Chromosome Walking in the Petunia Inflata Self-Incompatibility ( S -) Locus and Gene Identification in an 881-kb Contig Containing S 2 -RNase

Self-incompatibility (SI) in the Solanaceae, Rosaceae and Scrophulariaceae is controlled by the polymorphic S locus, which contains two separate genes encoding pollen and pistil determinants in SI interactions. The S-RNase gene encodes the pistil determinant, whereas the pollen determinant gene, named the pollen S gene, has not yet been identified. Here, we set out to construct an integrated genetic and physical map of the S locus of Petunia inflata and identify any additional genes located at this locus. We first conducted chromosome walking at the S 2 locus using BAC clones that contained either S 2 -RNase or one of the nine markers tightly linked to the S locus. Ten separate contigs were constructed, which collectively spanned 4.4 Mb. To identify additional genes located at the S 2 locus, a 328-kb region (part of an 881-kb BAC contig) containing S 2 -RNase was completely sequenced. Approximately 76% of the region contained repetitive sequences, including transposon-like sequences. Other than S 2 -RNase , an F-box gene, named PiSLF 2 ( S 2 -allele of P. inflata S -locus F-box gene), was the only predicted gene whose deduced amino acid sequence was similar to the sequences of known proteins in the database. Two different cDNA selection methods were used to identify additional genes in the 881-kb contig; 11 groups of cDNA clones were identified in addition to those for S 2 -RNase and PiSLF 2 . RT-PCR analysis of expression profiles and PCR analysis of BAC clones and genomic DNA confirmed that seven of these 11 newly identified genes were located in the 881-kb contig.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/43454/1/11103_2004_Article_DO00000142.pd

Identification and Characterization of Components of a Putative Petunia S-Locus F-Box–Containing E3 Ligase Complex Involved in S-RNase–Based Self-Incompatibility

Petunia inflata S-locus F-box (Pi SLF) is thought to function as a typical F-box protein in ubiquitin-mediated protein degradation and, along with Skp1, Cullin-1, and Rbx1, could compose an SCF complex mediating the degradation of nonself S-RNase but not self S-RNase. We isolated three P. inflata Skp1s (Pi SK1, -2, and -3), two Cullin-1s (Pi CUL1-C and -G), and an Rbx1 (Pi RBX1) cDNAs and found that Pi CUL1-G did not interact with Pi RBX1 and that none of the three Pi SKs interacted with Pi SLF(2). We also isolated a RING-HC protein, S-RNase Binding Protein1 (Pi SBP1), almost identical to Petunia hybrida SBP1, which interacts with Pi SLFs, S-RNases, Pi CUL1-G, and an E2 ubiquitin-conjugating enzyme, suggesting that Pi CUL1-G, SBP1, and SLF may be components of a novel E3 ligase complex, with Pi SBP1 playing the roles of Skp1 and Rbx1. S-RNases interact more with nonself Pi SLFs than with self Pi SLFs, and Pi SLFs also interact more with nonself S-RNases than with self S-RNases. Bacterially expressed S(1)-, S(2)-, and S(3)-RNases are degraded by the 26S proteasomal pathway in a cell-free system, albeit not in an S-allele–specific manner. Native glycosylated S(3)-RNase is not degraded to any significant extent; however, deglycosylated S(3)-RNase is degraded as efficiently as the bacterially expressed S-RNases. Finally, S-RNases are ubiquitinated in pollen tube extracts, but whether this is mediated by the Pi SLF–containing E3 complex is unknown