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Infection and Immune Response in the Nematode Caenorhabditis elegans Elicited by the Phytopathogen Xanthomonas
Infection and Immune Response in the Nematode Caenorhabditis elegans Elicited by the Phytopathogen Xanthomonas
Journal of Microbiology and Biotechnology. 2014. Sep, 24(9): 1269-1279
Copyright © 2014, The Korean Society For Microbiology And Biotechnology
  • Received : January 09, 2014
  • Accepted : May 18, 2014
  • Published : September 28, 2014
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About the Authors
Yanli Bai
Gansu Key Laboratory of Biomonitoring and Bioremediation for Environmental Pollution, School of Life Sciences, Lanzhou University, Lanzhou 730000, P. R. China
Dejuan Zhi
School of Pharmacy, Lanzhou University, Lanzhou 730000, P. R. China
Chanhe Li
Gansu Key Laboratory of Biomonitoring and Bioremediation for Environmental Pollution, School of Life Sciences, Lanzhou University, Lanzhou 730000, P. R. China
Dongling Liu
Gansu Key Laboratory of Biomonitoring and Bioremediation for Environmental Pollution, School of Life Sciences, Lanzhou University, Lanzhou 730000, P. R. China
Juan Zhang
Gansu Key Laboratory of Biomonitoring and Bioremediation for Environmental Pollution, School of Life Sciences, Lanzhou University, Lanzhou 730000, P. R. China
Jing Tian
School of Pharmacy, Lanzhou University, Lanzhou 730000, P. R. China
Xin Wang
School of Pharmacy, Lanzhou University, Lanzhou 730000, P. R. China
Hui Ren
School of Pharmacy, Lanzhou University, Lanzhou 730000, P. R. China
Hongyu Li
School of Pharmacy, Lanzhou University, Lanzhou 730000, P. R. China

Abstract
Xanthomonas oryzae pv. oryzae ( Xoo ) strains are plant pathogenic bacteria that can cause serious blight of rice, and their virulence towards plant host is complex, making it difficult to be elucidated. Caenorhabditis elegans has been used as a powerful model organism to simplify the host and pathogen system. However, whether the C. elegans is feasible for studying plant pathogens such as Xoo has not been explored. In the present work, we report that Xoo strains PXO99 and JXOIII reduce the lifespan of worms not through acute toxicity, but in an infectious manner; pathogens proliferate and persist in the intestinal lumen to cause marked anterior intestine distension. In addition, Xoo triggers (i) the p38 MAPK signal pathway to upregulate its downstream C17H12.8 expression, and (ii) the DAF-2/DAF-16 pathway to upregulate its downstream gene expressions of mtl-1 and sod-3 under the condition of daf-2 mutation. Our findings suggest that C. elegans can be used as a model to evaluate the virulence of Xoo phytopathogens to host.
Keywords
Introduction
Caenorhabditis elegans has been used as a favorable model organism to study the innate immunity of humans or other animal hosts, because the conserved host defense mechanisms are aroused when challenged by pathogenic bacteria [20 , 27] . In its natural habitat, C. elegans is confronted with the danger of diverse pathogens frequently, which could induce the host antimicrobial mechanism to alleviate or eliminate the infection effectively. Previous works have demonstrated that C. elegans can be infected by many kinds of pathogenic microbes, and investigated the virulence of pathogens and the worm host defense response [10 , 31] . When infection occurs, pathogenic bacteria colonize in the C. elegans intestine and lead to intestinal distention, which in turn affects the normal physiological function and immunity, and ultimately causes the death of infected worms [2 , 19] . To counter the infection, worm hosts employ specific signal transduction pathways such as p38 MAPK and DAF-2/DAF-16 to regulate the expressions of downstream defense effectors that allow hosts to survive [29 , 33] .
The DAF-2/DAF-16 insulin signaling pathway is involved in mediating the innate immune response in C. elegans [22] . The FOXO transcriptional factor homolog DAF-16, located downstream of DAF-2, is crucial for pathogen resistance [23] . Under normal physiological condition, DAF-2 induces cytosolic DAF-16 phosphorylation to keep it sequestered in the cytoplasm for maintaining the normal lifespan of the worm [3] . On the other hand, the inactivation of DAF-2 can induce DAF-16 nuclear translocation and thus initiate expression of immune-related genes for defending against pathogen infection and lengthening the worm lifespan [4] . Several immune-related genes that act downstream of DAF-16 have been identified, including the metallothionein homologu mtl-1 and the mitochondrial superoxide dismutase gene sod-3 , both of which contribute to robust immunity [25 , 28 , 32] .
