and afte... more Grape ripe rot is a fungal disease, which is almost inextinguishable particularly before and after harvest in most vineyards. The management of this disease largely depends on the breeding of resistant varieties. Thus, the search for resistant varieties should parallel the molecular analysis of the markers associated with disease resistance. Here, we tested the leaf inoculation assay of Colletotrichum acutatum and C. gloeosporioides by inoculating them into 350 grapevine varieties. As a result of the phenotypes common to both species, 119 varieties (34%) were classified as resistant. Consequently, we discovered the locus with nucleotide-binding sites and carboxyterminal leucine-rich repeat regions (NBS-LRR) associated with grape ripe rot resistance through genotyping-by-sequencing and genome-wide association study. Ultimately, we identified the Vitis vinifera Colletotrichum resistance protein (VvCRP) markers from chromosome 3, which belongs to the CC-NBS-LRR type, as disease-resistant proteins. These markers were verified as the melting peak, and their chromatograms of nucleotide sequences were visualized in each single nucleotide polymorphism (SNP) loci. All the SNP markers differentiated successfully the resistant from the susceptible cultivars. Interestingly, we also found that a resistant cultivar named Bailey Alicante A has a heterozygous genotype, substantiating its potential usefulness for crops such as grapevine with many hybrids. Altogether, our results indicate that VvCRP markers can be utilized to distinguish grape ripe rot-resistant cultivars, particularly in crossbreeding populations.
We previously isolated Nicotiana benthamiana matrix metalloprotease 1 (NMMP1) from tobacco leaves... more We previously isolated Nicotiana benthamiana matrix metalloprotease 1 (NMMP1) from tobacco leaves. The NMMP1 gene encodes a highly conserved, Zn-containing catalytic protease domain that functions as a factor in the plant's defense against bacterial pathogens. Expression of NMMP1 was strongly induced during interactions between tobacco and one of its pathogens, Phytophthora infestans. To elucidate the role of the NMMP1 in defense of N. benthamiana against fungal pathogens, we performed gain-of-function and loss-of-function studies. NMMP1-overexpressing plants had stronger resistance responses against P. infestans infections than control plants, while silencing of NMMP1 resulted in greater susceptibility of the plants to the pathogen. This greater susceptibility correlated with fewer NMMP1 transcripts than the non-silenced control. We also examined cell death as a measure of disease. The amount of cell death induced by the necrosis-inducing P. infestans protein 1, PiNPP1, was dependent on NMMP1 in N. benthamiana. Potato plants overexpressing NMMP1 also had enhanced disease resistance against P. infestans. RT-PCR analysis of these transgenic potato plants revealed constitutive upregulation of the potato defense gene NbPR5. NMMP1-overexpressing potato plants were taller and produced heavier tubers than control plants. We suggest a role for NMMP1in pathogen defense and development.
The objectives of this study were to 1) evaluate the efficiency of the protocol of Agrobacterium-... more The objectives of this study were to 1) evaluate the efficiency of the protocol of Agrobacterium-mediated transformation of cucumber to introduce phytoene synthase-2a carotene desaturase (PAC genes); 2) demonstrate the integration of PAC genes into the genome of putative transgenic cucumber based on growth on selection medium, PCR and Southern analysis; 3) evaluate the expression of PAC genes in transgenic cucumber based on the analysis of RT-PCR and Northern blot hybridization. Out of 5,945 cotyledonary-node explants inoculated with Agrobacterium, 65 (1.1%) explants produced 238 shoots. Integration of PAC genes into the genome of the cucumber was demonstrated based on the analysis of gDNA-PCR, 21 out of the 238 plants regenerated; while 6 plants proved positive for Southern blot hybridization. Transgene expression was demonstrated based on analysis of RT-PCR, 6 plants proved positive out of the 6 plants analyzed; while 4 plants out of 6 proved positive during Northern blot hy-bridization. This study successfully demonstrated the production of transgenic cucumber, integration, and expression of the PAC gene in cucumber.
Agrobacterium tumefaciens-mediated cotyledonary-node explants transformation was used to produce ... more Agrobacterium tumefaciens-mediated cotyledonary-node explants transformation was used to produce transgenic cucumber. Cotyledonary-node explants of cucumber (Cucumis sativus L. cv., Eunsung) were co-cultivated with Agrobacterium strains (EHA101) containing the binary vector (pPZP211) carrying with CaMV 35S promoter-nptII gene as selectable marker gene and 35S promoter-DQ gene (unpublished data) as target gene. The average of transformation efficiency (4.01%) was obtained from three times experiments and the maximum efficiency was shown at 5.97%. A total of 9 putative transgenic plants resistant to paromomycin were produced from the cultures of cotyledonary-node explants on selection medium. Among them, 6 transgenic plants showed that the nptII gene integrated into each genome of cucumber by Southern blot analysis.
