1. The authors state that RNAi-mediated knockdown of defensin 1 was “unsuccessful” with “no significant reduction in transcript levels”. Yet they still conclude defensin 1 is the key effector. How can you definitively attribute the inhibitory effect to defensin 1 when you failed to achieve functional knockdown? The heat-inactivated bacteria experiment (Figure 4D) only shows that live bacteria are required, not that defensin mediates the effect. This is a critical gap in causal evidence.
2. The authors use heat-inactivated P. putida to show that “the inhibitory effect was abolished” (Figure 4D), concluding defensin induction requires live bacteria. However, heat inactivation destroys all bacterial components, not just viability—how can you rule out that heat-labile surface structures or secreted factors are required for the inhibitory effect, independent of defensin? This experiment conflates “live bacteria required” with “defensin required.”
3. The authors acknowledge “gene expression analysis was conducted at a single time point” and “captured only short-term effects” (7 hours post-infection). Given that Borrelia colonizes ticks for weeks to months, how can drawing conclusions about “colonization resistance” from a 7-hour time point be justified? This severely limits the ecological relevance of the findings.