Tissue-specific gene expression in the pericarp of barley (Hordeum vulgare L.): Promoter cloning and analysis

There is a critical need for cereal promoters capable of targeting transgene expression in cereal spike tissues for grain quality and disease resistance applications. Expression of transgene-mediated Fusarium Head Blight (FHB) resistance in barley and wheat, for example, should be targeted to readily infected spike tissues including pericarp, lemma, and palea. In the search for such promoters, our differential display screening for barley genes that are expressed in the pericarp, but not in leaves, detected a novel lipid transfer protein encoding gene, Ltp6, and a germin-like protein encoding gene, GerB. Inverse PCR was used to derive 2345 bp of upstream Ltp6 sequence. Promoter deletion studies revealed that constructs containing at least 192 bp of Ltp6 upstream sequence and the 5'UTR conferred tissue-specific expression and retained most of the promoter strength. Deletion of 64 bp (-192/-128) from this upstream sequence reduced expression levels by 80%. Sequence and Southern blot analyses revealed that GerB has high sequence identity with GerF, its closest paralog. GerB and GerF individual expression patterns were determined using RNA gel blots coupled with single stranded conformation polymorphism (SSCP) analysis of RT-PCR products. This provided evidence of both overlapping redundancy and subfunctionalization. GerB is predominantly expressed in developing shoots, while GerF is predominantly expressed in seedling roots, developing spikes and pericarp/testa. Promoter deletion studies located a GerF region (-356/-97) responsible for high promoter activity, and showed the ability of GerB and GerF upstream sequences to drive gfp expression in coleoptiles, pericarps, and lemma/palea of developing spikes. The period of maximum susceptibility to FHB in spike tissues extends from pollination up to the early dough stage of seed development. Ltp6 -driven gfp expression in the pericarp at the early dough stage of seed development can be traced to fluorescent ovary initials in spikelet primordia of transgenic barley. Similarly, GerB and GerF promoter sequences are capable of driving gfp expression in pericarp, but also in lemma/palea of developing spikes. None of these three promoters drove gfp expression in mature barley leaves, as predicted by expression analyses. Thus, Ltp6, GerB, and GerF gene promoters are suitable candidates for targeting tissue-specific FHB-resistance in barley.