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Chronologically inappropriate morphogenesis (Chinmo) is required for maintenance of larval stages of fall armyworm
Wednesday, 2024/12/11 | 08:09:00

Xien ChenJinmo KooSurjeet Kumar Arya, and Subba Reddy Palli

PNAS; November 26 2024; 121 (49) e2411286121; https://doi.org/10.1073/pnas.2411286121

Significance

The fall armyworm (FAW) is threatening food security worldwide. CRISPR/Cas9-based genome editing was used to identify genes required for larval development in FAW. Development of Chinmo mutant (Chinmo-M), Kr-h1 mutant (Kr-h1-M), and double mutant (Kr-h1/Chinmo-M) was arrested during L2 to L3, L5 to L6, and L2 to L3 molt, respectively. The expression of metamorphosis genes (Br-C and E93) increased prematurely in L3, L5, and L2 in the Chinmo-M, Kr-h1-M, and Kr-h1/Chinmo-M, respectively. Chromatin modification genes are differentially regulated in Chinmo-M and Kr-h1/Chinmo-M but not in Kr-h1-M. Single-cell multiome ATAC analysis revealed that Chinmo is involved in chromatin modifications preventing the promoter accessibility of metamorphosis genes. Chinmo is required for early larval development, making it an ideal target for pest management.

Abstract

Broad complex (Br-C) and eip93F (E93) transcription factors promote insect metamorphosis from larva to pupa and from pupa to adult, respectively. Recently, chronologically inappropriate morphogenesis (Chinmo) has been proposed as a larval specifier in Drosophila melanogaster. However, whether Chinmo is required for larval maintenance in lepidopteran insects, the underlying mechanisms involved in maintaining the larval stage, and its interactions with the JH signaling pathway are not well understood. Here, we used a binary transgenic CRISPR/Cas9 system to knockout Chinmo and Kr-h1 (primary response gene in the JH signaling pathway) in the fall armyworm (FAW). Kr-h1 knockout induced premature metamorphosis only after L5 (penultimate), whereas Chinmo and Kr-h1 double knockout induced premature metamorphosis in L3. Sequencing and differential gene expression (DEG) analysis of RNA isolated from mutants and single-cell multiome ATAC analysis of ChinmoKr-h1, and Chinmo and Kr-h1 double knockout Sf9 cells revealed that Chinmo participates in chromatin modifications that prevent the promoter accessibility and expression of metamorphosis promoting genes. These results suggest that Chinmo is a larval specifier that plays a major role in preventing metamorphosis in early larval stages by controlling chromatin accessibility near the promoters of genes such as Br-C and E93 required for pupal and adult development.

 

See https://www.pnas.org/doi/10.1073/pnas.2411286121

 

Figure 1: Knockout (KO) or overexpression (OE) of Kr-h1 and Chinmo in Sf9 cells affects expression of Br-C and E93. (A) Genomic structures of Kr-h1 and Chinmo and sgRNA targeting sites. One sgRNA targeting site located in common Exon II of the α and β isoforms of Kr-h1 and one sgRNA target site in Exon IV of Chinmo were chosen according to the GN19NGG rule. The target and PAM sequences are highlighted in green and red, respectively. (B) Schematic representation of the KO and OE constructs of Kr-h1 and Chinmo used for establishing stable Sf9 cells. (C) Fluorescence images of stable cells. The transfected Sf9 cells were treated with 100 ng/μL puromycin until all the living cells exhibited RFP signals. (D) Br-C and E93 expression in stable Sf9 cells. Total RNA was extracted from Cas9 cells and from KO and OE cells. RT–qPCR was subsequently performed with four biological replicates to evaluate the relative expression levels of Br-C and E93. Asterisks indicate significant differences between treatment (KO or OE cells) and control (Cas9 cells) based on Student’s t test. *P < 0.05. Fold change is indicated on top of the bar. (E) Promoter activity assay. The promoter-pG5Luc construct was cotransfected with the Kr-h1 or Chinmo overexpression plasmids into Sf9 cells. The cells were harvested two days posttransfection, after which a luciferase activity assay was conducted. All the data are presented as the means ± SDs (n = 5). Asterisks indicate significant differences between the treatment (Kr-h1 or Chinmo OE) and control (promoter construct only) groups based on Student’s t test. *P < 0.05. Fold change is indicated on top of the bar.

 

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