First Report of Stunt Nematode, Tylenchorhynchus zeae, on Corn in Republic of Korea.

Corn (Zea mays), one of the major food sources it contains rich in fiber and many vitamins, is one of the most widely consumed cereal grains in Republic of Korea. A survey of plant-parasitic nematodes (PPNs) was carried out in corn fields in Goesan, Republic of Korea from August in 2021. PPNs were extracted from the corn roots and soil using modified Baermann funnel methods and were identified using morphological and molecular analyses. Among the roots and soil samples of 21 fields, 5 fields (23.8%) were infected with stunt nematodes. Tylenchorhynchus zeae was originally described in India from soil around corn and is reported to dwarf plants, yellow leaves (Sethi and Swarup, 1968). Morphologically, characteristics of females were very similar to T. zeae with cylindrical body and slightly ventrally arcuated after fixation. Lip region slightly offset from body with four annuli. Stylet with anteriorly flattened knobs, the vulva was located in the center of the body, didelphic-amphidelphic reproductive system and tail conoid, tail terminus with obtuse smooth, with four incisures areolated throughout body. Bodies of males were similar to females but with shaper tails, with relatively strong bursa and spicules (Fig. S1). The morphology of Korean populations was in agreement with the described populations of India and China (Alvani et al., 2017; Xu et al., 2020). Measurements and micrographs with the light-microscope (DM5000; Leica[Germany]) and camera (DFC450; Leica[Germany]) were taken from females (n=10) for mean, standard deviation and range of body length: 553.2 ± 41.2 (492.7-643.6) µm, maximum body width: 19.4 ± 1.0 (17.6-21.0) µm, stylet length: 18.1 ± 0.4 (17.5-18.7) µm, percent of distance from anterior end to vulva / body length: 58.5 ± 1.3 (56.1-60.9), tail length: 31.7 ± 1.2 (30.3-34.0) µm, and distance of anterior to excretory pore: 96.5 ± 1.8 (94.1-99.4) µm. In addition, PCR was performed for the 28S rDNA D2-D3 segments using the primers D2A and D3B, and ITS region with the primers TW81 and AB28. The newly obtained sequences were submitted to GenBank database under accession numbers ON909086, ON909087 and ON909088 of 28S rDNA D2-D3 segments, and ON909123, ON909124 and ON909125 of ITS region. The resulting 28S rDNA D2-D3 segment sequences were 100% identical to KJ461565 and the BLASTn search of the ITS region sequences was most similar to T. zeae (KJ461599), which is the species isolated from corn in Spain. The identities of ITS region sequences on these populations were 99.89% (893/894), with no insertions/deletions. The phylogenetic relationships of the population strongly support T. zeae (Fig. S2). Phylogenetic relation analysis based on the two genes was constructed using PAUP version 4.0 and MrBayes 3.1.2 programs. To confirm pathogenicity, a modified version of Koch's postulates was conducted in the greenhouse by inoculating 100 females and males onto each of five pots of seedling corn (cv. Daehakchal) filled with the sterilized sandy soil and maintained for 60 days at 25℃ under the conditions. Tylenchorhynchus zeae reproduction factor was 2.21 ± 0.37 was observed at the end of the trial in soil on pots. The stunted and swollen roots and dwarfed and yellowing leaf shoots symptoms in the greenhouse pots trial were confirmed the same as those typical damage symptoms. To the best of our knowledge, this is the first report of T. zeae in Republic of Korea. The host range of T. zeae includes some economic crops such as cabbage, cauliflower, grapevine, and olive (Chen at al., 2007; Handoo et al., 2014). It is necessary to investigate the damage to economic crops in the Republic of Korea to this nematode.

Comparative analysis of complete mitochondrial genome sequences confirms independent origins of plant-parasitic nematodes.

