Teh-hui Kao Professor of Biochemistry & Molecular Biology 403 Althouse Laboratory, University Park, PA 16802 Phone: (814) 863-1042 Fax: (814) 863-7024 E-mail: txk3@psu.edu B.S.. in chemistry, National Taiwan University Ph.D. in chemistry/physical biochemistry, Yale University Kao Lab Web Site

Biochemical and Molecular Bases of Self/Non-Self Recognition During Plant Reproduction

Our major research focus over the past two decades has been on self-incompatibility, a self/non-self recognition mechanism that operates during sexual reproduction in flowering plants.  In addition, we have studied the role of a pollen-specific receptor-kinase in pollen development.  Currently, we are continuing to investigate the biochemical and molecular bases of the type of self-incompatibility that has been found in three families of flowering plants, including the Solanaceae (nightshade) family.  We are using Petunia inflata (a wild species of garden petunia) in the Solanaceae family as a model.  

Self-incompatibility is an intraspecific reproductive barrier that prevents flowering plants from self-fertilizing and promotes out-crossing.  It is estimated that more than half of the flowering plant species possesses self-incompatibility.  To date, however, only a small number of families have been studied at the molecular level.  Among them, three families, the Solanaceae, Rosaceae and Scrophulariaceae, employ the same self-incompatibility mechanism (Kao and Tsukamoto 2004).  Here, the outcome of pollination is controlled by a highly polymorphic locus named the S-locus.  If the S-haplotype of pollen matches one of the two S-haplotypes carried by the pistil, the pollen is recognized by the pistil as self-pollen and the growth of self-pollen tubes in the style is inhibited.  If the S-haplotype of pollen is different from the two S-haplotypes carried by the pistil, the pollen is recognized as non-self pollen and the growth of the pollen tubes in the style is not inhibited.  The non-self-pollen tube grows down through the style to the ovary to effect fertilization.

We are interested in addressing two fundamental questions about self-incompatibility.  First, how does a pistil distinguish between self and non-self pollen?  Second, how does the recognition of self-pollen lead to growth arrest of self-pollen tubes?  We first identified a gene located at the S locus, named the S-RNase gene, and used gain-of-function and loss-of-function approaches to show that the S-RNase gene controls pistil function in recognition and rejection of self-pollen (Lee et al. 1994).  We subsequently showed that the RNase activity of S-RNases is essential for rejection of self-pollen (Huang et al. 1994), and that the recognition function of S-RNases lies in the protein backbone but not in the carbohydrate moiety (Karunanandaa et al. 1994).  Since the S-RNase gene does not control pollen function in self-incompatibility, it is imperative that the gene that controls pollen specificity in self-incompatibility interaction also be identified.  To achieve this end, we identified pollen-expressed genes that are linked to the S-locus (McCubbin et al. 2000a), constructed a BAC library of S2-haplotype (McCubbin et al. 2000b), and then isolated BAC clones that contained DNA fragments in the S-locus region (Wang et al. 2003).  From sequence analysis of a 328-kb S-locus region (constructed from three BAC clones), we identified a pollen-expressed gene, named PiSLF (Petunia inflata S-locus F-box), that is located 161 kb downstream of the S2-RNase gene (Wang et al. 2004).  We subsequently obtained in vivo evidence that PiSLF indeed encodes the pollen determinant in self-incompatibility (Sijacic et al. 2004).

