Regulatory Roles of Small Non-coding RNAs in Sugar Beet Resistance Against Beet curly top virus

Beet curly top virus (BCTV) mediated yield loss in sugar beets is a major problem worldwide. The circular single-stranded DNA virus is transmitted by the beet leafhopper. Genetic sources of BCTV resistance in sugar beet are limited and commercial cultivars rely on chemical treatments versus durable genetic resistance. Phenotypic selection and double haploid production have resulted in sugar beet germplasm (KDH13; 13 and KDH4-9; 4) that are highly resistant to BCTV. The molecular mechanism of resistance to the virus is unknown, especially the role of small non-coding RNAs (sncRNAs) during early plant–viral interaction. Using the resistant lines along with a susceptible line (KDH19-17; 19), we demonstrate the role of sugar beet microRNAs (miRNAs) in BCTV resistance during early infection stages when symptoms are not yet visible. The differentially expressed miRNAs altered the expression of their corresponding target genes such as pyruvate dehydrogenase (EL10Ac1g02046), carboxylesterase (EL10Ac1g01087), serine/threonine protein phosphatase (EL10Ac1g01374), and leucine-rich repeats (LRR) receptor-like (EL10Ac7g17778), that were highly expressed in the resistant lines versus susceptible lines. Pathway enrichment analysis of the miRNA target genes showed an enrichment of genes involved in glycolysis/gluconeogenesis, galactose metabolism, starch, and sucrose metabolism to name a few. Carbohydrate analysis revealed altered glucose, galactose, fructose, and sucrose concentrations in the infected leaves of resistant versus susceptible lines. We also demonstrate differential regulation of BCTV derived sncRNAs in the resistant versus susceptible lines that target sugar beet genes such as LRR (EL10Ac1g01206), 7-deoxyloganetic acid glucosyltransferase (EL10Ac5g12605), and transmembrane emp24 domain containing (EL10Ac6g14074) and altered their expression. In response to viral infection, we found that plant derived miRNAs targeted BCTV capsid protein/replication related genes and showed differences in expression among resistant and susceptible lines. The data presented here demonstrate the contribution of miRNA mediated regulation of metabolic pathways and cross-kingdom RNA interference (RNAi) in sugar beet BCTV resistance.

Diverse Regulations of Viral and Host Genes in Tomato Germplasms Responding to Tomato Yellow Leaf Curl Virus Inoculation

Tomato yellow leaf curl virus (TYLCV) is the dominating pathogen of tomato yellow leaf curl disease that caused severe loss to tomato production in China. In this study, we found that a TYLCV-resistant tomato line drastically reduced the accumulation of viral complementary-sense strand mRNAs but just moderately inhibit that of viral DNA and virion-sense strand mRNAs. However, two other resistant lines did not have such virus inhibition pattern. Analysis of differential expressed genes showed that the potential host defense-relevant processes varied in different resistant tomatoes, as compared to the susceptible line, suggesting a diversity of tomato TYLCV-resistance mechanisms.

Phenotypic and molecular screening of dry bean (Phaseolus vulgaris L.) breeding lines for resistance to bean common mosaic virus and bean common mosaic necrosis virus

Bean common mosaic virus (BCMV) and bean common mosaic necrosis virus (BCMNV) are among the most economically important virus species infecting common bean. The use of resistant plant cultivars is the most effective way to control these viruses. National dry bean breeding studies have been conducted by seven different governmental agricultural research institutes in Turkey, and advanced breeding lines have been developed by using the selected local dry bean populations and crossing studies. In this study, 204 breeding lines were tested for resistance levels to BCMV and BCMNV. Initially, BCMNV NL-3 and BCMV NL-4 strains were individually sap-inoculated onto the leaves of bean plants belonging to each breeding lines with 10 replications, and the reactions of plants were evaluated for symptomatic appearance of virus infection 21 days after inoculation. Additionally, phenotypic evaluation was confirmed by molecular markers linked to resistance genes. As a result of the study, 153 breeding lines were found to involve the dominant I gene whereas four and five of the tested lines had the recessive genes bc-1² and bc-2², respectively. In conclusion, it was emphasized that these breeding lines could be registered after evaluating them in terms of yield and quality. Also, the use of seeds of the resistant lines to supply the source of virus-resistance in breeding studies and maintaining their seeds at the national genebank were recommended.

