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with RIPA buffer and then solubilized in 8 M urea, 2% SDS, 50 mM DTT,
50 mM Tris pH 7.4 at room temperature. To test the LBP-2 protein level
in animals treated with RNAi targeting ECRs, synchronized L1 DCD130
worms were grown on RNAi plates at 25 °C until day 4 of adulthood. 100
worms for each RNAi treatment were collected in loading buffer with
reducing agent (NuPAGE, Thermofisher) and incubated at 70 °C for 10
min. Total protein staining was performed on the transfer blot using
REVERT Total Protein Stain Kit (Li-Cor 926-11010) with staining solution
diluted 2:1 with 100% methanol. The anti-tagRFP antibody (1:1,000,
Evrogen) was used to detect LBP-2::tagRFP protein with secondary
anti-rabbit antibody (1:10,000, IRDye680RD, Li-Cor). Detection was
performed with Image Studio software (v.4.0.21) on Li-Cor Odyssey
CLx imager in the 700 nm channel.
Co-purification
Cultures of C. elegans were performed as previously described^34. In
brief, DCD130 and DCD362 were grown as a synchronized population
at 25 °C in liquid culture until day 3. Worms were collected and washed
once in M9 plus 0.01% Triton, twice with M9, once with PBS before
being frozen in liquid nitrogen and ground in a mortar. One-hundred
milligrams of frozen ground worms were dissolved in 200 μl ice cold
DPBS (Sigma) supplemented with protease inhibitors (Serva; Mix M)
and syringed 20 times. The lysate was centrifuged for 5 min at 800g
at 4 °C. The liquid layer between lipid lid and pellet was collected
and centrifuged for 5 min at 2,200g at 4 °C. Then, 40 μl of HisTrap
HP Sepharose (GE Healthcare; 50% solution pre-washed three times
with DPBS supplemented with protease inhibitors) was added to the
supernatant and incubated for 2 h at 4 °C. Flow-through was collected
at 800g at 4 °C and beads were washed once with 500 μl DPBS with
protease inhibitors followed by further washing steps in 500 μl of
40 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole, pH 7.7
and 500 μl of 40 mM sodium phosphate, 300 mM NaCl, 70 mM imida-
zole, pH 7.7. After a final washing step with 500 μl of 40 mM sodium
phosphate, 300 mM NaCl, 10 mM imidazole, pH 7.7 the proteins bound
to the beads were eluted with 100 μl of 40 mM sodium phosphate,
300 mM NaCl, 500 mM imidazole, pH 7.7. The resulting protein solu-
tions were analysed by western blot. The target proteins were spe-
cifically detected with antibodies directed against tagRFP (1:4,000,
Evrogen) or the 6xHistidine tag (1:2000, anti-His H3 antibody, Santa
Cruz), followed by respectively anti-rabbit-IgG-POD conjugate anti-
body (Sigma) and anti-mouse-IgG-POD conjugate antibody (Sigma).
POD was visualized by spraying the membrane with WesternBrightTM
ECL-spray (Advansta) following the manufacturer`s instructions. The
luminescence signal was detected using an ImageQuant Las4000 sys-
tem (GE Healthcare, v.1.2).
