Nature - USA (2020-10-15)

Article

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P11 choice assay

Choice Index

n.s.

24 h OP50 trained

24 h PA14 trained

PA14 plate

minus

OP50 plate

−1.0 0

−0.7 5

−0.5 0

−0.2 5

0.00

0.25

0.50

0.75

Mean diff

erence

a b

24 h PA14 trained

24 h E. coli trained

24 h E. coli + sRNA trained

PA14 sRNA expression in E. coli

PA14

preference

E. coli

preferenc

e

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Choice Index

** ****

E. colisRNA overexpression strain

PA14E. co

li contro

l

P1^1 P2^4 P1^6 Sr32RsmZErSA

n.s.

n.s.

controlP 11

0

100

200

300

400

P0 training

condition

total brood size

n.s.

1234

0

50

100

150

200

250

Day post training

Progeny number

n.s. n.s.

n.s. n.s.

Progeny counts per day

E. coli control

E. coli expressing P11 small RNA

OP50

PA 14

PA 14 P1 1

c

f g

24 h E. coli control trained

24 h E. coli + sRNA trained

d

P11 sRNA

metabolites

innate immunity

Pseudomonas

exposure

daf-7

independent

(pyochelin/PCN)

(pmk-1 dependent,

pmk-1 independent/irg-1)

PA 14 P1 1

inactive pathway

active pathway

PA 14

avoidance

e Wild-type worms on PA 14 P11 lawn

P11-PA 14

isolated

small RNAs

on OP50

inactive pathway

inactive pathway

Wild-type worms on PA14 P11 sRNA

PA 14

attraction

P11 sRNA

metabolites

innate immunity

ASI daf-7p::GFP

ASJ daf-7p::GFP

(pyochelin/PCN)

(pmk-1 dependent,

pmk-1 independent/irg-1)

E.co

li

+ P1

1

pr efere

nce

OP

50

pref

erenc

e

ASI daf-7p::GFP

ASJ daf-7p::GFP

daf-7

independent

Extended Data Fig. 6 | Identif ication and testing of the differentially
regulated PA14 sRNA. a, P11 expressed in E. coli induces PA14 avoidance in a
choice assay between PA14 and E. coli strain MG1655. Worms were trained on
OP50, PA14, E. coli strain MG1655 containing empty vector (control), or E. coli
strain MG1655 expressing PA14 small RNAs (red). Following training, a choice
assay was performed between E. coli strain MG1655 and PA14. Similar to Fig. 3c,
worms trained on PA14 or E. coli expressing P11 exhibited PA14 avoidance
behaviour. One-way ANOVA with Tukey’s multiple comparison test. b, PA14
plate-trained worms do not avoid E. coli expressing P11 compared to OP50 in a
choice assay. Two-tailed Student’s t-test. c, PA14 ΔP11 bacteria grown on NGM
plates produce pyocyanin (blue pigment on plates) similar to PA14. d, e, Model
showing induced learning pathways for wild-type worms on isolated PA14 ΔP11
sRNA (d) or on PA14 ΔP11 bacteria lawns (e). f, Exposure to E. coli expressing P11

does not affect total brood size (left) (two-tailed Student’s t-test), or the

number of progeny hatched per day (right). Two-way ANOVA with Tukey’s

multiple comparison test. n = 15 worms were analysed per condition. g, Worms

exposed to control (top) or E. coli expressing P11 appear healthy after 24 h of

training. Biological replicates: 2 (a), 3 (b, c, g). For choice assays, each dot

represents an individual choice assay plate (average of 115 worms per plate)

with data shown from all replicates. For box plots, centre line is the median, box

extends from the 25th to the 75th percentile; whiskers denote minimum–

maximum values. **P ≤ 0.01, ****P < 0.0001, n.s., not significant. Mean

differences are shown using Cumming estimation plots^47 , with each graphed as

a bootstrap sampling distribution. Mean differences are depicted as dots; 95%

confidence intervals are indicated by the ends of the vertical bars. See

Supplementary Table 4 for exact sample sizes (n) and P values.