Gil Je Lee

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.

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.

Capsicum annuum WRKYb transcription factor that binds to the CaPR-10 promoter functions as a positive regulator in innate immunity upon TMV infection.

In plant, some WRKY transcription factors are known to play an important role in the transcriptional reprogramming associated with the immune response. By using WRKY-domain-specific differential display procedure, we isolated CaWRKYb gene, which is rapidly induced during an incompatible interaction between hot pepper and Tobacco mosaic virus (TMV) pathotype P(0) infection. The recombinant CaWRKYb bound to the W box-containing CaPR-10 promoter probes efficiently and the specificity of binding was confirmed by mutant study and competition with cold oligonucleotides. Also, in GUS reporter activity assay using Arabidopsis protoplasts with the CaPR-10 promoter, GUS activity was increased in the presence of CaWRKYb. And CaWRKYb-knockdown plant showed reduced number of hypersensitive response local lesions upon TMV-P(0) infection. Furthermore, CaWRKYb-knockdown plant exhibited compromised resistance to TMV-P(0) by accumulating more TMV, apparently through decreased expression of CaPR-10, CaPR-1, and CaPR-5. These results suggest that CaWRKYb is involved as a positive transcription factor in defense-related signal transduction pathways in hot pepper.

Capsicum annuum WRKY transcription factor d (CaWRKYd) regulates hypersensitive response and defense response upon Tobacco mosaic virus infection

WRKY transcription factors regulate biotic, abiotic, and developmental processes. In terms of plant defense, WRKY factors have important roles as positive and negative regulators via transcriptional regulation or protein-protein interaction. Here, we report the characterization of the gene encoding Capsicum annuum WRKY transcription factor d (CaWRKYd) isolated from microarray analysis in the Tobacco mosaic virus (TMV)-P(0)-inoculated hot pepper plants. CaWRKYd belongs to the WRKY IIa group, a very small clade in the WRKY subfamily, and WRKY IIa group has positive/negative regulatory roles in Arabidopsis and rice. CaWRKYd transcripts were induced by various plant defense-related hormone treatments and TMV-P(0) inoculation. Silencing of CaWRKYd affected TMV-P(0)-mediated hypersensitive response (HR) cell death and accumulation of TMV-P(0) coat protein in local and systemic leaves. Furthermore, expression of some pathogenesis-related (PR) genes and HR-related genes was reduced in the CaWRKYd-silenced plants compared with TRV2 vector control plants upon TMV-P(0) inoculation. CaWRKYd was confirmed to bind to the W-box. Thus CaWRKYd is a newly identified Capsicum annuum WRKY transcription factor that appears to be involved in TMV-P(0)-mediated HR cell death by regulating downstream gene expression.

Capsicum annuum transcription factor WRKYa positively regulates defense response upon TMV infection and is a substrate of CaMK1 and CaMK2

Plants are constantly exposed to pathogens and environmental stresses. To minimize damage caused by these potentially harmful factors, plants respond by massive transcriptional reprogramming of various stress-related genes via major transcription factor families. One of the transcription factor families, WRKY, plays an important role in diverse stress response of plants and is often useful to generate genetically engineered crop plants. In this study, we carried out functional characterization of CaWRKYa encoding group I WRKY member, which is induced during hypersensitive response (HR) in hot pepper (Capsicum annuum) upon Tobacco mosaic virus (TMV) infection. CaWRKYa was involved in L-mediated resistance via transcriptional reprogramming of pathogenesis-related (PR) gene expression and affected HR upon TMV-P0 infection. CaWRKYa acts as a positive regulator of this defense system and could bind to the W-box of diverse PR genes promoters. Furthermore, we found Capsicum annuum mitogen-activated protein kinase 1 (CaMK1) and 2 (CaMK2) interacted with CaWRKYa and phosphorylated the SP clusters but not the MAPK docking (D)-domain of CaWRKYa. Thus, these results demonstrated that CaWRKYa was regulated by CaMK1 and CaMK2 at the posttranslational level in hot pepper.

