Agriculture Reference
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gene is expressed in the tapetum and (importantly) in pollen, as predicted for the
pollen
S
-gene (Lai
et al.
, 2002).
Independent sequence analysis of a larger region of the
S
2
-locus of
P. inflata
(a 328-kb contig of three BAC clones containing
S-RNase
) has shown a similar
abundance of retrotransposons and, interestingly, also contains a pollen-expressed
gene similar to
SLF
though it lies approximately 167 kb from
S-RNase.
Sequence
analysis of
SLF
from additional
S
-haplotypes of
P. inflata
revealed
∼
90% amino
acid identity between three haplotypes and only
30% identity to
Antirrhinum
SLF-S
2
(Sijacic
et al.
, 2004). This sequence polymorphism combined with the fact
that the
SLF
gene appears to be the closest pollen expressed gene to
S-RNase
in
both
Antirrhinum
and
Petunia
, made
SLF
a prime candidate for being the pollen
S
-component of this SI system.
Confirmation that
SLF
is indeed the pollen
S
-gene has recently been achieved
using a transgenic approach (Sijacic
et al.
, 2004). The design of these experiments
required rather more thought than standard gain- or loss-of-function approaches. Re-
sults from X-ray mutagenesis studies had previously suggested that a loss of pollen
S
-function (i.e. self-compatibility) results from a phenomenon termed
competitive
interaction
(Golz
et al.
, 1999, 2001). Competitive interaction occurs when a plant
carries two different
S
-haplotypes as a result of the
S
-locus or some part of it being
duplicated, or as a result of tetraploidy. Of the pollen produced by such plants, the
pollen that carry two different pollen
S
-alleles (heteroalleic pollen), but not those that
carry two identical pollen
S
-alleles, fail to be rejected in the SI response. In a series of
experiments that capitalise on this information, the
SLF
2
allele of
P. inflata
has been
transformed into plants of
S
1
S
1
,
S
1
S
2
and
S
2
S
3
genotypes under regulation of its na-
tive promoter. It was found that
S
1
S
1
/
SLF
2
transgenic plants were self-compatible
and that all progeny resulting from self-pollination carried the
SLF
2
transgene.
S
1
S
2
/
SLF
2
and
S
2
S
3
/
SLF
2
transgenic plants were also self-compatible and analysis of
progeny generated by selfing these plants again demonstrated that all progeny carried
the transgene, but importantly no
S
2
S
2
progeny were found. Hence, the
SLF
2
trans-
gene causes a breakdown of SI only in heteroallelic pollen, precisely as the pollen
S
-
gene product is predicted to behave by competitive interaction (Sijacic
et al.
, 2004).
The possession of an F-box by SLF implicates a particular mode of action for
these proteins. F-box-containing proteins are components of ubiquitin ligase com-
plexes, which, together with ubiquitin-activating enzymes and ubiquitin-conjugating
enzymes, mediate protein degradation by the 26S proteosome (Bai
et al.
, 1996). Re-
cent results suggest that SLF is indeed part of an SCF complex (Qiao
et al.
, 2004).
Hence it is envisaged (though not proven) that SLF acts by mediating the degradation
of S-RNases in compatible pollinations, and that some form of specific interaction
between S-RNase and SLF of the same haplotype blocks ubiquitination in a self-
incompatible interaction. A question that remains unanswered with regards to SLF
and 'competitive interaction' is whether or not 'knocking out'
SLF
is pollen lethal,
as has been predicted. If SLF is indeed mediating degradation of S-RNase, this
would be expected, as the inability to neutralise cross S-RNases would lead to uni-
versally incompatible pollen. A further possibility is that SLF is essential to pollen
∼
