Kyung Hee Paek

Overexpression of the Tobacco Tsi1 Gene Encoding an EREBP/AP2-Type Transcription Factor Enhances Resistance against Pathogen Attack and Osmotic Stress in Tobacco

Using mRNA differential display analysis, we isolated a salt-induced transcript that showed a significant sequence homology with an EREBP/AP2 DNA binding motif from oilseed rape plants. With this cDNA fragment as a probe, cDNA clone Tsi1 (for Tobacco stress-induced gene1) was isolated from a tobacco cDNA library. RNA gel blot analysis indicated that transcripts homologous with Tsi1 were induced not only in NaCl-treated leaves but also in leaves treated with ethephon or salicylic acid. Transient expression analysis using a Tsi1::smGFP fusion gene in BY-2 cells indicated that the Tsi1 protein was targeted to the nucleus. Fusion protein of Tsi1 with GAL4 DNA binding domain strongly activated transcription in yeast, and the transactivating activity was localized to the 13 C-terminal amino acids of Tsi1. Electrophoretic mobility shift assays revealed that Tsi1 could bind specifically to the GCC and the DRE/CRT sequences, although the binding activity to the former was stronger than that to the latter. Furthermore, Agrobacterium-mediated transient expression and transgenic plants expressing Tsi1 demonstrated that overexpression of the Tsi1 gene induced expression of several pathogenesis-related genes under normal conditions, resulting in improved tolerance to salt and pathogens. These results suggest that Tsi1 might be involved as a positive trans-acting factor in two separate signal transduction pathways under abiotic and biotic stress.

The Cytokinin-Activated Transcription Factor ARR2 Promotes Plant Immunity via TGA3/NPR1-Dependent Salicylic Acid Signaling in Arabidopsis

Cytokinins affect plant immunity to various pathogens; however, the mechanisms coupling plant-derived cytokinins to pathogen responses have been elusive. Here, we found that plant-derived cytokinins promote resistance of Arabidopsis to Pseudomonas syringae pv. tomato DC3000 (Pst). Modulated cytokinin levels or signaling activity in CKX- or IPT-overexpressing plants or in ahk2 ahk3 mutants correlated with altered resistance. In fact, the cytokinin-activated transcription factor ARR2 contributes specifically to Pst resistance. The salicylic acid (SA) response factor TGA3 binds ARR2, and mutation of TGA-binding cis-elements in the Pr1 promoter abolished cytokinin- and ARR2-dependent Pr1 activation. Cytokinin treatment did not increase pathogen resistance in tga3 plants, as the cytokinin-dependent induction of Pr1 was eliminated. Moreover, SA signaling enhanced binding of ARR2/TGA3 to the Pr1 promoter. Taken together, these results show that cytokinins modulate the SA signaling to augment resistance against Pst, a process in which the interaction between TGA3 and ARR2 is important.

Induction of pepper cDNA encoding a lipid transfer protein during the resistance response to tobacco mosaic virus

Pepper (Capsicum annuum) plants exhibit hypersensitive response (HR) against infection by many tobamoviruses. A clone encoding a putative nonspecific lipid transfer protein (CaLTP1) was isolated by differential screening of a cDNA library from resistant pepper leaves when inoculated with tobacco mosaic virus (TMV) pathotype P0. The predicted amino acid sequence of CaLTP1 is highly similar to that of the other plant LTPs. Southern blot analysis showed that a small gene family of LTP-related sequences was present in the pepper genome. Transcripts homologous to CaLTP1 accumulated abundantly in old leaves and flowers. CaLTP1 expression was induced in the incompatible interaction with TMV-P0 but was not induced in the compatible interaction with TMV-P1.2. In correlation with the temporal progression of HR in the inoculated leaves, CaLTP1 transcripts started to accumulate at 24 h after TMV-P0 inoculation, reaching a maximal level at 48 h. A strain of Xanthomonas campestris pv. vesicatoria (Xcv) that carries the bacterial avirulence gene, avrBs2, was infiltrated into leaves of a pepper cultivar containing the Bs2 resistance gene. A marked induction of CaLTP1 expression was observed in Xcv-infiltrated leaves. Effects of exogenously applied abiotic elicitors on CaLTP1 expression were also examined. Salicylic acid caused a rapid accumulation of CaLTP1 transcripts in pepper leaves and ethephon treatment also induced the expression of the CaLTP1 gene. Transient expression in the detached pepper leaves by biolistic gene bombardment indicated that CaLTP1 is localized mostly at the plant cell surface, possibly in the cell wall. These results suggest possible role(s) for LTPs in plant defense against pathogens including viruses.

