Science - USA (2022-02-04)

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

RESEARCH | RESEARCH ARTICLE