split-green-fluorescent-protein-system-to-visualize-effectors

Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the ...

Seoul National University

Ascorbic acid (1), a natural antioxidant, was modified by employing transglycosylation activity of Bacillus stearothermophilus maltogenic amylase with maltotriose and acarbose as donor molecules to enhance its oxidative stability. The transglycosylation reaction with maltotriose as donor created mono- and di-glycosyl transfer products with an alpha-(1,6)-glycosidic linkage. In addition, two acarviosine-glucosyl transfer products were generated when transglycosylation was performed with acarbose as a donor. All transfer products were observed by TLC and HPLC, and purified by Q-sepharose anion exchange and Biogel P-2 gel permeation chromatographies. LC/MS and (13)C NMR analyses revealed that the structures of the transfer products were 6-O-alpha-D-glucosyl- (2) and 6-O-alpha-D-maltosyl-ascorbic acids (3) in the reaction of maltotriose, and 6-O-alpha-acarviosine-D-glucosyl- (4) and 2-O-alpha-acarviosine-D-glucosyl ascorbic acids (5) in the reaction of acarbose. The stability of the transglycosylated ascorbic acid derivatives was greatly enhanced against oxidation by Cu(2+) ion and ascorbate oxidase. Among them, compound 3 proved to be the most stable against in vitro oxidation. The antioxidant effects of glycosyl-derivatives of ascorbic acid on the lipid oxidation in cooked chicken breast meat patties indicated that they had antioxidant activities similar to that of ascorbic acid. It is suggested that the transglycosylated ascorbic acids can possibly be applied as effective antioxidants with improved stability in food, cosmetic, and other applications.

Modification of ascorbic acid using transglycosylation activity of Bacillus stearothermophilus maltogenic amylase to enhance its oxidative stability.

This study was done to modify erythritol to change its physicochemical and sensory properties. Erythritol, a four-carbon sugar alcohol, was transglycosylated by Bacillus stearothermophilus maltogenic amylase with maltotriose as a donor molecule. The presence of various transglycosylation products of erythritol was confirmed by TLC and high performance ion exchange chromatography (HPIC). The major transfer product was purified by gel filtration chromatography on Bio-Gel P-2. Examination by LC-MS, matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF-MS), and 13C NMR showed that the major transfer product was maltosyl-erythritol. Results of 13C NMR of maltosyl-erythritol suggested that linkage was formed between the C1 carbon of glucose unit in maltose and either one of the two carbon atoms of the terminal hydroxyl groups of erythritol, so that a mixture of 1-O- and 4-O-alpha-maltosyl-erythritol was produced. The sweetness of maltosyl-erythritol was about 40% that of sucrose, and its negative sensory properties were less than those of erythritol.

Maltosyl-erythritol, a major transglycosylation product of erythritol by Bacillus stearothermophilus maltogenic amylase.

Formaldehyde (FA) is classified as a human carcinogen and has been linked to increased leukemia rates in some epidemiologic studies. Inhalation of FA induces sensory irritation at relatively low concentrations. However, little is known concerning the cellular alterations observed after FA exposure in humans.

Identification of gene markers for formaldehyde exposure in humans.

We investigated whether a combination of recently introduced methods, total demineralization and ion-exchange columns, would increase DNA recovery from old bone. Ten bone samples taken after a burial period of approximately 60 years were used in this study. Bone powder was digested using total or incomplete demineralization. DNA was extracted by the standard organic method. The DNA extract was purified with ion-exchange columns or QIAquick spin columns. The efficiency of different DNA extraction methods was compared in terms of DNA concentration, inhibitors generated by real-time PCR, and conventional STR typing results. The mean DNA concentration using the total demineralization method is approximately 3 times higher than that using the incomplete demineralization method. For DNA purification, the method using QIAquick spin columns appeared to yield approximately double the DNA than the method using ion-exchange columns. Furthermore, 2 out of 10 samples showed higher levels of inhibition with C(T) values of IPC > or =30 cycles when using only ion-exchange columns. In STR results, total demineralization yielded more locus profiles by 4.2 loci than incomplete demineralization, and QIAquick spin columns also yielded more locus profiles by 3.5 loci than ion-exchange columns. Total demineralization of bone powder significantly increased DNA yield and improved STR typing results. However, the use of ion-exchange columns was not efficient when compared with the method using QIAquick spin columns. It is suggested that the combination of total demineralization and QIAquick spin columns lead to greatly improved STR typing results.

Technical note: Efficiency of total demineralization and ion-exchange column for DNA extraction from bone.