PiSLF contains an F-box domain, and most F-box proteins that have been characterized so far are involved in ubiquitin-mediated protein degradation.  Specifically, F-box proteins are a component of a type of E3 ubiquitin ligase called SCF complex, which, in conjunction with E1 ubiquitin-activating enzyme and E2 ubiquitin-conjugating enzyme, mediates the transfer of a polyubiquitin chain to protein substrates.  Each F-box protein interacts with specific proteins, and the ubiquitinated proteins are recognized by the 26S proteasome and degraded.  We propose that PiSLF functions as a conventional F-box protein, and a PiSLF mediates specific degradation of its non-self S-RNases inside a pollen tube.  Consequently, only self S-RNase is able to exert its cytotoxic effect to degrade pollen tube RNA, resulting in growth inhibition of self-pollen tubes in the pistil.  For example, when an S1 pollen tube is growing down through a pistil of S­1S2 genotype, both S1-RNase and S2-RNase produced in the pistil are taken up by the S1 pollen tube; however, PiSLF1 produced in the S1 pollen tube would mediate degradation of S2-RNase (a non-self S-RNase with respect to PiSLF1), but allow S1-RNase to degrade RNA inside the S1 pollen tube.  We have used a variety of molecular, biochemical, and genetic approaches to examine this hypothesis, and some of the key results obtained to date are summarized below (Hua and Kao 2006):  (1) PiSLF is a component of a novel E3 ubiquitin, which also contains a Cullin-1 (named PiCUL1-G) and a RING-finger protein (named PiSBP1); (2) S-RNases are ubiquitinated and degraded in pollen tube extracts; (3) a PiSLF interacts with its non-self S-RNases more strongly than with its self-S-RNase; (4) an S-RNase interacts with its non-self PiSLFs more strongly than with its self PiSLF.  These results are consistent with our hypothesis, as preferential interaction between a PiSLF and its non-self S-RNases would allow PiSLF to specifically mediate the ubiquitination and degradation of non-self S-RNases.      

The questions we are currently addressing include the following.  (1) Since there are a large number of F-box proteins in plants, we are interested in determining whether PiSLF is unique in its function in self-incompatibility, and if so, what features of PiSLF confer on it this unique function.  We have studied several F-box proteins that share a number of properties with PiSLF, and found that none of them function in self-incompatibility.  Sequence comparison between these PiSLF-like proteins and PiSLF has revealed regions that are unique to PiSLF.  Chimeric proteins between PiSLF and PiSLF-like proteins will be generated for the study of the role of these regions in vitro and in vivo (i.e., in transgenic plants).  (2) We are interested in determining the biochemical basis for the binding affinity differences between self-interactions and non-self interactions of PiSLF and S-RNase.  Sequence comparison of different allelic variants of PiSLF has identified regions that may be involved in general interactions with both self and non-self S-RNases, and regions that may be involved in specific interactions with self-S-RNase.  We will again use chimeric proteins to examine the role of these regions in vitro and in vivo.  (3) We are interested in reconstituting the complex that contains PiSLF, and examining whether it preferentially mediates ubiquitination of non-self S-RNases.     

Selective protein degradation via the ubiquitin-mediated pathway has rapidly emerged as an important regulatory mechanism for a variety of cellular and developmental processes in diverse organisms.  In plants, this mechanism has been implicated in regulating floral organ identity, circadian rhythm, and auxin and jasmonate responses.  PiSLF is among the very few F-box proteins whose substrates have been identified.  Moreover, because there are literally hundreds of S-RNases and their corresponding PiSLFs in species that possess S-RNase-mediated self-incompatibility, this self-incompatibility system will be useful for biochemical characterization of the interactions between an F-box protein and its substrates.  The information gained from our research will be valuable to understanding not only this self-incompatibility system, but also many cellular and developmental processes in a variety of organisms in which regulation of protein degradation has been implicated.

Representative Publications:

• Hua, Z. and Kao, T.-h. (2006). Identification and characterization of components of a putative PiSLF-containing E3 ligase complex involved in S-RNase-based self-incompatibility. Plant Cell 18, 2531-2553.

• Dowd, P. E., Coursol, S., Skirpan A. L., Kao, T.-h. and Gilroy, S. (2006). Petunia phospholipase C1 is involved in pollen tube growth. Plant Cell 18, 1438-1453.
• Skirpan, A.L., Dowd, P. E., Sijacic, P., Jaworski, C. J., Gilroy, S. and Kao, T.-h. (2006). Isolation and characterization of PiORP1, a Petunia oxysterol-binding-protein related protein involved in receptor-kinase mediated signaling in pollen, and analysis of the ORP gene family in Arabidopsis. Plant Mol. Biol. 61, 553-565.

