So Young Yi

The Pepper Transcription Factor CaPF1 Confers Pathogen and Freezing Tolerance in Arabidopsis

An ERF/AP2-type transcription factor (CaPF1) was isolated by differential-display reverse transcription-PCR, following inoculation of the soybean pustule pathogen Xanthomonas axonopodis pv glycines 8ra, which induces hypersensitive response in pepper (Capsicum annuum) leaves. CaPF1 mRNA was induced under conditions of biotic and abiotic stress. Higher levels of CaPF1 transcripts were observed in disease-resistant tissue compared with susceptible tissue. CaPF1 expression was additionally induced using various treatment regimes, including ethephon, methyl jasmonate, and cold stress. To determine the role of CaPF1 in plants, transgenic Arabidopsis and tobacco (Nicotiana tabacum) plants expressing higher levels of CaPF1 were generated. Gene expression analyses of transgenic Arabidopsis and tobacco revealed that the CaPF1 level in transgenic plants affects expression of genes that contain either a GCC or a CRT/DRE box in their promoter regions. Furthermore, transgenic Arabidopsis plants expressing CaPF1 displayed tolerance against freezing temperatures and enhanced resistance to Pseudomonas syringae pv tomato DC3000. Disease tolerance was additionally observed in CaPF1 transgenic tobacco plants. The results collectively indicate that CaPF1 is an ERF/AP2 transcription factor in hot pepper plants that may play dual roles in response to biotic and abiotic stress in plants.

Capsicum annuum WRKY protein CaWRKY1 is a negative regulator of pathogen defense

Plants respond to pathogens by regulating a network of signaling pathways that fine-tune transcriptional activation of defense-related genes. The aim of this study was to determine the role of Capsicum annuum WRKY zinc finger-domain transcription factor 1 (CaWRKY1) in defense. In previous studies, CaWRKY1 was found to be rapidly induced in C. annuum (chili pepper) leaves by incompatible and compatible pathogen inoculations, but the complexity of the network of the WRKY family prevented the function of CaWRKY1 in defense from being elucidated. Virus-induced gene silencing of CaWRKY1 in chili pepper leaves resulted in decreased growth of Xanthomonas axonopodis pv. vesicatoria race 1. CaWRKY1-overexpressing transgenic plants showed accelerated hypersensitive cell death in response to infection with tobacco mosaic virus and Pseudomonas syringe pv. tabaci. Lower levels of pathogenesis-related gene induction were observed in CaWRKY1-overexpressing transgenic plants following salicylic acid (SA) treatments. This work suggests that the newly characterized CaWRKY1, which is strongly induced by pathogen infections and the signal molecule SA, acts as a regulator to turn off systemic acquired resistance once the pathogen challenge has diminished and to prevent spurious activation of defense responses at suboptimal concentrations of SA.

PPI1: A Novel Pathogen-Induced Basic Region-Leucine Zipper (bZIP) Transcription Factor from Pepper

We have isolated a full-length cDNA, PPI1 (pepper-PMMV interaction 1), encoding a novel basic region-leucine zipper (bZIP) DNA-binding protein, from expressed sequence tags differentially expressed in Capsicum chinense P1257284 infected with Pepper mild mottle virus (PMMV). PPI1 encodes a predicted protein of 170 amino acids and contains a putative DNA-binding domain that shares significant amino acid identity with ACGT-binding domains of members of the bZIP DNA-binding protein family. PPI1 was localized in the nucleus and had transcriptional activation activity in yeast. Transcripts of the PPI1 gene were preferentially induced during an incompatible interaction by inoculation with PMMV, Pseudomonas syringae pv. syringae 61, and Xanthomonas campestris pv. vesicatoria race 3. However, the PPII gene was not induced by abiotic stressors that activate the plant defense-signaling pathway. Our data provide the first evidence that a bZIP transcription factor is preferentially induced by pathogen attack, suggesting that PPI1 may play a specific functional role in the regulation of expression of plant defense-related genes.

