microPublication Biology2578-9430Caltech Library10.17912/micropub.biology.000525Negative ResultNew FindingPhenotype DataC. elegansATFS-1 plays no repressive role in the regulation of epidermal immune responseMartineauCeline N1MaynardClaire A1PujolNathalie1§
Aix Marseille Univ, INSERM, CNRS, CIML, Turing Centre for Living Systems, Marseille, France
Correspondence to: Nathalie Pujol (
pujol@ciml.univ-mrs.fr
)
Fungal infection triggers the induction of antimicrobial peptide (AMP) genes in the epidermis (Pujol et al, 2008). We previously showed that this effect is suppressed by the mitochondrial unfolded protein response (UPR
mt
), which can be activated by knockdown of select genes including the mitochondrial metalloprotease
spg-7
(Zugasti et al, 2016). Here, we confirm that RNAi against
spg-7
triggers the UPR
mt
and blocks AMP induction during infection, whereas infection itself does not trigger the UPR
mt
. ATFS-1 is a key factor in the UPR
mt
, mediating much of the associated transcriptional response. We find that, surprisingly, ATFS-1 is not required for the suppression of AMP induction provoked by
spg-7(RNAi)
. These data show that the mitochondrial dysfunction that blocks the immune response upon infection or wounding is independent of ATFS-1.
(A-D) Quantification of relative green fluorescence (GFP/size) in young adult worms after infection (orange) or not (blue) with
the fungus
Drechmeria coniospora
treated with the either
sta-1
(control) or
spg-7
RNAi clones, with mean and SEM, **** p<0.0001. The induction of immune response was followed with a
nlp-29
p::GFP reporter using the
frIs7
transgene (A,C & D). The induction of UPR
mt
was followed with a
hsp-6
p::GFP reporter using the
zcIs13
transgene
(B). (A-B) Compared to
sta-1
control clone, the inactivation of
spg-7
induces the UPR
mt
(B) and blocks the induction of
nlp-29p
::GFP upon infection in the wild type (A), as previously shown (Zugasti
et al.
, 2014). (C-D) The same results were obtained in
atfs-1(gk3094)
mutant background (C) or in the background of an
atfs-1(tm4525)
mutant rescued with a form of ATFS-1 engineered to include a strong MTS signal (
bcSi81[atfs-1(NcATP9MTS)]
) (Rolland
et al.
, 2019) (D). (E) Representatives images of the induction of immune response upon wounding followed with the same
frIs7
reporter transgene. The induction of
nlp-29
p::GFP reporter is visualised simultanously with the constitutive expression of
col-12
p::RFP reporter in the epidermis with a GFP long pass filter. (F) Quantification of relative green fluorescence (GFP/size) in young adult worms after wounding (orange) or not (blue)
treated with the either
sta-1
(control) or
spg-7
RNAi clones, with mean and SEM, **** p<0.0001.
Description
Drechmeria coniospora
is a natural fungal pathogen of
Caenorhabditis elegans
. Fungal spores are able to pierce the cuticle of the worm, leading to hyphal growth in the entire organism.
C. elegans
counters the infection by triggering a rapid innate immune response. A hallmark of the response is the secretion of antimicrobial peptides (AMP) encoded by the
cnc
(caenacin) family and certain
nlp
(neuro-peptide-like protein) genes (Dierking
et al.
, 2011; Taffoni
et al.
, 2020). We previously showed that impairing mitochondrial function and inducing the mitochondrial unfolded protein response (UPR
mt
), blocks the expression of AMP genes after infection by
D. coniospora
, possibly through cross-tissue signaling between the intestine and the epidermis (Zugasti
et al.
, 2016; Ewbank & Pujol, 2016). The molecular pathways involved, however, remain unknown.
Here, we first confirmed that knock-down of the mitochondrial metalloprotease
spg-7
by RNAi activates the UPRmt through the induction of
hsp-6p
::GFP reporter (Figure 1A) (Yoneda
et al.
, 2004), and showed that infection by
D. coniospora
did not induce this reporter. Using a transcriptional reporter strain for AMP expression (
nlp-29
p::GFP) (Pujol
et al.
, 2008), we also confirmed that
spg-7
inactivation resulted in a robust block of AMP expression after infection compared to the
sta-1
control clone (Figure 1B) (Zugasti
et al.
, 2016).