• Kokubun, H., Nakano, M., Tsukamoto, T., Watanabe, H., Hashimoto, G., Marchesi, E., Bullrich, L., Basualdo, I. L., Kao, T.-h. and Ando, T. (2006). Distribution of self-compatible and self-incompatible populations of Petunia axillaris (Solanaceae) outside Uruguay. J. Plant Res. 119, 419-430.

• Tsukamoto, T., Ando. T., Watanabe, H., Marchesi, E. and Kao, T.-h. (2005). Duplication of the S-locus F-box gene is associated with breakdown of pollen function in an S-haplotype identified in a natural population of self-incompatible Petunia axillaries. Plant Mol. Biol. 57, 141-153.

• Kao, T.-h. and Tsukamoto, T. (2004). The molecular and genetic bases of S-RNase-based self-incompatibility. Plant Cell 16: S72-83.

• Sijacic, P, Wang, X., Skirpan, A. L., Wang, Y., Dowd, P. E., McCubbin, A. G., Huang, S. and Kao, T.-h. (2004). Identification of the pollen determinant of S-RNase-mediated self-incompatibility. Nature 429, 302-305.

• Wang, Y., Tsukamoto, T., Yi, K.-w., Wang, X., Huang, S., McCubbin, A. G. and Kao, T.-h. (2004). Chromosome walking in the Petunia inflata self-incompatibility (S-) locus and gene identification in an 881-kb contig containing S2-RNase. Plant Mol. Biol. 54, 727-742.

• Tsukamoto, T., Ando, T., Kokubun, H., Watanabe, H., Sato, T., Masada, M., Marchesi, E. and Kao, T.-h. (2003). Breakdown of self-incompatibility in a natural population of Petunia axillaris caused by a modifier locus that suppresses the expression of an S-RNase gene. Sex. Plant Reprod. 15, 255-266.

• Tsukamoto, T., Ando, T., Takahashi, K., Omori, T., Watanabe, H., Kokubun, H., Marchesi, E. and Kao, T.-h. (2003). Breakdown of self-Incompatibility in a natural population of Petunia axillaris caused by loss of pollen function.  Plant Physiol. 131, 1903-1912.

• Wang, Y., Wang, X., McCubbin, A. G. and Kao, T.-h. (2003). Genetic mapping and molecular characterization of the self-incompatibility (S-) locus in Petunia inflata. Plant Mol. Biol. 53, 565-580.

• Wang, Y., Wang, X., and Skirpan, A. L. and Kao, T.-h. (2003). S-RNase-mediated self-incompatibility. J. Exp. Bot. 54, 115-122.

• Fasano, J. M., Swanson, S. J., Blancaflor, E. B., Dowd, P. E., Kao, T.-h. and Gilroy, S. (2001). Changes in root cap pH are required for the gravity response of the Arabidopsis root. Plant Cell 13, 907-921.

• Skirpan, A. L., McCubbin, A. G., Ishimizu, T., Wang, X., Hu, Y., Dowd, P. E., Ma, H. and Kao, T.-h. (2001). Isolation and characterization of kinase interacting protein 1, a pollen protein that interacts with the kinase domain of PRK1, a receptor-like kinase of petunia. Plant Physiol. 126, 1480-1492.

• Wang, X., Hughes, A. L., Tsukamoto, T., Ando, T. and Kao, T.-h. (2001). Evidence that intragenic recombination contributes to allelic diversity of the S-RNase gene at the self-incompatibility (S) locus in Petunia inflata. Plant Physiol. 125, 1012-1022.

• Kao, T.-h. and McCubbin, A. G. (2000). A social stigma. Nature 403, 840-841.

• McCubbin, A. G. and Kao, T.-h. (2000). Molecular recognition and response in pollen and pistil interactions. Annu. Rev. Cell Dev. Biol. 16, 333-364.

• McCubbin, A. G., Wang, X. and Kao, T.-h. (2000a). Identification of self-incompatibility (S-) locus linked pollen cDNA markers in Petunia inflata. Genome 43, 619-627.

