Modified small-scale batch procedure for isolation of dsRNA from Cryphonectria parasitica

Cytoplasmically-transmissible viral double-stranded RNAs of the genus Hypovirus cause reduced virulence (hypovirulence) in the chestnut blight fungus Cryphonectria parasitica. Biological control of this fungal disease is done by inoculating selected artificial hypovirulent strains of C. parasitica in the wounds of attacked chestnut trees and is followed by testing the transmissibility of dsRNA to C. parasitica isolates affecting these trees. Here we present a modified protocol of isolation and detection of dsRNA. The proposed procedure requires smaller amounts of fungal material for dsRNA detection and uses less reagents, thus resulting in appreciable cost savings.Les ARN double brin (ARNdb) viraux du genre Hypovirus à transmission cytoplasmique réduisent la virulence (hypovirulence) du Cryphonectria parasitica, le champignon responsable de la brûlure du châtaigner. La lutte biologique contre cette maladie fongique se fait par l'inoculation, dans des blessures de châtaigniers atteints, de souches choisies de C. parasitica artificiellement hypovirulentes, suivie de l'analyse de la transmissibilité des ARNdb aux isolats de C. parasitica qui affectent ces arbres. Nous présentons ici un protocole modifié d'isolement et de détection d'ARNdb. La procédure proposée requiert une plus petite quantité de matériel fongique pour la détection d'ARNdb et demande moins de réactifs; il en découle donc des économies appréciables

Study on metal-triggered callose deposition in roots of maize and soybean

Abstract: Callose plays important roles in a variety of processes of plant development, and/or in a response to a range of biotic and abiotic stresses. In the current work we have studied and compared the effect of lead, cadmium and arsenic on accumulation of newly formed callose deposits in the roots of maize and soybean. We observed formation of characteristic callose deposits in the root cell walls, probably associated with plasmodesmata, depending on the type of metal and the plant species investigated. Further, the callose turnover was analysed by measuring of total callose content as well as activities of total β-(1,3)-glucanases in roots. The latter enzymes are responsible for callose depletion, and their possible role during metal stress has previously been proposed. However, neither of these biochemical values appeared to be sufficiently reliable for scoring the altered callose turnover (including local deposits) in plant tissue. The microscopical observations are discussed in light of the biochemical data obtained

Optimalizácia podmienok expresie droserovej chitinázy (Drosera rotundifolia L.) v troch expresných kmeňoch E. coli

Round-leaf sundew (Drosera rotundifolia L.), family Droseraceae, genus Drosera, is one of a few plant species with a strong antifungal potential. Chitinases of carnivorous plants play an important role in decomposition of chitin-containing cell structures of insect prey. The cell wall of many phytopathogenic fungi also contains chitin, which can be utilized by chitinases, thus round-leaf sundew represents an interesting gene source for plant biotechnology. The purpose of this study was to compare the suitability of 3 different E. coli expression strains (E. coli BL21- CodonPlus® (DE3)-RIPL, E. coli ArcticExpress (DE3)RIL and E. coli SHuffle® T7) for production and isolation of heterologous round-leaf sundew chitinase (DrChit). Results showed that the recombinant protein was successfully expressed in all three strains, but occurred in insoluble protein fraction. To get the DrChit protein into soluble protein fraction some modifications concerning to induction temperatures and concentration of the IPTG inductor were tested. In addition, composition of lysis buffer has been modified with supplementation of strong non-ionic detergents, Triton® X100 and Tween® 20, respectively. As these modifications didn’t increase the amount of the DrChit protein in soluble fraction, therefore, its isolation under denaturing conditions and subsequent refolding for activity assays is recommended.Rosička okrúhlolistá (Drosera rotundifolia L.), rodina Droseraceae, rod Drosera, je jednou z mála rastlinných druhov so silným antifungálnym potenciálom. Chitinázy mäsožravých rastlín zohrávajú dôležitú úlohu pri dekompozícií bunkových štruktúr obsahujúcih chitín z tela chyteného hmyzu. Bunkové steny viacerých fytopatogénnych húb obsahujú chitín, ktorý môže byť využívaný chitinázami, čo robí z rosičky okrúhlolistej zaujímavý genetický zdroj pre rastlinné biotechnológie. Cieľom práce bolo porovnanie vhodnosti použitia 3 rozdielnych expresných kmeňov E. coli (E. coli BL21-CodonPlus® (DE3)-RIPL, E. coli ArcticExpress (DE3)RIL a E. coli SHuffle® T7) pre produkciu a izoláciu heterológnej chitinázy rosičky okrúhlolistej (DrChit). Výsledky ukázali, že rekombinantný proteín bol úspešne exprimovaný vo všetkých 3 kmeňoch, ale vyskytoval sa v nerozpustnej proteínovej frakcii. Aby sa DrChit proteín dostal rozpustnej proteínovej frakcie, boli testované modifikácie teploty indukcie a koncentrácie induktora IPTG. Ďalej bolo modifikované zloženie lyzačného pufru pridaním silných neiónových detergentov, Triton® X100 a Tween® 20. Pretože tieto modifikácie nezvýšili množstvo získaného DrChit proteínu v rozpustnej frakcii, je odporúčaná jeho izolácia za denaturačných podmienok a následná obnova štruktúry proteínu