Exposure to Cry5B and pathogenic bacteria
For all experiments related to Cry5B and pathogenic bacteria, day 1
adult animals were transferred to lawns expressing Cry5B or contain-
ing pathogenic bacteria and maintained on these lawns at 20 °C for the
duration of the experiment. E. coli strain JM103 expressing pQE9-Cry5B
and E. coli strain JM103 containing empty vector pQE9 were cultured in
liquid LB medium containing carbenicillin and IPTG as described^37. Each
culture was diluted to 0.2 ± 0.05 OD by adding LB medium to generate
a stock solution for further dilutions. For 100% Cry5B expressing lawn,
100 μl of JM103 expressing Cry5B were spread on NGM plates with car-
benicillin and IPTG. To obtain diluted Cry5B expressing lawns, E. coli
JM103 containing empty vector was added to E. coli JM103 expressing
Cry5B and then spread on NGM plates with carbenicillin and IPTG. Plates
were incubated overnight at 25 °C. For Cry5B, we tested three differ-
ent dilutions: 100, 50 and 5% (Fig. 3a). All the tested concentrations
strongly reduced the aggregation of LBP-2 and for subsequent experi-
ments, we chose 5% to minimize death related to Cry5B (Fig. 3e). Bacillus
atrophaeus strain ATCC9372 (DSMZ Leibniz Institute), Microbacterium
nematophilum strain CBX102 (CGC) and control E. coli strain OP50
were cultured in liquid LB medium at 37 °C overnight. LB medium was
added to adjust each culture to the final concentration of 1 ± 0.05 OD
and 100 μl was spread on NGM plates.Lifespan and survival assays
For the lifespan assay, worms were grown on OP50-seeded NGM plates
at 20 °C and were scored every day for live animals, dead animals (no
longer responding to body touch) and censored animals (crawled off
plates, contaminated, ruptured and bag of worms). For survival analysis
in the presence of Cry5B toxin, worms were grown on OP50-seeded
NGM plates until day 1 at 20 °C and then transferred onto plates with 50%
E. coli strain JM103 expressing Cry5B or E. coli strain JM103 containing
empty vector, maintained at 20 °C. The 50% dilution was chosen as this
concentration allowed us to assess the effect of enhanced extracellular
proteostasis on Cry5B survival during chronic exposition. For survival
analysis with Cry5B and RNAi targeting ECRs, worms were grown on
plates seeded with RNAi clones until day 1 at 20 °C except for clec-1
knockdown where worms were grown on OP50 seeded NGM plates until
L4 and then transferred to plates seeded with clec-1 RNAi clone at 20 °C.
At day 1, worms were transferred onto plates with a mix of 25% E. coli
strain JM103 expressing Cry5B and 75% E. coli strain expressing RNAi.
Survival was scored daily for live animals, dead animals and censored
animals. For survival assays, bagged animals were included as dead.
The numbers of animals are reported in Extended Data Tables 1–3.qRT–PCR
Approximately 1,000 or more synchronized worms were used for
each qRT–PCR experiment. Worms were rinsed from plates with M9
and washed twice with M9. Total RNA was prepared from worms using
QIAZOL lysis reagent (Qiagen) and further purified with RNeasy Plus
Universal Mini kit (Qiagen). cDNA was prepared by reverse transcription
(SuperScript III, Invitrogen) using oligo-dT primers (Qiagen). qRT–PCR
was performed on an ABI 7000 Instrument using SYBR Green detec-
tion (Applied Biosystems). For Cry5B experiments, day 1 adults were
exposed for 3 h at 20 °C to either 100% E. coli strain JM103 expressing
Cry5B or E. coli strain JM103 containing empty vector, as previously
described^18. In the wild-type background, six independent experiments
were performed for each treatment. In the kgb-1 (km21) background,
five independent experiments were performed for each treatment. To
quantify LBP-2::tagRFP expression level on Cry5B, four independent
experiments were performed for each treatment. To test the expression
levels of ECRs with vhp-1 RNAi, synchronized L1 larvae were cultured at
20 °C until day 1 on vhp-1 RNAi or empty vector plates. For each condi-
tion, four independent experiments were performed. eft-2 was used as
the qRT–PCR normalization control^21. To test the expression levels of
ECRs with age, synchronized L1 larvae were cultured at 25 °C until days
2 or 8 of adulthood (sterile background fer-15(b26)II; fem-1(hc17)IV).
pgk-1 was used as the qRT–PCR normalization control. For each condi-
tion, four independent experiments were performed. Primers used are
listed in Supplementary Table 7.RNA-seq experiments
Approximately 250 worms were collected per sample. Day 1 adults were
exposed for 24 h at 20 °C to either 50% E. coli strain JM103 expressing
Cry5B or E. coli strain JM103 containing empty vector. Worms were
collected in M9, washed twice with M9 and frozen in liquid nitrogen.
Total RNA was isolated using Direct-zol RNA Mini-Prep Kit following
the manufacturers’ instructions (Zymo Research). RNA concentra-
tion was quantified using Qubit (Invitrogen Life Technologies) and
Nanodrop (PEQLAB Biotechnologie GmbH) measurements. RNA-seq
libraries were prepared using TruSeq RNA library preparation kit v2
(Illumina Inc.) according to the manufacturer’s instructions from 1 μg
of total RNA in each sample. Libraries were quantified using Qubit and
Bioanalyzer measurements (Agilent Technologies) and normalized
to 2.5 nM. Samples were sequenced as 150-bp paired-end reads on