The PMK-1 p38 mitogen-activated protein kinase pathway participates in the response to diverse physiological stimuli, especially pathogenic attack by intestinal infection [36] . This NSY-1-SEK-1-PMK-1 MAPK pathway plays an important role in protecting worm hosts against the infection of Yersinia pestis [6] , Salmonella enterica [1] , Enterococcus faecalis [14] , and other gram-positive and gram-negative pathogens. The survival rate of sek-1(km4) worms was notably decreased after P. aeruginosa infection, indicating that loss-of-function mutations in sek-1 enhanced susceptibility to the pathogen [21] . C17H12.8 locates downstream of the p38 MAPK pathway and acts as an antimicrobial effector, and its mRNA level can be significantly upregulated in response to infection [39] .
Xanthomonas oryzae pv. oryzae ( Xoo ) is a bacterial plant pathogen that causes serious blight of rice, which decreases the yield of rice sharply in most of the rice growing countries [17] . The exploitation of resistant cultivars is the only reliable method to battle against this destructive disease [34] . Thus, it becomes important and urgent to elucidate Xoo virulence and the host resistance mechanism. However, Xoo virulence is extremely complex [36] and efforts are urgently needed to explore this opening issue with a simpler, versatile, and time-saving model organism.
Previous work has proved that Xoo could kill worms faster than the control Escherichia coli OP50 [7] . Here we provide evidence that Xoo causes an infectious impact. It has been shown that Xoo strain PXO99 is more virulent among the Philippine race for rice [12] . Compared with Japonic race JXOIII, PXO99 exhibits stronger virulent activity in rice lines and causes serious lesion in the leaf [41] . Hence, both PXO99 and JXOIII strains were selected for the killing assay to test whether similar virulence in rice could be ranked in C. elegans . Pathogenic bacteria proliferation in the intestinal lumen was detected. Since the p38 MAPK and DAF-2/DAF-16 signal pathways participate in the anti-infection of C. elegans , we examined whether these two signal pathways are required in response to Xoo infection. Finally, whether expressions of immune-related genes are induced was determined. Our results suggest that C. elegans can serve as a useful model host to evaluate the virulence of Xoo phytopathogens.
Materials and Methods
- Bacteria and Nematode Strains
Wild-type Bristol N2; CB1370, daf-2(e1370)III ; CF1295, daf-16(mu86)I ; daf-2(e1370)III ; muEx108 ; TJ356, daf-16::GFP ( zIs356 IV ); and CF1038, daf-16(mu86)I were maintained on NGM (nematode growth medium) supplied with the standard food source E. coli OP50. All the strains were provided by the Caenorhabditis Genetics Center (CGC). KU4, sek-1(km4) X , was kindly gifted from the Ausubel Laboratory of the Department of Molecular Biology, Massachusetts General Hospital.
Xoo strain Philippine race PXO99 and Japonic race JXOIII were kindly supplied by Prof. J.S. Wang, Key Laboratory of Monitoring and Management of Plant Diseases and Pests, Ministry of Agriculture, Department of Plant Protection, Nanjing Agricultural University, China [24] . Strains were cultured by inoculating a single colony in nutrient agar (NA) liquid medium (0.5% peptone, 0.3% beef extract, 1% glucose, 0.1% yeast extract) until the logarithmic phase at 28℃.
- C. elegans Killing Assay
Plates for the killing assay were prepared as follows. First, 50 μl of bacteria cultures was spread onto NGM plates, and the plates were incubated at 28℃ for 24 h and then equilibrated to room temperature before assay. Worms were synchronized using alkaline hypochlorite and cultured at proper growth temperature until the L4 stage larvae [5] . Then the worms were washed twice in M9 buffer and transferred to assay plates. After 16 h of treatment, at least 25 worms were picked to fresh NGM plates supplied with OP50, and the death and total animals were scored every day. A worm was scored as “death” when it failed to respond to a gentle touch with a platinum wire. Nematodes that crawled away from the NGM plates were excluded from the assay. At the first three to seven days, live worms were transferred to fresh NGM plates every 48h to exclude the newborn larvae. Each assay was performed with three parallel plates in at least two independent trails. Assays were performed at 20℃. CB1370 ( daf-2 ) was maintained at 15℃ to exclude the dauer arrest phenotype. After the young adult stage, worms were moved back to 20℃ for assays in order to compare among different strains under the same temperature condition.
