The Self-Incompatibility Phenotype in Brassica Is Altered by the Transformation of a Mutant S Locus Receptor Kinase

Abstract

The self-incompatible (SI) Brassica napus line W1, which carries the 910 S allele, was transformed with an inactive copy of the 910 S locus receptor kinase ( SRK ) gene. Two transformed lines were analyzed based on their heritable ability to set self-seed. The first line was virtually completely self-compatible (SC), and reciprocal pollinations with the original W1 line demonstrated that only the stigma side of the SI phenotype was altered. An analysis of the expression of endogenous SRK-910 demonstrated that the mechanism of transgene action is via gene suppression. Furthermore, the expression of the S locus glycoprotein gene present in the 910 allele ( SLG-910 ), SLG-A10 , which is derived from a nonfunctional S allele, and an S locus-related gene were also suppressed. When the transgene was crossed into another SI line carrying the A14 S allele, it was also capable of suppressing the expression of the endogenous genes and of making this line SC. The second transgenic line studied was only partly SC. In this case as well, only the stigma phenotype was affected, although no gene suppression was detected for endogenous SRK-910 or SLG-910 . In this line, the expression of the transgene most likely was causing the change in phenotype, and no effect was observed when this transgene was crossed into the other SI line. Therefore, this work reinforces the hypothesis that the SRK gene is required, but only for the stigma side of the SI phenotype, and that a single transgene can alter the SI phenotype of more than one S allele.

Characterization of Brassica S‐haplotypes lacking S‐locus glycoprotein1

“…Previous attempts to change the SI phenotype of Brassica plants by the introduction of the SRK or SLG gene of a di¡erent S-haplotype often led to reduced expression of the endogenous SRK and SLG genes in transgenic plants, due to sense cosuppression by the SLG and/or SRK transgenes. As a result, the transgenic plants became self-compatible [24,25]. With the evidence we have presented in this work showing that SLG is not essential in SI, we could also conclude that down regulation of SRK is most likely the reason for the breakdown of SI in these transgenic plants.…”

The integral membrane S-locus receptor kinase of Brassica has serine/threonine kinase activity in a membranous environment and spontaneously forms oligomers in planta

“…In our study, we have shown that recombinant SRK autophosphorylates constitutively and exists as dimers in nonpollinated stigmas. The fact that a recombinant kinase-defective SRK expressed in transgenic Brassica plants acts as a dominant negative mutant further suggests that homooligomerization of fully functional SRK is necessary for the SI reaction (14). However, the detection of SRK dimers in nonpollinated stigmas indicates that the activation of SRK during the SI response requires an additional step after the formation of SRK homooligomers.…”

“…Moreover, attempts to overexpress SRK in Brassica have encountered problems because of cosuppression effects (14,33). To overcome this obstacle and to gain more insight into how SRK functions at the molecular level, we expressed recombinant SRK in the insect cell͞baculovirus system.…”

“…For example, SRK was shown to be mutated in two Brassica lines that exhibited a self-compatible phenotype (12,13). Moreover, Stahl and coworkers (14) recently have shown that the SI phenotype was altered in transgenic Brassica napus plants expressing a kinase-defective SRK that seems to act as a dominant negative mutant. Their results strongly indicate that SRK is a key component of the SI reaction.…”

Molecular Biology of Self-incompatibility in Brassica Species.

“…A pollination test indicated that the pollen of the transgenic B. napus did not gain the SI phenotype (Sasaki et al, 1998). Selfincompatible B. napus transformed with an inactive copy of SRK gene became self-compatible due to co-suppression and dominant-negative effects (Stahl et al, 1998). The transgenes led to a dramatic reduction in the expression of the endogenous S locus and related genes indicating homology-dependent silencing.…”