hexenal for a Michael addition reaction, the
product of which is aromatized to form CPH
(Fig. 4B).
CPH is responsible forEmpoascaresistance
To test whether CPH is responsible forEmpoasca
resistance, we fedE. decipienswith physiolog-
ically relevant concentrations of 1mM (esti-
mated from field-collected elicited leaves) of
NMR-confirmed CPH in diets containing 10%
glucose in vitro. After 6 hours of feeding, the
CPH treatment caused almost 100% mortal-
ity ofE. decipiens, in contrast to leafhopper
growth on control diets (P=3×10−^8 ; Student’s
ttest) (Fig. 4C). We further silencedNaAT1
expression inN. attenuataplants using VIGS,
which disrupted the production of both CP
and CPH ( 14 ) (Fig. 3D). In vivo choice assays
revealed thatNaAT1-silenced plants received
significantly moreEmpoascadamage and
higherEmpoascanumbers than EV plants
(Fig. 4C). Similarly, silencing eitherNaPPO1
orNaPPO2in plants abolished only the accu-
mulation of CPH in plants, without significant
alterations in other phenolamide pools (Fig. 3D
and fig. S16), and resulted in a clearEmpoasca
feeding preference (P= 0.013 andP= 0.022,
respectively) and greaterEmpoascadamage
(P= 0.008 andP= 0.008, respectively) com-
pared with that observed in EV plants (Fig. 4C).
Baiet al.,Science 375 , eabm2948 (2022) 4 February 2022 7of9
Fig. 4. Structural elucida-
tion, biosynthesis,
function, and engineering
ofm/z347.19 in vitro
and in planta.(A) In vitro
enzymatic assays for
m/z347.19 production
revealed that either NaPPO1
or NaPPO2 can catalyze
the condensation of CP and
(Z)-3-hexenal (Z3H) to
formm/z347.19. Extracted
ion chromatograms (EICs)
for doubly charged CP
dimer (m/z250.13) and
m/z347.19 inE. coliÐ
expressing NaPPO1 and
NaPPO2 with CP and
Z3H or (E)-2-hexenal (E2H).
Compounds 1, 2, 3, and
4 denote isomers of
CP dimer; compounds
5 and 6 denote isomers of
m/z347.19. (B) Proposed
enzymatic reactions
mediated by NaPPO1 or
NaPPO2 form/z347.19
synthesis: NaPPO1/2
oxidize CP to caffeoyl
quinone putrescine and
activate Z3H for the Michael
addition reaction, followed
by aromatization to yield
m/z347.19. (C) Mortality
rates of in vitroEmpoasca
nonchoice assays (n=4,25
Empoascaleafhoppers
per replicate) in which
Empoascawere fed for
6 hours either with 1mMm/z
347.19 synthesized by using
NaPPO1/2, CP, and Z3H
and diluted in 10% glucose
solution or with 10%
glucose as control (top).
Empoascain planta choice assays (n= 8) conducted with VIGS plants of EV, PPO1, PPO2, and AT1 (bottom). (D) Genes involved in the biosynthesis ofm/z347.19.
(E) Engineering of biosynthetic pathways for the production ofm/z347.19 in plants. NeitherV. fabanor the wild tomatoS. chilenseaccumulatem/z347.19 in
response to elicitation.S. chilenseaccumulates CP after MeJA elicitation, whereasV. fabadoes not (inserted heatmaps).S. chilensecan be engineered to producem/z
347.19 by expressing NaPPO1, NaPPO2, and NaBBL2 with Z3H infiltrations in MeJA-elicitedS. chilenseleaves.m/z347.19 accumulates only inS. chilenseexpressing
NaPPO1/2 together with NaBBL2, but not NaPPO1/2 alone.V. fabacan be engineered to producem/z347.19 by expressing NaPPO1, NaPPO2, and NaBBL2 with
Z3H and CP infiltrations in leaves. Also shown are mortality rates of in vivoEmpoascafeeding assays (n= 4, 25Empoascaleafhoppers per replicate) in which
Empoascawere fed for 10 hours on leaves of reconstitutedS. chilenseandV. faba, respectively.
A
B
D
100
75
50
25
0
Mortality (%)
***
10%Glucose10%Glucose
+m/z 347.19
HO
HO
N
H
O
NH 2 O
O
N
H
O
NaPPO1/NaPPO2 NH^2
O
HO
N
H
NH 2
O
H
O
H
HO
HO
N
H
O
NH 2
O
H
Michael addition
Aromatization
oxidation
quinone
Z3H
N-Caffeoylputrescine
NaPPO1/NaPPO2
(5, 6) m/z 347.19
O
0
2
4
EV PPO1 PPO2 AT1
Damage area (%)
b
aaa
0.0
2.5
5.0
7.5
Empoasca
number
b
a a a
C
Phenylalanine
O
NH 2
OH
Caffeoyl-CoA
CoA
O
S
OH
OH
m/z 347.19
O
NH NH^2
OH
OH
CH 3
O
O
NH
NH 2
OH
OH
NaPALNaC4H Na4CLNaHCTNaC3HNaHCTNaAT1 NaPPO1NaPPO2NaBBL2
Z3H
NaODC NaHPL
NH 2
H 2 N
Phenylpropanoid pathway Polyamine pathway GLV pathway Present study
Putrescine
N-Caffeoylputrescine
E
Solanum
chilense
3 4 5
0
2
4
6
8
Intensity (
x10
3 )
m/z 347.19
Retention time (min)
NaPPO1&2
+NaBBL2+Z3H
NaPPO1&2+Z3H
EV+Z3H
EICm/z 347.19
m/z 347.19
CP
MeJAC
Absent Present
Untransformed WT
1 2 3 4 5 6
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Intensity (
x10
4 )
1 2 34 5 6
m/z 250.13 m/z 347.19
[M+2H]2+ [M+H]+
NaPPO1+CP+Z3H
NaPPO2+CP+Z3H
NaPPO1+CP+E2H
NaPPO2+CP+E2H
CP+Z3H
m/z 250.13
m/z 347.19
EIC
Retention time (min)
Vicia faba
0
200
400
600
800
4 5
Retention time (min)
Intensity
EV+CP+Z3H
NaPPO1&2
+NaBBL2+CP+Z3H
EICm/z 347.19 m/z 347.19
m/z 347.19
CP
MeJAC
Absent Present
NaPPO1&2+NaBBL2+CP
Untransformed WT
0
25
50
75
100
Treatment
Mortality (%)
0
25
50
75
100
Treatment
Mortality (%)
EV+Z3H
NaPPO1+NaBBL2+Z3H
NaPPO2+NaBBL2+Z3H
EV+Z3H
NaPPO1+NaBBL2+CP+Z3H
NaPPO2+NaBBL2+CP+Z3H
EV+CP
b b
a a a
b b
EV+Z3HNaPPO1
+NaBBL2+Z3H
NaPPO2
+NaBBL2+Z3H
EV+Z3HNaPPO1
+NaBBL2
+CP+Z3H
NaPPO2
+NaBBL2+CP+Z3H
EV+CP
O
O
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