HD-ZIP III Activity Is Modulated by Competitive Inhibitors via a Feedback Loop in Arabidopsis Shoot Apical Meristem Development

Shoot apical meristem (SAM) development is coordinately regulated by two interdependent signaling events: one maintaining stem cell identity and the other governing the initiation of lateral organs from the flanks of the SAM. The signaling networks involved in this process are interconnected and are regulated by multiple molecular mechanisms. Class III homeodomain-leucine zipper (HD-ZIP III) proteins are the most extensively studied transcription factors involved in this regulation. However, how different signals are integrated to maintain stem cell identity and to pattern lateral organ polarity remains unclear. Here, we demonstrated that a small ZIP protein, ZPR3, and its functionally redundant homolog, ZPR4, negatively regulate the HD-ZIP III activity in SAM development. ZPR3 directly interacts with PHABULOSA (PHB) and other HD-ZIP III proteins via the ZIP motifs and forms nonfunctional heterodimers. Accordingly, a double mutant, zpr3-2 zpr4-2, exhibits an altered SAM activity with abnormal stem cell maintenance. However, the mutant displays normal patterning of leaf polarity. In addition, we show that PHB positively regulates ZPR3 expression. We therefore propose that HD-ZIP III activity in regulating SAM development is modulated by, among other things, a feedback loop involving the competitive inhibitors ZPR3 and ZPR4.

Ectopic expression of Tsi1 in transgenic hot pepper plants enhances host resistance to viral, bacterial, and oomycete pathogens.

In many plants, including hot pepper plants, productivity is greatly affected by pathogen attack. We reported previously that tobacco stress-induced gene 1 (Tsi1) may play an important role in regulating stress responsive genes and pathogenesis-related (PR) genes. In this study, we demonstrated that overexpression of Tsi1 gene in transgenic hot pepper plants induced constitutive expression of several PR genes in the absence of stress or pathogen treatment. The transgenic hot pepper plants expressing Tsi1 exhibited resistance to Pepper mild mottle virus (PMMV) and Cucumber mosaic virus (CMV). Furthermore, these transgenic plants showed increased resistance to a bacterial pathogen, Xanthomonas campestris pv. vesicatoria and also an oomycete pathogen, Phytophthora capsici. These results suggested that ectopic expression of Tsi1 in transgenic hot pepper plants enhanced the resistance of the plants to various pathogens, including viruses, bacteria, and oomycete. These results suggest that using transcriptional regulatory protein genes may contribute to developing broad-spectrum resistance in crop plants.

Isolation of pepper mRNAs differentially expressed during the hypersensitive response to tobacco mosaic virus and characterization of a proteinase inhibitor gene

Abstract Pepper ( Capsicum annuum L. cv. Bugang) displays a hypersensitive response (HR) upon infection by tobacco mosaic virus (TMV) pathotype P 0 . To elucidate molecular mechanisms underlying this response, a cDNA library was constructed and differentially screened between TMV-P 0 -inoculated and uninoculated leaves. Of 3260 clones analyzed by reverse RNA slot-blot hybridization, 22 showed up-regulated expression upon TMV-P 0 challenge. Among these, 16 clones showed significantly higher levels of expression in response to the avirulent pathotype TMV-P 0 than to the virulent pathotype TMV-P 1.2 . Nucleotide sequence analysis and database searches revealed that the polypeptides deduced from the selected clones displayed significant sequence homology to various known proteins, indicating conservation of the wide range of up-regulated proteins during HR against viral challenge in higher plants. A cDNA clone encoding a putative proteinase inhibitor II ( CaPinII ) was selected and further analyzed. Upon TMV-P 0 inoculation, the CaPinII transcript accumulated maximally at 72 h post-inoculation and declined afterwards. In contrast, inoculation with TMV-P 1.2 did not induce CaPinII expression to any detectable level. The CaPinII gene was also expressed in response to the treatment of various chemicals, as well as wounding. Southern blot analysis showed that a small family of genes homologous to the CaPinII gene is present in the pepper genome.

