Engineered Phomopsis liquidambaris with Fhb1 and Fhb7 Enhances Resistance to Fusarium graminearum in Wheat.

Fusarium head blight is one of the most serious diseases caused by Fusarium graminearum in wheat. Here, we developed a new way to prevent and control Fusarium head blight by introducing the resistance genes Fhb1 and Fhb7 into the endophytic fungus Phomopsis liquidambaris, named PL-Fhb1 and PL-Fhb7, respectively, which could colonize wheat. The wheat seedlings were preinoculated with PL-Fhb1 and PL-Fhb7 to enhance the resistance against deoxynivalenol (DON) and PL-Fhb1 and PL-Fhb7 inhibited the growth of F. graminearum by 73% and 49%, respectively. The incidence rate of diseased spikes decreased to 35.2% and 45.4%, and the corresponding DON levels for wheat grains decreased from 13.2 to 1.79 µg/g and from 13.2 µg/g to 0.39 µg/g when the leaves were preinoculated with PL-Fhb1 and PL-Fhb7 after overwintering, respectively. The incidence rates of diseased spikes decreased to 25.7% and 34.7%, and the DON levels for wheat grains decreased from 17.48 µg/g to 1.23 µg/g and from 17.48 µg/g to 0 µg/g when the wheat flowers were inoculated with PL-Fhb1 and PL-Fhb7, and the wheat flowers were subsequently infected with F. graminearum, respectively. It was confirmed that DON was transformed into DON-glutathione (GSH) by PL-Fhb7 using high-performance liquid chromatography-mass spectrometry (HPLC-MS). However, PL-Fhb1 may have increased plant immunity and enhanced the resistance to F. graminearum. This study indicates that engineered endophytes can improve the resistance to Fusarium head blight and presents a new method for the biological control of Fusarium head blight.

Overexpression of chitinase in the endophyte Phomopsis liquidambaris enhances wheat resistance to Fusarium graminearum.

Fusarium head blight (FHB) is a disease that affects wheat crops worldwide and is caused by Fusarium graminearum. Effective and safe strategies for the prevention and treatment of the disease are very limited. Phomopsis liquidambaris, a universal endophyte, can colonize wheat. Two engineered strains, Phomopsis liquidambaris OE-Chi and IN-Chi, were constructed by transformation with a plasmid and integration of a chitinase into the genome, respectively. The OE-Chi and IN-Chi strains could inhibit the expansion of Fusarium sp. in plate confrontation assays in vitro. Colonization of the OE-Chi strain in wheat showed better effects than colonization of the IN-Chi strain and alleviated the inhibition of wheat growth caused by F. graminearum. The shoot length, root length and fresh weight of infected wheat increased by 164.9%, 115.4%, and 190.7%, respectively, when the plants were inoculated with the OE-Chi strain. The peroxidase (POD) activity in the wheat root increased by 38.0%, and it was maintained at a high level in the shoot, which suggested that the OE-Chi strain could enhance the resistance of wheat to F. graminearum. The root and shoot superoxide dismutase (SOD) activities were decreased by 11.8% and 19.0%, respectively, which may be helpful for colonization by the OE-Chi strain. These results suggested that the Phomopsis liquidambaris OE-Chi strain may be a potential endophyte in the biocontrol of FHB.

Co-opting the fermentation pathway for tombusvirus replication: Compartmentalization of cellular metabolic pathways for rapid ATP generation.

The viral replication proteins of plus-stranded RNA viruses orchestrate the biogenesis of the large viral replication compartments, including the numerous viral replicase complexes, which represent the sites of viral RNA replication. The formation and operation of these virus-driven structures require subversion of numerous cellular proteins, membrane deformation, membrane proliferation, changes in lipid composition of the hijacked cellular membranes and intensive viral RNA synthesis. These virus-driven processes require plentiful ATP and molecular building blocks produced at the sites of replication or delivered there. To obtain the necessary resources from the infected cells, tomato bushy stunt virus (TBSV) rewires cellular metabolic pathways by co-opting aerobic glycolytic enzymes to produce ATP molecules within the replication compartment and enhance virus production. However, aerobic glycolysis requires the replenishing of the NAD+ pool. In this paper, we demonstrate the efficient recruitment of pyruvate decarboxylase (Pdc1) and alcohol dehydrogenase (Adh1) fermentation enzymes into the viral replication compartment. Depletion of Pdc1 in combination with deletion of the homologous PDC5 in yeast or knockdown of Pdc1 and Adh1 in plants reduced the efficiency of tombusvirus replication. Complementation approach revealed that the enzymatically functional Pdc1 is required to support tombusvirus replication. Measurements with an ATP biosensor revealed that both Pdc1 and Adh1 enzymes are required for efficient generation of ATP within the viral replication compartment. In vitro reconstitution experiments with the viral replicase show the pro-viral function of Pdc1 during the assembly of the viral replicase and the activation of the viral p92 RdRp, both of which require the co-opted ATP-driven Hsp70 protein chaperone. We propose that compartmentalization of the co-opted fermentation pathway in the tombusviral replication compartment benefits the virus by allowing for the rapid production of ATP locally, including replenishing of the regulatory NAD+ pool by the fermentation pathway. The compartmentalized production of NAD+ and ATP facilitates their efficient use by the co-opted ATP-dependent host factors to support robust tombusvirus replication. We propose that compartmentalization of the fermentation pathway gives an evolutionary advantage for tombusviruses to replicate rapidly to speed ahead of antiviral responses of the hosts and to outcompete other pathogenic viruses. We also show the dependence of turnip crinkle virus, bamboo mosaic virus, tobacco mosaic virus and the insect-infecting Flock House virus on the fermentation pathway, suggesting that a broad range of viruses might induce this pathway to support rapid replication.

Effects of hydrothermal temperature and time on hydrothermal synthesis of colloidal hydroxyapatite nanorods in the presence of sodium citrate.

In this paper, colloidal hydrophilic hydroxyapatite nanorods were synthesized in the presence of sodium citrate via thermal-decomplexing method. The influences of hydrothermal temperature and time on the synthesis of HA nanorods were characterized in terms of structure, size, morphology, and colloidal stability through TEM, XRD, zeta potential, DLS and long-term standing test. Results show that increasing hydrothermal temperature and prolonging hydrothermal time would evidently improve crystallinity and enlarge size of HA nanorods but decrease the colloidal stability of nanorods. It is worth noting that the effect of raising the hydrothermal temperature and time on diameter increase is far greater than that on length increase; meanwhile, the colloidal stability would be seriously deteriorated when the hydrothermal temperature is over 180 °C for 24 h or when the hydrothermal temperature is 150 °C for over 48 h, in these cases, dispersion of HA nanorods would apparently settle within 2 months. The origin responding to the results is that although the charge density of HA nanorods is not obviously affected, the dynamic diameters of HA particles increase greatly, which reduces colloidal stability of the dispersion. This work provides new insights into the role of hydrothermal temperature and time on tailoring morphology, crystallinity and colloidal stability of HA nanorods. Moreover, it would be helpful to optimize the experimental procedure both on scientific and industrial applications related to HA. For example, on the premise of satisfying the necessary requirements including crystallinity, size, morphology and colloid stability, it is feasible to compress the consumption of experimental time through raising the hydrothermal temperature, or vice versa.