Determination of Fire Blight Susceptibility on Wild Rosaceae Plants in Korea by Artificial Inoculation

The fire blight caused by Erwinia amylovora (Ea) is a devastating disease of Rosaceae plants, including commercially important apple and pear trees. Since the first report in Korea in May 2015, it has been spreading to neighboring regions gradually. Host plants can be infected by pollinators like bees, rainfall accompanied by wind, and cultural practices such as pruning. Many studies have revealed that wild Rosaceae plants such as Cotoneaster spp., Crataegus spp., Pyracantha spp., Prunus spp., and Sorbus spp. can be reservoirs of Ea in nature. However, wild Rosaceae plants in Korea have not been examined yet whether they are susceptible to fire blight. Therefore, the susceptibility to fire blight was examined with 25 species in 10 genera of wild Rosaceae plants, which were collected during 2020–2022, by artificial inoculation. Bacterial suspension (108 cfu/ml) of Ea type strain TS3128 was inoculated artificially in flowers, leaves, stems, and fruits of each plant species, and development of disease symptoms were monitored. Moreover, the presence of Ea bacteria from inoculated samples were checked by conventional polymerase chain reaction. Total 14 species of wild Rosaceae plants showed disease symptoms of fire blight, and Ea bacteria were detected inside of inoculated plant parts. These results suggest that wild Rosaceae plants growing nearby commercial apple and pear orchards in Korea can be Ea reservoirs, and thus they should be monitored regularly to minimize the damage by Ea infection and spreading

Combining ChIP-chip and Expression Profiling to Model the MoCRZ1 Mediated Circuit for Ca2+/Calcineurin Signaling in the Rice Blast Fungus

Significant progress has been made in defining the central signaling networks in many organisms, but collectively we know little about the downstream targets of these networks and the genes they regulate. To reconstruct the regulatory circuit of calcineurin signal transduction via MoCRZ1, a Magnaporthe oryzae C2H2 transcription factor activated by calcineurin dephosphorylation, we used a combined approach of chromatin immunoprecipitation - chip (ChIP-chip), coupled with microarray expression studies. One hundred forty genes were identified as being both a direct target of MoCRZ1 and having expression concurrently differentially regulated in a calcium/calcineurin/MoCRZ1 dependent manner. Highly represented were genes involved in calcium signaling, small molecule transport, ion homeostasis, cell wall synthesis/maintenance, and fungal virulence. Of particular note, genes involved in vesicle mediated secretion necessary for establishing host associations, were also found. MoCRZ1 itself was a target, suggesting a previously unreported autoregulation control point. The data also implicated a previously unreported feedback regulation mechanism of calcineurin activity. We propose that calcium/calcineurin regulated signal transduction circuits controlling development and pathogenicity manifest through multiple layers of regulation. We present results from the ChIP-chip and expression analysis along with a refined model of calcium/calcineurin signaling in this important plant pathogen

Isolation and Characterization of the Genes Involved in the Berberine Synthesis Pathway in Asian Blue Cohosh, <i>Caulophyllum robustum</i>

Caulophyllum robustum, commonly named Asian blue cohosh, is a perennial herb in the family Berberidaceae. It has traditionally been used for folk medicine in China. We isolated berberine from the leaves, stem, roots, and fruits of C. robustum, and this is the first report on berberine in this species. Transcriptome analysis was conducted for the characterization of berberine biosynthesis genes in C. robustum, in which, all the genes for berberine biosynthesis were identified. From 40,094 transcripts, using gene ontology (GO) analysis, 26,750 transcripts were assigned their functions in the categories of biological process, molecular function, and cellular component. In the analysis of genes expressed in different tissues, the numbers of genes in the categories of intrinsic component of membrane and transferase activity were up-regulated in leaves versus stem. The berberine synthesis genes in C. robustum were characterized by phylogenetic analysis with corresponding genes from other berberine-producing species. The co-existence of genes from different plant families in the deepest branch subclade implies that the differentiation of berberine synthesis genes occurred early in the evolution of berberine-producing plants. Furthermore, the copy number increment of the berberine synthesis genes was detected at the species level

Dynamic Chloroplast Genome Rearrangement and DNA Barcoding for Three Apiaceae Species Known as the Medicinal Herb “Bang-Poong”

Three Apiaceae species Ledebouriella seseloides, Peucedanum japonicum, and Glehnia littoralis are used as Asian herbal medicines, with the confusingly similar common name &#8220;Bang-poong&#8221;. We characterized the complete chloroplast (cp) genomes and 45S nuclear ribosomal DNA (45S nrDNA) sequences of two accessions for each species. The complete cp genomes of G. littoralis, L. seseloides, and P. japonicum were 147,467, 147,830, and 164,633 bp, respectively. Compared to the other species, the P. japonicum cp genome had a huge inverted repeat expansion and a segmental inversion. The 45S nrDNA cistron sequences of the three species were almost identical in size and structure. Despite the structural variation in the P. japonicum cp genome, phylogenetic analysis revealed that G. littoralis diverged 5&#8722;6 million years ago (Mya), while P. japonicum diverged from L. seseloides only 2&#8722;3 Mya. Abundant copy number variations including tandem repeats, insertion/deletions, and single nucleotide polymorphisms, were found at the interspecies level. Intraspecies-level polymorphism was also found for L. seseloides and G. littoralis. We developed nine PCR barcode markers to authenticate all three species. This study characterizes the genomic differences between L. seseloides, P. japonicum, and G. littoralis; provides a method of species identification; and sheds light on the evolutionary history of these three species