Correct DNA quantification is an essential part to obtain reliable STR typing results. Forensic DNA analysts often use commercial kits for DNA quantification; among them, real-time-based DNA quantification kits are most frequently used. Incorrect DNA quantification due to the presence of PCR inhibitors may affect experiment results. In this study, we examined the alteration degree of DNA quantification results estimated in DNA samples containing a PCR inhibitor by using a Quantifiler® Human DNA Quantification kit. For experiments, we prepared approximately 0.25 ng/μl DNA samples containing various concentrations of humic acid (HA). The quantification results were 0.194-0.303 ng/μl at 0-1.6 ng/μl HA (final concentration in the Quantifiler reaction) and 0.003-0.168 ng/μl at 2.4-4.0 ng/μl HA. Most DNA quantity was undetermined when HA concentration was higher than 4.8 ng/μl HA. The C                 (T) values of an internal PCR control (IPC) were 28.0-31.0, 36.5-37.1, and undetermined at 0-1.6, 2.4, and 3.2 ng/μl HA. These results indicate that underestimated DNA quantification results may be obtained in the DNA sample with high C                 (T) values of IPC. Thus, researchers should carefully interpret the DNA quantification results. We additionally examined the effects of HA on the STR amplification by using an Identifiler® kit and a MiniFiler™ kit. Based on the results of this study, it is thought that a better understanding of various effects of HA would help researchers recognize and manipulate samples containing HA.

Effects of humic acid on DNA quantification with Quantifiler® Human DNA Quantification kit and short tandem repeat amplification efficiency.

We investigated several methods for obtaining successful short tandem repeat (STR) results from high-humic acid (HA)-content samples. DNA purification efficiency was tested for QIAquick(®) PCR Purification, QIAamp(®) DNA Investigator and Prepfiler™ Forensic DNA Extraction kits. HA-removal capacity of Inhibitor Remover and InhibitEX(®) Tablet was tested. Experiments on overcoming HA effects on STR amplification were conducted using an AmpliTaq Gold(®) DNA Polymerase and a TaKaRa Ex Taq™ Hot Start Version (Ex Taq HS) with BSA addition. QIAquick kit was most efficient in HA removal and Ex Taq HS showed high resistance to HA. Increasing the amounts of Taq polymerases and BSA addition were shown to be efficient in overcoming PCR inhibition, but BSA addition was superior to the former method. Inhibitor Remover and InhibitEX(®) Tablet did not positively affect the STR results. This study will help achieve better STR results with high-HA-content samples. 

Improvement of short tandem repeat analysis of samples highly contaminated by humic acid.

Hot pepper (Capsicum annuum), one of the oldest domesticated crops in the Americas, is the most widely grown spice crop in the world. We report whole-genome sequencing and assembly of the hot pepper (Mexican landrace of Capsicum annuum cv. CM334) at 186.6× coverage. We also report resequencing of two cultivated peppers and de novo sequencing of the wild species Capsicum chinense. The genome size of the hot pepper was approximately fourfold larger than that of its close relative tomato, and the genome showed an accumulation of Gypsy and Caulimoviridae family elements. Integrative genomic and transcriptomic analyses suggested that change in gene expression and neofunctionalization of capsaicin synthase have shaped capsaicinoid biosynthesis. We found differential molecular patterns of ripening regulators and ethylene synthesis in hot pepper and tomato. The reference genome will serve as a platform for improving the nutritional and medicinal values of Capsicum species. 

Genome sequence of the hot pepper provides insights into the evolution of pungency in Capsicum species.

The estimation of age from biological samples (i.e., remains) at crime scenes could provide useful information about both victims and other persons related to criminal activities. Signal-joint T cell receptor excision circle (sjTREC) levels in peripheral blood decline with age, and negative correlations between sjTREC levels and age have been demonstrated in several ethnic groups. To validate the utility of sjTREC for age estimation in Koreans, Taqman qPCR was used to quantify the sjTREC level in samples obtained from 172 individuals ranging from 16 to 65 years old. We modified the previously reported method by using a shorter amplicon and confirmed the efficiency and utility of this method in this report. Our results showed that the linear negative regression curve between sjTREC levels and age was characterized by r=-0.807 and a standard error of 8.49 years. These results indicate that sjTREC level is an effective age estimation method in Koreans. The value of the standard error of quantification was not different from previous reports for other population groups.

Age estimation via quantification of signal-joint T cell receptor excision circles in Koreans.

