A small asparagine-rich protein required for S-allele-specific pollen rejection in Nicotiana

Although S-locus RNases (S-RNases) determine the specificity of pollen reje ction in self-incompatible (SI) solanaceous plants, they alone are not suff icient to cause S-allele-specific pollen rejection. To identify non-S-RNase sequences that are required for pollen rejection, a Nicotiana alata cDNA l ibrary was screened by differential hybridization, One clone, designated MT , hybridized strongly to RNA from N. alata styles but not to RNA from Nicot iana plumbaginifolia, a species known to lack one or more factors necessary for S-allele-specific pollen rejection. Sequence analysis revealed a 101-r esidue ORF including a putative secretion signal and an asparagine-rich dom ain near the C terminus. RNA blot analysis showed that the MT-transcript ac cumulates in the stigma and style before anthesis, The timing of MT-express ion lags slightly behind S-C10-RNase in SI N, alata SC10SC10 and is well co rrelated with the onset of S-allele-specific pollen rejection in the style. An antisense-MT construct was prepared to test for a role in pollen reject ion. Transformed (N, plumbaginifolia x SI N, alata SC10SC10) hybrids with r educed levels of MT-protein continued to express SC10-RNase but failed to r eject SC10-pollen. Control hybrids expressing both SC10-RNase and MT-protei n showed a normal S-allele-specific pollen rejection response. We conclude that MT-protein is directly implicated in pollen rejection.