Download Supporting Information Legends Supplementary Table S1

Supporting Information Legends
Supplementary Table S1: Complete list of cDNAs identified by the yeast two hybrid split
ubiquitin system using SlSUT2 cloned in met25pXCgate as bait. The accession number of
proteins with highest homology are given and the percentage of sequence identity. In
addition EST numbers from Solanum tuberosum are given.
Supplementary Table S2: Quantification of interaction strength by photometric ßgalactosidase assay. StDev was calculated from three independent experiments. Strongest
interaction is detectable between SlSUT2 and the BAK1-like and the MSBP1 prey protein.
Quantification of the interaction between StSUT1 and StSUT4 with the PDI protein published
earlier (Krügel et al., 2012) was performed as positive control of the assay. Cotransformation of the bait constructs together with the empty prey vector served as negative
control.
Supplementary Figure S1. Subcellular localization of SUT2 in yeast and in plants. a.
StSUT2-GFP fusion protein expressed in the yeast mutant strain SuSy7 under control of the
Adh1 promoter in the yeast expression vector 112A1NE (Reinders et al., 2002). b. SlSUT2YFP fusion protein expressed under control of the 35S promoter in the plant binary
GATEWAY vector pK7GW2.0 in transiently expressing leaf epidermis cells of Nicotiana
tabacum transiently transformed by Agrobacterium infiltration. c. The same SlSUT2-YFP
fusion construct expressed in stably transformed Nicotiana tabacum plants. Note that SUT2
proteins do not efficiently reach the plasma membrane of overexpressed heterologously.
Supplementary Figure S2. Yeast two hybrid split ubiquitin system
a.
Yeast growth on SD medium without HULT in the presence of 15 mM 3-AT of yeast
strain L40ccU transformed with SlSUT2 in Cub as a bait and with the empty Nub vector as a
control. Full length cDNAs of PDI, MSBP1 and BAK1-like were cloned into the Nub vector to
confirm interaction with SlSUT2.
b.
Quantification of interaction by photometric ß-galactosidase assay. Strongest
interaction with SlSUT2 was observed for MSBP1 and BAK1-like.
Supplementary Figure S3. Tissue-specific expression pattern of genes encoding
SlSUT2-interacting proteins as determined by qPCR.
Total RNA was extracted from different flower organs and from sink and source leaves and
used for qRT-PCR. Transcript accumulation of the StMSBP1 gene (a) and the StBAK1-like
gene (b) is calculated using ubiquitin as internal reference gene and standard deviation is
given. Note that both genes coding for SlSUT2-interacting proteins show main expression in
anthers of mature flowers as previously shown for SlSUT2 (Hackel et al. 2006) indicating
that interaction partners are co-expressed.
Supplementary Figure S4. Sequence alignment of BAK1-like receptor kinases and BRI1
receptor kinases. The BAK1-like protein from potato (XP_006342998) shows 51% identity to
BAK1 from Ricinus communis (XP_002510316) and 75% identity to a receptor kinase from
Arabidopsis (At3g13690). Alignment was performed with full length amino acid sequences
from BAK1 from Arabidopsis thaliana (NP_001190904), Nicotiana attenuata (ADT91693),
Sacharum officinarum (ABX83681), Saccharum hybrid cultivar (AHF18212), Ricinus
communis (XP_002510316), Solanum lycopersicon (XP_004235609) and with the BRI1
Sequences
from
Arabidopsis
thaliana
(NP_195650),
Nicotiana
benthamiana
(ABO27628)and Solanum tuberosum (ABO27627) according to Clustal W using the
DNASTAR Laser Gene MegAlign software version 7.1.0
Supplementary Figure S5. Co-expression analysis of At3g13690 reveals co-expression
with the Arabidopsis cell elongation protein Dwarf1/DIMINUTO (At3g19820). Co-expression
analysis was performed with the ATTED-II software (http://atted.jp/top_search.shtml)
Supplementary Figure S6. Hypothetical model illustrating SlSUT2-brassinosteroid
interaction. The model illustrates a potential sucrose efflux into the periarbuscular space
from where it is retrieved by SlSUT2 back into the plant root cells. Efflux might potentially be
mediated by still uncharacterized SWEET proteins which are known to act as sugar efflux
carrier (Chen et al., 2010). Alternatively, sucrose is cleaved by the cell wall invertase LIN6
that is inducible by brassinosteroids and cleavage products are taken up by a
monosaccharide transporter into the AM fungus. SlSUT2 forms a complex with MSBP1 and
the receptor like kinase BAKL. Interaction between MSBP1 and BAKL is assumed to prevent
BR signaling via the BR receptor BRI1. BR and BR signaling positively affect LIN6 activity
and inhibit defense responses against biotrophic AM fungi. The model is based on
experimental data in this work and on published data from tomato, Rhizophagus irregularis
and Arabidopsis (Goetz et al., 2000, Song et al., 2009).