- Brood Size
Brood size is a good indicator of diet quality in C. elegans [35] . The worms were treated with PXO99 and JXOIII for 16 h and then transferred to OP50 plates. Brood sizes were determined as previously described [15] . Total progenies (unhatched eggs and larvae) were counted. This continued for about one week until adult worms no longer laid eggs.
- Virulence of Bacterial Metabolism Products on C. elegans
To confirm whether diffusible toxins were present during the killing processes, bacteria were spread to NA agar plates covered by a sterile 0.22 μm nitrocellulose (NC) filter. The NA medium was modified by the addition of 0.15 M sorbitol [26] to satisfy the osmolarity requirement for fast killing. After being incubated at 28℃ for 24 h, the plates were cooled to room temperature and the filter membranes were removed, 20-30 worms (L4 stage larvae) were transferred to the plates, and the death and total worms were counted at 6, 12, and 24 h. Each assay was carried out in triplicate and the mean death rate was computed.
- C. elegans Infected by Xoo Is Examined Under Differential Interference Contrast (DIC) Microscopy
C. elegans infected by pathogen was characterized by anterior intestine distension [14] . Whether there is intestine distension after treating animals with Xoo was tested in the present work. Wild-type N2 was treated for 16 h as described above in the killing assay and transferred to fresh NGM plates; OP50-treated worms were employed as the control. Sodium azide was used to anesthetize the worms. Intestinal tracts were examined at 1, 5, and 7 days using DIC microscopy. Considering that Enterococcus faecalis infected C. elegans and the killing was not dependent on secreted toxins [14] , worms infected by E. faecalis were used as the positive control.
- C. elegans Bacterial CFU Analysis
The colony-forming unit (CFU) analysis was modified from a previous work [30] . Briefly, wild-type N2 was exposed to PXO99 and JXOIII in the same conditions as the killing assay. At 1, 3, 5, and 7 days, infected worms were washed thoroughly for five times with M9 buffer containing 1 mM sodium azide, and then a 50 μl suspension was plated to determine the external CFU. Ten worms were transferred to a tissue grinder containing 150 μl of 10 mM MgSO 4 , and the worms were disrupted until no intact worms could be observed under a dissection microscope. The resultant suspension was diluted with 10 mM MgSO 4 and plated onto NA plates. Colonies were counted to determine the CFU per worm a fter overnight incubation a t 28 ℃ for PXO99 and JXOIII, and at 37℃ for E. coli OP50.
- Quantitative RT-PCR Analyses
Nematodes were treated as described for the killing assays above. After 16 h, the worms were washed three times in M9 buffer to exclude the interference of bacterial RNA. Then the total RNA was extracted using RNAiso Plus Reagent (TaKaRa), and cDNA was generated using the Primescript RT reagent kit with gDNA Eraser (TaKaRa). The qRT-PCR was carried out with the SYBR Green methodology using SYBR Premix Ex Taq II (TaKaRa) and performed on a BIO-RAD S1000 Thermal Cycler. Cycle threshold (Ct) values were normalized against the control gene act-3 . All the tests were repeated at least twice, and each replicate was measured in triplicate. Fold change was calculated using the 2 -ΔΔCt method.
Primers of mtl-1 , sod-3 , and C17H12.8 were graciously provided by Troemol [39] . Other primers were designed using Primer Premier 5.0, and the amplification specificity against the C. elegans genome was checked and the efficiency was tested with a dilution series of template. All the primers used in our research are listed in Table 3 .
- DAF-16 Nuclear Localization
TJ356, daf-16::GFP ( zIs356 IV ) were treated as for the N2 described for killing assays. At 8 and 16 h, worms were observed under a fluorescence microscope (Nikon) to determine whether the transcriptional factor DAF-16 had translocated to the nucleus. Worms treated by E. faecalis were used as the positive control.