Tobacco Tsip1, a DnaJ-Type Zn Finger Protein, Is Recruited to and Potentiates Tsi1-Mediated Transcriptional Activation

Tobacco stress-induced1 (Tsi1) is an ethylene-responsive-element binding protein/APETALA2–type transcription factor that plays an important role in both biotic and abiotic stress signaling pathways. We show that Tsi1-interacting protein1 (Tsip1), a DnaJ-type Zn finger protein, interacts with Tsi1 in vitro and in yeast (Saccharomyces cerevisiae). The transcript level of Tsip1 in tobacco (Nicotiana tabacum) increased upon treatment with salicylic acid (SA), ethylene, gibberellic acid, NaCl, and virus challenge. Tsip1 appeared to be physically associated with the chloroplast surface but dissociated from it after SA treatment. Tsip1 colocalized and coimmunoprecipitated with Tsi1 in plant cells following SA treatment. Tsip1 expression increased Tsi1-mediated transcription and was able to functionally compensate for loss of the Tsi1 transcriptional activation domain through a direct interaction with Tsi1. Transgenic plants simultaneously coexpressing Tsi1 and Tsip1 displayed stronger pathogen resistance and salt tolerance than did transgenic plants expressing either Tsi1 or Tsip1 alone. Concurrent with this, the expression of a subset of stress-related genes was induced in a cooperative manner in Tsi1/Tsip1 transgenic plants. These results together implied that Tsi1 recruits Tsip1 to the promoters of stress-related genes to potentiate Tsi1-mediated transcriptional activation.

A hot pepper gene encoding WRKY transcription factor is induced during hypersensitive response to Tobacco mosaic virus and Xanthomonas campestris

Plant WRKY transcription factors were previously implicated in the alteration of gene expression in response to various pathogens. The WRKY proteins constitute a large family of plant transcription factors, whose precise functions have yet to be elucidated. Using a domain-specific differential display procedure, we isolated a WRKY gene, which is rapidly induced during an incompatible interaction between hot pepper and Tobacco mosaic virus (TMV) or Xanthomonas campestris pv . vesicatoria (Xcv). The full-length cDNA of CaWRKY-a (Capsicum annuum WRKY-a) encodes a putative polypeptide of 546 amino acids, containing two WRKY domains with a zinc finger motif. The expression of CaWRKY-a could be rapidly induced by not only chemical elicitor such as salicylic acid (SA) or ethephon but also wounding treatments. The nuclear localization of CaWRKY-a was determined in transient expression system using tobacco BY-2 cells by polyethylene glycol (PEG)-mediated transformation experiment. With oligonucleotide molecules containing the putative W-box sequences as a probe, we confirmed that CaWRKY-a protein had W-box-binding activity. These results suggest that CaWRKY-a might be involved as a transcription factor in plant defense-related signal transduction pathways.

Satellite-RNA-mediated resistance to cucumber mosaic virus in transgenic plants of hot pepper (Capsicum annuum cv. Golden Tower)

Abstract We previously established a system of in vitro regeneration and Agrobacterium-mediated transformation for hot pepper plants. The level of protection against cucumber mosaic virus in the progeny of the transgenic hot pepper plants that express cucumber mosaic virus (CMV) satellite RNA was investigated. The transgenic hot pepper plants were self-fertilized, and their progeny were tested for stable inheritance and expression of the cDNA of CMV satellite RNA. Polymerase chain reaction and RNA gel blot analyses showed that the introduced gene was stably transmitted and expressed in the progeny. Symptom attenuation in the offspring was confirmed upon inoculation with CMV-Y or CMV-Korea (CMV-Kor) strains.

SRD1 is involved in the auxin-mediated initial thickening growth of storage root by enhancing proliferation of metaxylem and cambium cells in sweetpotato (Ipomoea batatas)

A sweetpotato (Ipomoea batatas cv. ‘Jinhongmi’) MADS-box protein cDNA (SRD1) has been isolated from an early stage storage root cDNA library. The role of the SRD1 gene in the formation of the storage root in sweetpotato was investigated by an expression pattern analysis and characterization of SRD1-overexpressing (ox) transgenic sweetpotato plants. Transcripts of SRD1 were detected only in root tissues, with the fibrous root having low levels of the transcript and the young storage root showing relatively higher transcript levels. SRD1 mRNA was mainly found in the actively dividing cells, including the vascular and cambium cells of the young storage root. The transcript level of SRD1 in the fibrous roots increased in response to 1000 μM indole-3-acetic acid (IAA) applied exogenously. During the early stage of storage root development, the endogenous IAA content and SRD1 transcript level increased concomitantly, suggesting an involvement of SRD1 during the early stage of the auxin-dependent development of the storage root. SRD1-ox sweetpotato plants cultured in vitro produced thicker and shorter fibrous roots than wild-type plants. The metaxylem and cambium cells of the fibrous roots of SRD1-ox plants showed markedly enhanced proliferation, resulting in the fibrous roots of these plants showing an earlier thickening growth than those of wild-type plants. Taken together, these results demonstrate that SRD1 plays a role in the formation of storage roots by activating the proliferation of cambium and metaxylem cells to induce the initial thickening growth of storage roots in an auxin-dependent manner.