BACKGROUND: The nematode infraorder Tylenchomorpha (Class Chromadorea) includes plant parasites that are of agricultural and economic importance, as well as insect-associates and fungal feeding species. Among tylenchomorph plant parasites, members of the superfamily Tylenchoidea, such as root-knot nematodes, have great impact on agriculture. Of the five superfamilies within Tylenchomorpha, one (Aphelenchoidea) includes mainly fungal-feeding species, but also some damaging plant pathogens, including certain Bursaphelenchus spp. The evolutionary relationships of tylenchoid and aphelenchoid nematodes have been disputed based on classical morphological features and molecular data. For example, similarities in the structure of the stomatostylet suggested a common evolutionary origin. In contrast, phylogenetic hypotheses based on nuclear SSU ribosomal DNA sequences have revealed paraphyly of Aphelenchoidea, with, for example, fungal-feeding Aphelenchus spp. within Tylenchomorpha, but Bursaphelenchus and Aphelenchoides spp. more closely related to infraorder Panagrolaimomorpha. We investigated phylogenetic relationships of plant-parasitic tylenchoid and aphelenchoid species in the context of other chromadorean nematodes based on comparative analysis of complete mitochondrial genome data, including two newly sequenced genomes from Bursaphelenchus xylophilus (Aphelenchoidea) and Pratylenchus vulnus (Tylenchoidea). RESULTS: The complete mitochondrial genomes of B. xylophilus and P. vulnus are 14,778 bp and 21,656 bp, respectively, and identical to all other chromadorean nematode mtDNAs in that they contain 36 genes (lacking atp8) encoded in the same direction. Their mitochondrial protein-coding genes are biased toward use of amino acids encoded by T-rich codons, resulting in high A+T richness. Phylogenetic analyses of both nucleotide and amino acid sequence datasets using maximum likelihood and Bayesian methods did not support B. xylophilus as most closely related to Tylenchomorpha (Tylenchoidea). Instead, B. xylophilus, was nested within a strongly supported clade consisting of species from infraorders Rhabditomorpha, Panagrolaimomorpha, Diplogasteromorpha, and Ascaridomorpha. The clade containing sampled Tylenchoidea (P. vulnus, H. glycines, and R. similis) was sister to all analyzed chromadoreans. Comparison of gene arrangement data was also consistent with the phylogenetic relationships as inferred from sequence data. Alternative tree topologies depicting a monophyletic grouping of B. xylophilus (Aphelenchoidea) plus Tylenchoidea, Tylenchoidea plus Diplogasteromorpha (Pristionchus pacificus), or B. xylophilus plus Diplogasteromorpha were significantly worse interpretations of the mtDNA data. CONCLUSIONS: Phylogenetic trees inferred from nucleotide and amino acid sequences of mtDNA coding genes are in agreement that B. xylophilus (the single representative of Aphelenchoidea) is not closely related to Tylenchoidea, indicating that these two groups of plant parasites do not share an exclusive most recent common ancestor, and that certain morphological similarities between these stylet-bearing nematodes must result from convergent evolution. In addition, the exceptionally large mtDNA genome size of P. vulnus, which is the largest among chromadorean nematode mtDNAs sequenced to date, results from lengthy repeated segments in non-coding regions.

Haplotype diversity of Heterodera koreana (Tylenchida: Heteroderidae), affecting bamboo in Korea.

In a survey of plant-parasitic nematodes in agricultural fields, cyst-forming nematodes were found in soil planted bamboo in Korea. The aim of this study was to identify the cyst nematodes based on morphological and molecular characteristics. As the results, the morphology and morphometrics of cysts and second-stage juveniles (J2s) were consistent with those of previous descriptions of Heterodera koreana. In phylogenetic analyses based on DNA sequences, these cyst nematodes were clustered together with clade of H. koreana in internal transcribed spacer (ITS) region, and large subunit D2-D3 segments (LSU D2-D3). These nematodes were clustered together with clade of H. koreana in cytochrome c oxidase subunit I (COI) gene, but a haplotype was different when compared with previous reported haplotypes (haplotype A-C) in Japan. This study showed these cyst nematodes were identified as H. koreana, and a new haplotype of H. koreana is distributed in Korea. We suggest that the new haplotype of H. koreana name as haplotype D.