- Immunoblot Analysis
To prepare total protein lysates for western blotting, worms were harvested using M9 buffer as described above for RNA isolation. After being frozen with liquid nitrogen, samples were added to the same volume of SDS buffer (4% SDS, 100 mM Tris-HCl, pH 6.8, and 20% glycerol) and boiled for 15 min by vortexing the samples once after 7-8 min. Supernatants were collected and total protein concentrations were measured using a BCA Protein Assay Kit (TaKaRa). Each sample was analyzed using three different antisera: anti-phospho-p38 (Thr 180 /Tyr 182 ) antibody (Cell Signaling Technology), anti-p38 MAPK antibody (Cell Signaling Technology), and anti-GAPDH antibody (Sigma). For the quantification of PMK-1 activity, the band intensity of phospho-p38 blots was normalized using the anti-GAPDH blots and quantified using ImageJ software.
- Statistical Analysis
SPSS ver. 17.0 was used for data processing to calculate the TD 50 (time at which half of the population is dead) by the Kaplan-Meier method, and the log-rank test was adopted for statistical analysis. RT-PCR statistical tests were calculated from OP50-normalized cycle threshold values prior to conversion to relative fold changes. The normalized values for induction expression for the three replicates were compared using a 1-sample t-test. ImageJ was employed to measure the width of intestinal region after infection by Xoo and E. faecalis . Mean values were obtained from at least 10 individuals in each group. Immunoblot data were also processed by ImageJ software for quantification of PMK-1 activity.
Results
- Xoo Kills C. elegans
A wide variety of bacterial pathogens kill C. elegans by drastically shortening the lifespan of the worms [9] . We therefore first tested the effect of Xoo strains PXO99 and JXOIII on the lifespan of nematodes by replacing E. coli OP50 with these phytopathogenic bacteria as food supply. The results showed that the lifespan of N2 fed with PXO99 and JXOIII significantly decreased compared with the control ( Fig. 1 A).
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Kaplan-Meier survival plots of C. elegans mutant strains and wild-type N2. L4 worms were transferred on PXO99 and JXOIII for 16 h, and then transferred back to E. coli OP50; nematodes fed on E. coli OP50 acted as controls. (A) N2 surviving fraction. (B) KU4, sek-1(km4)X surviving fraction. (C) CB1370, daf-2(e1370)III surviving fraction. (D) CF1295, daf-16(mu86)I; daf-2(e1370)III surviving fraction. (E) CF1038, daf-16(mu86)I surviving fraction. PXO99 group (dark gray solid line), JXOIII group (black dotted line), OP50 (black solid line). Graphed: ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
We suspected that the shortened lifespan is due to starvation, so we evaluated the food nutrition by index of brood size. In contrast to the control g roup fed on OP50, the brood size of wild-type N2 that ingested PXO99 was significantly reduced, and only tended to decrease when fed on JXOIII ( Fig. 2 ). It seemed that PXO99 was not a proper food resource and JXOIII was a less nutritional food resource than OP50. When wild-type N2 worms were fed with heat-killed PXO99 and JXOIII, they exhibited the normal lifespan as that fed with E. coli OP50 (data not shown), indicating that the dead PXO99 and JXOIII contain the same nutrition level as E. coli OP50. We therefore concluded that it is most likely that live Xoo kills the nematode C. elegans in a manner of infection.
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PXO99 and JXOIII decreased the total brood size in wild-type N2. All values are represented by means ± SD of three replicates, n = 15. Significant differences compared with the control group OP50 are indicated by asterisks (**p < 0.01).
- Xoo Proliferates and Establishes a Persistent Infection in the Intestine of C. elegans
Pathologic change is often characterized by anterior intestine distension after infection by pathogens such as E. faecalis in C. elegans [14] . Similar to E. faecalis , significant anterior intestine distentions were also clearly observed after infection by PXO99 and JXOIII for 1, 5, and 7 days ( Figs. 3 and 4 ). CFU analysis showed that PXO99 and JXOIII could escape the pharyngeal grinding and proliferate in the C. elegans intestine ( Fig. 5 ). No visible bacterial clone was observed after spreading the supernatant liquid on NA plates just before crashing the worms ( Fig. 5 ), suggesting that the CFU results can exclude the interference from nematodes having been washed incompletely.
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Differential interference contrast images showing anterior intestine distention after infection by PXO99 and JXOIII for 1, 5, and 7 days. Nematodes fed on E. coli OP50 served as the negative control and E. faecalis as the positive control. Red arrows show the pharyngeal grinder organ, and yellow arrows point to the intestinal lumen.