• McCubbin, A. G., Zuniga, C. and Kao, T.-h. (2000b). Construction of a binary artificial chromosome library of Petunia inflata and the identification of large genomic fragments linked to the self-incompatibility (S-) locus. Genome 43, 820-826.

• McCubbin, A. G. and Kao, T.-h. (1999). The emerging complexity of self-incompatibility(S-) loci. Sex. Plant. Reprod. 12, 1-5.

• Lee, H.-S., Chung, Y.-Y., Das, C., Karunanandaa, B., van Went, J.-L. Mariani, C. and Kao, T.-h. (1997). Embryo sac development is affected in Petunia inflata plants transformed with an antisense gene encoding the extracellular domain of receptor kinase PRK1. Sex. Plant Reprod. 10, 341-350.

• McCubbin, A. G., Chung, Y.-Y. and Kao, T.-h. (1997). A mutant S3 RNase of Petunia inflata lacking RNase activity has an allele-specific dominant negative effect on self-incompatibility interactions. Plant Cell 9, 85-95.

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

• Lee, H.-S., Karunanandaa, B., McCubbin, A., Gilroy, S. and Kao, T.-h. (1996). PRK1, a receptor-like kinase of Petunia inflata, is essential for post-meiotic development of pollen. Plant J. 9, 613-624.

• Richman, A. D., Kao, T.-h., Schaeffer, S. W. and Uyenoyama, M. K. (1995). S-allele sequence diversity in natural populations of Solanum carolinense, Horsenettle. Heredity 75, 405-415.

• Huang, S., Lee, H.-S., Karunanandaa, B. and Kao, T.-h. (1994). Ribonuclease activity of Petunia inflata S proteins is essential for rejection of self-pollen. Plant Cell. 6, 1021-1028.

• Karunanandaa, B., Huang, S. and Kao, T.-h. (1994). Carbohydrate moiety of the Petunia inflata S3 protein is not required for self-incompatibility interactions between pollen and pistil. Plant Cell 6, 1933-1940.

• Lee, H.-S., Huang, S. and Kao, T.-h. (1994). S proteins control rejection of incompatible pollen in Petunia inflata. Nature 367, 560-563.

• Mu, J.-H., Lee, H.-S. and Kao, T.-h. (1994). Characterization of a pollen-expressed receptor-like kinase gene of Petunia inflata and the activity of its encoded kinase. Plant Cell 6, 709-721.

• Coleman, C. E. and Kao, T.-h. (1992). The flanking regions of two Petunia inflata S-alleles are heterogeneous and contain repetitive sequences. Plant Mol. Biol. 18, 725-737.

• Lee, H.-S., Singh, A. and Kao, T.-h. (1992). RNase X2, a pistil-specific ribonuclease from Petunia inflata shares sequence similarity with solanaceous S-proteins. Plant Mol. Biol. 20, 1131-1141.

• Clark, A. G. and Kao, T.-h. (1991). Excess nonsynonymous substitution at shared polymorphic sites among self-incompatibility alleles of Solanaceae. Proc. Natl. Acad. Sci. USA 88, 9823-9827.

• Ioerger, T. R., Gohlke, J. R., Xu, B. and Kao, T.-h. (1991). Primary structural features of the self-incompatibility protein in Solanaceae. Sex. Plant Reprod. 4, 81-87.

• Singh, A., Ai, Y. and Kao, T.-h. (1991). Characterization of ribonuclease activity of three S-allele-associated proteins of Petunia inflata. Plant Physiol. 96, 61-68.

• Ai, Y., Singh, A., Coleman, C. E., Ioerger, T. R., Kheyr-Pour, A. and Kao T-h (1990). Self-incompatibility in Petunia inflata: isolation and characterization of cDNAs encoding three S-allele-associated proteins. Sex. Plant Reprod. 3, 130-138.

• Ioerger, T. R., Clark, A. G. and Kao, T.-h. (1990). Polymorphism at the self-incompatibility locus in Solanaceae predates speciation. Proc. Natl. Acad. Sci. USA 87, 9732-9735.

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