Investigating the causal role of MRE11A p.E506* in breast and ovarian cancer.

The nuclease MRE11A is often included in genetic test panels for hereditary breast and ovarian cancer (HBOC) due to its BRCA1-related molecular function in the DNA repair pathway. However, whether MRE11A is a true predisposition gene for HBOC is still questionable. We determined to investigate this notion by dissecting the molecular genetics of the c.1516G > T;p.E506* truncating MRE11A variant, that we pinpointed in two unrelated French-Canadian (FC) HBOC patients. We performed a case-control study for the variant in ~ 2500 breast, ovarian, and endometrial cancer patients from the founder FC population of Quebec. Furthermore, we looked for the presence of second somatic alterations in the MRE11A gene in the tumors of the carriers. In summary, these investigations suggested that the identified variant is not associated with an increased risk of developing breast or ovarian cancer. We finally performed a systematic review for all the previously reported MRE11A variants in breast and ovarian cancer. We found that MRE11A germline variants annotated as pathogenic on ClinVar often lacked evidence for such classification, hence misleading the clinical management for affected patients. In summary, our report suggests the lack of clinical utility of MRE11A testing in HBOC, at least in the White/Caucasian populations.

Point-of-care diagnostic (POCD) method for detecting Bursaphelenchus xylophilus in pinewood using recombinase polymerase amplification (RPA) with the portable optical isothermal device (POID).

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is a causative agent of pine wilt disease (PWD). To date, although several molecular diagnostic methods have been developed, rapid on-site diagnostic tools for detecting PWN in pinewood are limited. In this study, a point of care diagnostic (POCD) method for detecting PWN in pinewood using recombinase polymerase amplification (RPA) assay was developed. This method comprises quick gDNA extraction buffer (DAP buffer) for the direct extraction of gDNA of PWN from pinewood and a battery-mounted portable optical isothermal device (POID) for the detection of PWD in the field. The RPA assay can distinguish between the PWN and its conspecies which exist in pinewood and can complete diagnostic procedures within 25 min in the field. Moreover, the RPA assay can detect PWN in old wood samples in both natural and stored conditions. The POCD-RPA assay to detect PWN will be useful for epidemiological investigations in the field as well as for quarantine processes in the wood trade.

Comparative Transcriptome Analysis of Pinus densiflora Following Inoculation with Pathogenic (Bursaphelenchus xylophilus) or Non-pathogenic Nematodes (B. thailandae).

Pinus densiflora (Korean red pine) is a species of evergreen conifer that is distributed in Korea, Japan, and China, and of economic, scientific, and ecological importance. Korean red pines suffer from pine wilt disease (PWD) caused by Bursaphelenchus xylophilus, the pinewood nematode (PWN). To facilitate diagnosis and prevention of PWD, studies have been conducted on the PWN and its beetle vectors. However, transcriptional responses of P. densiflora to PWN have received less attention. Here, we inoculated Korean red pines with pathogenic B. xylophilus, or non-pathogenic B. thailandae, and collected cambium layers 4 weeks after inoculation for RNA sequencing analysis. We obtained 72,864 unigenes with an average length of 869 bp (N50 = 1,403) from a Trinity assembly, and identified 991 differentially expressed genes (DEGs). Biological processes related to phenylpropanoid biosynthesis, flavonoid biosynthesis, oxidation-reduction, and plant-type hypersensitive response were significantly enriched in DEGs found in trees inoculated with B. xylophilus. Several transcription factor families were found to be involved in the response to B. xylophilus inoculation. Our study provides the first evidence of transcriptomic differences in Korean red pines inoculated with B. xylophilus and B. thailandae, and might facilitate early diagnosis of PWD and selection of PWD-tolerant Korean red pines.

