CRISPR/Cas9-Mediated Knockout of <i>PxPGRP</i>4 Influences Midgut Microbial Homeostasis and Immune Responses in <i>Plutella xylostella</i>
Shuzhong Li | Xiaoxia Xu | Dongran Fu | Mingyou Liu | Congjing Feng | Fengliang Jin
Peptidoglycan recognition proteins (PGRPs) are essential for innate immune recognition and regulation from insects to mammals. However, the specific role of PGRPs in responding to <i>Bacillus thuringiensis</i> (Bt) infection and maintaining midgut microbial homeostasis in <i>Plutella xylostella</i> remains poorly understood. In this study, we identified and characterized a PGRP gene from <i>P. xylostella</i>, designated <i>PxPGRP</i>4. The spatiotemporal expression analysis revealed that <i>PxPGRP</i>4 is predominantly expressed in the midgut of naïve larvae and at adult stages. A homozygous mutant strain featuring a four-base pair nucleotide deletion was successfully generated through CRISPR/Cas9-mediated knockout of <i>PxPGRP</i>4. The bioassay results indicated that the susceptibility of <i>P. xylostella</i> larvae to Cry1Ac protoxin was significantly increased by the loss of <i>PxPGRP</i>4 expression. Furthermore, 16S rRNA sequencing and qPCR analysis revealed that the <i>PxPGRP</i>4 mutants exhibited a significantly reduced total bacterial load and altered microbiota composition in the midgut compared to the wild-type strain, with a shift in the dominant bacterial family from Enterobacteriaceae to Enterococcaceae. Additionally, the knockout of <i>PxPGRP</i>4 resulted in significant alterations in the expression of midgut immune-related genes. These findings highlight the crucial role of <i>PxPGRP</i>4 as a modulator of midgut microbiota and immune responses and provide valuable insights into <i>Bt</i> resistance management.
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