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Quantitative result of intestine distention after infection by Xoo and E. faecalis. At least 10 samples were measured in each group. All values are represented by means ± SD. Significant differences compared with the control group are indicated by asterisks (**p < 0.01).
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Quantification of live PXO99, JXOIII, and E. coli OP50 in nematode intestine. CFU per worm was determined after exposure to PXO99 and JXOIII for 16 h, and then transfer to E. coli OP50 for 1, 3, 5, and 7 days. Each symbol represents the average of 10 worms. Error bars equal standard deviation (**p < 0.01).
It can be noted that the intestine distention was also observed when fed with E. coli OP50 until 5 days, and that the intestine proliferation increased with aging of the worms in our experiment ( Figs. 3 and 5 ). This result identified with previous research [12] , and it can be explained by that immunosenescence exists in C. elegans [40] .
- Xoo Killing C. elegans Is Independent of Bacterial Metabolic Products
Pathogenic effects in C. elegans are usually classified into slow-killing dependent on live microbes and fast-killing based on secreted microbial products [9] . To determine whether Xoo can kill worms by a fast-killing way, the acute toxicity of PXO99 and JXOIII was also tested in our experiment. The results summarized in Table 2 show that dead worms were less than 2% in all treatments after 24 h. Thus Xoo barely had acute toxicity on wild-type N2 or mutant strains, suggesting that PXO99 and JXOIII reduced the lifespan of C. elegans via a toxicity-independent mechanism.
Effect ofXoostrains PXO99 and JXOIII on the lifespan ofC. elegans.
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ap-value (log rank test) compared with control group of OP50.
Death rate ofC. eleganstreated with PXO99 and JXOIII metabolic products.
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Death rate of C. elegans treated with PXO99 and JXOIII metabolic products.
Because secreted bacterial products may diffuse slowly in solid plate, we also used a liquid assay by 96-well plate to address this issue. Similar results were obtained, in that no significant dead N2 worms were found ( Fig. 8 ).
- p38 MAPK Signal Pathway Is Required in Defending Against Xoo Infection
sek-1 , an essential gene in the p38 MAPK pathway, mediates the nematodes to defend against pathogenic bacteria, and mutation of sek-1 compromises the host immunity [18] . Our results showed that the sek-1 mutant displayed much more susceptibility to both PXO99 ( p < 0.0001) and JXOIII ( p < 0.0001) than that in N2 ( Fig. 1 B and Table 1 ). Unlike the sek-1 mutant, PMK-1 downstream of sek-1 was significantly activated by PXO99 and JXOIII in the N2 worms ( Fig. 9 ). The CUB-like gene C17H12.8 locates downstream of the p38 MAPK pathway, and is regulated by sek-1 [24] . As expected, C17H12.8 expression was dramatically increased in wild-type N2, and was completely suppressed in sek-1 mutant strain KU4 ( Fig. 6 D).
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qRT-PCR results in wild-type N2 and mutant strains after treatment with Xoo for 16 h. (A and B) mtl-1 and sod-3 transcript levels in CB1370 (daf-2); CF1295 (daf-2; daf-16); and KU4 (sek-1); the two genes are upregulated by daf-16. (C) sek-1 fold change in CB1370 (daf-2) and CF1295 (daf-2; daf-16). (D) CUB-like gene C17H12.8, regulated by the p38 MAPK pathway, was measured in CB1370 (daf-2), CF1295 (daf-2; daf-16), and KU4 (sek-1). (E) Expression level of daf-2 and daf-16 in mutant sek-1 background, with N2 as control. Results are obtained from two independent biological replicates and normalized to the control gene act-3. Error bars represent SD. *t-test, p < 0.05 in comparison with N2 fed on OP50.
- DAF-2/DAF-16 Signal Pathway Is Required in Defending Against Xoo Infection Under daf-2 Mutation
CB1370 is a mutant strain in daf-2 , and CF1295 is a mutant strain in daf-2 and daf-16 . The killing assay results showed that the lifespan of CB1370 was only slightly shortened by treating with PXO99, but not by JXOIII. In contrast, the lifespan of CF1295 was significantly shortened by treating with either PXO99 or JXOIII. Thus, the mutation in daf-2 appeared to render worms more resistant to Xoo infection than that in N2, and the double mutation in daf-2 and daf-16 appeared to abrogate this effect ( Figs. 1 C and 1 D and Table 1 ). Furthermore, CF1038 ( daf-16 ) was used to test whether DAF-16 is always required in a daf-2 wild-type background. A negative result is shown in Fig. 1 E and Table 1 , suggesting that daf-16 was only required for mutant daf-2 nematodes to resist Xoo challenges.
