LETTER TO THE EDITOR

LETTER TO THE EDITOR

In a recent research article published in THE PLANT CELL ("Hypervariable Domains of Self-Incompatibility RNases Mediate Allele-Specific Pollen Recognition"), Matton et al.1997 show that the pollen recognition function of the Solanum chacoense S₁₁ RNase can be converted to that of the S₁₃ RNase byreplacing the two hypervariable (HV) regions of S₁₁ RNase with thoseof S₁₃ RNase. From these results, Matton et al. conclude that, "one allelic form of the S RNase molecule can be converted into another by modification of the HV domains alone and that allelic specificity can be determined by the HV domains alone."

Although in this case the differences in allelic specificity between these two very closely related S RNases (the S₁₁ and S₁₃ RNases differ in only 10 out of 190 amino acids, with four differences in the exchanged HV regions and six elsewhere in the molecules [ Saba-El-Leil et al. 1994 ; Figure 1]) can be localized to the HV regions alone, it should be emphasized that this is not necessarily the case for all S RNases. Indeed, domainswap experiments performed by our group and that of Bruce McClure (S. Huang, A.G. McCubbin, and T.-h. Kao, unpublished results cited in Kao and McCubbin 1996 ; Zurek et al. 1997 ) suggest that regions outside theHV regions are involved in pollen recognition.

For example, when the HV regions of Petunia inflata S₃ RNase were replaced with those of S₁ RNase, the resulting chimeric protein, which had normal RNase activity, was no longer able to reject S₃ pollen. However, it did not gain the ability to reject S₁ pollen (Kao and McCubbin 1996 ). In a more extensive study, nine chimeric S RNases were constructed by swapping the HV regions, as well as other regions, between the Nicotiana alata S_A2 and S_C10 RNases. Again, none of the resulting chimeric S RNases was able to reject either S_A2 or S_C10 pollen, despite the fact that they all had normal RNase activity (Zurek et al. 1997 ). These results suggest that allele-specific pollen recognition byS RNases depends on amino acids located both in the HV regions and in other parts of the molecule.

Matton et al.'s conclusion that the HV regions are required for allele-specific pollen recognition supports the results of the previous studies (Kao and McCubbin 1996 ; Zurek et al. 1997 ). However, because the role of amino acids that are the same in the S₁₁ and S₁₃ RNases cannot be addressed in these experiments, their findings do not contradict the other conclusion drawn from domain swap experiments with more divergent S RNases—that allele-specific interactions between pollen and S RNase also requires amino acids that fall outside the HV regions.

There are some clues as to the possible locations of these amino acids. Sequence comparisons among theS RNases have revealed that there are nine scattered HV amino acids which couldpotentially play a role in pollen recognition (Tsai et al. 1992 ; Figure 1). The fact that these nine amino acids happen to be identical in S₁₁ and S₁₃ RNases does not preclude their potential importance and should not be taken to mean that the HV regions alone mediate S allele–specific pollen recognition in general. Indeed, there is no particular requirement for allelic variability at these amino acid positions; they could be identical, as they are in highly similar S RNases, or different, as they are in divergent S RNases.

To reconcile the difference between their finding and the previous results,Matton et al. suggest that one explanation for the inability of the chimeric S RNases to reject pollen in the previous studies may be that using pairs of S RNases with highly divergent sequences causes protein folding problems for the chimeric RNases (Matton et al. 1997 ). However, despite the overall sequence divergence between the S_A2 and S_C10 RNases, one of thechimeric S RNases constructed by Zurek et al., in which a relatively short region outside the HV regions was exchanged, is quite similar to the S_A2 RNase (only eight amino acid changes). In addition, the observation that all of the chimeric S RNases tested in the domain swap studies possessed normal RNase activity suggests that their overall conformation was unaffected (McCubbin et al., 1996; Zurek etal. 1997 ). These results lend further support to the hypothesis that residuesoutside the two HV regions are also involved in S allele specificity.

There are a number of ways in which the role of amino acids inside and outside the HV domains could be tested in the future. For example, it would be interesting to evaluate whether altering any of the conserved amino acids outside the S₁₁ and S₁₃ HV regions in positions that correspond to the nine scattered HV residues (see Figure 1) results in loss of the pollen recognition function for one S RNase without affecting the other. The role of identical residues within the HV regions of the S₁₁ and S₁₃ RNases could be assayed in a similar fashion. Moreover, the domain swapping approach could be extended to include regions from a more divergent S. chacoenseS RNase such as the S₂ RNase (see Figure 1). Does exchanging regions that are identical in the S₁₁ and S₁₃ RNases with the corresponding (divergent) regions of the S₂ RNase have an effect on pollen recognition?

In conclusion, any domain swap experiment between a pair of S RNases only demonstrates the role of those exchanged amino acids that differ between the two S RNases understudy; it cannot address the role of amino acids that are conserved between the S RNases. Ultimately, determining precisely which amino acids in a specific S RNase are involved in pollen recognition will require elucidation of the crystal structure of the S RNase and identification of the pollen S alleleproducts.

REFERENCES

Kao, T.-h., and McCubbin, A.G. (1996) How flowering plants discriminate between self andnon-self pollen to prevent inbreeding. Proc. Natl. Acad. Sci. USA 93:12059-12065.

Matton, D.P., Maes, O., Laublin, G., Xike, Q., Bertrand, C., Morse, D., and Cappadocia, M. (1997) Hypervariable domains of self-incompatibility RNases mediate allele-specificpollen recognition. Plant Cell 9:1757-1766.

Saba-El-Leil, M., Rivard, S., Morse, D., and Cappadocia, M. (1994) The S₁₁ and S₁₃ self-incompatibility alleles in Solanum chacoense Bitt. are remarkably similar. Plant Mol. Biol. 24:571-583.

Tsai, D.-S., Lee, H.-S., Post, L.C., Kreiling, K.M., and Kao, T.-h. (1992) Sequence of an S-protein of Lycopersicon peruvianum and comparison with other solanaceous S-proteins. Sex. Plant Reprod. 5:256-263.

Xu, B., Mu, J., Nevins, D.L., Grun, P., and Kao, T.-h. (1990) Cloning and sequencing of cDNAs encoding two self-incompatibility associated proteins in Solanum chacoense.. Mol. Gen. Genet. 224:341-346.

Zurek, D.M., Mou, B., Beecher, B., and McClure, B. (1997) Exchanging domains between S-RNases from Nicotiana alata disrupts pollen recognition. Plant J. 11:797-808.

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