Nature - USA (2020-10-15)

Nature | Vol 586 | 15 October 2020 | 447

(DCR-1)^12 ,^13 is required for the avoidance induced by lawn exposure
(Extended Data Fig. 2a, b), avoidance induced by sRNA specifically
requires SID-2 and DCR-1^12 ,^13 (Fig. 2a–c, Extended Data Fig. 2c).
SID-2 is expressed in the intestine^11 ,^14 and is required for learning
induced by sRNA (Fig. 2b), whereas other components—includ-
ing DCR-1—are expressed more broadly^15. Intestinal rescue of dcr-1
restored sRNA-mediated learning (Fig. 2a, c, Extended Data Fig. 2d),

which suggests that the uptake and processing of bacterial sRNA by SID-2 and DCR-1, respectively, are initiated in the intestine.

Aberrant sRNA responses in RNAi mutants Although DCR-1 and SID-2 are required for PA14 sRNA-mediated learning, we identified a class of RNAi mutants with aberrant PA14 and/or

b 24 h OP50 24 h PA 14

–0.6

–0.4

–0.2

0

0.2

0.4

0.6

0.8

1.0

Ch

oice

ind

ex

**** NS

N2

(^0) daf-7p prg-1;
2,000
4,000
6,000
8,000
10,000
12,000
14,000
16,000
ASI mean intensity (AU)
***** NS

cd
0
2,000
4,000
6,000
8,000
10,000
12,000
ASI mean intensity (AU)
OP50 PA14
NS
prg-1;
NS
e
24 h OP50
24 h PA 14
–0.8
–0.6
–0.4
–0.2
0
0.2
0.4
0.6
0.8
Choice Index
NS
NS
N2 hpl-2(ok916)
24 h OP50 training (P 0 only) 24 h PA14 training (P 0 only)
** NS NS
–1.0
–0.8
–0.6
–0.4
–0.2
0
0.2
0.4
0.6
0.8
1.0
Choice index
L sRNA
P 0 F 1 F 2 F 3 F 4 F 5
Generation
PA14 sRNA – control
PA14 lawn – control
LawnsRNA
0
0.2
0.4
0.6
0.8 P^0
Lear
ning index

–0.8
–0.6
–0.4
–0.2
0
0.2
0.4
0.6
0.8
Choice index
**** NS
–0.8
–0.6
–0.4
–0.2
0
0.2
0.4
0.6
0.8
1.0
Choice index

N2 glp-1(e2141)
NS NS
–1.0
–0.8
–0.6
–0.4
–0.2
0
0.2
0.4
0.6
0.8
Choice index
NS
NS
N2 sid-2(qt42)
–0.6
–0.4
–0.2
0
0.2
0.4
0.6
0.8
Choice index
NS
NS
N2 meg-3(tm4259)
meg-4(ax2026)
PA14
prefer
ence
OP50
prefer
ence
a
f
–0.8
–0.6
–0.4
–0.2
0
0.2
0.4
0.6
0.8
1.0
N2

** NS
–0.6
–0.4
–0.2
0
0.2
0.4
0.6
0.8
Choice index
N2 rde-4(ne305)
***** NS
ijk
lm n o
h
PA14
prefer
ence
OP50
prefer
ence
PA
14
preference
OP50
prefe
re nce
PA
14
prefe
rence
OP
50
prefer
ence
24 h OP50 24 h PA 14 24 h OP50 24 h PA 14
24 h OP50 24 h PA 14
N2 prg-1
GL::prg-1
24 h OP50 24 h PA 14
(n4357)
Choice index
1.0
0.8
0.6
0.4
0.2
0
–0.2
–0.4
–0.6
–0.8
24 h OP50 24 h PA 14
–0.8
–0.6
–0.4
–0.2
0
0.2
0.4
0.6
0.8 NS
N2 dcr-1 dcr-1;
(mg375)int::dcr-1
NSNS
NS
NSNS
–0.6
–0.4
–0.2
0
0.2
0.4
0.6
0.8
Choice index
**** NS
N2 rrf-3(pk1426)
NS
24 h OP50 24 h PA 14 24 h OP50 24 h PA 14
N2 glp-1(e2141)
L sRNA LsRNALsRNA LsRNALsRNA
::gfp daf-7p::gfp
–0.1 F 1 F 2 F 3 F 4 F 5
0
0.1
0.2
0.3
0.4
0.5
0.6
P 0
Generation
Lear
ning index
P 0 training condition
pmk-1
alg-1
drh-1
dcr-1 int rescue
prg-1 GL rescue
vhp-1
sid-2
dcr-1
prg-1
rrf-1
rrf-3
hpl-2
glp-1
meg-3 meg-4
rde-1
rde-2
rde-4
mut-7
ASI ablation
sid-1
maco-1