Amphotericin B resistance in Leishmania mexicana: Alterations to sterol metabolism, lipid transport and oxidative stress response

Amphotericin B is increasingly used in treatment of leishmaniasis. Here, fourteen independent lines of Leishmania mexicana and one L. infantum line were selected for resistance to either amphotericin B or the related polyene antimicrobial, nystatin. Sterol profiling revealed that, in each line, the predominant ergostane-type sterol of wild-type cells was replaced by other sterol species. Broadly, two different profiles emerged among the resistant lines. Whole genome sequencing then showed that these distinct profiles were due either to mutations in the sterol methyl transferase (C24SMT) gene locus or the sterol C5 desaturase (C5DS) gene. In three lines an additional deletion of the miltefosine transporter was found. Differences in sensitivity to amphotericin B were apparent, depending on whether cells were grown in HOMEM, supplemented with foetal bovine serum, or a serum free defined medium (DM). These differences appeared to relate to the presence of lipids in the former. Metabolomic analysis after exposure to AmB showed that a large increase in glucose flux via the pentose phosphate pathway preceded cell death in cells sustained in HOMEM but not DM, indicating the oxidative stress was more significantly induced under HOMEM conditions. Several of the lines were tested for ability to infect macrophages and replicate as amastigote forms, alongside their ability to establish infections in mice. While several lines showed reduced virulence, at least one AmB resistant line displayed heightened virulence in mice whilst retaining its resistance phenotype, emphasising the risks of resistance emerging to this critical drug.

Strain-, Sex-, and Time-Dependent Antidepressant-like Effects of Cannabidiol

Cannabidiol (CBD) is a non-intoxicating compound extracted from Cannabis sativa, showing antidepressant-like effects in different rodent models. However, inconsistent results have been described depending on the species and the strain used to assess depressive-like behavior. Moreover, only a few studies investigated the effect of CBD in female rodents. Therefore, we aimed to (i) investigate the effects of CBD in two different strains of mice (Swiss and C57BL/6) and a rat model of depression based on selective breeding (Flinders Sensitive and Resistant Lines, FSL and FRL) subjected to tests predictive of antidepressant-like effects and (ii) investigate the influence of sex in the effects of CBD in both mice and rats. CBD induced an antidepressant-like effect in male Swiss but not in female Swiss or C57BL/6 mice in the tail suspension test (TST). In male FSL rats, CBD produced an antidepressant-like effect 1 h post injection. However, in female FSL, CBD induced a bimodal effect, increasing the immobility time at 1 h and decreasing it at 2 h. In conclusion, strain, sex, and administration time affect CBD’s behavioral response to rodents exposed to tests predictive of antidepressant effects.