The Activated SA and JA Signaling Pathways Have an Influence on flg22-Triggered Oxidative Burst and Callose Deposition

The first line of defense in plants against pathogens is induced by the recognition of microbe-associated molecular patterns (MAMP). Perception of bacterial flagellin (flg22) by the pattern recognition receptor flagellin-sensing 2 (FLS2) is the best characterized MAMP response, although the underlying molecular mechanisms are not fully understood. Here we studied the relationship between salicylic acid (SA) or jasmonic acid (JA) signaling and FLS2-mediated signaling by monitoring flg22-triggered responses in known SA or JA related mutants of Arabidopsis thaliana (L.) Heynh. The sid2 mutant, impaired in SA biosynthesis, had less basal FLS2 mRNA accumulation than the wild type, which correlated with suppression of early flg22 responses such as ROS production and induction of marker genes, WRKY29 and FRK1. The JA-signaling mutants, jar1 and coi1, exhibited an enhanced flg22-triggered oxidative burst and more callose accumulation than the wild type, and pretreatment with SA or coronatine (COR), a structural mimic of JA-isoleucine, altered these flg22-induced responses. Nonexpressor of pathogenesis-related genes 1 (NPR1) acted downstream of SID2 and required SA-dependent priming for the enhanced flg22-triggered oxidative burst and callose deposition. Activation of JA signaling by COR pretreatment suppressed the flg22-triggered oxidative burst and callose accumulation in a coronatine insensitive 1 (COI1) dependent manner. COR had a negative effect on flg22 responses but only the flg22-triggered oxidative burst depended on SA-JA/COR signaling antagonism. Thus the activated SA and JA signaling pathways have an influence on flg22-triggered oxidative burst and callose deposition. These results may explain how SA and JA signaling are cross talked for regulation of flg22-triggered responses.

Ectopic expression of pepper CaPF1 in potato enhances multiple stresses tolerance and delays initiation of in vitro tuberization

Ethylene-responsive factors (ERFs) are plant-specific transcription factors, many of which have been linked to plant defense responses. However, little is known about the functional significance of ERF genes in potato plants compared to the model plant species Arabidopsis. We show here that overexpression of CaPF1, an ERF/AP2-type pepper transcription factor gene, effectively increased tolerance to freezing, heat, heavy metal, and oxidative stress in potatoes. Interestingly, CaPF1 was involved in tuber formation in potato plants. The time course of microtuber formation was significantly retarded in potato plants that overexpressed CaPF1 compared with wild-type potato plants. Overall, the results of the present study indicate that the pepper transcription factor gene, CaPF1, is involved in promotion of multiple stress tolerance and retardation of in vitro tuberization in potato plants.

Pathogen-induced expression of plant ATP:citrate lyase1

Hypersensitive response (HR) in plants is generally characterized by a rapid and localized cell death, cell wall strengthening and synthesis of phytoalexins and PR proteins [1]. In order to understand the molecular and cellular defense mechanisms better during HR that results from the interaction between plant pathogen and its non-host, we inoculated hot pepper (Capsicum annuum cv. Pukang) leaves with soybean pustule pathogen, Xanthomonas campestris pv. glycines8ra. A pool of genes induced or repressed by infection of the pathogen has been isolated by using a diierential displaypolymerase chain reaction (DD-PCR) technique (Yi et al., unpublished data). Approximately one half of the isolated genes had no sequence similarity in existing databases and the major portion of identi¢ed DNA fragments consisted of enzymes involved in primary or secondary metabolic pathways. One of the fragments was found to have signi¢cant sequence homology with ATP:citrate lyase (ACL) known from diverse organisms including alga, fungi, and mammals. ACL is known to catalyze the following reaction: citrate+ CoA+ATPCoxaloacetate+ADP+Pi+acetyl-CoA. In the cytosol of animals, oleaginous yeast, and fungi, acetyl-CoA produced by the action of ACL is the major precursor for fatty acid and sterol biosynthesis [2]. However, in plants, the biosynthetic origin of cytosolic acetyl-CoA that is needed for biosynthesis of phytochemicals is not established. In sweet potato, ACL activity has been observed to increase 10-fold in root tissues response to infection by the fungal pathogen, Ceratocystis ¢mbriata, and to correspond to the accumulation of phytoalexin ipomeamarone [3], but the molecular characterization of a plant ACL has not yet been reported. In order to isolate a full-length cDNA clone encoding ACL, a cDNA library was constructed from hot pepper leaves inoculated with X. campestris pv. glycines8ra. After screening of the cDNA library by using a cDNA fragment of ACL produced by DD-PCR as a probe, a cDNA clone with an open reading frame containing 608 amino acids was ¢nally isolated and designated as Ca-ACL1 (GenBank accession number: AF290958). The deduced amino acid of Ca-ACL1 gene has signi¢cant sequence homology with the C-terminal part of known animal ACLs. Calculated molecular mass of hot pepper ACL polypeptide was 66 kDa, which is only half of the animal ACLs. The ACL catalytic center, GHAGA, and the histidine residue that is autophosphorylated by ATP during catalysis are highly conserved in Ca-ACL1, but none of the three additional phosphorylation sites (Tyr446, Ser450, Ser454) that are involved in the regulation of rat ACL activity exists [2]. To investigate the expression kinetics of the Ca-ACL1 transcripts during inoculation with X. campestris pv. glycines8ra, total RNA was isolated from hot pepper leaves 0, 4, 8, and 12 h after inoculation, and Northern blot analysis was performed by using 32P-labeled Ca-ACL1 as a probe. HR cell death on pepper leaves appeared at approximately 15 h post-in¢ltration, whereas no visible symptom was detected from the leaves in¢ltrated with buier (0.9% NaCl). Pathogenesis-related protein 4 (PR4) probe was included as a positive marker of pathogen inoculation. Approximately 2 kb mRNA corresponding for Ca-ACL1 was signi¢cantly induced 4^8 h after inoculation of X. campestris pv. glycines8ra, but not in buierin¢ltrated tissues. In the duplicated blots, PR4 transcript was only expressed in leaf tissues inoculated with the pathogen (Fig. 1A). To analyze whether induction of Ca-ACL1 mRNA level is speci¢c to incompatible plant^microbe interaction, we inoculated two nearly isogenic pepper lines, C. annuum cv. ECW2DR (BS2) and C. annuum cv. ECW (bs2) with a natural pepper pathogen X. campestris pv. vesicatoria race3 (avrBS2). In Fig. 1B, signi¢cant accumulation of the Ca-ACL1 transcript was observed in the resistant (C. annuum cv. ECW2DR) but not in the susceptible (C. annuum cv. ECW) strain. In the identical blot, a higher level of PR4 mRNA was only detected in the resistant strain. In both cases, induced levels of Ca-

