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 rejection in self-incompatible (SI) solanaceous plants, they alone are not sufficient to cause S-allele-specific pollen rejection. To identify non-S-RNase sequences that are required for pollen rejection, a Nicotiana alata cDNA library was screened by differential hybridization, One clone, designated MT, hybridized strongly to RNA from N. alata styles but not to RNA from Nicotiana plumbaginifolia, a species known to lack one or more factors necessary for S-allele-specific pollen rejection. Sequence analysis revealed a 101-residue ORF including a putative secretion signal and an asparagine-rich domain near the C terminus. RNA blot analysis showed that the MT-transcript accumulates in the stigma and style before anthesis, The timing of MT-expression lags slightly behind S-C10-RNase in SI N, alata SC10SC10 and is well correlated 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 rejection. Transformed (N, plumbaginifolia x SI N, alata SC10SC10) hybrids with reduced levels of MT-protein continued to express SC10-RNase but failed to reject SC10-pollen. Control hybrids expressing both SC10-RNase and MT-protein showed a normal S-allele-specific pollen rejection response. We conclude that MT-protein is directly implicated in pollen rejection.