Targeted expression of redesigned and codon optimised synthetic gene leads to recrystallisation inhibition and reduced electrolyte leakage in spring wheat at sub-zero temperatures

Abstract Antifreeze proteins (AFPs) adsorb to ice crystals and inhibit their growth, leading to non-colligative freezing point depression. Crops like spring wheat, that are highly susceptible to frost damage, can potentially be made frost tolerant by expressing AFPs in the cytoplasm and apoplast where ice recrystallisation leads to cellular damage. The protein sequence for HPLC-6 α-helical antifreeze protein fromwinter flounderwas rationally redesigned after removing the prosequences in the native protein. Wheat nuclear gene preferred amino acid codons were used to synthesize a recombinant antifreeze gene, rAFPI. Antifreeze protein was targeted to the apoplast using a Murine leader peptide sequence from the mAb24 light chain or retained in the endoplasmic reticulum using C-terminus KDEL sequence. The coding sequences were placed downstream of the rice Actin promoter and Actin-1 intron and upstream of the nopaline synthase terminator in the plant expression vectors. Transgenic wheat lines were generated through micro projectile bombardment of immature embryos of spring wheat cultivar Seri 82. Levels of antifreeze protein in the transgenic lines without any targeting peptide were low (0.06–0.07%). The apoplast-targeted protein reached a level of 1.61% of total soluble protein, 90% of which was present in the apoplast. ER-retained protein accumulated in the cells at levels up to 0.65% of total soluble proteins. Transgenic wheat line T-8 with apoplast-targetedantifreeze protein exhibited the highest levels of antifreeze activity and provided significant freezing protection even at temperatures as low as −7◦C

The complex of Diplodia species associated with Fraxinus and some other woody hosts in Italy and Portugal

Studies on the taxonomy and phylogeny of Diplodia have been hampered by the lack of an ex-type culture linked to the holotype of D. mutila, which is the type of the genus. In this study a large collection of Diplodia strains, obtained from ash and other woody hosts showing V-shaped cankers and branch dieback, were identified based on morphological characters and DNA sequence data from ITS and EF1-α loci. Results of combined morphological and phylogenetic analyses showed that the Fraxinus isolates from Italy, the Netherlands, Portugal and Spain belong to three distinct species namely Diplodia fraxini, Diplodia mutila and Diplodia subglobosa sp. nov. An epitype was designated for Diplodia mutila, with associated ex-epitype cultures. The name D. fraxini is re-instated and a neotype designated. Two species, Diplodia seriata and Diplodia pseudoseriata were reported for the first time on Fraxinus spp