RESUMO
BACKGROUND: When creating plant transformation vectors, full control of nucleotides flanking the insert in the final construct may be desirable. Modern ligase-independent methods for DNA-recombination are based on linearization by classical type II restriction endonucleases (REs) alone or in combination with nicking enzymes leaving residual nucleotides behind in the final construct. We here explore the use of type IIS and type IIB REs for vector linearization that combined with sequence and ligase-independent cloning (SLIC) overcomes this problem and promotes seamless gene-insertion in vectors. Providing the basis for a collection of biolistic plant transformation vectors ready to be cloned with different genes-of-interest, we present two vectors, where promoter and terminator are joined by a spacer. During spacer-removal linearization (SRL), type IIS and type IIB REs remove their own recognition sequences from the vector leaving no undesired, short sequences behind. RESULTS: We designed two plant transformation vectors prepared for SRL in combination with SLIC, pAUrumII and pAUrumIII, harboring a spacer with recognition sites for a type IIS and IIB RE, respectively. The gene for a green fluorescent protein, gfp, was successfully cloned into both vectors; traces of pAUrumIII, however, contaminated the transformation due to incomplete linearization, an issue not encountered with the type IIS linearized pAUrumII. Both constructs, pAUrumII-gfp and pAUrumIII-gfp, were functional, when tested in vitro on wheat and barley endosperm cells for transient gfp expression. CONCLUSIONS: All nucleotides flanking an insert in a biolistic plant transformation vector can be customized by means of SRL in combination with SLIC. Especially type IIS REs promote an efficient cloning result. Based on our findings, we believe that the SRL system can be useful in a series of plant transformation vectors, favoring the presence of functional sequences for optimal expression over redundant cloning-site remnants.
RESUMO
Pathogens induce the expression of many genes encoding plant transcription factors, though specific knowledge of the biological function of individual transcription factors remains scarce. NAC transcription factors are encoded in plants by a gene family with proposed functions in both abiotic and biotic stress adaptation, as well as in developmental processes. In this paper, we provide convincing evidence that a barley NAC transcription factor has a direct role in regulating basal defence. The gene transcript was isolated by differential display from barley leaves infected with the biotrophic powdery mildew fungus, Blumeria graminis f.sp. hordei (Bgh). The full-length cDNA clone was obtained using 5'-RACE and termed HvNAC6, due to its high similarity to the rice homologue, OsNAC6. Gene silencing of HvNAC6 during Bgh inoculation compromises penetration resistance in barley epidermal cells towards virulent Bgh. Complementing the effect of HvNAC6 gene silencing, transient overexpression of HvNAC6 increases the occurrence of penetration resistant cells towards Bgh attack. Quantitative RT-PCR shows the early and transient induction of HvNAC6 in barley epidermis upon Bgh infection. Additionally, our results show that the Arabidopsis HvNAC6 homologue ATAF1 is also induced by Bgh and the ataf1-1 mutant line shows decreased penetration resistance to this non-host pathogen. Collectively, these data suggest a conserved role of HvNAC6 and ATAF1 in the regulation of penetration resistance in monocots and dicots, respectively.
