Many flowering plants prevent inbreeding and increase genetic diversity by a process called self-incompatibility, in which pollination fails to set seed if the pollen is identified as its own by the pistil. A research team, led by Teh-hui Kao, Professor of Biochemistry and Molecular Biology at Penn State, has announced, in a paper published in the May 20 issue of Nature, the discovery of a gene of petunias that controls pollen function in self-incompatibility. This discovery completes a critical missing link in the understanding of how self-incompatibility works. Ten years ago, Kao announced, in another paper published in Nature, the identification of the gene, called the S-RNase gene (S for self-incompatibility), that controls pistil function in self-incompatibility. "This male component turned out to be much more elusive than the pistil component," says Kao. "Our team, as well as others, has worked for the past ten years to find it." The recently identified gene, named PiSLF (for Petunia inflata S-locus F-box), encodes a new member of a large family of F-box proteins that are known to mediate protein degradation in diverse organisms, including animals, plants and yeast.

While a species may have as many as 50 or 60 different S-alleles, each plant has only two of them, one inherited from each parent. An allele is one of a number of possible variants of a particular gene; for example, two alleles exist for each of the three genes that determine eye color in humans. Pollen grains are haploid, meaning that they contain only a single set of chromosomes, and thus each pollen grain contains only one of the two S-alleles of the parent plant. The pistil, on the other hand, is diploid, meaning that it has two sets of chromosomes (one from each parent) and therefore has both S-alleles of the parent plant. During pollination, if the S-allele of the pollen does not match either of the two S-alleles in the pistil, the pollen will germinate on the surface of the pistil to produce pollen tubes, which will then grow through the pistil to the ovary to effect fertilization. However, if the S-allele of the pollen matches either of the two S-alleles in the pistil, growth of the pollen tube is stopped about one third of the way to the ovary, preventing fertilization. Triggering this self-incompatibility response requires an interaction between the product of an S-allele produced in pollen and the product of a genetic counterpart produced in the pistil. To identify the pollen component in self-incompatibility, the team examined the DNA sequence of a chromosomal region containing the S2-allele of the S-RNase gene (the previously identified pistil component for plants containing the specific S-locus allele that is labeled S2). "The gene controlling the pollen function must be very closely linked to the S-RNase gene to prevent recombination," says Kao. "Otherwise, recombination between these two genes would cause the breakdown of self-incompatibility, which has never been observed in nature"

After identifying the PiSLF gene, located approximately 161 kb from the S-RNase gene, Kao's team had to demonstrate that the gene was indeed the pollen component of self-incompatibility. "Other labs have found similar genes in the vicinity of the S-RNase gene in various other species" he says. "But proximity alone is insufficient to show the relationship." They took advantage of a phenomenon known as competitive interaction to demonstrate the function of the PiSLF gene in self-incompatibility. It has been known for some time that if pollen has two different S-alleles (which could result when the chromosomal region containing the pollen S-allele is duplicated in a plant), the pollen fails to function in self-incompatibility and thus cannot be rejected by any plant pistil. However, pollen with two identical S-alleles (again resulting from duplication of the pollen S-allele) remains functional in self-incompatibility. The team carried out three sets of experiments. In one set, the S2-allele of PiSLF was introduced into plants of S1S1 genotype – plants containing two identical S-locus genes of a type labeled S1 - via standard plant transformation techniques. For each transgenic plant generated, half of the pollen produced contained the endogenous (originating from within the plant) pollen S1-allele plus the PiSLF2 transgene (a gene that is introduced from a source outside the plant), whereas the other half only contained the endogenous pollen S1-allele. If PiSLF is the pollen component, the pollen that contained PiSLF2 should contain two different pollen S-alleles, S1 from the endogenous gene and S2 from the transgene, and based on competitive interaction, should fail to function in self-incompatibility. However, the pollen that contained only the endogenous pollen S1-allele should function normally. Thus, the prediction was that the transgenic plants would set seeds upon self-pollination (i.e., becoming self-compatible) and that all the resulting progeny should inherit the PiSLF2 transgene. The results from this set of experiments, as well as from two other sets using different genotypes of plants as recipient of PiSLF2, were completely in agreement with the prediction based on competitive interaction and based on the assumption that PiSLF is the pollen component.

The team that made this discovery consisted of five graduate students, Paja Sijacic, Xi Wang, Andrea L. Skirpan, Yan Wang and Peter E. Dowd, and a postdoctoral scholar, Andrew G. McCubbin. In addition, a research scientist, Shihshieh Huang, at Monsanto (a former graduate student of Kao's group) participated in the project as a collaborator.

This discovery could have commercial application for hybrid seed production in crop plants, such as corn and soy bean, that have lost self-incompatibility. Raising hybrid seed has been one of the major goals of horticultural and agricultural practice, because hybrid plants are more productive (due to hybrid vigor) and more uniform in quality than plants derived from self-pollination or random pollination. To raise hybrid seed, self-pollination and sib-pollination (pollination by a plant of the same hybrid) must be circumvented. One method is hand emasculation of the line used as female parent, which is then naturally cross-pollinated by pollen from the line serving as male parent and planted in an adjacent row. However, this process is very labor intensive and invariably expensive. If the crop plants can be made self-incompatible by the introduction of the genes controlling self-incompatibility, then all seeds produced will be hybrids resulting from cross-pollination between two different lines. This would facilitate the production and increase the yield of hybrid seed and, at the same time, reduce the labor costs.

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Contact: Barbara K. Kennedy, science@psu.edu, 814-863-4682, Penn State

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