While the study provides compelling evidence for the effectiveness of Nb4-11 in conferring herbicide resistance, certain aspects merit further discussion. For instance, the reported IC50 of the nanobody expressed in E. coli (29.3 ng/mL) is significantly lower than the IC50 of the nanobody expressed in transgenic plants (184.6 ng/mL). Could the authors clarify whether the reduced binding affinity in plants is due to post-translational modifications, expression levels, or structural changes in the nanobody? Furthermore, the herbicide resistance observed at 2 g ai/ha is below the typical application rates of 50–500 g ai/ha in agricultural settings. What strategies could be explored to enhance resistance to field-level herbicide concentrations?
I also would like to know whether the resistance mechanism conferred by Nb4-11 in transgenic Arabidopsis thaliana is primarily due to direct sequestration of 2,4-D or if it involves an indirect modulation of endogenous hormone pathways. Were the expression levels of Nb4-11 uniform across plant tissues, and how might tissue-specific expression influence the observed resistance?