Most flowering plants produce bisexual flowers containing both male and female reproductive organs, and they would have a strong tendency to self-fertilize, if no mechanisms prevent them from doing so. Since inbreeding is deleterious to any organism, flowering plants have evolved a variety of reproductive strategies, including self-incompatibility (SI), to prevent inbreeding and promote out-crossing. SI allows pistils to reject self-pollen and accept non-self pollen for fertilization. This project uses Petunia inflata to study one type of SI mechanism, and has previously identified the S-RNase expressed by the pistil and PiSLF (P. inflata S-locus F-box) that is expressed by the pollen. During growth of a pollen tube in a pistil, the PiSLF in the pollen tube only allows self S-RNase to inhibit pollen tube growth. Several hypotheses that explain how PiSLF and S-RNase interact to elicit specific rejection of self pollen tubes will be tested. The project will use in vivo approaches to test these hypotheses by dissecting the functions of three domains of PiSLF in interactions with S-RNase, studying the biochemical function of a potential component of the PiSLF-containing complex, and examining the role of glycosylation of S-RNase in SI. SI provides a model for studying self/non-self recognition, so completion of this project will have wider implications for biological sciences. PiSLF and S-RNase could be used for restoring SI to crop species to facilitate hybrid seed production, which will have tremendous agronomic benefits. The project will engage three graduate and three undergraduate students to prepare them for future careers in research. The researchers in this project will continue to participate in an annual biology fair at Penn State to educate the attendees, particularly K-12, about plant reproductive biology and SI.

Project Report

?Self-incompatibility is a reproductive strategy adopted by many flowering plants to prevent inbreeding and promote outcrosses. It was discovered in the 18th century and was documented by Charles Darwin in a monograph published in 1865. Genetic studies conducted in the early to mid 20th century on the self-incompatible species in the Solanaceae (nightshade) family established that self/non-self recognition between pollen and pistil is controlled by a single polymorphic locus, named the S-locus. For a given species, there may be over 100 different variants of the S-locus, referred to as S-haplotypes. Pollen is recognized by the pistil as self-pollen if its S-haplotype is present in the pistil, and pollen is recognized by the pistil as non-self pollen if its S-haplotype is not present in the pistil. Self-incompatibility has long attracted the interest of biologists from many disciplines such as genetics, evolutionary biology, population biology, cell biology, physiology, etc., but up until the early 1980s, nothing was known about the genes involved. Since 1986, the PI’s lab has been using Petunia inflata, a wild species of Petunia in the Solanaceae family, as a model to investigate how pistils distinguish between self and non-self pollen, and then specifically reject self-pollen to prevent inbreeding. Prior to the start of this project, the PI’s lab had identified the S-RNase gene as the gene that controls pistil specificity, and the PiSLF (P. inflata S-locus F-box) gene as the gene that controls pollen specificity. In this project, the PI’s lab used several in vivo approaches to test the hypotheses it had formulated to explain how PiSLF and S-RNase interact inside a pollen tube to elicit specific rejection of self pollen tubes. A region of PiSLF involved in interactions with S-RNase was identified, the biochemical function of a potential component of the PiSLF-containing complex was studied, and the S-RNase was found to exert its cytotoxic function in the cytoplasm of pollen tubes. Most significantly, in collaboration with Professor Seiji Takayama’s lab in Nara, Japan, it was discovered that PiSLF is not the only gene that controls pollen specificity. PiSLF was renamed Type-1 SLF or SLF1. To date, a total of 10 types of SLF genes have been identified, seven first by the PI’s lab and three first by Professor Takayama’s lab. A new model, named collaborative non-self recognition, was proposed to explain the biochemical basis of self-incompatibility. The unexpected finding about the involvement of multiple SLF genes in controlling pollen specificity has raised many new questions about the evolution and operation of the self-incompatibility system in Petunia, some of which are being pursued in the new phase of the PI’s research. The self-incompatibility mechanism in Petunia has parallels in other self/non-self discrimination systems involved in host defense, most notably, adaptive immunity of vertebrates. Many T-cell receptors (analogous to SLF proteins) are required to collectively recognize a wide variety of foreign antigens (analogous to non-self S-RNases) in order to mount the immune response to destroy them (analogous to the degradation of non-self S-RNases). No T-cell receptors should recognize self-antigens, lest autoimmune disease would result, and similarly, no SLF proteins should recognize self S-RNase, lest SI would break down. Thus, the results obtained in this project have wider implications for the self/non-self recognition process, a fundamental process in biology. The self-incompatibility system also provides a model for F-box protein-mediated protein degradation, a key regulatory mechanism in many cellular and developmental processes in eukaryotes. From a practical point of view, SI could be used to facilitate hybrid-seed production. As most crop species are self-compatible, making production of hybrid seed labor intensive, costly and inefficient, understanding the biochemical basis of the interaction between different members of the SLF family and S-RNases will help design strategies for using their genes to restore self-incompatibility back to crop species. If accomplished, this will have tremendous agronomic benefits. This project provided research training for six graduate students (four female and two male) and thirteen undergraduate students (eight female and five male). Two of the graduate students have received a Ph.D. degree and one has received a Master’s degree. All but two of the undergraduate students conducted research under the REU support. All the graduate and undergraduate students have gained valuable experience in the design and execution of experiments that will be valuable for their pursuit of future research. The PI’s lab participated in an annual biology fair at Penn State in 2009, 2010 and 2012, using posters and live demonstration to educate the attendees, particularly K-12, about plant reproductive biology and self-incompatibility. A graduate student and an undergraduate student teamed up with two undergraduate students majoring in Telecommunications to produce a video about self-incompatibility, which was posted on YouTube.

National Science Foundation (NSF)
Division of Integrative Organismal Systems (IOS)
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Amy Litt
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Pennsylvania State University
University Park
United States
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