Regulation Of C. Elegans Gene And Behavior By An Endogenous E. Coli Noncoding RNA OxyS

Objective:Regulation of the chemosensasory gene che-2and behavior of C. elegans by anendogenous RNA OxyS feeding on E. coli was investigated.Methods:Behavioral assays were carried out to investigate the effects of the E. coliendogenous RNA OxyS on C. elegans; Semi-quantitative RT-PCR and real-time PCRwere performed to semi-quantify or quantify che-2mRNA level; Western blot wasemployed to investigate the quantification of CHE-2::GFP protein level; The GFPintensity of CHE-2::GFP protein in the Pche-2::che-2::gfp transgenic line wascompared by confocal microscopy.Results:1. In the food competition assay, the number of worms staying on OxyR or OxySstrains was decreased gradually, but the number of worms didn’t change obviously onΔOxyS strain. The decease was greater on OxyS strain than on OxyR strain. In thefood searching ability assay, within90minutes worms feeding on OxyR or OxySstrains for one week exhibited lower ability in food searching,80%and75%respectively, compared to95%for the N2worms. While only10%of che-2mutantswere able to find OP50strain in the same period.2. In the chemo-attractive assay to NaCl, the number of worms finding NaClreduced significantly within90minutes after feeding on OxyR or OxyS for a week,40%and25%respectively, compared to95%for the N2worms. The decreasingnumber was larger on OxyS than on OxyR. While only10%of che-2mutants were able to find NaCl in the same period. In the chemo-avoidance assay to copper acetate,the number of worms which crossed the copper acetate line increased obviouslywithin90minutes after feeding on OxyR or OxyS strains for one week,20%and35%respectively, compared to5%for the N2worms. The increasing number was larger onOxyS than on OxyR. While around45%of che-2mutants were able to cross thecopper acetate line in the same period.3. In the food competition assay, OxyS E. coli repelled C. elegans but not C.briggsae, while OxyS-cb-expressing E. coli repelled C. briggsae but not C. elegans.4. In the food competition assay, the number of alg-2,sid-1, sid-2mutants and N2worms staying on OxyR strain was descending, among which the degree ofdcr-1,rde-1,rde-2mutants was relatively small, and the smallest degree was observedin either alg-1or rde-4mutants.5. In the food competition assay, the number of N2worms feeding on OxyS,che-2RNAi1or che-2RNAi2strains was decreased. While decreasing number onOxyS was less than that on the latter two. In the food searching ability assay, thenumber of worms was decreased after feeding on OxyS, che-2RNAi1or che-2RNAi2for a week, while decreasing number was larger on OxyS than on che-2RNAistrains.6. Confocal imaging showed that intensity of GFP signal in Pche-2::che-2::gfptransgenic line feeding on OxyR or OxyS strains was weaker than the worms feedingon K12,and intensity of GFP siganl in Pche-2::che-2::gfp transgenic line withinjection of OxyS RNA was decreased too. Injection of the17nt-complementarysingle-stranded small RNA (ssRNA) into Pche-2::che-2::gfp transgenic line alsocaused reduction of GFP signal, although to a less degree than that by the injection offull length OxyS or feeding on OxyS-expressing E. coli. Western blot confirmed thisreduction; furthermore, decline in che-2mRNA level was also detected in N2wormsfeeding on OxyS-expressing E. coli by semi-quantitative RT-PCR and real-time PCR. Conclusions:1. OxyS-expressing E. coli repells C. elegans by inhibiting its chemosensoryability, which implicates its protective effects against being hunted by C. elegans.2. Endogenous noncoding RNA, OxyS, downregulates C. elegans chemosensorygene che-2in mRNA level.3. Noncoding RNA OxyS might play an interspecies ecological role, and therecould be a co-evolutionary relationship between predators (C. elegans) and preys (E.coli),which highlights an elementary principle on the interaction between theenvironment (such as food) and gene expression of animals.