The tuf gene of “Candidatus Phytoplasma mali”, the causal agent of apple proliferation was PCR cloned in an expression vector and expressed in Escherichia coli. First, phytoplasma DNA extracted from periwinkle was amplified using primers designed on the basis of the tuf gene and the PCR product was cloned into pGEM-T (Promega). In the next step specific primers were constructed containing some plasmid sequences and restriction enzyme sites. With this primers the sequence in pGEM-T was amplified, the product was digested with restriction enzymes, and inserted into the pQE40 expression vector (Qiagen). In this plasmid the tuf gene was fused to the 6xHIS tag, and DHFR. The production of 6xHIS-DHFR-Tu fusion protein protein was induced with IPTG and expressed in E. coli M15. The new fusion protein was found in the insoluble fraction of the bacterium. The identity of the protein was verified with polyacrylamid gel-electrophoresis and Western blot analysis using antiserum raised against the 6xHIStag of the fusion protein.
Authors:B. Barna, B. Harrach, O. Viczián, and J. Fodor
Heat shock treatment of near isogenic barley lines induced susceptibility against powdery mildew (Blumeria graminis f. sp. hordei, Bgh). When barley lines were immersed into hot water (48–49 °C) for 20 seconds one day before inoculation with Bgh race A6, the heat treatment increased susceptibility in susceptible barley cv. Ingrid and in its near-isogenic barley lines carrying different effective resistance genes. Microscopic investigations indicated vigorous development of the pathogen not only on heat treated susceptible Ingrid and resistant Mla, but also on Mlg-resistant and even mlo-resistant lines. However, when longer heat stress was used, infection density increased gradually on the susceptible Ingrid leaves, and the 40–50 sec heat treatment induced the development of visible powdery mildew colonies even on mlo leaves. Heat stress significantly increased leakage of ions from leaf segments from all barley lines with or without specific resistance genes and caused a late decrease of SOD and a slight increase in CAT enzyme activities, which correlated with the slightly down-regulated levels of hydrogen peroxide in the heat treated barley leaves. Significant increase of RNase activities was found after heat stress, and there was a slight degradation of total DNA as a consequence of heat pretreatment in all barley lines.
Authors:O. Viczián, A. Künstler, Y. Hafez, and L. Király
High concentrations of the reactive oxygen species (ROS) superoxide (O2•−) and hydrogen peroxide (H2O2) contribute to the induction of plant cell and tissue death (necrosis). In an effort to create transgenic plants with high antioxidant capacity that could resist necrotic symptoms we produced two transgenic tobacco (Nicotiana tabacum cv. SR1) lines (S1 and S2) overexpressing a tomato chloroplast superoxide dismutase (SlChSOD). SOD genes encode for antioxidant enzymes that dismutate superoxide to hydrogen peroxide. Therefore, SOD-overproducing plants may contain high levels of hydrogen peroxide and are sensitive to stress-related necrosis unless sufficient degradation of hydrogen peroxide is conferred by elevated expression of antioxidants like e.g. catalases and peroxidases. Indeed, line S1 displayed elevated expression of a glutathione peroxidase (NtGPX) and a glutathione S-transferase (NtGSTU1b), as compared to wild type plants. Interestingly, however, expression of a catalase (NtCAT1) was repressed in both SOD-overexpressing lines. This predicts that such plants could be sensitive to localized necrosis (HR) caused by virus infection, since repression of NtCAT1 has been shown to occur during virus-induced HR (e.g. Dorey et al., 1998; Künstler et al., 2007). To elucidate whether other catalases might play a role in resistance to virus induced HR-type necrotic symptoms, a maize catalase (ZmCat2) was transiently overexpressed in Nicotiana edwardsonii and N. edwardsonii var. Columbia plants by agroinfiltration. Inoculation of agroinfiltrated plants with Tobacco mosaic virus (TMV) revealed that ZmCat2 confers enhanced resistance to HR-type necrosis during TMV infection. It seems that catalases may play different roles in influencing resistance to virus-induced hypersensitive necrosis.