Structure and function of class III pistil-specific extensin-like protein in interspecific reproductive barriers

Background: The transmitting tissue of the style is the pathway for pollen tube growth to the ovules and has components that function in recognizing and discriminating appropriate pollen genotypes. In Nicotiana tabacum, the class III pistil extensin-like (PELPIII) arabinogalactan protein is essential for the inhibition of N. obtusifolia pollen tube growth. The transmitting tissue-specific (TTS) arabinogalactan protein amino acid sequence and expression pattern is similar to PELPIII, but it facilitates self-pollinated N. tabacum. The TTS and PELPIII arabinogalactan protein can be divided into the less conserved N-terminal (NTD) and the more conserved C-terminal (CTD) domains. This research tested whether the NTD is the key domain in determining PELPIII function in the inhibition of interspecific pollen tube growth. Three variant PELPIII gene constructs were produced where the PELPIII NTD was exchanged with the TTS NTD and a single amino acid change (cysteine to alanine) was introduced into the PELPIII NTD. The PELPIII variants of N. tabacum were tested for activity by measuring the inhibition N. obtusifolia pollen tube growth by using them to complement a 3'UTR RNAi transgenic line with reduced PELPIII mRNA. Results: The RNAi N. tabacum line had reduced PELPIII mRNA accumulation and reduced inhibition of N. obtusifolia pollen tube growth, but had no effect on self-pollen tube growth or pollen tube growth of 12 other Nicotiana species. The NTD of PELPIII with either the PELPIII or TTS CTDs complemented the loss PELPIII activity in the RNAi transgenic line as measured by inhibition of N. obtusifolia pollen tube growth. The TTS NTD with the PELPIII CTD and a variant PELPIII with a cysteine to alanine mutation in its NTD failed to complement the loss of PELPIII activity and did not inhibit N. obtusifolia pollen tube growth. Conclusion: The NTD is a key determinant in PELPIII's function in regulating interspecific pollen tube growth and is a first step toward understanding the mechanism of how PELPIII NTD regulates pollen tube growth.