( bserved in different cell ty...)Single-Cell Complementation of Barley Mlo Mutants Using a PDS-1000/He Hepta System

Detached leaf sections were heavily inoculated with spores of B. graminis f. sp. hordei isolate K1 approximately 24 hr after bombardment. Microscopic fungal growth was visible 34 days after inoculation as the formation of aerial hyphae and conidiophores. In the vast majority of sections, formation of fungal colonies originated from single infected cells expressing GFP (Figure 3). Because plasmid pUGLUM contains both GFP and Mlo, our data suggest that transient expression of wild-type Mlo complements mlo resistance at the single cell level. This is consistent with results obtained after cobombardment of 2 plasmids containing GFP or Mlo (Shirasu et al. 1999).

A particle inflow gun with a single outlet and a much smaller target area was used in a previous study (Shirasu et al. 1999). Under those conditions, leaf sections of 3 cm length could be bombarded in a single shot. About 20 leaf segments and several bombardments were necessary to obtain 100300 GFP expressing cells (Shirasu et al. 1999).

Modification of the experimental setup described here generated a comparable number of cells expressing the GFP reporter gene after a single bombardment. Application of the Hepta adaptor resulted in consistently high numbers of transfected cells in several independent experiments. Generally, we found multiple clusters of transfected epidermal cells in bombarded leaf segments as shown in Figure 2. Thus, the Hepta adaptor is useful for experiments in which several independent constructs need to be tested or in which high numbers of transformed cells are desired.
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