Genome-wide analysis-based single nucleotide polymorphism marker sets to identify diverse genotypes in cabbage cultivars (Brassica oleracea var. capitata).

Plant variety protection is essential for breeders' rights granted by the International Union for the Protection of New Varieties of Plants. Distinctness, uniformity, and stability (DUS) are necessary for new variety registration; to this end, currently, morphological traits are examined, which is time-consuming and laborious. Molecular markers are more effective, accurate, and stable descriptors of DUS. Advancements in next-generation sequencing technology have facilitated genome-wide identification of single nucleotide polymorphisms. Here, we developed a core set of single nucleotide polymorphism markers to identify cabbage varieties and traits of test guidance through clustering using the Fluidigm assay, a high-throughput genotyping system. Core sets of 87, 24, and 10 markers are selected based on a genome-wide association-based approach. All core markers could identify 94 cabbage varieties and determine 17 DUS traits. A genotypes database was validated using the Fluidigm platform for variety identification, population structure analysis, cabbage breeding, and DUS testing for plant cultivar protection.

Identification of Plant Viruses Infecting Pear Using RNA Sequencing.

Asian pear (Pyrus pyrifolia) is a widely cultivated and commercially important fruit crop, which is occasionally subject to severe economic losses due to latent viral infections. Thus, the aim of the present study was to examine and provide a comprehensive overview of virus populations infecting a major pear cultivar ('Singo') in Korea. From June 2017 to October 2019, leaf samples (n = 110) of pear trees from 35 orchards in five major pear-producing regions were collected and subjected to RNA sequencing. Most virus-associated contigs matched the sequences of known viruses, including apple stem grooving virus (ASGV) and apple stem pitting virus (ASPV). However, some contigs matched the sequences of apple green crinkle-associated virus and cucumber mosaic virus. In addition, three complete or nearly complete genomes were constructed based on transcriptome data and subjected to phylogenetic analyses. Based on the number of virus-associated reads, ASGV and ASPV were identified as the dominant viruses of 'Singo.' The present study describes the virome of a major pear cultivar in Korea, and looks into the diversity of viral communities in this cultivar. This study can provide valuable information on the complexity of genetic variability of viruses infecting pear trees.

Characterization of Type VI Secretion System in Xanthomonas oryzae pv. oryzae and Its Role in Virulence to Rice.

Type VI secretion system (T6SS) is a contact-dependent secretion system, employed by most gram-negative bacteria for translocating effector proteins to target cells. The present study was conducted to investigate T6SS in Xanthomonas oryzae pv. oryzae (Xoo), which causes bacterial blight in rice, and to unveil its functions. Two T6SS clusters were found in the genome of Xoo PXO99A. The deletion mutants, Δhcp1, Δhcp2, and Δhcp12, targeting the hcp gene in each cluster, and a double-deletion mutant targeting both genes were constructed and tested for growth rate, pathogenicity to rice, and inter-bacterial competition ability. The results indicated that hcp in T6SS-2, but not T6SS-1, was involved in bacterial virulence to rice plants. However, neither T6SS-1 nor T6SS-2 had any effect on the ability to compete with Escherichia coli or other bacterial cells. In conclusion, T6SS gene clusters in Xoo have been characterized, and its role in virulence to rice was confirmed.

Unraveling the role of quorum sensing-dependent metabolic homeostasis of the activated methyl cycle in a cooperative population of Burkholderia glumae.

The activated methyl cycle (AMC) is responsible for the generation of S-adenosylmethionine (SAM), which is a substrate of N-acylhomoserine lactone (AHL) synthases. However, it is unknown whether AHL-mediated quorum sensing (QS) plays a role in the metabolic flux of the AMC to ensure cell density-dependent biosynthesis of AHL in cooperative populations. Here we show that QS controls metabolic homeostasis of the AMC critical for AHL biosynthesis and cellular methylation in Burkholderia glumae, the causal agent of rice panicle blight. Activation of genes encoding SAM-dependent methyltransferases, S-adenosylhomocysteine (SAH) hydrolase, and methionine synthases involved in the AMC by QS is essential for maintaining the optimal concentrations of methionine, SAM, and SAH required for bacterial cooperativity as cell density increases. Thus, the absence of QS perturbed metabolic homeostasis of the AMC and caused pleiotropic phenotypes in B. glumae. A null mutation in the SAH hydrolase gene negatively affected AHL and ATP biosynthesis and the activity of SAM-dependent methyltransferases including ToxA, which is responsible for the biosynthesis of a key virulence factor toxoflavin in B. glumae. These results indicate that QS controls metabolic flux of the AMC to secure the biosynthesis of AHL and cellular methylation in a cooperative population.

Rapid and Specific Detection of Apple stem grooving virus by Reverse Transcription-recombinase Polymerase Amplification.

