DELLAs in a loss-of-functionslr1mutant (Fig.
3E) ( 11 ) or in the presence of the high-level
DELLA accumulation conferred by theSlr1-d6
gain-of-function mutation (Fig. 3F) ( 35 ). Al-
though the mutant SLR1 DELLA encoded by
Slr1-d6was relatively resistant to gibberellin-
mediated destruction, NGR5-HA was still de-
stabilized by exogenous gibberellin treatment
(Fig. 3F). Thus, gibberellin-promoted destabi-
lization of NGR5, although dependent on GID1,
is neither dependent on nor downstream of
gibberellin-induced destruction of DELLAs. Wetherefore explored the possibility of an alterna-
tive, previously unknown, DELLA-independent
mechanism whereby the SCFGID2E3 ubiquitin
ligase directly mediates gibberellin-promoted
destruction of NGR5, finding that GID1 in-
teracts directly with NGR5 [as assayed by
both split firefly luciferase complementation
(SFLC) and Co-IP; Fig. 3, G and H] and that
the strength of this interaction is potentiated
by increasing concentrations of gibberellin
(Fig.3,HandI).Thus,aswiththeDELLAs,
gibberellin enhances the interaction between
NGR5 and GID1, thereby identifying NGR5
as a potential alternative substrate for GID1-
promoted polyubiquitination.
Although NGR5 lacks the specific DELLA
motif that enables the GID1-DELLA interaction
( 33 , 34 ), we found a motif within the AP2-R2
(repeated units 2) ( 18 )domainofNGR5toen-
able the GID1-NGR5 interaction (fig. S10). Fur-
thermore, NGR5 also interacted with the GID2
F-box component of the SCFGID2E3 ubiquitin
ligase that normally targets SLR1 for destruc-
tion in the 26Sproteasome(Fig.3,JandK).
Accordingly, an in vitro ubiquitination assay
showed that GST (glutathioneS-transferase)–
NGR5 fusion protein is polyubiquitinated by
GID2-Flag (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys
peptide) fusion protein in the presence of
ubiquitin-activating enzyme E1, ubiquitin-
conjugating enzyme E2, and ubiquitin, but not
in the absence of GID2-Flag (Fig. 3L), which
suggests that NGR5 is a substrate of the SCFGID2
E3 ubiquitin ligase. Additional time-course ex-
periments showed that gibberellin promotes
the progressive degradation of GST-NGR5
but that this degradation is inhibited both by
MG132 and ingid1-c1, a CRISPR/Cas9-generated
GID1loss-of-function mutant (Fig. 3M and fig.
S8B). Finally, lack of GID2 function (in agid2-c1
mutant; fig. S8C) also inhibits gibberellin-
mediated degradation of GST-NGR5 (Fig. 3M).
We conclude that the gibberellin-mediated reg-
ulation of NGR5 is not due to gibberellin-
promoted destruction of DELLAs, but is due to
a previously unknown direct and gibberellin-
potentiated interaction of NGR5-GID1, lead-
ing to polyubiquitination of NGR5 by the
SCFGID2E3 ubiquitin ligase and subsequent
destruction in the proteasome.DELLA-NGR5 modulation of tiller N response
We next found that NGR5 interacts directly
with SLR1 [in yeast two-hybrid screens (table
S2) and in BiFC and Co-IP assays (fig. S11, A
and B)]. Nonetheless, we additionally found
that the LC2-NGR5 interaction is not inhib-
