Supplementary informationfor
Phosphorylation of S344 in the calmodulin-binding domain negatively
regulates CCaMK function during bacterial and fungal symbioses
Pratyush Routray1,4,a, J. Benjamin Miller2,a, Liqun Du1,3,a, Giles Oldroyd2 and B.W.
Poovaiah1,b
Supplementary Figure Legends:
Figure S1.Identification of S9 as autophosphorylation site of MtCCaMK by
LC-MS/MS.
The figure represents the MS2spectrum of peptide KLSPDEYEVSEILGR and their
relative abundance. The top panel shows the possible b- and y- ion fragment of
KLSPDEYEVSEILGR. The table shows the predicted masses of different MS 2
ionized peptides fragment, with product ion identified by LC-MS/MS marked in
red color. At the bottom is the graphical representation of relative abundance of
different ionized peptides identified through LC-MS/MS. The Y-axis shows the
intensity as arbitrary units and X-axis represents the mass to charge ratio. The
presence of y-98 ions after y12 suggests S9 of MtCCaMK as the phosphorylation
site.
1
Figure S2. Identification of S344 as autophosphorylation site of MtCCaMK
by LC-MS/MS.
The figure represents the MS2 spectrum of peptide AAAIASVWSPSTIFLR and
their relative abundance.
The top panel shows the possible b- and y-ion
fragments of AAAIASVWSPSTIFLR. The table shows the predicted masses of
different MS2 ionized peptides fragment, with product ion identified by LC-MS/MS
marked in red color. The bottom panel shows the graphical representation of
relative abundance of different ionized peptides identified through LC-MS/MS.
The Y-axis shows the intensity as arbitrary units and X-axis represents the mass
to charge ratio. The spectrum showing 800 m/z unit shift from the expected
unmodified peptide ion in y-ion masses beginning with the y6 indicates S344 as
the phosphorylation site of MtCCaMK.
Figure S3.Complementation of ccamk-1 with S344 mutants for RNS.
ccamk-1plants transformed with different CCaMKmutants were examined for
mature nodules 28 days after the inoculation of Sinorhizobiummeliloti1021
carrying hemA::LacZreported gene (a). Roots and nodules of these transformed
plants were stained for β-galactosidase activity (b) where dark blue color
represents the presence of rhizobia.
For further confirmation, nodules were
sectioned and stained with toluidine blue to observe the bacterial presence (c).
The dark blue color shows the presence of rhizobia. As expected, ccamk-1 plants
transformed with S344A produced normal nodules (a) and stained dark blue
color (b) confirming the presence of rhizobia inside the nodule. In addition, the
2
dark blue color of the nodule cross section further supports the presence of
rhizobia (c). Similar phenotypes are also observed for T271A-S344A constructs
(a, b, c).
However, S344D and T271A-S344D constructs failed to produce
nodules, and roots did not stained dark blue. This clearly indicates that S344D
mutation alone or on T271A background failed to complement ccamk-1 plants for
both root nodule organogenesis and bacterial entry. Scale bar for Figure a, bis
500 µm and for Figurec is 50 µm.
FigureS4.Complementation of ccamk-1 with different S344 mutants for
AMS.
ccamk-1plants transformed with different CCaMK mutants in S344 and T271A
positions were grown in soil containingRhizophagusirregularis. The transgenic
roots were stained with black ink to identify the fungal structures.
Plants
transformed with WT CCaMK, T271A, S344A and T271A-S344A showed fungal
vesicles and arbuscles indicating the ability of these mutants to complement
ccamk-1 plants.
However, as expected, very little if any fungal structure is
observed in S344D and T271A-S344D transformed plants indicating their failure
to complement ccamk-1 for AMS. Scale bar 100 µm.
FigureS5.Multiple sequence alignment of calcium/calmodulin-dependent
protein kinase from different plants showing conservation of S9 and S344.
3
Multiple sequence alignment (MSA) of CCaMK of different legume and nonlegume plants was performed using ClustalW2 software from EMBL-EBI website.