Sequence information for primers used in qRT-PCR.
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Sequence information for primers used in qRT-PCR.
The DAF-16 intracellular location was easily observed under fluorescence microscopy after fusing GFP as a reporter gene to the daf-16 gene in worm strain TJ356. In normal condition, inactive DAF-16 in the cytosol will exhibit a diffuse homogenous fluorescence, and active DAF-16 will transfer to the nucleus in a form of dotted fluorescence [15] .
To further verify whether DAF-16 is involved in C. elegans defense responses to PXO99 and JXOIII under wild-type daf-2 context, TJ356 was used in the present work to test the nuclear translocation of DAF-16. After being exposed to PXO99 and JXOIII for 8 and 16 h, DAF-16 nuclear translocation did not occur, but it did happen in the positive control E. faecalis group ( Fig. 7 ). We also tested whether DAF-16 translocated into the nucleus after being treated with PXO99 and JXOIII for 24 h. No positive result was observed (data not shown).
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DAF-16 nuclear localization by DAF-16::GFP fluorescent image analysis. TJ356, daf-16::GFP (zIs356 IV) was exposed to PXO99 and JXOIII for 8 and 16 h, and nuclear translocation did not occur. E. coli OP50 and E. faecalis served as the negative and positive control, respectively.
MTL-1 and SOD-3 serve as defensive effectors downstream of the insulin-like pathway and arm worms with an immune response to the pathogenic bacteria [39] . In the present work, mtl-1 and sod-3 expressions were significantly induced in CB1370 ( daf-2 ) when treated with PXO99 and JXOIII ( p < 0.05) ( Figs. 6 A and 6 B). In contrast, such elevation was abolished in daf-2/daf-16 double-mutant worms ( p < 0.05) ( Figs. 6 A and 6 B).
Discussion
C. elegans can be infected by phytopathogens of Erwinia chrysanthemi , Erwinia carotovora subsp. carotovora , and Agrobacterium tumefaciens [8] . In the present work, our results revealed that PXO99 and JXOIII could kill C. elegans in a slow-killing manner dependent on proliferation in the intestine rather than the acute toxicity of bacterial extracellular products. Moreover, inactive bacteria after treatment at 60℃ for 1 h failed to kill the host (data not shown), indicating that live bacteria were indispensable in the process of infection. Additionally, PXO99 exhibited much stronger virulence to C. elegans than JXOIII from the results of the killing assay, consistent with the virulent ranking of those two bacterial strains in rice lines [41] . These observations suggested that C. elegans can serve as a useful host model for these phytopathogens.
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Liquid assay of fast-killing by bacterial metabolic products. Xoo did not kill C. elegans during the infection process.
Similar to E. faecalis [14] , PXO99 and JXOIII could also survive the digestion and proliferate in the intestine lumen ( Fig. 5 ), which then led to the anterior intestine distension ( Figs. 3 and 4 ). Both bacterial strains significantly shortened the worm lifespan as compared with the control group fed on E. coli OP50. The difference is that the CFU for PXO99 and JXOIII reached, respectively, 10 6 and 10 3 in the host intestine ( Fig. 5 ), with a TD 50 of 9.6 and 12.5 days. In contrast, the CFU and TD 50 for E. faecalis treated worms were, respectively, 10 4 and 5.0 days [14] . Other research work on the identification of traditional Chinese medicine to promote innate immunity of C. elegans against E. faecalis in our lab gave a TD 50 of 9.1 days (personal communication). As more Xoo cells accumulated in the intestine, but did not result in faster killing, our results suggested that PXO99 and JXOIII are less virulent to nematodes than E. faecalis . This could be due to differences in virulent factors such as extracellular enzymes or extracellular polysaccharides used by these plant pathogens [36] .
