Isolation of S-RNase binding proteins from Solanum chacoense: identification of an SBP1 (RING finger protein) orthologue

Abstract

Currently, the most attractive working model of gametophytic self-incompatibility (SI) involving SRNases postulates the presence of an inhibitor protein or complex expressed in pollen tubes that would counteract the cytotoxic effect of the ribonuclease activity of the SRNase. Since it has been previously shown that allelespecific recognition is mediated through the hypervariable domain sequence of the S-RNase, we have targeted this region to isolate pollen-expressed interacting proteins in the yeast two-hybrid system. One of the isolated proteins corresponds to a RING finger protein highly similar to the previously isolated SBP1 protein from Petunia hybrida. This protein is postulated to be part of the RING finger E3 ligase family. The ScSBP1 gene is expressed in almost all tissues tested, suggesting a more general role than only being involved in SI. Although the ScSBP1 gene is polymorphic, linkage analysis showed that it was unlinked to the S-locus. The isolation of this S-RNase-binding protein in two different species and with four different SRNase sequences as bait, strengthens its putative involvement in the SI response. Furthermore, comparison of the bait sequences used suggests that the SBP1 protein interacts with conserved sequences located between the HVa and HVb domains.

Protein Interactions in S-RNase-Based Gametophytic Self-Incompatibility

“…Similarly Hua & Kao (2006) used a partial bait (HVa-HVb-C3) of the Petunia inflata S2-RNase to screen a two-hybrid library and isolated PiSBP1. Similar to other reports (O'Brien et al, 2004;Sims & Ordanic, 2001) PiSBP1 did not interact with full-length S-RNase, with non-specific controls, or importantly, with a non-Slocus ribonuclease. Hua & Kao (2006) further showed that SBP1 interacted with PiSLF 2 and PiSLF 1 in both two-hybrid and pull-down assays, as well as with Cullin-1 and PhUBC1 (Sims, unpublished), an E2 conjugation enzyme protein from Petunia hybrida.…”

“…Lee et al (2008) used C-terminal domains of the style-transmitting-tract proteins TTS and 120K to screen a pollen two-hybrid library from Nicotiana alata, and also pulled out NaSBP1 from this screen. All of these reports (Hua & Kao, 2006;Lee et al, 2008;O'Brien et al, 2004;Sims & Ordanic 2001) showed that SBP1 was not pollen-specific, but was expressed in all tissues examined. SBP1 is non-allelic as well, as SBP1 isolated from S 1 S 1 and S 3 S 3 lines of Petunia hybrida are 100% identical.…”

“…Figure 2) nor with an unrelated bait protein, P53. ScSBP1 was isolated from Solanum chacoense (O'Brien et al, 2004), using a yeast two-hybrid screen with the HVa+HVb regions of the S 11 -Rnase and the S 13 -RNase as bait. Both the S 11 and S 13 baits interacted with ScSBP1, however a full-length S-RNase with a single amino acid change (H144L) at one of the activesite histidines failed to interact with SBP1.…”

Pollen Proteins Bind to the C-terminal Domain of Nicotiana alata Pistil Arabinogalactan Proteins

“…Others have shown that SBP1-like proteins bind to unrelated proteins (43). This, together with the ubiquitous expression of NaSBP1 (16,44) and SBP1-like proteins, suggests that their function is not restricted to pollination. The expression and binding results are consistent with housekeeping functions such as degradation of misfolded protein or targeting proteins to various cellular locations.…”

Identification of an S-RNase binding protein1 (SBP1) homolog of apple (Malus^|^times;domestica)

“…The petunia SBP1 included a RING-HC domain and interacted with S-RNase, and showed no S haplotype-specific sequence polymorphism. SBP1 homologs have been identified in Solanum chacoense (O'Brien et al 2004) and Nicotiana alata ; however, SBP1-like protein has not yet been characterized outside Solanaceae. Here, we isolated an apple homolog of SBP1 from pollen RNA and named it MdSBP1.…”