Apple stem grooving virus (ASGV) is considered to cause the most economically important viral disease in pears in Korea. The current PCR-based methods used to diagnose ASGV are time-consuming in terms of target detection. In this study, a novel assay for specific ASGV detection that is based on reverse transcription-recombinase polymerase amplification is described. This assay has been shown to be reproducible and able to detect as little as 4.7 ng/µl of purified RNA obtained from an ASGV-infected plant. The major advantage of this assay is that the reaction for the target virus is completed in 1 min, and amplification only requires an incubation temperature of 42°C. This assay is a promising alternative method for pear breeding programs or virus-free certification laboratories.

Complete Genome Sequence of Ornithogalum Mosaic Virus Infecting Gladiolus spp. in South Korea.

We report here the first complete genome sequence of Ornithogalum mosaic virus (OrMV) isolated from Taean, South Korea, in 2011, which was obtained by next-generation sequencing and Sanger sequencing. The sequence information provided here may serve as a potential reference for other OrMV isolates.

Bacterial quorum sensing and metabolic slowing in a cooperative population.

Acyl-homoserine lactone (AHL)-mediated quorum sensing (QS) controls the production of numerous intra- and extracellular products across many species of Proteobacteria. Although these cooperative activities are often costly at an individual level, they provide significant benefits to the group. Other potential roles for QS include the restriction of nutrient acquisition and maintenance of metabolic homeostasis of individual cells in a crowded but cooperative population. Under crowded conditions, QS may function to modulate and coordinate nutrient utilization and the homeostatic primary metabolism of individual cells. Here, we show that QS down-regulates glucose uptake, substrate level and oxidative phosphorylation, and de novo nucleotide biosynthesis via the activity of the QS-dependent transcriptional regulator QsmR (quorum sensing master regulator R) in the rice pathogen Burkholderia glumae. Systematic analysis of glucose uptake and core primary metabolite levels showed that QS deficiency perturbed nutrient acquisition, and energy and nucleotide metabolism, of individuals within the group. The QS mutants grew more rapidly than the wild type at the early exponential stage and outcompeted wild-type cells in coculture. Metabolic slowing of individuals in a QS-dependent manner indicates that QS acts as a metabolic brake on individuals when cells begin to mass, implying a mechanism by which AHL-mediated QS might have evolved to ensure homeostasis of the primary metabolism of individuals under crowded conditions.

A simple and sensitive biosensor strain for detecting toxoflavin using ß-galactosidase activity.

In this study, we developed a simple and sensitive biosensor for the determination of toxoflavin (which is toxic to various plants, fungi, animals, and bacteria) in natural samples based on ß-galactosidase activity. The proposed toxoflavin detection method for toxin-producing bacteria or toxin-contaminated foods is simple and cost effective. Burkholderia glumae, a species known to cause rice grain rot and wilt in various field crops, produces toxoflavin under the control of a LysR-type transcriptional regulator ToxR and its ligand toxoflavin. As the expression of toxoflavin biosynthetic genes requires toxoflavin as a co-activator of ToxR, a novel biosensor stain was constructed based on lacZ reporter gene integration into the first gene of the toxoflavin biosynthesis operon, toxABCDE of B. glumae. The biosensor was composed of a sensor strain (COK71), substrates (X-gal or ONPG), and culture medium, without any complex preparation process. We demonstrated that the biosensor strain is highly specific to toxoflavin, and can quantify relative amounts of toxoflavin compared with known concentrations of toxoflavin. The proposed method was reliable and simple; samples containing 50-500 nM of toxoflavin could be analyzed. More importantly, the proposed biosensor strain could identify toxoflavin-producing bacteria in real samples. The excellent performance of this biosensor is useful for diagnostic purposes, such as detecting toxoflavin-contaminated foods and environmental samples.

Bacterial quorum sensing, cooperativity, and anticipation of stationary-phase stress.

Acyl-homoserine lactone-mediated quorum sensing (QS) regulates diverse activities in many species of Proteobacteria. QS-controlled genes commonly code for production of secreted or excreted public goods. The acyl-homoserine lactones are synthesized by members of the LuxI signal synthase family and are detected by cognate members of the LuxR family of transcriptional regulators. QS affords a means of population density-dependent gene regulation. Control of public goods via QS provides a fitness benefit. Another potential role for QS is to anticipate overcrowding. As population density increases and stationary phase approaches, QS might induce functions important for existence in stationary phase. Here we provide evidence that in three related species of the genus Burkholderia QS allows individuals to anticipate and survive stationary-phase stress. Survival requires QS-dependent activation of cellular enzymes required for production of excreted oxalate, which serves to counteract ammonia-mediated alkaline toxicity during stationary phase. Our findings provide an example of QS serving as a means to anticipate stationary phase or life at the carrying capacity of a population by activating the expression of cytoplasmic enzymes, altering cellular metabolism, and producing a shared resource or public good, oxalate.