Nature - USA (2020-05-14)

202 | Nature | Vol 581 | 14 May 2020

Article

f

a c

de

180°

K9 5

K170 K186

K286 K337

K358

+

+

• 
  


• 
  

+

+

• 
  

• 
  

+

• 
  

• 
  

• 
  

+

• 
  

• 
  


• 
  

RHA3A

BIK1-HA

UBQ

BIK1(9KR)-HA

IB: anti-RHA3A

BIK1

IB: anti-UBQ

Ub-BIK1

Ubn-RHA3A

100

150

75

50

250

75

RHA3A

IB: anti-HA

20

25075 Autorad.

BIK1BIK1(9KR)

GST BAK1

K

GST BAK1

K

CBB

GST

BAK1K

BIK1

4658

32

4658 BIK1

32

BAK1K

Quant. Ub-BIK1^0

1.0

2.0abdbcdcede

BIK1BIK1(N5KR)

BIK1(C4KR)BIK1(9KR)

----

----

RK^41

K^4141

R 41

KR 31

K^3131

R 31

KR 186

K^186186

R 186

KR 95

K^9595

R 95

KR 170

K^170170

R 170

KK 337

R^337337

R 337

KK 358

R^358358

R 358

KK 286

R^286286

R 286

KK 366

R^366366

R 366

----

----

----

----

----

----

----

----

----

----

----

----

----

----

g22

BIK1(

9KR)

BIK1(

C4KR)

BIK1(N5

KR)

BIK1

FLAG–UBQ

• 
  
  
  
  • –+ –+ –+
  
  
  
  

Ub-BIK1

IP: anti-FLAG

IB: anti-HA

75

50

IB: anti-HA^50 pBIK1

BIK1^

b

MS/MS scan (m/z)

50

100

Relative abundance 500 1,0001,500

ALLQQ QDNLGK(GG)PSQTNPVK

b 3 b 4 b^7 b 9 b^10 b^11

b 11 2+

y 3 y^8 2+y^9 2+ y 7

y 10 2+

y 11 2+y

12 2+

y 13 2+

y 14 2+y 15 2+

y 16 2+

y 17 2+

y 4 b^5

y 8

y 6 2+ b 8

R.K 31 SSSTVAAAQK.T

K.SSSTVAAAQK 41 TEGEILSSTPVK.S

K.GWLDESTLTPTK 95 PGTGLVIAVK.K

K.K 106 LNQEGFQGHR.E

R.GAYFK 170 PLPWFLR.V

R.VNVALDAAK 186 GLAFLHSDPVK.V

R.ALDHNRPAK 286 EENLVDWAR.P

R.MASVAVQCLSFEPK 337 SR.P

R.ALQQLQDNLGK 358 PSQTNPVK.D

R.ALQQLQDNLGKPSQTNPVK 366 DTK.K

Fig. 3 | Identification of sites of RHA3A-mediated BIK1 ubiquitination.
a, BIK1 is ubiquitinated by RHA3A at multiple lysine residues. Ubiquitinated lysine
residues with a diglycine remnant identified by LC–MS/MS analysis are shown in
red with amino-acid positions. b, MS/MS spectrum of the peptide containing K 358.
c, Structure of BIK1 with six lysines identified as ubiquitination sites shown.
Structural information was obtained from the Protein Data Bank (PDB ID: 5TOS)
and analysed by PyMOL. d, BIK1(9KR) shows compromised flg22-induced
ubiquitination. FLAG–UBQ and HA-tagged BIK1 mutants were expressed in
protoplasts followed by treatment with 100 nM flg22 for 30 min. Quantification of

fold change in BIK1 ubiquitination is shown mean ± s.e.m. overlaid on dot plot

(middle). Different letters indicate significant difference with others (for

example, the rightmost bar is significantly different from those marked a, b, and c

but not d or e) (P < 0.05, one-way ANOVA, n = 3). Lysines mutated in BIK1 mutants

are shown in red (bottom). e, RHA3A cannot ubiquitinate BIK1(9KR). The assay

was performed as in Fig. 2d. f, BIK1(9KR) exhibits normal in vitro kinase activity.

