Biochemistry and Molecular Biology
Penn State Science
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Teh-hui Kao

Teh-hui Kao

Main Content

  • Distinguished Professor of Biochemistry and Molecular Biology
333 South Frear Laboratory
University Park, PA 16802
Email: txk3@psu.edu
Phone: (814) 863-1042

Research Interests

Biochemical and molecular bases of self/non-self recognition during plant reproduction

Graduate Programs

BMMB, PLBIO

Research Summary

Mechanism of self/non-self recognition between pollen and pistil in self-incompatible plants  Self-incompatibility (SI) is a self/non-self recognition mechanism that allows the pistil of flowering plants producing bisexual flowers to distinguish between self (genetically related) and non-self (genetically unrelated) pollen to prevent inbreeding and promote out-crossing.  We use Petunia inflata (a wild species, one of the progenitor species of garden petunia) as a model to study the SI mechanism possessed by Solanaceae and two other families of flowering plants.  Here, SI is controlled by the highly polymorphic S-locus.  If the S-haplotype of haploid pollen matches either S-haplotype of the diploid pistil, the pollen is recognized as self-pollen and the growth of self-pollen tubes in the style is inhibited; if the S-haplotype of pollen is different from both S-haplotypes of the pistil, the pollen is recognized as non-self pollen and their tubes are allowed to grow down through the style to the ovary to effect fertilization.

 

We are interested in two fundamental questions: How does a pistil distinguish between self and non-self pollen?  How does the self and non-self recognition lead to growth arrest of self-pollen tubes in the style?  Over the past more than three decades of research, we have used a number of approaches, including an in vivo functional approach, to understand the biochemical and molecular bases of SI.  Some of the key findings are listed below: (1) using gain-of-function and loss-of-function approaches, the polymorphic S-RNase gene was identified as the gene that regulates pistil specificity in SI (Lee et al., Nature 367: 560-563, 1994); (2) using site-directed mutagenesis, it was shown that the RNase activity of S-RNase is essential for the function of S-RNase in rejection of self-pollen (Huang et al., Plant Cell 6: 1021-1028, 1994); (3) identification of an S-locus F-box (SLF) gene, PiSLF1 (now named SLF1), from sequencing Bacterial Artificial Chromosome (BAC) clones spanning a 328-kb region containing S2-RNase (McCubbin et al., Genome 43: 820-826, 2000; Wang et al., Plant Mol Biol 54: 724-742, 2004), and using an in vivo functional approach, SLF1 was shown to be involved in regulating pollen specificity (Sijacic et al., Nature 429: 302-305, 2004); (4) using RNAseq and pollen transcriptome analyses, a total of 17 pollen-specific polymorphic SLF genes (including SLF1) have been identified in both S2-haplotype and S3-haplotype (Williams et al., Plant Cell 26: 2873-2888, 2014); (5) using coimmunoprecipitation and mass spectrometry, all 17 SLF proteins of S2 and S3 pollen were shown to be assembled into similar SCF complexes, which also contain PiSSK1 (a pollen-specific Skp1-like protein), PiRBX1 (a RING-finger protein) and PiCUL1-P (a pollen-specific Cullin1) (Li et al., Plant Reprod 27: 31-45, 2014; Plant J 87: 606-616, 2016); (6) discovery that SLF proteins are themselves subject to ubiquitin-mediated degradation by the 26S proteasome (Sun et al., Plant J 83: 213-223, 2015), and
identification of pollen proteins that may regulate the dynamic life cycle of SCFSLF complexes.

 

A current focus is on testing the predictions of the collaborative non-self recognition model formulated after the discovery that more than one SLF gene regulates pollen specificity (Kubo et al., Science 330: 796-799, 2010).  According to this model, for a given S-haplotype, each SLF interacts with a subset of its non-self S-RNases, and all SLF proteins that constitute the pollen specificity determinant collectively interact with the entire suite of their non-self S-RNases to mediate ubiquitination and degradation, allowing cross compatible pollination.  However, none of the SLF proteins interact with their self S-RNase, allowing the self S-RNase to inhibit pollen tube growth.  Ongoing projects include the following: (1) using an in vivo functional assay to establish comprehensive interaction relationships between SLF proteins of S2-haplotype and S3-haplotype, and 11 S-RNases, and so far the results support the model (Sun and Kao, Plant Cell 25: 470-485, 2013; Williams et al., Mol Plant 7: 567-569, 2014); (2) using the chimeric gene approach to identify amino acids of SLF proteins involved in differential interactions with S-RNases (Wu et al., Plant Cell Physiol doi.org/10.1093/pcp/pcx176); (3) using CRISPR/Cas9 genome editing technology to knock out genes encoding the pollen-specific components of SCFSLF complexes (i.e., PiSSK1, PiCUL1-P, and various SLF proteins) to determine whether they are specifically involved in SI and whether SCFSLF complexes are indeed required for cross-compatible pollination (Sun and Kao, Plant Reprod doi.org/10.1007/s00497-017-0314-1).  The information gained will be valuable for ultimate understanding of the biochemical, molecular, and structural bases of this complex and fascinating self/non-self recognition system, which is thought to have played a vital role in the evolutionary success of flowering plants.

chart describing the pollination process.
Figure 1: The pistil of a self-incompatible flowering plant can recognize pollen, which has landed on, or been brought to, its stigmatic surface as self-pollen or non-self pollen, based on whether the S-haplotype of the pollen is present or not present in the pistil.  For the pollinations depicted, S1 and S2 pollen are recognized as self pollen by the pistil of S1S2 genotype and the growth of their tubes is arrested in the upper segment of the style; S4 pollen is recognized as non-self pollen and its tube is allowed to grow down to the ovary to effect fertilization.  This reproductive trait allows flowering plants to prevent inbreeding and promote out-crossing.