Previous work has reported that both the PMK-1 p38 MAPK pathway and DAF-2/DAF-16 pathway are activated in C. elegans during infection by pathogenic bacteria, which then confer pathogenic resistances via downstream genes involved in host survival [38] . In this study, we found that the p38 MAPK pathway specifically participated in the innate immunity response to Xoo pathogens. With regard to the DAF-2/DAF-16 pathway, we found that DAF-16 functioned only under the daf-2 mutant background, but not under the wild-type daf-2 background. Thus, we noted that expressions of sek-1 and C17H12.8 were not induced in CB1370 ( daf-2 ), but were significantly elevated in CF1295 ( daf-2 ; daf-16 ) after infection by Xoo ( Figs. 6 C and 6 D). These findings suggest that there is no need to elicit the expression of sek-1 and C17H12.8 in the daf-2 mutant with DAF-16 activated, indicating that a compensation effect of the molecular pathway may exist in C. elegans innate immunity. Because the sek-1 expression can always be detected, it is reasonable to hypothesize that the p38 MAPK pathway may play a more specific role than the DAF-2/DAF-16 pathway in the Xoo infection process. Our results support that the p38 MAPK pathway mediates responses specifically to pathogen attack [39] , whereas the DAF-2/DAF-16 pathway functions in mutant daf-2 background. Additional transcriptomic analysis is needed for a further understanding of the interplay between these two pathways.
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p38 MAPK signal pathway was activated in response to Xoo infection. (A) PMK-1 activation was measured in wild-type N2 and sek-1(km4) mutant animals by immunoblotting using a phospho-specific p38 (P-p38) antibody. Anti-GAPDH was used as a control. (B) Quantification of phospho-PMK-1 band intensities in wild-type N2 worms as in (A). No activated phospho-PMK-1 was detected in sek-1(km4) mutant animals in our research. For the quantification of PMK-1 activity, the band intensity of phospho-p38 blots was normalized using the anti-GAPDH blots. The data are representative of three independent experiments. **t-test, p < 0.01 in comparison with OP50.
It is worthy to pursue the reason why DAF-16 does not translocate to the nucleus in TJ356 worms when treated with Xoo ( Fig. 7 ). Previous studies have shown that P. aeruginosa can inhibit host defensive responses by activating the DAF-2 insulin-like signaling pathway to establish a persistent infection [11] . In the present work, pathogens of PXO99 and JXOIII may have an inhibitory effect on the host immune system and lead to no immune-related gene expression. However, considering our killing assay result and RT-PCR result that sek-1 is always required for Xoo resistance, except for CB1370 ( daf-2 ), then it is reasonable to speculate that the hosts integrate all information input, and finally exhibit resistance or susceptibility to Xoo pathogens.
In conclusion, our findings revealed that PXO99 and JXOIII proliferated in the intestine lumen of Caenorhabditis elegans , which led to intestine distention and shortened the nematode lifespan. These findings suggest that C. elegans could be a useful host model to mimic host-pathogen interaction for evaluating the virulence of Xoo phytopathogens. Xoo consistently triggered the p38 MAPK signal pathway and only selectively operated the DAF-2/DAF-16 pathway. Upregulation of downstream genes of these pathways functioned as immune response effectors for resisting the Xoo pathogens. Our study also suggests that the p38 MAPK pathway may be more important than the DAF-2/DAF-16 pathway in the C. elegans innate immune response to Xoo infection. It is also possible that the p38 MAPK pathway and DAF-2/DAF-16 pathway can exert a compensation effect under a certain condition.
Acknowledgements
The authors wish to thank Dr. K. Y. Chen, Rutgers University, for his valuable suggestions and kind help in checking the grammar and syntax of this manuscript. This work was supported by the National Natural Science Foundation of China (Grant No. 31071335); the Special International Cooperation Project of MOST (Grant No. 2012DFA30480); the Key Project of Gansu Province Science and Technology (Grant No. 1002WKDE55); Gansu Province International Science and Technology Cooperation Project (Grant No. 090WCGA900); the Fundamental Research Funds for the Central Universities of China (Grant No. lzujbky-2013-71); the Fundamental Research Funds for the Central Universities of China (lzujbky-2014-147); the Fundamental Research Funds for the Central Universities of China (lzujbky-2014-149); the Fundamental Research Funds for the Central Universities of China (lzujbky-2014-150); and the Fundamental Research Funds for the Central Universities (Grant No. lzujbky-2013-75). The nematode strains used in our work were provided by theCaenorhabditisGenetics Center (CGC).
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