The kinase assay was performed using GST–BIK1 or GST–BIK1(9KR) as the kinase

and GST or GST–BAK1K (kinase domain) as the substrate. All experiments except

MS analyses were repeated three times with similar results.

bc

WTbik1BIK1BIK1(9KR)

bik1

Total

photon

count (×10

4 )

(^012)
2
4
8
6
P=0.0271
P=0.0008
P=0.0018
P=0.0310
P=0.0021
P=0.0009
2
WTbik1BIK1BIK1(9KR)
bik1
1
10
102
103
104
Bacterial
number
(CFU
cm
–2)
P=0.035 3
P=0.018 1
P=0.008 1
P=0.039 0
P=0.008 6
P=0.021 6
a
Total
photon
count (×10
4 ) P=0.0090
WT
amiR-RHA3A/B
(^012)
1
2
3
4
5 P=0.0376
d
100–120
20–3 0
10–2 0
0
g22
(min)
BIK1–RFP+FLS2–YFP BIK1(9KR)-RFP+FLS2–YFP
RFP YFP Merge RFP YFP Merge
0
10
20 P<0.0001
BIK1–RFP +FLS2–YFP
Puncta
per
1,000
μm
2
0
5
10 P=0.0002
BIK1(9KR)–RFP+FLS2–YFP
g22
(min)
P=0.0008
0
10
0
10–2
0
20–3
0
30–4
0
40–5
0
50–6
0
100–120
0
10–2
0
2 0–3030–
40
40–
50
5 0–
60
100–
120
0
20
40
efg
WT
amiR-RHA3A/B
1
105
106
107
108
P<0.0001
P=0.0002
Bacterial
number
(CFU
cm
–2)
2
IB: anti-FLAG
IB: anti-HA
IB: anti-FLAG
IB: anti-HA
BIK1(9KR)Ctrl
FLS2-HA
BIK1
g22–+ – + –
175 FLS2
46
BIK1
pBIK 1
46
BIK1
pBIK 1
(^175) FLS2
Co-IP
efˆcienc
yo
f
FLS2–BIK
1
Enhanced
Reduced
10
1

0. 1

P=0.017 4

P=0.011 0

P=0.0083

P=0.672 1

IP: anti-FLAG

Inpu

t

Fig. 4 | RHA3A/B-mediated monoubiquitination of BIK1 contributes to its
function in immunity and endocytosis. a, pBIK1::BIK19KR-HA/bik1 transgenic
plants (lines 1 and 2) cannot complement bik1 for flg22-induced ROS
production. One-way ANOVA; wild-type, BIK1/bik1: n = 53; bik1: n = 54;
BIK1(9KR)/bik1: n = 55. In all panels, data are shown as mean ± s.e.m. overlaid on
dot plot; lines beneath P values indicate relevant pairwise comparisons. b, The
pBIK1::BIK19KR-HA/bik1 transgenic plants show increased bacterial growth of Pst
DC3000 hrcC–. Plants were spray-inoculated and bacterial growth was measured
at four days post-inoculation (dpi). One-way ANOVA, n = 6. CFU, colony-forming
units. c, amiRNA-RHA3A/B plants show reduced flg22-induced ROS production.
One-way ANOVA, n = 51. d, amiRNA-RHA3A/B plants show increased bacterial
growth of Pst DC3000. Plants were hand-inoculated and bacterial growth was
measured at 2 dpi. One-way ANOVA, n = 5. e, f, Flg22-induced endocytosis of

BIK1, BIK1(9KR), and FLS2 in N. benthamiana leaf epidermal cells. e, BIK1–

TagRFP or BIK1(9KR)–TagRFP was co-expressed with FLS2–YFP followed by

treatment with 100 μM flg22 and then imaged at the indicated time points by

confocal microscopy. Scale bars, 20 μm. f, Quantification of BIK1–TagRFP

(magenta) and FLS2–YFP (green) puncta. One-way ANOVA, additional images

and n values shown in Extended Data Fig. 9c. g, BIK1(9KR) does not enable

flg22-induced dissociation of BIK1 from FLS2. Top, co-IP was performed using

protoplasts expressing FLS2–HA and BIK1–FLAG or BIK1(9KR)–FLAG, followed

by treatment with 1 μM flg22 for 15 min. Bottom, the interaction of BIK1 with

FLS2 was quantified as intensity from IP: anti-FLAG, IB: anti-HA divided by

intensity from IP: anti-FLAG, IB: anti-FLAG. Mean ± s.e.m. fold change (BIK1 no

treatment = 1.0; one-way ANOVA, n = 3). All experiments were repeated three

times with similar results.