Youn-Sig Kwak

Large‐scale insertional mutagenesis using the Tnt1 retrotransposon in the model legume Medicago truncatula

Medicago truncatula is a fast-emerging model for the study of legume functional biology. We used the tobacco retrotransposon Tnt1 to tag the Medicago genome and generated over 7600 independent lines representing an estimated 190,000 insertion events. Tnt1 inserted on average at 25 different locations per genome during tissue culture, and insertions were stable during subsequent generations in soil. Analysis of 2461 Tnt1 flanking sequence tags (FSTs) revealed that Tnt1 appears to prefer gene-rich regions. The proportion of Tnt1 insertion in coding sequences was 34.1%, compared to the expected 15.9% if random insertions were to occur. However, Tnt1 showed neither unique target site specificity nor strong insertion hot spots, although some genes were more frequently tagged than others. Forward-genetic screening of 3237 R(1) lines resulted in identification of visible mutant phenotypes in approximately 30% of the regenerated lines. Tagging efficiency appears to be high, as all of the 20 mutants examined so far were found to be tagged. Taking the properties of Tnt1 into account and assuming 1.7 kb for the average M. truncatula gene size, we estimate that approximately 14,000-16,000 lines would be sufficient for 90% gene tagging coverage in M. truncatula. This is in contrast to more than 500,000 lines required to achieve the same saturation level using T-DNA tagging. Our data demonstrate that Tnt1 is an efficient insertional mutagen in M. truncatula, and could be a primary choice for other plant species with large genomes.

Accumulation of the Antibiotic Phenazine-1-Carboxylic Acid in the Rhizosphere of Dryland Cereals

ABSTRACT Natural antibiotics are thought to function in the defense, fitness, competitiveness, biocontrol activity, communication, and gene regulation of microorganisms. However, the scale and quantitative aspects of antibiotic production in natural settings are poorly understood. We addressed these fundamental questions by assessing the geographic distribution of indigenous phenazine-producing (Phz+) Pseudomonas spp. and the accumulation of the broad-spectrum antibiotic phenazine-1-carboxylic acid (PCA) in the rhizosphere of wheat grown in the low-precipitation zone (<350 mm) of the Columbia Plateau and in adjacent, higher-precipitation areas. Plants were collected from 61 commercial wheat fields located within an area of about 22,000 km2. Phz+ Pseudomonas spp. were detected in all sampled fields, with mean population sizes ranging from log 3.2 to log 7.1 g−1 (fresh weight) of roots. Linear regression analysis demonstrated a significant inverse relationship between annual precipitation and the proportion of plants colonized by Phz+ Pseudomonas spp. (r 2 = 0.36, P = 0.0001). PCA was detected at up to nanomolar concentrations in the rhizosphere of plants from 26 of 29 fields that were selected for antibiotic quantitation. There was a direct relationship between the amount of PCA extracted from the rhizosphere and the population density of Phz+ pseudomonads (r 2 = 0.46, P = 0.0006). This is the first demonstration of accumulation of significant quantities of a natural antibiotic across a terrestrial ecosystem. Our results strongly suggest that natural antibiotics can transiently accumulate in the plant rhizosphere in amounts sufficient not only for inter- and intraspecies signaling but also for the direct inhibition of sensitive organisms.

Recent insights into the diversity, frequency and ecological roles of phenazines in fluorescent Pseudomonas spp.

Phenazine compounds represent a large class of bacterial metabolites that are produced by some fluorescent Pseudomonas spp. and a few other bacterial genera. Phenazines were first noted in the scientific literature over 100 years ago, but for a long time were considered to be pigments of uncertain function. Following evidence that phenazines act as virulence factors in the opportunistic human and animal pathogen Pseudomonas aeruginosa and are actively involved in the suppression of plant pathogens, interest in these compounds has broadened to include investigations of their genetics, biosynthesis, activity as electron shuttles, and contribution to the ecology and physiology of the cells that produce them. This minireview highlights some recent and exciting insights into the diversity, frequency and ecological roles of phenazines produced by fluorescent Pseudomonas spp.

Microbial and biochemical basis of a Fusarium wilt-suppressive soil

Crops lack genetic resistance to most necrotrophic pathogens. To compensate for this disadvantage, plants recruit antagonistic members of the soil microbiome to defend their roots against pathogens and other pests. The best examples of this microbially based defense of roots are observed in disease-suppressive soils in which suppressiveness is induced by continuously growing crops that are susceptible to a pathogen, but the molecular basis of most is poorly understood. Here we report the microbial characterization of a Korean soil with specific suppressiveness to Fusarium wilt of strawberry. In this soil, an attack on strawberry roots by Fusarium oxysporum results in a response by microbial defenders, of which members of the Actinobacteria appear to have a key role. We also identify Streptomyces genes responsible for the ribosomal synthesis of a novel heat-stable antifungal thiopeptide antibiotic inhibitory to F. oxysporum and the antibiotic’s mode of action against fungal cell wall biosynthesis. Both classical- and community-oriented approaches were required to dissect this suppressive soil from the field to the molecular level, and the results highlight the role of natural antibiotics as weapons in the microbial warfare in the rhizosphere that is integral to plant health, vigor and development.