Molecular Markers For Selection of Resistance to Facial Eczema

<p>The disease facial eczema is caused by the fungal metabolite sporidesmin which produces photosensitisation of animals whose liver and biliary tract have been damaged by the toxin. Sporidesmin is produced by the pasture fungus Pithomyces chartarum and affects ruminant animals that graze on contaminated pasture. Previous studies have shown that sporidesmin is metabolised in the liver and have suggested that the toxin is metabolically inactivated by enzymes in the glutathione S-transferase and cytochrome P-450 families. The activities of these enzymes were therefore measured in liver extracts from Romneys that had been selected for resistance or susceptibility to sporidesmin - induced liver damage. Although there were no differences in cytochrome P-450 CO binding spectra or cytochrome c reductase between the selection lines, resistant Romneys had greater nitroanisole O-demethylase activity and this difference was apparently enhanced two days after dosing with sporidesmin. Dose-dependent differences occurred in the absence of major hepatocellular injury suggesting that they reflected changes in enzyme activity rather than changes in tissue mass. Aminopyrine N-demethylase did not vary significantly between the selection lines. Some differences in GSH-dependent metabolism were also observed. Undosed resistant Romneys showed greater GSH-dependent metabolism of sporidesmin in a spectrophotometric assay. It is possible that glutathione S-transferase Mu or Theta isoforms had greater activity in the resistant lines as differences were observed using p-nitrobenzyl chloride and 1,2 epoxy-3-p-nitrophenoxypropanol but not with 1-chloro-2,4-dinitrobenzene or 1,2-dichloro-4-nitrobenzene that are good substrates for these isoforms. 2-D PAGE was applied to the separation of whole homogenate and soluble proteins. Variations in expression of some proteins including GST Mu isoforms were found between the selection lines. Roles of cytochrome P-450 and glutathione S-transferase in the hepatic detoxication of sporidesmin have previously been demonstrated. Results obtained in this study suggest that resistant Romneys may have greater cytochrome P-450 O-demethylase and glutathione S-transferase activities that could be responsible for increased metabolic inactivation of sporidesmin. These differences may in the future be of use in design of DNA probes to enhance detection and selection of facial eczema resistant livestock.</p>

Molecular Markers For Selection of Resistance to Facial Eczema

<p>The disease facial eczema is caused by the fungal metabolite sporidesmin which produces photosensitisation of animals whose liver and biliary tract have been damaged by the toxin. Sporidesmin is produced by the pasture fungus Pithomyces chartarum and affects ruminant animals that graze on contaminated pasture. Previous studies have shown that sporidesmin is metabolised in the liver and have suggested that the toxin is metabolically inactivated by enzymes in the glutathione S-transferase and cytochrome P-450 families. The activities of these enzymes were therefore measured in liver extracts from Romneys that had been selected for resistance or susceptibility to sporidesmin - induced liver damage. Although there were no differences in cytochrome P-450 CO binding spectra or cytochrome c reductase between the selection lines, resistant Romneys had greater nitroanisole O-demethylase activity and this difference was apparently enhanced two days after dosing with sporidesmin. Dose-dependent differences occurred in the absence of major hepatocellular injury suggesting that they reflected changes in enzyme activity rather than changes in tissue mass. Aminopyrine N-demethylase did not vary significantly between the selection lines. Some differences in GSH-dependent metabolism were also observed. Undosed resistant Romneys showed greater GSH-dependent metabolism of sporidesmin in a spectrophotometric assay. It is possible that glutathione S-transferase Mu or Theta isoforms had greater activity in the resistant lines as differences were observed using p-nitrobenzyl chloride and 1,2 epoxy-3-p-nitrophenoxypropanol but not with 1-chloro-2,4-dinitrobenzene or 1,2-dichloro-4-nitrobenzene that are good substrates for these isoforms. 2-D PAGE was applied to the separation of whole homogenate and soluble proteins. Variations in expression of some proteins including GST Mu isoforms were found between the selection lines. Roles of cytochrome P-450 and glutathione S-transferase in the hepatic detoxication of sporidesmin have previously been demonstrated. Results obtained in this study suggest that resistant Romneys may have greater cytochrome P-450 O-demethylase and glutathione S-transferase activities that could be responsible for increased metabolic inactivation of sporidesmin. These differences may in the future be of use in design of DNA probes to enhance detection and selection of facial eczema resistant livestock.</p>

