Choline and folate are interrelated methyl donors. Previous studies showed that folate prevents genomic damage in human lymphocytes in vitro; however, the association between choline and human genomic stability is uncertain. To explore the genotoxicity, cytotoxicity, and cytostatic effects and possible interactions of choline and/or folate deficiency on the human genome, lymphocytes from 6 volunteers were cultured in 18 combinations of choline (CC) and folic acid (FA) media for 9 days. The genotoxicity was evaluated by micronuclei, nucleoplasmic bridges, and nuclear buds in the binucleated cell; the cytotoxicity indices included apoptosis and necrosis, and the cytostatic effects were indicated by nuclear division index (NDI). Across all choline concentrations, the frequencies of all biomarkers except NDI were diminished when FA concentration was more than or equal to 120 nmol/L. The frequencies of micronuclei, buds, and necrosis were significantly higher at lower levels of CC (0–6 μmol/L) compared with higher concentrations of CC (12–21.5 μmol/L) while maintaining the same FA concentration. We concluded that both choline and folate significantly impact genomic stability and cell death, although effects of folate were 2.5- to 6.2-fold greater, depending on the biomarker and dose. A combination of 12 μmol/L CC and 120 nmol/L FA appears to be optimal for genomic integrity in vitro.
Rhizoctonia cerealis is the causal pathogen of the devastating disease, sharp eyespot, of the important crop wheat (Triticum aestivum L.). In phytopathogenic fungi, several M36 metalloproteases have been implicated in virulence, but pathogenesis roles of M35 family metalloproteases are largely unknown. Here, we identified four M35 family metalloproteases from R. cerealis genome, designated RcMEP2–RcMEP5, measured their transcriptional profiles, and investigated RcMEP2 function. RcMEP2-RcMEP5 are predicted as secreted metalloproteases since each protein sequence contains a signal peptide and an M35 domain that includes two characteristic motifs HEXXE and GTXDXXYG. Transcription levels of RcMEP2-RcMEP5 markedly elevated during the fungus infection to wheat, among which RcMEP2 expressed with the highest level. Functional dissection indicated that RcMEP2 and its M35 domain could trigger H2O2 rapidly-excessive accumulation, induce cell death, and inhibit expression of host chitinases. This consequently enhanced the susceptibility of wheat to R. cerealis and the predicated signal peptide of RcMEP2 functions required for secretion and cell death-induction. These results demonstrate that RcMEP2 is a virulence factor and that its M35 domain and signal peptide are necessary for the virulence role of RcMEP2. This study facilitates a better understanding of the pathogenesis mechanism of metalloproteases in phytopathogens including R. cerealis.
Dendrobium officinale Kimura et Migo is a commercially and pharmacologically highly prized species widely used in Western Asian countries. In contrast to the extensive genomic and transcriptomic resources generated in this medicinal species, detailed metabolomic data are still missing. Herein, using the widely targeted metabolomics approach, we detect 649 diverse metabolites in leaf and stem samples of D. officinale. The majority of these metabolites were organic acids, amino acids and their derivatives, nucleotides and their derivatives, and flavones. Though both organs contain similar metabolites, the metabolite profiles were quantitatively different. Stems, the organs preferentially exploited for herbal medicine, contained larger concentrations of many more metabolites than leaves. However, leaves contained higher levels of polyphenols and lipids. Overall, this study reports extensive metabolic data from leaves and stems of D. officinale, providing useful information that supports ongoing genomic research and discovery of bioactive compounds.