We then investigated whether ATFS-1, a master regulator of the UPR
mt
, is required for the
spg-7
suppression. As the responsiveness of ATFS-1 is dependent on its relatively weak mitochondrial targeting sequence (Rolland
et al.
, 2019), its function can be impaired not only by loss-of function mutations but also by alterations that strengthen the mitochondrial targeting sequence (MTS). To examine AMP expression under both of these conditions, we crossed the
nlp-29p
::GFP reporter to the
atfs-1(gk3094)
loss of function mutant and to the
atfs-1(tm4525)
loss of function mutant expressing an allele with a strong MTS (
bcSi81[atfs-1(NcATP9MTS)]
), known to be incapable of triggering the normal UPR
mt
-associated transcriptional response (Rolland
et al.
, 2019) and assayed the level of suppression of
nlp-29
p::GFP expression in response to
spg-7(RNAi)
. In our hands, loss of
atfs-1
did not change the induction of
nlp-29
expression associated with infection by
D. coniospora
. Further, there was a clear suppression of AMP reporter gene expression in both mutant backgrounds upon
spg-7(RNAi)
following infection (Figures 1C and 1D). We also showed that
atfs-1
is not required for the induction of the same immune response upon wounding (Figure 1E), nor for its suppression by
spg-7
RNAi (Figure 1F). We conclude that
atfs-1
is not required for the normal epidermal response to fungal infection or wounding and that the signal that provokes the repression of
nlp-29
expression upon mitochondrial dysfunction is independent of the key UPR
mt
transcription factor ATFS-1.
Methods
RNA interference
RNAi bacterial clones were obtained from the Ahringer library and verified by sequencing (Kamath
et al.
, 2003). RNAi bacteria were seeded on NGM plates supplemented with 100 μg/ml ampicillin and 1 mM Isopropyl-β-D-thiogalactopyranoside (IPTG). Worms were transferred onto RNAi plates as L3 larvae to avoid the lethality associated with
spg-7
(RNAi) and cultured at 25 °C until young adult stage.
D. coniospora
infection & wounding
Infections with
D. coniospora
were carried out at 25 °C as described (Pujol
et al.
, 2008). Briefly, synchronized L4 worms obtained following treatment with an alkaline hypochlorite solution, were infected by adding 100 μl of a fresh spore solution to RNAi plates and then incubated at 25 °C for 18h. Wounding was performed with a microinjection needle in the tail region of Day1 adult worms and analysed after 4h at 25 °C as described (Taffoni
et al.
, 2020).
Analyses with the Biosort worm sorter
Fluorescent protein expression of reporter strains was quantified with the COPAS (Complex Object Parametric Analyzer and Sorter) Biosort system (Union Biometrica; Holliston, MA) as described (Labed
et al.
, 2008). For each strain, a minimum of 50 synchronized young adult worms were analyzed for length (assessed as TOF, time of flight), optical density (assessed as extinction) and Green and/or Red fluorescence (GFP/Red). Raw data were filtered on the TOF for adult worms (400 ≤ TOF ≤ 1000). Fluorescent ratio (Green/TOF) is presented for each worm with mean and SEM for each conditions. Statistical analyses were performed in Graphpad Prism software using one-way ANOVA with Bonferroni correction.
Reagents
IG274
frIs7[nlp-29p::GFP, col-12p::DsRed] IV
(Pujol
et al.
, 2008)
IG1825
atfs-1(gk3094) V; frIs7[nlp-29p::GFP, col-12p::DsRed] IV
(This study)
SJ4100
zcIs13[hsp-6::GFP] V
(Yoneda
et al.
, 2004)
MD4323
bcSi81[atfs-1(NcATP9MTS)] II; unc-119(ed3) III; atfs-1(tm4525) zcIs9[hsp-60p::GFP] V
(Rolland
et al.
, 2019)
VC3201
atfs-1(gk3094) V (C. elegans
Deletion Mutant Consortium, 2012)
spg-7
RNAi clone sjj_Y47G6A_247.f
sta-1
RNAi clone sjj_Y51H4A.o
Acknowledgments
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Funding
This work was funded by the French National Research Agency (ANR-16-CE15-0001-01, ANR-16-CONV-0001) and institutional grants from CNRS, Aix Marseille University, National institute of Health and Medical Research (Inserm) to the CIML.
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