Cryptochromes are flavoproteins that play a central role in the circadian oscillations of all living organisms except archaea. Cryptochromes are clustered into three subfamilies: plant-type cryptochromes, animal-type cryptochromes and cryptochrome-DASH proteins. These subfamilies are composed of photolyase/cryptochrome superfamily with 6-4 photolyase and cyclobutane pyrimidine dimer photolyase. Cryptochromes have conserved domain architectures with two distinct domains, an N-terminal photolyase-related domain and a C-terminal domain. Although the molecular function and domain architecture of cryptochromes are conserved, their molecular mechanisms differ between plants and animals. Thus, cryptochromes are one of the best candidates for comparative and evolutionary studies. Here, we have developed a Web-based platform for comparative and evolutionary studies of cryptochromes, dbCRY (http://www.dbcryptochrome.org/). A pipeline built upon the consensus domain profile was applied to 1438 genomes and identified 1309 genes. To support comparative and evolutionary genomics studies, the Web interface provides diverse functions such as (i) browsing by species, (ii) protein domain analysis, (iii) multiple sequence alignment, (iv) homology search and (v) extended analysis opportunities through the implementation of 'Favorite Browser' powered by the Comparative Fungal Genomics Platform 2.0 (CFGP 2.0; http://cfgp.snu.ac.kr/). dbCRY would serve as a standardized and systematic solution for cryptochrome genomics studies. Database URL: http://www.dbcryptochrome.org/ 

dbCRY: a Web-based comparative and evolutionary genomics platform for blue-light receptors.

Potyviruses are one of the most destructive viral pathogens of Solanaceae plants. In Capsicum annuum landrace CM334, a broad-spectrum gene, Pvr4 is known to be involved in resistance against multiple potyviruses, including Pepper mottle virus (PepMoV), Pepper severe mosaic virus (PepSMV), and Potato virus Y (PVY). However, a potyvirus avirulence factor against Pvr4 has not been identified. To identify the avirulence factor corresponding to Pvr4 in potyviruses, we performed Agrobacterium-mediated transient expressions of potyvirus protein coding regions in potyvirus-resistant (Pvr4) and -susceptible (pvr4) pepper plants. Hypersensitive response (HR) was observed only when a RNA-dependent RNA polymerase (NIb) of PepMoV, PepSMV, or PVY was expressed in Pvr4-bearing pepper leaves in a genotype-specific manner. In contrast, HR was not observed when the NIb of Tobacco etch virus (TEV), a virulent potyvirus, was expressed in Pvr4-bearing pepper leaves. Our results clearly demonstrate that NIbs of PepMoV, PepSMV, and PVY serve as avirulence factors for Pvr4 in pepper plants. 

RNA-dependent RNA polymerase (NIb) of the potyviruses is an avirulence factor for the broad-spectrum resistance gene Pvr4 in Capsicum annuum cv. CM334.

Nonhost resistance, a resistance of plant species against all nonadapted pathogens, is considered the most durable and efficient immune system of plants but yet remains elusive. The underlying mechanism of nonhost resistance has been investigated at multiple levels of plant defense for several decades. In this review, we have comprehensively surveyed the latest literature on nonhost resistance in terms of preinvasion, metabolic defense, pattern-triggered immunity, effector-triggered immunity, defense signaling, and possible application in crop protection. Overall, we summarize the current understanding of nonhost resistance mechanisms. Pre- and postinvasion is not much deviated from the knowledge on host resistance, except for a few specific cases. Further insights on the roles of the pattern recognition receptor gene family, multiple interactions between effectors from nonadapted pathogen and plant factors, and plant secondary metabolites in host range determination could expand our knowledge on nonhost resistance and provide efficient tools for future crop protection using combinational biotechnology approaches. [Formula: see text] Copyright © 2017 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Current Understandings of Plant Nonhost Resistance.

Pathogenic gram-negative bacteria cause serious diseases in animals and plants. These bacterial pathogens use the type III secretion system (T3SS) to deliver effector proteins into host cells; these effectors then localize to different subcellular compartments to attenuate immune responses by altering biological processes of the host cells. The fluorescent protein (FP)-based approach to monitor effectors secreted from bacteria into the host cells is not possible because the folded FP prevents effector delivery through the T3SS Therefore, we optimized an improved variant of self-assembling split super-folder green fluorescent protein (sfGFP) system to investigate the spatiotemporal dynamics of effectors delivered through bacterial T3SS into plant cells. In this system, effectors are fused to 11th β-strand of super-folder GFP (sfGFP11), and when delivered into plant cells expressing sfGFP1-10 β-strand (sfGFP1-10), the two proteins reconstitute GFP fluorescence. We generated a number of  transgenic lines expressing sfGFP1-10 targeted to various subcellular compartments to facilitate localization of sfGFP11-tagged effectors delivered from bacteria. We demonstrate the efficacy of this system using  effectors AvrB and AvrRps4 in  and transgenic Arabidopsis plants. The versatile split sfGFP system described here will facilitate a better understanding of bacterial invasion strategies used to evade plant immune responses.