TRPV4 and KRAS and FGFR1 gain-of-function mutations drive giant cell lesions of the jaw.

Giant cell lesions of the jaw (GCLJ) are debilitating tumors of unknown origin with limited available therapies. Here, we analyze 58 sporadic samples using next generation or targeted sequencing and report somatic, heterozygous, gain-of-function mutations in KRAS, FGFR1, and p.M713V/I-TRPV4 in 72% (42/58) of GCLJ. TRPV4 p.M713V/I mutations are exclusive to central GCLJ and occur at a critical position adjacent to the cation permeable pore of the channel. Expression of TRPV4 mutants in HEK293 cells leads to increased cell death, as well as increased constitutive and stimulated channel activity, both of which can be prevented using TRPV4 antagonists. Furthermore, these mutations induce sustained activation of ERK1/2, indicating that their effects converge with that of KRAS and FGFR1 mutations on the activation of the MAPK pathway in GCLJ. Our data extend the spectrum of TRPV4 channelopathies and provide rationale for the use of TRPV4 and RAS/MAPK antagonists at the bedside in GCLJ.

Comparison of complete mitochondrial genomes of pine wilt nematode Bursaphelenchus xylophilus and Bursaphelenchus mucronatus (Nematoda: Aphelenchoidea) and development of a molecular tool for species identification.

We determined the complete mitochondrial genome sequences for Bursaphelenchus mucronatus, one species of pinewood nematode. The genome is a circular-DNA molecule of 14,583 bp (195 bp smaller than its congener Bursaphelenchus xylophilus) and contains 12 protein-coding genes (lacking atp8), 22 tRNA genes, and 2 rRNA genes encoded in the same direction, consistent with most other nematodes. Based on sequence comparison of mtDNA genomes, we developed a PCR-based molecular assay to differentiate B. xylophilus (highly pathogenic) and B. mucronatus (relatively less virulent) using species-specific primers. The molecular identification system employs multiplex-PCR and is very effective and reliable for discriminating these Bursaphelenchus species, which are economically important, but difficult to distinguish based on morphology. The comparison of the mitochondrial genomes and molecular identification system of the two species of Bursaphelenchus spp. should provide a rich source of genetic information to support the effective control and management (quarantine) of the pine wilt disease caused by pinewood nematodes.

Growth and density-dependent regulation of NO synthase by the actin cytoskeleton in pulmonary artery endothelial cells.

We previously reported association of eNOS with actin increases eNOS activity. In the present study, regulation of activity of eNOS by actin cytoskeleton during endothelial growth was studied. We found eNOS activity in PAEC increased when cells grew from preconfluence to confluence. eNOS activity was much greater in PAEC in higher density than those in lower density, suggesting increase in eNOS activity during cell growth is caused by increase in cell density. Although eNOS protein contents were also increased when endothelial cells grew from preconfluence to confluence, magnitude of increase in eNOS activity was much higher than increase in eNOS protein content, suggesting posttranslational mechanisms played an important role in regulation of eNOS activity during endothelial growth. Confocal fluorescence microscopy revealed eNOS was colocalized with G-actin in preconfluent cells in perinuclear region, with both G-actin in perinuclear area and cortical F-actin in plasma membrane in confluent cells. There was more beta-actin coimmunoprecipitated with eNOS in Triton X-100-soluble fraction in confluent cells in later growth phase and in high density. Decrease in eNOS association with beta-actin by silencing beta-actin expression using beta-actin siRNA causes inhibition of eNOS activity, NO production, and endothelial monolayer wound repair in PAEC. Moreover, PAEC incubation with cytochalasin D and jasplakinolide resulted in increases in eNOS/actin association and in eNOS activity without changes in eNOS protein content. Yeast two-hybrid experiments suggested strong association between eNOS oxygenase domain and beta-actin. These results indicate increase in eNOS association with actin is responsible for greater eNOS activity in confluent PAEC.