ABSTRACT Plant receptor-like kinases (RLKs) are proteins that are involved in the regulation of d... more ABSTRACT Plant receptor-like kinases (RLKs) are proteins that are involved in the regulation of development, hormone signaling, abiotic, and biotic stress responses. It has been suggested that cysteine-rich receptor-like kinases (CRKs), which are one of the largest RLK groups, is significant in pathogen defense and programmed cell death. The CRK1 gene is isolated and characterized from tomato (Solanum lycopersicum L.). The SlCRK1 has two C-X8-C-X2-C motifs: a trans-membrane region and a kinase domain similar to other CRKs. The semi-quantitative RT-PCR exhibits the specific expression of SlCRK1 in the flower, but not in the root, leaf, seed, and fruit of the tomato. In addition, SlCRK1 exhibits pollen-specific expression in the floral organ. SlCRK1 has pollen-specific cis-acting elements in the promoter region, and its promoter has pollen-specific activity in the homozygous transgenic plants of tomato and Arabidopsis as confirmed through histochemical GUS assays. Moreover, the expression of SlCRK1 is not detected via stress treatment or hormone treatment. In this study, SlCRK1 from tomato is characterized and its promoter can be useful in developing transgenic plants with foreign genes that should be expressed in pollens.
Oh, S.-K., Jang, H. A., Kim, J., Choi, D., Park, Y.-I. and Kwon, S.-Y. 2014. Expression of cucumb... more Oh, S.-K., Jang, H. A., Kim, J., Choi, D., Park, Y.-I. and Kwon, S.-Y. 2014. Expression of cucumber LOX genes in response to powdery mildew and defense-related signal molecules. Can. J. Plant Sci. 94: 845–850. The cucumber genome contains 23 lipoxygenase (LOX) genes. The expression of seven type-I and six type-II LOX genes was induced when cucumber leaves were challenged with Sphaerotheca fuliginea and treated with salicylic acid, methyl jasmonate, and abscisic acid. These 13 CsLOX genes were differentially regulated during biotic and abiotic stresses.
Kim, W. B., Lim, C. J., Jang, H. A., Yi, S. Y., Oh, S.-K., Lee, H. Y., Kim, H. A., Park, Y.-I. an... more Kim, W. B., Lim, C. J., Jang, H. A., Yi, S. Y., Oh, S.-K., Lee, H. Y., Kim, H. A., Park, Y.-I. and Kwon, S.-Y. 2014. SlPMEI, a pollen-specific gene in tomato. Can. J. Plant Sci. 94: 73–83. Pectin is one of the main components of plant cell walls, and its biosynthesis is controlled by pectin methylesterase (PME). Pectin methylesterase inhibitors (PMEIs) are key regulators of PME. We report here the cloning and characterization of a novel Solanum lycopersicum L. PMEI gene, SlPMEI. RT-PCR studies of leaf, seed, fruit, flower, and flower organs confirmed that SlPMEI is expressed specifically in pollen. Promoter analysis of SlPMEI revealed pollen-specific cis-acting elements (pollen lat52 and g10). In addition, SlPMEI is expressed independently of abiotic stress, pathogen exposure, and growth stage in tomato, and a histochemical analysis of promoter activity revealed pollen-specific expression in both Arabidopsis and tomato. Under the microscope, we observed pollen-specific GUS expressio...
Homologs of the cytoplasmic protein kinase Pti1 are found in diverse plant species. A clear role ... more Homologs of the cytoplasmic protein kinase Pti1 are found in diverse plant species. A clear role of Pti1 in plant defense response has not been established. We identified a Pti1 homolog in cucumber (CsPti1-L). CsPti1-L expression was induced when cucumber plants were challenged with the fungal pathogen Sphaerotheca fuliginea or with salt treatment. CsPti1-L expression in cucumber leaves also was induced by methyl jasmonate, salicylic acid, and abscisic acid. CsPti1-L exhibited autophosphorylation activity and was targeted to the cytoplasm. Transgenic Nicotiana benthamiana expressing CsPti1-L exhibited greater cell death and increased ion leakage in response to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000, resistance to Botrytis cinerea infection, and higher tolerance to salt stress. RT-PCR analysis of transgenic N. benthamiana overexpressing CsPti1-L revealed constitutive upregulation of multiple genes involved in plant-defense and osmotic-stress responses. Our results suggest a functional role for CsPti1-L as a positive regulator of pathogen-defense and salt-stress responses.
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Papers by Hyun-A Jang