NPR1 is Instrumental in Priming for the Enhanced flg22-induced MPK3 and MPK6 Activation

Pathogen-associated molecular patterns (PAMPs) activate mitogen-activated protein kinases (MAPKs), essential components of plant defense signaling. Salicylic acid (SA) is also central to plant resistance responses, but its specific role in regulation of MAPK activation is not completely defined. We have investigated the role of SA in PAMP-triggered MAPKs pathways in Arabidopsis SA-related mutants, specifically in the flg22-triggered activation of MPK3 and MPK6. cim6, sid2, and npr1 mutants exhibited wild-type-like flg22-triggered MAPKs activation, suggesting that impairment of SA signaling has no effect on the flg22-triggered MAPKs activation. Pretreatment with low concentrations of SA enhanced flg22-induced MPK3 and MPK6 activation in all seedlings except npr1, indicating that NPR1 is involved in SA-mediated priming that enhanced flg22-induced MAPKs activation.

Microarray Analysis of bacterial blight resistance 1 mutant rice infected with Xanthomonas oryzae pv. oryzae

We analyzed the transcriptional profile of the Xoo infected bbr1 mutant using a commercial rice gene chip containing 51,279 transcripts. Microarray revealed 92 genes with increased levels of expression and 22 genes with decreased levels of expression in bbr1. Some of the differentially expressed genes were validated by qRT-PCR. Higher expression of defense-related genes and AP2 domain containing transcription factors along with lower expression of reactive oxygen scavenging enzymes may be responsible for defense signaling in the bbr1 upon Xoo infection. The putative target genes of AP2 domain containing transcription factors also showed differential gene expression during Xoo infection, some of which encoded bacterial pathogen resistance-related protein. Induction of AP2 domain containing transcription factors along with up-regulation of their putative target genes during Xoo infection may inhibit pathogen spread in the bbr1. This observation supports the hypothesis that AP2 domain containing transcription factors is involved in the regulation of differentially expressed genes in bbr1.

How does SA signaling link the Flg22 responses

Salicylic acid (SA) has a central role in activating plant resistance to pathogens. SA levels increase in plant tissue following pathogen infection and exogenous SA enhances resistance to a broad range of pathogens. To study the relevance of the SA signaling in the flg22 response, we investigated the responses of SA-related mutants to flg22, a 22-amino acid peptide of the flagellin bacterial protein. We identified SA as an important component of the flg22-triggered oxidative burst, a very early event after flg22 detection, and gene induction, an early event. SA acted partially by enhancing accumulation of FLS2 mRNA. We also provide new evidence that NPR1 play a role in SA-induced priming event that enhances the flg22-triggered oxidative burst, which is correlated with enhancement of the flg22-induced callose deposition. Based on these observations, we conclude that SA signaling is required for early as well as late flg22 responses.

SlPMEI, a pollen-specific gene in tomato

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 expression in the stamen of transgenic tomato plant. These results indicate that the promoter of SlPMEI has strong pollen-specific activity, and could therefore be useful for development of industrially and agronomically important transgenic plants.