Wheat (Triticum aestivum L.) is a staple food of more than 50% of global population. Rhizoctonia cerealis is the causal agent of sharp eyespot, a devastating disease of cereal crops including wheat. Cutinases produced by fungal pathogens play important roles in host-pathogen compatible interactions, but little is known about cutinases in R. cerealis. In this study, we identified a total of six cutinase encoding genes from R. cerealis genome, designated as RcCUT1-RcCUT6, analyzed their expression patterns during the infection, and determined virulence role for RcCUT1. All the proteins, RcCUT1-RcCUT6, contain a highly conserved GYSKG motif and another conserved C-x(3)-D-x(2)-C-x(2)-[GS]-[GSD]-x(4)-[AP]-H motif in the carbohydrate esterase 5 domain. The RcCUT1, RcCUT2, RcCUT4, and RcCUT5 are predicted to be secreted proteins containing four cysteine residues. These six cutinase genes had different expression patterns during the fungal infection process to wheat, among which RcCUT1 was highly expressed across all the infection time points but RcCUT6 was not expressed at all and the others were expressed only at certain time points. Further, RcCUT1 was heterologously expressed in Escherichia coli to obtain a purified protein. The purified RcCUT1 was shown to possess the cutinase activity and be able to induce necrosis, H2O2 accumulation, and expression of defense-related genes when infiltrated into wheat and Nicotiana benthamiana leaves. In contrast, RcCUT1 protein with serine mutation at the first motif had no cutinase activity, consequently lost the ability to induce necrosis. Noticeably, application of the purified RcCUT1 with R. cerealis led to significantly higher levels of the disease in wheat leaves than application of the fungus alone. These results strongly suggest that RcCUT1 serves as a virulence factor for the fungus. This is the first investigation of the cutinase genes in R. cerealis and the findings provide an important insight into pathogenesis mechanisms of R. cerealis on wheat.
Objective To study whether human lymphoblast cell line cultured by choline deficient medium had effects on DNA damage despite of sufficient folic acid.Method Lymphoblast cells were cultured in RPMI-1640 medium which was modified with 24 different combination of choline(0,3,6,12,18,21.5 μmol/L),folic acid(FA,30,60,120,240 nmol/L) and methionine(50 μmol/L).After 9 d cytokinesis-block micronucleus cytome assay(CBMN Cyt) was used to evaluate the frequencies of genotoxic indices including micronucleated binucleated cell(MNed BNC),nucleoplasmic bridge(NPB) and nuclear bud(NBUD).Results The statistic significance of MNed BNC‰ and NBUD‰ was found between low level(0-6 μmol/L) and high level of choline(12-21.5 μmol/L) in all concentrations of folic acid(P0.001).In 30 and 60 nmol/L FA,NPB was significantly decreased when the choline concentrations were 12μmol/L(P0.001),but in 120 and 240 nmol/L FA,the frequencies of NPB among six groups of choline were insignificantly different.Choline and folic acid also had significant impacts on variation of all genotoxic indices(P0.001).Conclusion Choline deficiency could bring DNA damage in human lymphoblast cell line.This preliminary investigation suggested that one should consider not only supplying adequate amounts of folic acid(120nmol/L),but also maintaining choline concentration 12 μmol/L.
In this study, we produced a specific monoclonal antibody (mAb) against triadimefon (TDF) and triadimenol (TDN). The half-maximal inhibitory concentration for TDF and TDN was 5.25 and 9.98 ng/mL, respectively, with a linear detection range (IC20–IC80) of 1.09–25.35 ng/mL and 2.29–43.61 ng/mL, respectively. Subsequently, we developed a colloidal gold immunochromatography test strip for the analysis of TDF and TDN in tomato. The results obtained from the colloidal gold immunochromatography test strip were in accordance with those obtained from the indirect competitive enzyme-linked immunosorbent assay. The colloidal gold immunochromatography test strip is efficient and suitable for on-site detection of TDF and TDN.
The rice blast disease (caused by Magnaporthe oryzae) is a devastating disease in China. Understanding the molecular mechanisms of interaction for the cognate avirulence (AVR) gene with host resistance (R) genes, as well as their genetic evolution is essential for sustainable rice production. In the present study, we conducted a high-throughput nucleotide sequence polymorphism analysis of the AVR-Pi9 gene that was amplified from the rice-growing regions of the Yunnan Province in China. We detected the presence of seven novel haplotypes from 326 rice samples. In addition, the sequences of AVR-Pi9 were also obtained from two non-rice hosts, Eleusine coracana and Eleusine indica. The sequence analysis revealed the insertions and deletions in the coding and non-coding regions of the gene. The pathogenicity experiments of these haplotypes on previously characterized monogenic lines showed that the newly identified haplotypes are virulent in nature. The breakdown of resistance was attributed to the development of new haplotypes. Our results suggest that the mutation in the AVR-Pi9 gene is an alarming situation in the Yunnan province and thus needs attention.
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