Transforming Leaf strips from Petunia inflata.

Figure 2: Leaf strips of Petunia inflata are transformed with Agrobacterium tumefaciens carrying a transgene construct, and the transformed tissues are cultured for regeneration of transgenic plants.  The ability to introduce genes into transgenic P. inflata plants allows in vivo studies of the function and structure/function relationships of the genes involved in self-incompatibility.

Selected Publications

  • Wu, L., Williams, J.S., Wang, N., Khatri, W.A., San Román, D., Kao, T.-h. (2017). Use of domain-swapping to identify candidate amino acids involved in differential interactions between two allelic variants of type-1 S-locus F-box protein and S3-RNase of Petunia inflata. Plant Cell Physiol. doi.org/10.1093/pcp/pcx176.
  • Sun, L., Kao, T.-h. (2017). CRISPR/Cas9-mediated knockout of PiSSK1 reveals essential role of S-locus F-box protein-containing SCF complex in recognition of non-self S-RNases during cross-compatible pollination in self-incompatible Petunia inflata. Plant Reprod. doi.org/10.1007/s00497-017-0314-1.
  • Li, S., Williams, J.S., Sun, P., Kao, T.-h. (2016). All 17 S-locus F-box proteins of S2- and S3-haplotypes of Petunia inflata are assembled into similar SCF complexes with specific function in self-incompatibility. Plant J. 87: 606-616.
  • Sun, P., Li, S., Lu, D., Williams, J.S., Kao, T.-h. (2015). Pollen S-locus F-box proteins of Petunia involved in S-RNase-based self-incompatibility are themselves subject to ubiquitin-mediated degradation. Plant J. 83: 213-223.
  • Williams, J.S., Wu, L., Li, S., Sun, P., Kao, T.-h. (2015). Insight into S-RNase-based self-incompatibility in Petunia: recent findings and future directions. Front. Plant Sci. doi:10.3389/fpls.2015.00041.
  • Williams, J.S., Der, J.P., dePamphilis, C.W., Kao, T.-h. (2014). Transcriptome analysis reveals the same 17 S-locus F-box genes in two haplotypes of the self-incompatibility locus of Petunia inflata. Plant Cell 26: 2873-2888.
  • Li, S., Sun P, Williams, J.S., Kao, T.-h. (2014). Identification of the self-incompatibility locus F-box protein-containing complex in Petunia inflata. Plant Reprod. 27: 31-45.
  • Williams, J.S., Natale, C.A., Wang, N., Li, S., Brubaker, T.R, Sun, P., Kao, T.-h. (2014). Four previously identified Petunia inflata S-locus-F-box genes are involved in pollen specificity in self-incompatibility. Mol. Plant 7: 567-569.
  • Sun, P., Kao, T.-h. (2013). Self-incompatibility in Petunia inflata: the relationship between a self-incompatibility locus F-box protein and its non-self S-RNases. Plant Cell 25: 470-485.
  • Meng, X., Hua, Z., Sun, P., Kao, T.-h. (2011). The amino terminal F-box domain of Petunia inflata S-locus F-box protein is involved in self-incompatibility mechanism. AoB Plaints doi:10.1093/aodpla/plr016.
  • Kubo, K.-I., Entani, T., Takara, A., Wang, N., Fields, A.M., Hua, Z., Toyoda, M., Kawashima, S.-i., Ando, T., Isogai, A., Kao, T.-h., Takayama, S. (2010). Collaborative non-self recognition in S-RNase-based self-incompatibility. Science 330: 796-799.
  • Hua, Z., Kao, T.-h. (2008). Identification of major lysine residues of S3-RNase of Petunia inflata involved in ubiquitin-26S proteasome-mediated degradation in vitro. Plant J. 54: 1094-1104.
  • Hua, Z., Meng, X., Kao, T.-h. (2007). Comparison of Petunia inflata S-locus F-box protein (Pi SLF) and Pi SLF-like proteins reveals its unique function in S-RNase-based self-incompatibility. Plant Cell 19: 3593-3609.
  • Hua, Z., 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.
  • Sijacic, P., Wang, X., Skirpan, A.L., Wang, Y., Dowd, P.E., McCubbin, A.G., Huang, S., 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., 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., Takahashi, K., Omori, T., Watanabe, H., Kokubun, H., Marchesi, E., 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, X., Hughes, A.L., Tsukamoto, T., Ando, T., 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.
  • McCubbin, A.G., Zuniga, C., Kao, T.-h. (2000). Construction of a bacterial 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., Wang, X., Kao, T.-h. (2000). Identification of self-incompatibility (S-) locus linked pollen cDNA markers in Petunia inflata. Genome 43: 619-627.
  • Huang, S., Lee, H.-S., Karunanandaa, B., Kao, T.-h. (1994). Ribonuclease activity of Petunia inflata S-proteins is essential for rejection of self-pollen. Plant Cell 6: 1021-1028.
  • Lee, H.-S., Huang, S., Kao, T.-h. (1994). S proteins control rejection of incompatible pollen in Petunia inflata. Nature 367: 560-563.
  • Ioerger, T.R., Gohlke, J.R., Xu, B., Kao, T.-h. (1991). Primary structural features of the self-incompatibility protein in Solanaceae. Sex. Plant Reprod. 4: 81-87.
  • Ioerger, T.R., Clark, A.G., Kao, T.-h. (1990). Polymorphism at the self-incompatibility locus in Solanaceae predates speciation. Proc. Natl. Acad. Sci. USA 87: 9732-9735.