ited by the presence of SLR1 (fig. S12), which
suggests that the SLR1-NGR5 interaction does
not directly interfere with the LC2-NGR5 inter-
action that determines NGR5 function. Further
experiments showed that the LHR1 (leucine
heptad repeat 1) motif of the DELLA protein
is necessary for the NGR5-SLR1 interactionWuet al.,Science 367 , eaaz2046 (2020) 7 February 2020 4of9
05101525
20NJ6 NJ6NJ6NJ6-sd1
NJ6-sd1+ GA3NJ6-sd1+ GA3+ MG132
Anti-HSP90NJ6 + GA3
NJ6-sd1 NJ6-sd1
NJ6-sd1+ GA3
NJ6-gid1-10
NJ6-gid1-10NJ6-gid1-10+ GA3NJ6-sd1- ngr5
NJ6-sd1- ngr5
+ GA3NJ6-sd1 p35S::GID1
p35S::NGR5-HANJ6-gid1-10
NJ6-slr1
NJ6-slr1
+ GA3
NJ6-gid1-10
+ GA3NJ6-sd1p35S::GID1+ GA3A B LN HNCEDFIJKMNGR5-HA0 2510
GST-GID1NJ6
GST-NGR5NJ6 + GA 3 + MG132
GST-NGR5NJ6 + GA 3
GST-NGR5NJ6-gid1-c1
GST-NGR5NJ6-gid2-c1
GST-NGR5NJ6-gid1-c1 + GA 3
GST-NGR5GST
pull-downGA 3 (μM)
++++
++++
Anti-HA
Anti-GSTAnti-GST
Anti-HSP90
Anti-GST
Anti-HSP90
Anti-GST
Anti-HSP90
Anti-GST
Anti-HSP90Anti-HSP90Anti-GSTAnti-GST
Anti-HSP90Anti-HAAnti-HAAnti-HSP90Anti-HAAnti-HA
Anti-SLR1
Anti-HSP90nLUCnLUC
-GID2nLUC
-GID2nLUCcLUCcLUC
cLUC
-NGR5cLUC
-NGR5GID2-Flag
NGR5-HA
InputIP: HA++
+-
Anti-Flag
Anti-HA
Anti-FlagSlr1-d6 p35S::NGR5-HA036 9 (min)LN HN LN HN LN HNGL E1
E2
GID2-Flag
Ub
GST-NGR5+ +
++
-+
+ +
+++ +
+ +
-+
+ +
+ +Anti-GSTAnti-UbiqMockGA3abcd ddae
f faa aaaa aaaa++ ++nLUCnLUC
-GID1nLUC
-GID1nLUCcLUCcLUC
cLUC
-NGR5cLUC
-NGR5++ ++NJ6-gid2-c1 + GA 3
GST-NGR5
Anti-HSP90Anti-GSTp35S::NGR5-HATiller numbers per plantNJ6-sd1IP: HAAnti-HA
Anti-HSP90IB: UbiqNJ6-sd1
p35S::NGR5-HAGID1-Flag
NGR5-HAInputIP: FlagGA 3 (1 μM)+
++++
+
+
Anti-Flag
Anti-HA
Anti-HAHFig. 3. Gibberellin-GID1-SCFGID2targets NGR5 for destruction.(A) Mature plants grown in low
(90 kg/ha; LN) versus high (180 kg/ha; HN) nitrogen supply. Scale bar, 20 cm. (B) Tiller number.
Data are means ± SE (n=20).(C) Immunodetection of NGR5-HA. (D) Immunodetection of poly-
ubiquitinated NGR5-HA. (E) Accumulation of NGR5-HA. (F) Effects of gibberellin on NGR5-HA and
SLR1 accumulation. In (C) to (F), total protein was extracted from tiller buds of 3-week-old plants
treated for 4 hours with 1mMGA 3 and/or 100mM MG132; HSP90 serves as loading control.
(GandJ) SFLC assays. nLUC-tagged GID1 (G) or nLUC-tagged GID2 (J) was co-transformed into
tobacco leaves along with cLUC-targeted NGR5. (HandK) Co-IP assays. Flag-tagged GID1 (H) or
Flag-tagged GID2 (K) was co-transformed into rice protoplasts along with HA-targeted NGR5.
(I) Pull-down assays. (L) In vitro ubiquitination assay. The immunoprecipitated GID2-Flag proteins
transiently expressed in rice protoplasts were used in an in vitro ubiquitination reaction in the presence
of E1, E2 (UbcH5B), His-Ub, and GST-NGR5. (M) Gibberellin-GID1-SCFGID2destabilizes GST-NGR5.
Lysates from NJ6, NJ6-gid1-c1,andNJ6-gid2-c1plants were co-incubated with GST-NGR5 in
the presence or absence of 100mMGA 3 and 100mM MG132. The lysates were harvested at various
incubation times and immunoblotted to assess the accumulation of NGR5 and HSP90.
RESEARCH | RESEARCH ARTICLE