Inhibition of the UFD-1-NPL-4 complex reduces survival of C. elegans on P. aeruginosa

(A) Representative survival plots of N2 animals on P. aeruginosa PA14 at 25°C after treatment with the empty vector (EV) control, ufd-1, and npl-4 RNAi. P<0.001 for ufd-1 and npl-4 RNAi compared to EV control.

(B) Representative survival plots of N2 animals grown on bacteria for RNAi against ufd-1 and npl-4 along with the EV control at 20°C. Day 0 represents young adults. P<0.001 for ufd-1 and npl-4 RNAi compared to EV control.

(C) Representative fluorescence (top) and the corresponding brightfield (bottom) images of N2 animals incubated on P. aeruginosa-GFP for 24 hours at 25°C after growth on the EV control, ufd-1, and npl-4 RNAi bacteria. Scale bar = 200 μm.

(D) Quantification of GFP levels of N2 animals incubated on P. aeruginosa-GFP for 24 hours at 25°C after growth on the EV control, ufd-1, and npl-4 RNAi bacteria. ***P < 0.001 via the t-test (n = 16 worms each).

(E) Colony-forming units (CFU) per animal of N2 worms incubated on P. aeruginosa-GFP for 24 hours at 25°C after growth on the EV control, ufd-1, and npl-4 RNAi bacteria. **P < 0.01 via the t-test (n = 6 biological replicates).

Reduced colonization with P. aeruginosa upon ufd-1 knockdown is independent of the ER-UPR pathways

(A) Representative survival plots of xbp-1(tm2482) animals on P. aeruginosa PA14 at 25°C after treatment with the empty vector (EV) control and ufd-1 RNAi. The difference between the EV and ufd-1 RNAi survival plots is nonsignificant.

(B) Representative fluorescence images of xbp-1(tm2482) animals incubated on P. aeruginosa-GFP for 24 hours at 25°C after growth on the EV control and ufd-1 RNAi bacteria. Scale bar = 200 μm.

(C) Quantification of GFP levels of xbp-1(tm2482) animals incubated on P. aeruginosa-GFP for 24 hours at 25°C after growth on the EV control and ufd-1 RNAi bacteria. **P < 0.01 via the t-test (n = 16 worms each).

(D) Representative survival plots of atf-6(ok551) animals on P. aeruginosa PA14 at 25°C after treatment with the EV control and ufd-1 RNAi. P<0.001.

(E) Representative fluorescence images of atf-6(ok551) animals incubated on P. aeruginosa-GFP for 24 hours at 25°C after growth on the EV control and ufd-1 RNAi bacteria. Scale bar = 200 μm.

(F) Quantification of GFP levels of atf-6(ok551) animals incubated on P. aeruginosa-GFP for 24 hours at 25°C after growth on the EV control and ufd-1 RNAi bacteria. ***P < 0.001 via the t-test (n = 16 worms each).

(G) Representative survival plots of pek-1(ok275) animals on P. aeruginosa PA14 at 25°C after treatment with the EV control and ufd-1 RNAi. P<0.001.

(H) Representative fluorescence images of pek-1(ok275) animals incubated on P. aeruginosa-GFP for 24 hours at 25°C after growth on the EV control and ufd-1 RNAi bacteria. Scale bar = 200 μm.

(I) Quantification of GFP levels of pek-1(ok275) animals incubated on P. aeruginosa-GFP for 24 hours at 25°C after growth on the EV control and ufd-1 RNAi bacteria. ***P < 0.001 via the t-test (n = 24–25 worms each).

Reduced colonization with P. aeruginosa upon ufd-1 knockdown is independent of the major immunity pathways

(A) Representative survival plots of pmk-1(km25) animals on P. aeruginosa PA14 at 25°C after treatment with the empty vector (EV) control and ufd-1 RNAi. The difference between the EV and ufd-1 RNAi survival plots is nonsignificant.

(B) Representative fluorescence images of pmk-1(km25) animals incubated on P. aeruginosa-GFP for 24 hours at 25°C after growth on the EV control and ufd-1 RNAi bacteria. Scale bar = 200 μm.

(C) Quantification of GFP levels of pmk-1(km25) animals incubated on P. aeruginosa-GFP for 24 hours at 25°C after growth on the EV control and ufd-1 RNAi bacteria. ***P < 0.001 via the t-test (n = 16 worms each).

(D) Representative survival plots of dbl-1(nk3) animals on P. aeruginosa PA14 at 25°C after treatment with the EV control and ufd-1 RNAi. The difference between the EV and ufd-1 RNAi survival plots is nonsignificant.

