Tapetum-specific expression of harpin<sub>Pss</sub> causes male sterility in transgenic tobacco

Harpin, an elicitor molecule of bacterial origin induces hypersensitive response (HR) in non-host plants. In an attempt to induce male sterility, harpin was tagged with a signal peptide and expressed downstream to tapetum-specific TA29 promoter resulting in extracellular secretion, subsequent degeneration of tapetum and development of male sterility in tobacco. Putative transgenics were analyzed by PCR amplification of transgene, semiquantitative RT-PCR analysis from total RNA extracts from anther tissue with transgene specific probe, Western blotting using polyclonal antibody raised against harpin, by transmission and scanning electron microscopy, and by confocal microscopy of anthers and pollen at various stages of development. Varying degrees of male sterility (30-100 %) was observed with plants showing complete and partial male sterility as well as several morphological variations were seen especially in leaves and flowers. Further, some of the transgenics showed un-induced of HR-like local lesions in the vegetative tissues. HarpinPss got deposited on the pollen grains upon tapetal degeneration resulting in significant alterations in the morphology of pollen cell wall. However, megagametogenesis was not affected in complete and partial male sterile plants and female gametes were completely fertile. The complete male sterility was attributed to premature tapetal cell death due to sufficient extracellular harpinPss accumulation whereas insufficient protein content might be the reason for partial male sterility. These findings indicate the possible use of cytotoxic harpinPss for the development of male sterile plants.