Ribonuclease activity of Petunia inflata S proteins is essential for rejection of self-pollen

S proteins, pistil-specific ribonucleases that cosegregate with S alleles, have previously been shown to control rejection of self-pollen in Petunia inflata and Nicotiana alata, two solanaceous species that display gametophytic self-incompatibility. The ribonuclease activity of S proteins was thought to degrade RNA of self-pollen tubes, resulting in the arrest of their growth in the style. However, to date no direct evidence has been obtained. Here, the ribonuclease activity of S₃ protein of P. inflata was abolished, and the effect on the pistil's ability to reject S₃ pollen was examined. The S₃ gene was mutagenized by replacing the codon for His-93, which has been implicated in ribonuclease activity, with a codon for asparagine, and the mutant S₃ gene was introduced into P. inflata plants of S₁S₂ genotype. Two transgenic plants produced a level of mutant S₃ protein comparable to that of the S₃ protein produced in self-incompatible S₁S₃ and S₂S₃ plants, yet they failed to reject S₃ pollen. The mutant S₃ protein produced in these two transgenic plants did not exhibit any detectable ribonuclease activity. We have previously shown that transgenic plants (S₁S₂ plants transformed with the wild-type S₃ gene) producing a normal level of wild-type S₃ protein acquired the ability to reject S₃ pollen completely. Thus, the results reported here provide direct evidence that the biochemical mechanism of gametophytic self-incompatibility in P. inflata involves the ribonuclease activity of S proteins.