Diversity, Virulence, and 2,4-Diacetylphloroglucinol Sensitivity of Gaeumannomyces graminis var. tritici Isolates from Washington State

We determined whether isolates of the take-all pathogen Gaeumannomyces graminis var. tritici become less sensitive to 2,4-diacetylphloroglucinol (2,4-DAPG) during wheat monoculture as a result of exposure to the antibiotic over multiple growing seasons. Isolates of G. graminis var. tritici were baited from roots of native grasses collected from noncropped fields and from roots of wheat from fields with different cropping histories near Lind, Ritzville, Pullman, and Almota, WA. Isolates were characterized by using morphological traits, G. graminis variety-specific polymerase chain reaction and pathogenicity tests. The sensitivity of G. graminis var. tritici isolates to 2,4-DAPG was determined by measuring radial growth of each isolate. The 90% effective dose value was 3.1 to 4.4 microg ml(-1) for 2,4-DAPG-sensitive isolates, 4.5 to 6.1 microg ml(-1) for moderately sensitive isolates, and 6.2 to 11.1 microg ml(-1) for less sensitive isolates. Sensitivity of G. graminis var. tritici isolates to 2,4-DAPG was normally distributed in all fields and was not correlated with geographic origin or cropping history of the field. There was no correlation between virulence on wheat and geographical origin, or virulence and sensitivity to 2,4-DAPG. These results indicate that G. graminis var. tritici does not become less sensitive to 2,4-DAPG during extended wheat monoculture.

Saccharomyces cerevisiae Genome-Wide Mutant Screen for Sensitivity to 2,4-Diacetylphloroglucinol, an Antibiotic Produced by Pseudomonas fluorescens

ABSTRACT 2,4-Diacetylphloroglucinol (2,4-DAPG), an antibiotic produced by Pseudomonas fluorescens, has broad-spectrum antibiotic activity, inhibiting organisms ranging from viruses, bacteria, and fungi to higher plants and mammalian cells. The biosynthesis and regulation of 2,4-DAPG in P. fluorescens are well described, but the mode of action against target organisms is poorly understood. As a first step to elucidate the mechanism, we screened a deletion library of Saccharomyces cerevisiae in broth and agar medium supplemented with 2,4-DAPG. We identified 231 mutants that showed increased sensitivity to 2,4-DAPG under both conditions, including 22 multidrug resistance-related mutants. Three major physiological functions correlated with an increase in sensitivity to 2,4-DAPG: membrane function, reactive oxygen regulation, and cell homeostasis. Physiological studies with wild-type yeast validated the results of the mutant screens. The chemical-genetic fitness profile of 2,4-DAPG resembled those of menthol, sodium azide, and hydrogen peroxide determined in previous high-throughput screening studies. Collectively, these findings indicate that 2,4-DAPG acts on multiple basic cellular processes.

Proteomic analyses of the interaction between the plant-growth promoting rhizobacterium Paenibacillus polymyxa E681 and Arabidopsis thaliana

Plant growth‐promoting rhizobacteria (PGPR) facilitate the plant growth and enhance their induced systemic resistance (ISR) against a variety of environmental stresses. In this study, we carried out integrative analyses on the proteome, transcriptome, and metabolome to investigate Arabidopsis root and shoot responses to the well‐known PGPR strain Paenibacillus polymyxa (P. polymyxa) E681. Shoot fresh and root dry weights were increased, whereas root length was decreased by treatment with P. polymyxa E681. 2DE approach in conjunction with MALDI‐TOF/TOF analysis revealed a total of 41 (17 spots in root, 24 spots in shoot) that were differentially expressed in response to P. polymyxa E681. Biological process‐ and molecular function‐based bioinformatics analysis resulted in their classification into seven different protein groups. Of these, 36 proteins including amino acid metabolism, antioxidant, defense and stress response, photosynthesis, and plant hormone‐related proteins were up‐regulated, whereas five proteins including three carbohydrate metabolism‐ and one amino acid metabolism‐related, and one unknown protein were down‐regulated, respectively. A good correlation was observed between protein and transcript abundances for the 12 differentially expressed proteins during interactions as determined by qPCR analysis. Metabolite analysis using LC‐MS/MS revealed highly increased levels of tryptophan, indole‐3‐acetonitrile (IAN), indole‐3‐acetic acid (IAA), and camalexin in the treated plants. Arabidopsis plant inoculated P. polymyxa E681 also showed resistance to Botrytis cinerea infection. Taken together these results suggest that P. polymyxa E681 may promote plant growth by induced metabolism and activation of defense‐related proteins against fungal pathogen.

