Figure 11. RNAi (MMP-1) knock-down Tribolium imagoes are more susceptible to infection by the fungal entomopathogen B. bassiana. Injection of Beauveria bassiana spores into the hemocoel of Tribolium imagoes resulted in killing within 4 to 5 days. MMP-1 knock- down animals died significantly faster than wild type animals. Values of seven independent trials including 10–15 animals per trial are shown for control (RNAi) animals and MMP-1 (RNAi) animals. Mean values of surviving control (RNAi) animals are shown in green and of surviving MMP-1 (RNAi) animals in red. Statistically significant differences were determined using Student’s t-test and are indicated (*, p,0.05). Ns, not significant. doi:10.1371/journal.pone.0004751.g011 In Vivo MMP Functions gen), were reared on whole-grain flour with 5% yeast powder at 32uC in darkness as described previously [56]. Ambion MEGA- script T7 kit (Applied Biosystems, Austin, TX) was used to prepare dsRNA according to the manufacturers’ protocol. Appropriate templates for Tribolium MMP-1 (XP_968822), Tribolium MMP-2 (XP_969495), Tribolium MMP-3 (XP_972146), a virus-derived MMP (MMP-4, Glean 10983), Tribolium TIMP (XP_001810132), and Tribolium RECK (XP_974450) were generated by PCR using gene specific primers containing a T7 polymerase promoter sequence at their 59 end (Table 1) and purified with the Macherey&Nagel (Germany) NucleoSpin Extract II Kit prior use. Injection of dsRNA into animals was performed with a micromanipulator under a dissecting stereomicroscope. Animals were anaesthetized for 5 min on ice before they were affixed to double-stick tape. About 0.2 mg dsRNA was injected dorsolaterally into the second/third abdominal segment of beetles. Controls received the same volume of dsRNA of the lepidopteran Galleria mellonella insect metalloproteinase inhibitor gene (AY330624) [57], which is not present in T. castaneum. Infection experiments with the entomopathogenic fungus Beauveria bassiana (BoverolH, Fytovita, Ostrozˇska´ Lhota, Czech Republic) were performed by injecting 104 conidia spores into the hemocoel of imagoes, and survival rate was determined every 24 hours at 25uC. For septic injury experiments we injected 104 heat-killed BL21(DE3) Escherichia coli cells (Invitrogen, Carlsbad, CA) into the hemocoel. Sequence alignments and phylogenetic analyses Sequences were aligned using the blosum62 program [58]. Accession numbers of MMPs used for sequence alignment and phylogenetic analyses are as follows: Tribolium castaneum (Tribolium MMP-1, XP_968822; Tribolium MMP-2, XP_969495; Tribolium MMP-3, XP_972146; virus-derived MMPs: Glean 10983, Glean 00226, Glean 06957, Glean 09406, Glean 10302, Glean 03344, Glean 06957, Glean 01812, Glean 16133, and Glean 04281), Anopheles gambiae (Agam MMP-1, XP_001688107; Agam MMP-2, XP_320653; Agam MMP-3, XP_554330), Drosophila melanogaster (Dmel MMP-1, NP_523852; Dmel MMP-2, NP_995788), Homo Figure 12. Purification and refolding of recombinant Tribolium MMP-1 and determination of its collagenolytic activity. (A) MMP-1 (lane 2) was purified to apparent homogeneity from the urea-soluble E. coli inclusion bodies fraction (lane 1) as observed by SDS–PAGE analysis. After refolding in appropriate buffer, we observed the MMP-1 protein band (according to the calculated molecular mass of about 30.3 kDa) and an additional band with an estimated two-fold molecular weight that may correspond to a dimer of MMP-1 (indicated by an asterisk). Molecular mass standards are indicated in kDa. (B) Collagen-IV degrading activity of recombinant Tribolium MMP-1 was monitored using DQTM collagen (type IV from human placenta). The reaction was found to be linear for at least 100 min under examined conditions. This activity was abolished in the presence of 10 mM GM6001. doi:10.1371/journal.pone.0004751.g012 PLoS ONE | www.plosone.org 11 March 2009 | Volume 4 | Issue 3 | e4751