– – – – – – – + + + +

+ + + +

– – – – – –

+ + +

±

Normal naive preference (lawn)

PA 14 lawn learning

Normal control sRNA response

PA 14 sRNA learning

Choice index Choice index

g

24 h OP50 24 h PA 14 24 h OP50 24 h PA 14 24 h OP50 24 h PA 14

P11 dependent

P11 independent

sid-1 (pk3321)

rrf-1 (pk1417)

Fig. 2 | dsRNA transport, processing machinery, the PRG-1 piRNA pathway,
and the germline are required for avoidance induced by bacterial sRNA.
a, Summary of genes required for naive preference, PA14 lawn learning and
sRNA learning. +, functions similarly to wild type; −, defective (required for)
naive choice or learning; ±, statistically borderline between the two. Int,
intestinal rescue; GL, germline rescue. b, dsRNA transporter s id-2 is required
for sRNA-induced learning. c, Intestinal rescue of dcr-1 restores sRNA-mediated
learning. d, e, sid-1(pk3321) dsRNA transporter (d) and rde-4(ne305) dsRNA
binding protein (e) mutants exhibit an abnormal sRNA response. f, Germline
prg-1 rescue restores sRNA-induced learning. g–i, RNA-dependent RNA
polymerase rrf-1(pk1417) (g), rrf-3(pk1426) (h), and heterochromatin protein
hpl-2 (ok 916) (i) mutants are deficient in PA14 sRNA-induced avoidance.
j, prg-1(n4 3 57) mutants do not induce da f-7p:: g f p expression in ASI neurons
upon PA14 training (n = 46, 44, 58 and 55 neurons, from left to right). AU,
arbitrary units. k, Germline-less glp-1(e2141) mutants learn to avoid PA14 after
lawn training (left), but are defective in sRNA-induced learning (right). l, glp-1

mutants do not induce da f-7p:: g f p expression in ASI neurons upon PA14 exposure (n = 60 (OP50), 54 (PA14) neurons, two-sided Student’s t-test). m, Germline P-granule mutants exhibit defective sRNA-induced learning. n, Untrained progeny of PA14 lawn (L) and sRNA-trained mothers avoid PA14 from generation F 1 to F 4 ; generation F 5 resumes PA14 attraction. o, Top, learning index for each generation, mean ± s.e.m. Bottom, P 0. Two-sided Student’s t-test. Each data point represents the learning index from an independent experiment of about 7–10 choice assay plates; each dot represents an individual choice assay plate (average of 115 worms per plate) from all replicates. b–k, m, Tw o - w a y ANOVA, Tukey’s multiple comparison test. Biological replicates: 3 (c, f–m), 4 (b, e), 5 (sRNA in n), 6 (d, lawn in n). Box plots: centre line, median; box range, 25–75th percentiles; whiskers denote minimum–maximum values. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P < 0.0001, NS, not significant. Estimation plots are provided in the Supplementary Information; see Supplementary Table 4 for exact sample sizes (n) and P values.

Nature - USA (2020-10-15)

Get our desktop app

Company

Features

Documentation

Resources