Water stress resistance of the new self-pollinated maize lines and hybrids

The directions of the economic use of maize are different, namely fodder, food, technical. Considering that maize belongs to mesophytes, the most harmful stress factor for it is drought. The breeding maize for drought tolerance is considered the most important direction for this crop. The purpose of the current paper was to present study results of the drought resistance of the initial material, namely the self-pollinated maize lines and hybrids. The study was carried out at the Agricultural Research Center “Donskoy”, located in the zone of insufficient moisture in 2018–2020. Drought tolerance was determined by the method of residual water deficit (RWD). As an initial material, there were used 24 self-pollinated maize lines and 50 test-cross hybrids. There have been identified the new drought-resistant middle-early and middle-ripening self-pollinated lines ‘KS 317 A’,’ KV 240’, ‘LSh 16’, ‘S 86’, ‘LSh 17’ and ‘LSh 2’, ‘SP 246 / 276-2’, ‘DS 498 / 203-4’, ‘DS 298 / 203-3’, ‘DS 257 / 85-0’, ‘SP 280-3’, ‘KB 373’, which had a low residual water deficit in the flowering phase (7.7–10.4%) and did not significantly increase it during the period from vegetation to milky-wax ripeness (up to 10,3–12.6%). The drought-resistant hybrids include ‘Stepnyak MV’, ‘GK 26 AM × DS 257 / 85-0’, ‘P 101 × Zp 498 A’, ‘KB 399 × S 232’, ‘GK 26 AM × KB 373’, ‘GK 26 AM × SP 246 / 276-2’, ‘C 204 × KS 318’. They were characterized by a low water deficit during the flowering period (7.4–10.4%) and its low increase (1.4–3.7%) during the growing season. Drought-resistant hybrids, as a rule, included drought-resistant lines. According to the results of the State Variety Testing, the new middle-ripening three-line maize hybrid ‘Stepnyak MV’ ((KB 262 M × KB 326 ZM) × KB 498 MV)), developed on the basis of the drought-resistant lines ‘KB 262 M’ and ‘KB 498 MV’, has been included into the State Register since 2019. The hybrid possessed a high grain yield (4.55 t/ha) in the dry years of 2018–2020. it was characterized by high values of the main economically valuable traits, such as high resistance to lodging (0.5% of lodged plants), high starch content in grain (72.0%), optimum grain moisture (14.0%) by the harvesting time.

Potency and preclinical evidence of synergy of oral azole drugs and miltefosine in an ex vivo model of Leishmania (Viannia) panamensis infection

Failure of treatment of cutaneous leishmaniasis with antimonial drugs and miltefosine is frequent. Use of oral combination therapy represents an attractive strategy to increase efficacy of treatment and reduce the risk of drug resistance. We evaluated the potency of posaconazole, itraconazole, voriconazole and fluconazole, and the potential synergy of those demonstrating the highest potency, in combination with miltefosine (HePC), against infection with Leishmania (Viannia) panamensis . Synergistic activity was determined by isobolograms and calculation of Fractional Inhibitory Concentration Index (FICI), based on parasite quantification using an ex vivo model of human PBMCs infected with a luciferase-transfected, antimony and miltefosine sensitive line of L. panamensis . The drug combination and concentrations that displayed synergy were then evaluated for anti-leishmanial effect in 10 clinical strains of L. panamensis by qRT-PCR of Leishmania 7SLRNA. High potency was substantiated for posaconazole and itraconazole against sensitive as well as HePC and antimony resistant lines of L. panamensis , whereas fluconazole and voriconazole displayed low potency. HePC combined with posaconazole (Poz) demonstrated evidence of synergy at free drug concentrations achieved in plasma during treatment (2 μM HePC + 4 μM Poz). FICI, based on 70% and 90% reduction of infection, was 0.5 for the sensitive line. Combination of 2 μM HePC + 4 μM Poz effected significantly greater reduction of infection by clinical strains of L. panamensis than individual drugs. Orally administrable miltefosine/posaconazole combinations demonstrated synergistic anti-leishmanial capacity ex vivo against L. panamensis , supporting their potential as a novel therapeutic strategy to improve efficacy, and effectiveness of treatment.