Suppression of UDP-glycosyltransferase-coding arabidopsis thaliana UGT74E2 gene expression leads to increased resistance to psuedomonas syringae pv. tomato DC3000 infection

Plants possess multiple resistance mechanisms that protect themselves against pathogen attack. To identify unknown components of the defense machinery in Arabidopsis, gene-expression changes were monitored in Arabidopsis thaliana under 18 different biotic or abiotic conditions using a DNA microarray representing approximately 25% of all Arabidopsis thaliana genes (www.genevestigator.com). Seventeen genes which are early responsive to salicylic acid (SA) treatment as well as pathogen infection were selected and their T-DNA insertion mutants were obtained from SALK institute. To elucidate the role of each gene in defense response, bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000 was inoculated onto individual T-DNA insertion mutants. Four mutants exhibited decreased resistance and five mutants displayed significantly enhanced resistance against Pst DC3000-infection as measured by change in symptom development as compared to wild-type plants. Among them, member of uridin diphosphate (UDP)-glycosyltransferase (UGT) was of particular interest, since a UGT mutant (At1g05680) showed enhanced resistance to Pst-infection in Arabidopsis. In systemic acquired resistance (SAR) assay, this mutant showed enhanced activation of SAR. Also, the enhanced SAR correlated with increased expression of defenserelated gene, AtPR1. These results emphasize that the glycosylation of UGT74E2 is a part of the SA-mediated disease-resistance mechanism.

Erratum: Induction of a pepper cDNA encoding SAR8.2 protein during the resistance response to tobacco mosaic virus (Molecules and Cells (2001) 12 (250-256)

Retraction Note: Mol. Cells 12 (2001) 250–256 Members of the editorial board have unanimously agreed to retract the article [Mol. Cells 12 (2001) 250–256] for potential misconducts mainly concerning manipulation and repeated uses of photomicrographs of control data internally along with mislabeling and/or externally in multiple publications. As specified in the “Instructions to Authors”, Molecules and Cells (Mol. Cells) explicitly prohibits mis-representation or falsification of experimental data including duplication of previously published data. In the article, lanes 1-4 and lanes 5–7 of rRNA gels in Fig. 2B are identical to lanes 1–4 and lanes 10–12 in Fig. 3A, respectively; two EtBr gels of rRNA in Figs. 4A and 4B are identical, and rRNA gels in Figs. 5A, 5B, and 5C are identical as well.

Erratum: A hot pepper cDNA encoding ascorbate peroxidase is induced during the incompatible interaction with virus and bacteria (Molecules and Cells (2002) 14 (75-84)

Retraction Note: Mol. Cells 14 (2002) 75–84 Members of the editorial board have unanimously agreed to retract the article [Mol. Cells 14 (2002) 75–84] for potential misconducts mainly concerning manipulation and repeated uses of photomicrographs of control data internally along with mislabeling and/or externally in multiple publications. As specified in the “Instructions to Authors”, Molecules and Cells (Mol. Cells) explicitly prohibits mis-representation or falsification of experimental data including duplication of previously published data. In the article, lanes 1-6 and Lanes 10–15 of EtBr gel of rRNA in Fig. 5 are mirror images of each other; a part of this image has been previously used in Fig. 4B of Mol. Cells 11 (2001) 122–127; this image has also been used in Fig. 2D of Plant Physiol. 135 (2004) 561–573; rRNA gels in Figs. 6A, 6B, and 6C are identical, and rRNA gels in Figs. 7A and 7B are identical as well.

Erratum: A hot pepper cDNA encoding a pathogenesis-related protein 4 is induced during the resistance response to tobacco mosaic virus (Molecules and Cells (2001) 11 (122-127))

Retraction Note: Mol. Cells 11 (2001) 122–127 Members of the editorial board have unanimously agreed to retract the article [Mol. Cells 11 (2001) 122–127] for potential misconducts mainly concerning manipulation and repeated uses of photomicrographs of control data internally along with mislabeling and/or externally in multiple publications. As specified in the “Instructions to Authors”, Molecules and Cells (Mol. Cells) explicitly prohibits mis-representation or falsification of experimental data including duplication of previously published data. In the article, lanes 1–6 and lanes 9–11 of rRNA gel in Fig. 3A are identical to lanes 1–6 and lanes 7–9 in Fig. 3C, respectively; two EtBr gels of rRNA in Fig. 4A are identical, and lanes 1–6 and lanes 10–15 in the EtBr gel of rRNA in Fig. 4B are mirror images of each other.