Proteomic analysis of Rhizoctonia solani AG-1 sclerotia maturation

Rhizoctonia solani (R. solani), a soil-borne necrotrophic pathogen, causes various plant diseases. Rhizoctonia solani is a mitosporic fungus, the sclerotium of which is the primary inoculum and ensures survival of the fungus during the offseason of the host crop. Since the fungus does not produce any asexual or sexual spores, understanding the biology of sclerotia is important to examine pathogen ecology and develop more efficient methods for crop protection. Here, one- and two-dimensional gel electrophoresis (1-DE and 2-DE, respectively) were used to examine protein regulation during the maturation of fungal sclerotia. A total of 75 proteins (20 proteins from 1-DE using matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry (MS) and 55 proteins from 2-DE using MALDI-TOF MS or MALDI-TOF/TOF MS) were differentially expressed during sclerotial maturation. The identified proteins were classified into ten categories based on their biological functions, including genetic information processing, carbohydrate metabolism, cell defense, amino acid metabolism, nucleotide metabolism, cellular processes, pathogenicity and mycotoxin production, and hypothetical or unknown functions. Interestingly, two vacuole function-related proteins were highly up-regulated throughout sclerotial maturation, which was confirmed at the transcript level by reverse transcriptase polymerase chain reaction (RT-PCR) analysis. These findings contribute to our understanding of the biology of R. solani sclerotia.

A Detail Investigation of Major Diseases Occurrence and Pathogen Population on Turfgrass Cultivation in Nationwide

We investigated turfgrass diseases and inoculum density at nationwide turfgrass cultivation sites in year of 2013. Occurrences of large patch and rust disease appeared in September. Brown patch recorded in September to October at Namhea and Pythium blight disease occurred outbreaks in early July at Namhea site. Some sites in Namhea damaged 3% area of total cultivation field by Sclerotinia homoeocarp. In Daepyeong (Gyeongnam), Fairy ring and large patch were recorded. Severe takeall and fairy ring have been observed in Gochang-si. Multi-site in Cheongju-si, brown patch was observed in pandemic level. Interesting enough, a cool-season turfgrass cultivate sites in Pyeongtaek-si brown patch, leaf blast, summer patch, and Curvularia leaf spot were investigated during the surveys period. Inoculum densities (Rhizoctonia spp.) at turfgrass cultivations sites were increased as cumulatively in all survey sites. The investigation result indicated that the disease occurrence and pathogens are similar as diseases in golf courses.

Functional characterization of orchardgrass cytosolic Hsp70 (DgHsp70) and the negative regulation by Ca2+/AtCaM2 binding

When plants are exposed to extreme temperature, stress-inducible proteins are highly induced and involved in subcellular defence mechanisms. Hsp70, one of stress-inducible proteins, functions as an ATP-dependent molecular chaperone in broad organisms to process such as the inhibition of protein denaturation, promotion of protein folding, and renaturation of denatured proteins. In this study, we isolated a heat-inducible orchardgrass Hsp70 (DgHsp70) that is a homolog of cytosolic Hsp70 that possesses a CaM-binding domain. Purified DgHsp70 protein displayed dose-dependent ATPase, holdase, and ATP-dependent foldase activities. To investigate functional roles of DgHsp70 by the association of Arabidopsis calmodulin-2 (AtCaM2), showing heat-sensitive reduction on transcription, we first characterized the binding activity by gel-overlay assay. DgHsp70 binds to AtCaM2 in the presence of Ca(2+) via a conserved CaM-binding domain. Ca(2+)/AtCaM2 binding decreased ATPase activity of DgHsp70, and concomitantly, reduced foldase activity. Based on the protein structure of bovine Hsc70, which is the closest structural homolog of DgHsp70, a CaM-binding domain is located near the ATP-binding site and CaM may span the ATP-binding pocket of Hsp70. Its decreased functional foldase activity may be caused by blocking ATP hydrolysis after Ca(2+)/AtCaM2 binding. It may associate with inhibition of functional activity of DgHsp70 in the absence of stress and/or de novo protein synthesis of DgHsp70 in the presence of thermal stress condition.