(E) Representative fluorescence images of dbl-1(nk3) animals incubated on P. aeruginosa-GFP for 24 hours at 25°C after growth on the EV control and ufd-1 RNAi bacteria. Scale bar = 200 μm.

(F) Quantification of GFP levels of dbl-1(nk3) animals incubated on P. aeruginosa-GFP for 24 hours at 25°C after growth on the EV control and ufd-1 RNAi bacteria. ***P < 0.001 via the t-test (n = 16 worms each).

(G) Representative survival plots of hlh-30(tm1978) animals on P. aeruginosa PA14 at 25°C after treatment with the EV control and ufd-1 RNAi. P<0.001.

(H) Representative fluorescence images of hlh-30(tm1978) animals incubated on P. aeruginosa-GFP for 24 hours at 25°C after growth on the EV control and ufd-1 RNAi bacteria. Scale bar = 200 μm.

(I) Quantification of GFP levels of hlh-30(tm1978) animals incubated on P. aeruginosa-GFP for 24 hours at 25°C after growth on the EV control and ufd-1 RNAi bacteria. ***P < 0.001 via the t-test (n = 16 worms each).

Inhibition of the UFD-1-NPL-4 complex improves survival of severely immunocompromised C. elegans on P. aeruginosa

(A) Representative survival plots of sek-1(km4) animals on P. aeruginosa PA14 at 25°C after treatment with the empty vector (EV) control, ufd-1, and npl-4 RNAi. P<0.001 for ufd-1 and npl-4 RNAi compared to EV control.

(B) Representative fluorescence images of sek-1(km4) animals incubated on P. aeruginosa-GFP for 12 hours at 25°C after growth on the EV control, ufd-1, and npl-4 RNAi bacteria. Scale bar = 200 μm.

(C) Quantification of GFP levels of sek-1(km4) animals incubated on P. aeruginosa-GFP for 12 hours at 25°C after growth on the EV control, ufd-1, and npl-4 RNAi bacteria. ***P < 0.001 via the t-test (n = 19–20 worms each).

(D) Representative survival plots of sek-1(km4) animals grown on bacteria for RNAi against ufd-1 and npl-4 along with the EV control at 20°C. Day 0 represents young adults. P<0.001 for ufd-1 and npl-4 RNAi compared to EV control.

(E) Representative survival plots of dbl-1(nk3);pmk-1(km25) animals on P. aeruginosa PA14 at 25°C after treatment with the EV control and ufd-1 RNAi. P<0.001.

(F) Representative fluorescence images of dbl-1(nk3);pmk-1(km25) animals incubated on P. aeruginosa-GFP for 12 hours at 25°C after growth on the EV control and ufd-1 RNAi bacteria. Scale bar = 200 μm.

(G) Quantification of GFP levels of dbl-1(nk3);pmk-1(km25) animals incubated on P. aeruginosa-GFP for 12 hours at 25°C after growth on the EV control and ufd-1 RNAi bacteria. ***P < 0.001 via the t-test (n = 24 worms each).

Knockdown of ufd-1 results in the upregulation of protease and intracellular pathogen response genes

(A) Volcano plot of upregulated and downregulated genes in ufd-1 RNAi versus empty vector (EV) control RNAi N2 animals. Orange and green dots represent significantly upregulated and downregulated genes, respectively, while the grey dots represent the genes that are not differentially regulated

(B)-(D) Gene ontology enrichment analysis for ufd-1 RNAi upregulated genes for biological processes (B), cellular component (C), and molecular function (D).

(E) Venn diagram showing the overlap between genes upregulated upon ufd-1 RNAi and upregulated upon Nematocida parisii infection (Bakowski et al., 2014). The P-value for the overlap between the data is 8.2×10-110.

(F) Venn diagram showing the overlap between genes upregulated upon ufd-1 RNAi and upregulated upon Orsay virus infection (Sarkies et al., 2013). The P-value for the overlap between the data is 2.7×10-62.

GATA transcription factor ELT-2 mediates the ufd-1 knockdown phenotypes

(A) Venn diagram showing the overlap between genes upregulated upon ufd-1 RNAi and upregulated in wt versus elt-2(-) larvae (Dineen et al., 2018). The P-value for the overlap between the data is 9.5×10-52.

(B) Venn diagram showing the overlap between genes upregulated upon ufd-1 RNAi and the low-complexity ELT-2 target genes (Mann et al., 2016). The P-value for the overlap between the data is 1.5×10-34.