Lichen Secondary Metabolites in <i>Flavocetraria cucullata</i> Exhibit Anti-Cancer Effects on Human Cancer Cells through the Induction of Apoptosis and Suppression of Tumorigenic Potentials

<div><p>Lichens are symbiotic organisms which produce distinct secondary metabolic products. In the present study, we tested the cytotoxic activity of 17 lichen species against several human cancer cells and further investigated the molecular mechanisms underlying their anti-cancer activity. We found that among 17 lichens species, <i>F. cucullata</i> exhibited the most potent cytotoxicity in several human cancer cells. High performance liquid chromatography analysis revealed that the acetone extract of <i>F. cucullata</i> contains usnic acid, salazinic acid, Squamatic acid, Baeomycesic acid, d-protolichesterinic acid, and lichesterinic acid as subcomponents. MTT assay showed that cancer cell lines were more vulnerable to the cytotoxic effects of the extract than non-cancer cell lines. Furthermore, among the identified subcomponents, usnic acid treatment had a similar cytotoxic effect on cancer cell lines but with lower potency than the extract. At a lethal dose, treatment with the extract or with usnic acid greatly increased the apoptotic cell population and specifically activated the apoptotic signaling pathway; however, using sub-lethal doses, extract and usnic acid treatment decreased cancer cell motility and inhibited <i>in</i><i>vitro</i> and <i>in</i><i>vivo</i> tumorigenic potentials. In these cells, we observed significantly reduced levels of epithelial-mesenchymal transition (EMT) markers and phosphor-Akt, while phosphor-c-Jun and phosphor-ERK1/2 levels were only marginally affected. Overall, the anti-cancer activity of the extract is more potent than that of usnic acid alone. Taken together, <i>F. cucullata</i> and its subcomponent, usnic acid together with additional component, exert anti-cancer effects on human cancer cells through the induction of apoptosis and the inhibition of EMT.</p></div

Reduction of phosphor-Akt level by the acetone extract of <i>F. cucullata</i> and usnic acid in sub-lethal concentrations.

<p>Phosphoprotein analysis for p(Ser⁶³)-c-jun, p(Ser⁴⁷³)-Akt, and p-(Thr²⁰²/Tyr²⁰⁴, Thr¹⁸⁵/Tyr¹⁸⁷)-ERK1/2 in A549 cells treated with the <i>F. cucullata</i> extract, usnic acid, or lichesterinic acid.</p

Activation of apoptosis pathway on human cancer cells by the acetone extract of <i>F. cucullata</i> and usnic acid in lethal concentrations.

<p>(A and D) Western blot analysis of poly (ADP-ribose) polymerase (PARP) and caspase-3 in cells treated with the <i>F. cucullata</i> (A) or usnic acid (D). Arrowheads indicate cleaved fragments of each protein. (B–C and E–F) Quantificational analysis of Bax (B and E) and Bcl-xL (C and F) protein expression levels in cells treated with the F. cucullata or usnic acid, respectively. Data represent mean ± S.E.M. (standard error of the mean). *p<0.05; **p<0.01; ***p<0.001; NS, no significant difference compared to the dimethylsulfoxide-treated group.</p

Induction of nuclear condensation of human cancer cells by the acetone extract of <i>F. cucullata</i> and its main component, usnic acid in lethal concentrations.

<p>(A) Hoechst 33258 staining of AGS (human gastric cancer cell line) cells treated with the <i>F. cucullata</i> extract or its subcomponents, usnic acid and lichesterinic acid. Arrows indicate cells showing condensed or fragmented nuclear morphology. Representative images are shown from three independent experiments. (B) Quantificational analysis of condensed or fragmented nuclear morphology in various cells treated with <i>F. cucullata</i> extract or its subcomponents. Data represent mean ± S.E.M. (standard error of the mean), n = 3. **p<0.01; ***p<0.001; NS, no significant difference compared to the dimethylsulfoxide-treated group.</p

Inhibition of <i>in</i><i>vivo</i> tumorigenicity of A549 cancer cells pretreated by the acetone extract of F. cucullata and usnic acid in sub-lethal concentrations.

<p>A549 cells were pretreated with indicated concentration of <i>F. cucullata</i>, usnic acid, or lichesterinic acid before subcutaneous injection into Balb/c nude mouse (n = 8) and tumor free survival number in each group during 4 weeks were measured.</p