Here only N terminal region and calmodulin binding region of CCaMK were
shown. The newly identified autophosphorylation sites from this study (S9 and
S344) are marked with red. From this MSA it is clear that S9 is conserved in
both legume and non-legumes CCaMK. S344 is also conserved in all CCaMK
used in this MSA except rice (OsCCaMK). In OsCCaMK cysteine is present at
the corresponding site (highlighted with yellow).
4
m/Z
Suppl. Figure1. Identification of the S9 as in vitro autophosphorylation site of
MtCCaMK by LC-MS/MS.
5
m/Z
Suppl. Figure2. Identification of the S344 as in vitro autophosphorylation site of
MtCCaMK by LC-MS/MS.
ow the data are interpreted
6
a
500 µm
ccamk-1 + CCaMK
500 µm
500 µm
ccamk-1 + S344A
ccamk-1 + T271A
500 µm
ccamk-1 + T271A-S344A
b
500 µm
WT + EV
500 µm
500 µm
ccamk-1 + EV
500 µm
ccamk-1 + S344D
ccamk-1 + CCaMK
ccamk-1 + S344A
500 µm
500 µm
ccamk-1 + T271A
500 µm
ccamk-1 + T271A-S344A
500 µm
ccamk-1 + T271A-S344D
C
500 µm
50 µm
50 µm
ccamk-1 + CCaMK
500 µm
500 µm
50 µm
50 µm
ccamk-1 + T271A
ccamk-1 + S344A
500 µm
50 µm
ccamk-1 + S344A
ccamk-1 + T271A-S344A
Suppl. Figure3.Complementation of ccamk-1 with S344 mutants for RNS.
500 µm
500 µm
500 µm
7
500 µm
WT + EV
ccamk-1 + EV
ccamk-1 + CCaMK
ccamk-1 + S344A
ccamk-1 + S344D
ccamk-1 + T271A
ccamk-1 + T271A-S344A
ccamk-1 + T271A-S344D
Suppl. Figure4.Complementation of ccamk-1 with different S344 mutants for AMS
8
LjCCaMK
MtCCaMK
NtCCaMK
OsCCaMK
LlCCaMK
MGYD-QTRKLSDEYEISEILGRGGFSVVRKGTKKSGNE----KTQVAIKTLRRLGSS--MGY--GTRKLSDEYEVSEILGRGGFSVVRKGTKKSSIEEEKSQSQVAIKTLRRLGASNNMGQREDGKTLSDEYEVTDILGRGGFSVVRRGTRRRTLHSGQHHEVVAIKTLRRFGPP--MSKT-ESRKLSDDYEVVDVLGRGGFSIVRRGVSKSEE-----KTQVAIKTLRRLGPAMAMSRH-ESRKLSDDYEVVDVLGKGGFSVVRRGISKSRGK----NNDVAIKTLRRYGYTLPG
*.
: ***:**: ::**:****:**:* :
: ******** * .
52
57
57
53
55
LjCCaMK
MtCCaMK
NtCCaMK
OsCCaMK
LlCCaMK
SAQELLSHPWVRGDKAKDEQMDPEIVSRLQSFNARRKLRAAAIASVWSSTIFLRTKKLRS
SALELLSDPWVKGEKAKDVQMDPEIVSRLQSFNARRKLRAAAIASVWSSTIFLRTKKLKS
TAQEILEHPWVTGDLAKQEQMDAEIVSRLQSFNSRRKFRAAAMASVLSSSFSLRTKKLKK
TASDLLRHPWVIGDCAKQDLMDAEVVSKLQKFNARRKLRAAAIASVLSCKVALRTKRLRN
TANDLLKHPWVIGDSAKQELIEPEVVSRLRSFNARRKLRAAAIASVLSSKVLLRTKKLKN
:* ::* .*** *: **: ::.*:**:*: **:***:****:*** *... ****:*:.
349
355
348
347
351
Suppl. Figure 5.Multiple sequence alignment of calcium/calmodulin-dependent protein
kinase from different plants showing conservation of S9 and S344.
9