(C) Representative fluorescence (top) and the corresponding brightfield (bottom) images of N2 animals incubated on P. aeruginosa-GFP for 24 hours at 25°C after growth on the empty vector (EV) control, ufd-1, EV+elt-2, EV+ufd-1, and ufd-1+elt-2 RNAi bacteria (see Methods for the details). Scale bar = 200 μm.

(D) Quantification of GFP levels of N2 animals incubated on P. aeruginosa-GFP for 24 hours at 25°C after growth on the EV control, ufd-1, EV+elt-2, EV+ufd-1, and ufd-1+elt-2 RNAi bacteria. ***P < 0.001 via the t-test (n = 20 worms each).

(E) Representative fluorescence (top) and the corresponding brightfield (bottom) images of sek-1(km4) animals incubated on P. aeruginosa-GFP for 12 hours at 25°C after growth on the EV control, ufd-1, EV+elt-2, EV+ufd-1, and ufd-1+elt-2 RNAi bacteria. Scale bar = 200 μm.

(F) Quantification of GFP levels of sek-1(km4) animals incubated on P. aeruginosa-GFP for 12 hours at 25°C after growth on the EV control, ufd-1, EV+elt-2, EV+ufd-1, and ufd-1+elt-2 RNAi bacteria. ***P < 0.001 and **P < 0.01via the t-test (n = 16–19 worms each).

(G) Representative survival plots of sek-1(km4) animals on P. aeruginosa PA14 at 25°C after treatment with the EV control, ufd-1, EV+elt-2, EV+ufd-1, and ufd-1+elt-2 RNAi bacteria. P<0.001 for ufd-1+elt-2 RNAi compared to EV+ufd-1 RNAi.

Inhibition of the UFD-1-NPL-4 complex does not affect the pharyngeal pumping rate

(A) Pharyngeal pumps per 30 sec of N2 animals grown on ufd-1 and npl-4 RNAi along with the empty vector (EV) control RNAi. *P < 0.05 via the t-test. n.s., nonsignificant (n = 45 worms each).

(B) Pharyngeal pumps per 30 sec of N2 animals grown on EV control, ufd-1, and npl-4 RNAi followed by incubation on P. aeruginosa PA14 at 25°C for 12 hours. ***P < 0.001 and **P < 0.01 via the t-test (n = 30 worms each).

Reduced gut colonization upon ufd-1 knockdown is independent of the tir-1 immunity pathway

(A) Representative survival plots of tir-1(qd4) animals on P. aeruginosa PA14 at 25°C after treatment with the empty vector (EV) control and ufd-1 RNAi. The difference between the EV and ufd-1 RNAi survival plots is nonsignificant.

(B) Representative fluorescence images of tir-1(qd4) animals incubated on P. aeruginosa-GFP for 24 hours at 25°C after growth on the EV control and ufd-1 RNAi bacteria. Scale bar = 200 μm.

(C) Quantification of GFP levels of tir-1(qd4) animals incubated on P. aeruginosa-GFP for 24 hours at 25°C after growth on the EV control and ufd-1 RNAi bacteria. ***P < 0.001 via the t-test (n = 20 worms each).

Knockdown of ufd-1 dictates survival of different worm strains on P. aeruginosa

(A) Representative survival plots of worm strains on P. aeruginosa PA14 at 25°C after treatment with the empty vector (EV) control RNAi.

(B) Representative survival plots of worm strains on P. aeruginosa PA14 at 25°C after treatment with ufd-1 RNAi.

The data in (A) and (B) is pooled from the data from Figures 1-3 and Figure S2.

Gene ontology enrichment analysis for ufd-1 RNAi downregulated gene

(A)-(C) Gene ontology enrichment analysis for ufd-1 RNAi downregulated genes for biological processes (A), cellular component (B), and molecular function (C).

Individual protease or intracellular pathogen response genes are not involved in reduced colonization of ufd-1 RNAi animals

(A) Quantification of GFP levels of N2 animals incubated on P. aeruginosa-GFP for 24 hours at 25°C after growth on indicated RNAi bacteria. P-values in comparison to EV and EV+ufd-1 RNAi are indicated. ***P < 0.001 and **P < 0.01 via the t-test. n.s., nonsignificant (n = 18–19 worms each).

(B) Quantification of GFP levels of N2 animals incubated on P. aeruginosa-GFP for 24 hours at 25°C after growth on indicated RNAi bacteria. P-values in comparison to EV are shown. P-values for all the co-RNAi treatments in comparison to EV+ufd-1 RNAi are nonsignificant. n = 19–20 worms each.