Spatiotemporal Monitoring of  Effectors via Type III Secretion Using Split Fluorescent Protein Fragments.

Transposable elements are major evolutionary forces which can cause new genome structure and species diversification. The role of transposable elements in the expansion of nucleotide-binding and leucine-rich-repeat proteins (NLRs), the major disease-resistance gene families, has been unexplored in plants.

New reference genome sequences of hot pepper reveal the massive evolution of plant disease-resistance genes by retroduplication.

The pepper Pvr4 encoding coiled-coil (CC) nucleotide-binding (NB) leucine-rich repeat (LRR) protein (CC-NLR) confer hypersensitive resistance (HR) to potyviruses, including Pepper mottle virus (PepMoV) by recognizing the viral avirulence protein, NIb. To figure out the Pvr4-mediated HR mechanism, we analyzed the signaling component genes and structure-function relationships of Pvr4 using chimeras and deletion mutants in Nicotiana benthamiana. Molecular chaperone components including HSP90, SGT1 and RAR1 were required, while plant hormones and mitogen-activated protein (MAP) kinase signaling components had little effect on Pvr4-NIb-mediated HR cell death. Domain swap analyses indicated that the LRR domain of Pvr4 determines recognition of PepMoV-NIb. Our deletion analysis further revealed that CC domain or CC-NBARC domain alone can trigger autoactive cell death in N. benthamiana. However, the fragments having only LRR domain could suppress CC-NBARC domain-induced cell death in trans. Further, C-terminal truncation analysis of Pvr4 revealed that minimum three out of five LRR exons showing high similarity was essential for Pvr4 function. The LRR domain may maintain Pvr4 in an inactive state in the absence of NIb. These results provide further insight into the structure and function of NLR protein signaling in plants.

Article	Year
Modification of ascorbic acid using transglycosylation activity of Bacillus stearothermophilus maltogenic amylase to enhance its oxidative stability. Journal of agricultural and food chemistry\| PubMed ID: 12010003	2002
Maltosyl-erythritol, a major transglycosylation product of erythritol by Bacillus stearothermophilus maltogenic amylase. Bioscience, biotechnology, and biochemistry\| PubMed ID: 12723599	2003
Identification of gene markers for formaldehyde exposure in humans. Environmental health perspectives\| PubMed ID: 17938736	2007
Technical note: Efficiency of total demineralization and ion-exchange column for DNA extraction from bone. American journal of physical anthropology\| PubMed ID: 19845025	2010
Effects of humic acid on DNA quantification with Quantifiler® Human DNA Quantification kit and short tandem repeat amplification efficiency. International journal of legal medicine\| PubMed ID: 21912926	2012
Improvement of short tandem repeat analysis of samples highly contaminated by humic acid. Journal of forensic and legal medicine\| PubMed ID: 24112347	2013
Genome sequence of the hot pepper provides insights into the evolution of pungency in Capsicum species. Nature genetics\| PubMed ID: 24441736	2014
Age estimation via quantification of signal-joint T cell receptor excision circles in Koreans. Legal medicine (Tokyo, Japan)\| PubMed ID: 24524944	2014
dbCRY: a Web-based comparative and evolutionary genomics platform for blue-light receptors. Database : the journal of biological databases and curation\| PubMed ID: 24816342	2014
RNA-dependent RNA polymerase (NIb) of the potyviruses is an avirulence factor for the broad-spectrum resistance gene Pvr4 in Capsicum annuum cv. CM334. PloS one\| PubMed ID: 25760376	2015
Current Understandings of Plant Nonhost Resistance. Molecular plant-microbe interactions : MPMI\| PubMed ID: 27925500	2017
Spatiotemporal Monitoring of Effectors via Type III Secretion Using Split Fluorescent Protein Fragments. The Plant cell\| PubMed ID: 28619883	2017
New reference genome sequences of hot pepper reveal the massive evolution of plant disease-resistance genes by retroduplication. Genome biology\| PubMed ID: 29089032	2017
Coiled-coil and leucine-rich repeat domain of the potyvirus resistance protein, Pvr4, has distinct role in signaling and pathogen recognition. Molecular plant-microbe interactions : MPMI\| PubMed ID: 29663867	2018

Hye-Young Lee

Biography

JoVE Journal Publications

Other Publications