Plant Molecular Biology 45: 365–376, 2001.
© 2001 Kluwer Academic Publishers. Printed in the Netherlands.
Further analysis of the interactions between the Brassica S receptor kinase
and three interacting proteins (ARC1, THL1 and THL2) in the yeast
, Waheeda Sulaman
, Helen Elina
and Daphne R.
Biology Department, York University, 4700 Keele Street, Toronto, Ontario M3J 1P3, Canada (
author for corre-
spondence; e-mail: firstname.lastname@example.org)
Laboratoire de Reproduction et D´eveloppement des Plantes, UMR 5667,
Centre National de la Recherche Scientiﬁque, Institut National de la Recherche Agronomique,
Sup´erieure, 46, all´ee d’Italie, 69364 Lyon Cedex 07, France; Present addresses:
Department of Biochemistry,
University of Missouri-Columbia, Columbia, MO, USA;
Biology Department, McGill University, Montreal PQ,
Received 24 July 2000; accepted in revised form 30 November 2000
Key words: ARC1, Brassica self-incompatibility, RLK5, S receptor kinase, thioredoxin h
The yeast two-hybrid system was used to further characterize the interactions between the Brassica S receptor ki-
nase (SRK) and three putative substrates, ARC1 and the two thioredoxinh proteins, THL1 and THL2. Interactions
were generally detectable with kinase domains of both Class I and Class II SRKs. Chimeric constructs were made
between the SRK
kinase domain and the non-interacting Arabidopsis RLK5 kinase domain. Only one chimeric
construct, SRR2, interacted with THL1 and THL2, while none of the chimeras were able to interact with ARC1.
SRR2 is largely made up of RLK5 kinase domain with the N-terminal end being derived from the SRK
domain and was the only chimeric construct that retained kinase activity. Deletion or substitution of a conserved
cysteine at the N-terminal end of the SRK
kinase domain resulted in loss of interaction with THL1 and THL2,
while the addition of this cysteine to a related receptor kinase, SFR1, conferred the ability to interact with the
thioredoxin h proteins. In addition, substitution of the cysteines in the THL1 active site abolished the interaction.
Lastly, the two Arabidopsis thioredoxin h clones most closely related to THL1 and THL2 were found to interact
with the SRK kinase domains. Thus, the nature of the interaction of the thioredoxin h clones with SRK involves
the reducing activity of these proteins and is restricted to the class of thioredoxin h proteins which have the variant
CPPC active site.
In self-incompatible Brassica, pollination is regulated
by the self-incompatibility system which acts to pre-
vent self-pollination, as well as pollination between
two plants carrying the same S allele (for review, see
De Nettancourt, 1997; Suzuki et al., 1997; Cock,
2000). While classically the self-incompatibility sys-
tem has been deﬁned to be controlled by a single
geneticS locus, molecular studies haveled to the char-
acterization of a number of genes tightly linked to the
S locus (Cui et al., 1999; Suzuki et al., 1999; Cas-
selman et al., 2000). Of these, the S receptor kinase
(SRK) gene was found to encode the female deter-
minant of self-incompatibility (Nasrallah et al., 1991;
Takasaki et al., 2000) while the S locus cysteine-rich
protein (SCR) was found to encode the male determi-
nant (Schopfer et al., 1999; Takayama et al., 2000).
The S locus glycoprotein (SLG), long thought to be
involved in Brassica self-incompatibility, is not essen-
tial and while Takasaki et al. (2000) found that SLG
enhancedthe self-incompatibilityresponsein the pistil