The discovery in the mid-1980s that the S-RNase
gene (initially simply referred to as the pistil S
-gene) encoded the pistil determinant of the Solanaceae SI system (Anderson et al., 1986
) marked the dawn of the molecular genetic/biochemical studies of this type of SI mechanism. Since then, there have been several major breakthroughs that led to new directions of research and enhanced our understanding of the SI mechanism. The demonstration that S-RNases have RNase activity (McClure et al., 1989
) and that the RNase activity is required for the function of S-RNase (Huang et al., 1994
) provided clues as to how the pistil might inhibit growth of self-pollen tubes at the biochemical level. The finding that uptake of S-RNases by the pollen tube is not S
-haplotype-specific (Luu et al., 2000
; Goldraij et al., 2006
) suggested that specific rejection of self-pollen tubes by the pistil most likely lies in differential fates of self and non-self S-RNases inside the pollen tube. Finally, the recent identification of the SLF
gene as the pollen determinant (Lai et al., 2002
; Entani et al., 2003
; Ushijima et al., 2003
; Qiao et al., 2004a
; Sijacic et al., 2004
; Ushijima et al., 2004
; Wang et al., 2004
; Sonneveld et al., 2005
; Tsukamoto et al., 2005
; Hauck et al., 2006a
; Vilanova et al., 2006
; Sassa et al., 2007
) allowed the formulation of new hypotheses and models for the biochemical basis of S
-haplotype-specific rejection of pollen tubes.
The S-RNase gene has been very extensively studied over the past quarter century, but there are still some aspects of S-RNase that remain unknown. For example, what specific amino acid residues of each S-RNase determine its S-allele specificity? What is the role, if any, of the glycan chain(s)? What is the mechanism for the uptake of S-RNases by pollen tubes? How might S-RNases sequestered in a vacuole-like compartment in the pollen tube be specifically released into the cytoplasm of an incompatible pollen tube to exert their cytotoxic effect?
The transgenic approach used to establish the function of SLF
in P. inflata
(Sijacic et al., 2004
) and Antirrhinum
(Qiao et al., 2004b
) is a robust assay for testing whether a candidate for the pollen S
-gene is indeed involved in controlling pollen specificity, provided that competitive interaction has been observed in the species under study. The S
genes, that share similar properties with SLF
but whose function has not been examined, will be good candidates for this in vivo
functional assay. This assay is based on whether a particular F-box protein produced in the pollen from a transgene can cause breakdown of SI in transgenic pollen, which, based on our model, reflects whether the F-box protein can interact with the S-RNases taken up into the transgenic pollen tubes to mediate their degradation. For example, if the S1
-allelic variant of SLF, when expressed in S2
pollen, causes breakdown of SI in S2
pollen, and if the S1
-allelic variant of an SLF-like protein fails do so, the results would be interpreted to mean that this SLF-like protein, unlike SLF1
, cannot interact with S2
-RNase to mediate its degradation. As more and more SLF-like proteins of the S1
-haplotype are tested, one can compare the sequences of those that do not cause breakdown of SI in S2
pollen with the sequences of those that do, if any, and SLF1
to identify any amino acid residues that might be responsible for the functional and biochemical differences between these two groups of F-box proteins.
The in vivo functional assay can also be used for structure/function studies to identify specific amino acids and domains of SLF that are involved in a particular function. For example, one can address the question of whether the F-box domain of SLF is required for its function by expressing a truncated SLF in pollen carrying a different S-allele and examining whether the truncated SLF can cause competitive interaction in transgenic pollen. Another key question about SLF is how the S-specificity is determined. FD1 and FD3 of PiSLF have been identified as the putative S-specificity determinant from in vitro protein-binding assays. One can test this model by expressing chimeric SLF proteins between two allelic variants in transgenic plants of the appropriate S-genotype, and examining the effect of the chimeric proteins on the SI behaviour of transgenic pollen. For example, based on our model, the chimeric protein containing FD1 and FD3 from PiSLF2 and FD2 from PiSLF3 would possess S2-allele specificity and would cause breakdown of SI in S3 pollen (heteroallelic), but not in S2 pollen (homoallelic), when introduced into plants of S2S3 genotype. Once a particular domain is found to possess a particular function, site-directed mutagenesis can be used to narrow down specific amino acids involved.
The tenet of the protein-degradation model proposed by our lab is that an SLF protein preferentially interacts with all its non-self S-RNases in the pollen tube to mediate their ubiquitination and ultimate degradation. This model predicts that absence of SLF in pollen would lead to the inability of the pollen to detoxify any S-RNase (self or non-self), and as a result, the pollen would be rejected by pistils of any S-genotype. One direct approach to test this prediction is to use the methodology of RNA interference (RNAi) to suppress the production of SLF in pollen, and to determine whether the transgenic pollen becomes incompatible with pistils that should normally recognize it as non-self pollen. For example, if our model is correct, then suppression of SLF1 in S1 pollen would result in the inability of the transgenic pollen to detoxify any S-RNase that it takes up, and consequently, the transgenic pollen would be rejected by pistils of any S-genotype. The same approach can be used to examine the involvement of all other genes that have been implicated in SI. For example, if PiSBP1 is indeed an essential component of the PiSLF-containing E3 ligase complex, suppression of its production in pollen would result in the inability of the E3 ligase complex to be assembled and consequently, the inability of PiSLF to mediate degradation of non-self S-RNases. The prediction is that the transgenic pollen, in which the expression of PiSBP1 is suppressed, would be rejected by pistils of any S-genotype.
In conclusion, tremendous progress has been made towards understanding the Solanaceae SI system since the cloning of the S-RNase gene was first reported many years ago, but there remain many key questions, as any new discovery invariably leads to new questions and new avenues of research. The availability of robust in vivo functional assays for S-RNase and SLF in P. inflata has made this species a good model system, and it would be of interest to determine how much of the information obtained is applicable to other taxa possessing the Solanaceae-type SI system, particularly those in subfamily Prunoideae of Rosaceae.