Science - USA (2022-02-04)

Baiet al.,Science 375 , eabm2948 (2022) 4 February 2022 4of9

A

0

5

10

15

20

25

30

0 0.5 1 2 3 6 9 24

0

20

40

60

80

100

120

140

160

180

NaJAZi

NaJAZh

Relative expression

M. sexta feeding

Empoasca feeding

Time after herbivory (h)

0 0.5 1 2 3 6 9 24

PCC

-1 1

P<0.05

Coumaroylputrescine(CoP)

Caffeoylputrescine(CP)

Feruloylputrescine(FP)

Coumaroylspermidine

Caffeoylspermidine

Feruloylspermidine

Coumaroyl,caffeoylspermidine

Coumaroyl,feruloylspermidine

Caffeoyl,feruloylspermidine

Di-caffeoylspermidine

Di-feruloyl-spermidine

Nicotine

Nicotinic acid

Chlorogenic acid

Coumaroylquinic acid

Feruloylquinic acid

Rutin

Kaempferol-glucose-rhamnose

Lyciumoside I

Attenoside

Lyciumoside IV

Nicotianoside I

Nicotianoside II

Nicotianoside IV

Nicotianoside V

Nicotianoside VI

Nicotianoside VII

O-acyl sugar #2, class 2

O-acyl sugar #3, class 2

O-acyl sugar #4, class 2

O-acyl sugar #5, class 2

O-acyl sugar #6, class 2

O-acyl sugar #7, class 3

O-acyl sugar #8, class 3

O-acyl sugar #9, class 3

O-acyl sugar #10, class 3

O-acyl sugar #11, class 3

O-acyl sugar #15, class 4

Phenylalanine

Tryptophan P<0.05

Tyrosine

# Empoasca

%Damage area

0

1

2

Log

2

Si

MS/MS

Metabolic

specificity

Metabolites

Phenolamides

Alkaloids

Quinate conjugates

Flavonoid glycosides

HGL-DTGs

O-acyl sugars

Amino acids

Others

Unknowns

ovJAZi

EV irMYC2

irMYB8

5.0 5.5 6.0 6.5 7.0

0.3

0.4

0.5

M. sexta feeding

M. sexta control

Empoasca feeding

Empoasca

control

Diversity (Hj)

Specialization (δj)

5.0 5.5 6.0 6.5 7.0

0.3

0.4

0.5

B

C D

irJAZc irJAZeirJAZgirJAZhovJAZiovJAZLEV

irMYC2 ovJAZiirMYB8irCV86irDH29 EV

0.0

2.5

5.0

7.5

10.0

Empoasca

number

Damage area (%)

irMYC2irMYB8ov

JAZiovJAZ

l

irJAZ irJAZh irDH29

e

irJAZc irJAZg irJAZiirJA

ZL

irCV8

(^6) EV
2
4
6
0
b
**

---

**

---

0
0
40
NaJAZi
NaJAZh
asc
LET
STT
ANT
NEC
OVA
ROT
LEC
SNP
OFL
SED
PED
COE
STI
FLB
NaJAZhNaJAZi
−2
−1
0
1
2
3
TPM (Z-score scaled)
b
b
a a
a
aa a
aaa
a
aa a
a
aa
a
a
a a
b b b
1.5
2.0
2.5
3.0
3.5
4.0
DMSO CoP+
DMSO
CP+
DMSO
FP+
DMSO
2
4
6
8
Damage area (%)
Empoasca
number
E
a
a
a a
aaaa
0
2
1
3
4
5
10%
Glucose
CoP+10%
Glucose
CP+10%
Glucose
FP+10%
Glucose
Mortality rate (%)
a
a
a
a
In vivo Empoasca
choice assay in irMYC2
In vitro Empoasca feeding assay
Herbivory-induced metabolome reconfigurations
irJAZcirJAZeirJAZgirJAZhovJAZiovJAZLEV
irMYC 2 ovJAZiirMYB 8 irCV 86 irDH29 EV
Fig. 2. Reverse genetics coupled with information theoryÐbased unbiased
metabolomics reveal anEmpoasca-elicited JA-JAZi module regulating induced
unknown putrescine-containing phenolamides correlated withEmpoasca
nonhost resistance.(A) Phenotypes ofEmpoasca-damaged leaves on transgenic
N. attenuatalines. Plants (n= 10) were randomly placed in an open-choice glasshouse
environment containingEmpoascaleafhoppers (fig. S4). Representative leaves are
shown for each genotype, with insets highlighting the damage of ovJAZiand irMYB8
lines. Different letters indicate significant differences [P< 0.001, one-way analysis of
variance (ANOVA) followed by Tukey’s post hoc multiple comparisons]. (B) Tissue-
specific expression profiles ofNaJAZhandNaJAZiinN. attenuataWT (left); heatmap
coloring depicts the Z-score–scaled transcripts per million (TPM). Kinetics of
relative transcript accumulations ofNaJAZhandNaJAZiin leaves ofN. attenuata
(n= 3) in response to continuousE. decipiensandM. sextafeeding with samples
harvested at 0 to 24 hours after the start of feeding. FLB, flower bud; STI, stigma; COE,
corolla early; PED, pedicel; SED, seed; OFL, opening flower; SNP, style; LEC, leaf
control; ROT, roots from OS-treated plants; OVA, ovary; NEC, nectary; ANT, anther;
STT, stem treated; LET, leaf treated. (C) Scatterplots of specialization (dj) versus
diversity (Hj) indices for specialized metabolomes of leaves (n=4)after72hoursof
feeding byE. decipiensandM. sextaon four transgenic lines selected from a set of
16 transgenic lines (see fig. S9 for the complete dataset). An increase in metabolome
specialization (dj) indicates that, on average, more herbivory or genotype-specific
metabolites are produced, whereas an increase in metabolome diversity (Hj) indicates
that either qualitatively more metabolites are produced or that quantitatively
the global metabolic frequency profile is more uniformly distributed. Colors denote
different insects, and symbols denote different treatments—triangles indicate insect
feeding, and circles indicate untreated controls. (D) A ranked metabolite specificity
(Si) index distribution plot was calculated for each metabolite on the basis of
E. decipiensspecifically elicited metabolomes from the four transgenic lines shown in
(C) (left) and further linked with a PCC coexpression heatmap among metabolites
andE. decipiensnumber and damage phenotypes (right) for which the data from all
reverse-genetics lines shown in (A) were used to enhance the statistical power of
the PCC calculations. Dots are colored on the basis of compound class annotations.
Only significant correlations withP<0.05areshownwithcolors.(E)InvivoEmpoasca
choice assays (n= 8) conducted by infiltrating synthetic CoP, CP, or FP diluted in 0.1%
dimethyl sulfoxide (DMSO) solutions into irMYC2leaves (top) and in vitroEmpoasca
nonchoice assays (n= 3, 25Empoascaleafhoppers per replicate) by feedingEmpoasca
with synthetic CoP, CP, or FP diluted in 10% glucose solutions (bottom) revealed that
these phenolamides were not affecting leafhopper behavior or performance.
RESEARCH | RESEARCH ARTICLE