Functional study of th antagonistic HLH/bHLH transcription factor IBH1 in Triticum turgidum ssp. durum via CRISPR-Cas9 madiated knock out.
Author(s)
Date Issued
2025
Type
conferenceObject
Abstract
Durum wheat (Triticum turgidum spp. durum) is a staple crop of fundamental relevance in the Mediterranean region. In recent years, durum wheat cultivation has been increasingly threatened by the increasing adverse climatic conditions such as droughts, heat waves and extreme weather events related to climate change. These abiotic stresses are significantly reducing crop yields and grain quality, necessitating the development of new resilient genotypes.
This study aims to improve the resilience of durum wheat cv. Svevo by modulating the brassinosteroid (BR) phytohormone signaling pathway, which plays a key role in regulating plant development and responses to stress. Specifically, a CRISPR/Cas9 genome editing approach was employed to knock out IBH1 (ILI1 Binding bHLH 1) transcription factor, a negative regulator of BR signaling, impacting on plant morphology and stress adaptation.
Immature embryos of cv. Svevo were transformed using Agrobacterium tumefaciens carrying constructs assembled with the Golden Gate modular cloning strategy, which offers high modularity and assembly efficiency. Two independent transformation experiments were carried out using different combinations of guide RNAs: the first with sgRNA-1 and sgRNA-3, and the second with sgRNA-2 and sgRNA-4. These guide RNAs were selected based on predicted on-target score and specificity within both homeologs of the tetraploid durum wheat genome.
Transformation efficiency was assessed via TaqMan quantitative PCR, which allowed estimation of construct copy number in the regenerated T₀ plants. Out of 43 T₀ positive events, 18 plants were found to contain a single-copy insertion. Subsequent next-generation sequencing (NGS) of the target regions revealed that five independent lines exhibited editing events in both homeohologs of IBH1. Among the four guide RNAs, sgRNA-3 showed the highest editing efficiency, consistent with computational predictions. All detected editing events consisted of single-base deletions at the expected cut site, leading to frameshift mutations.
To further investigate the functional consequences of the induced mutations, an alignment analysis using T-Coffee was performed. This revealed that the single-nucleotide deletions cause a disruption of the key domain necessary for DNA binding and interaction with partner proteins, suggesting a high probability of complete loss of function.
Moreover, in silico prediction of the tertiary protein structure indicated a drastic conformational change, especially in the protein product of the homeolog A, reinforcing the hypothesis of structural and functional loss of the protein.
Edited lines were advanced to the T₁ and T₂ offsprings, where they are currently undergoing molecular and phenotypic characterization. In the T₂ progeny, gene expression analyses are being conducted to determine the transcriptional impact of IBH1 knock-out. A STRING functional network analysis was carried out to reveal the correlation between IBH1 and other BR-regulated transcription factors, such as ILI and PRE genes. These downstream components are known to regulate plant architecture and hormone-mediated stress tolerance, suggesting that IBH1 disruption may significantly affect the broader BR signaling network.
Furthermore, phenotyping analysis and oxidative stress assays will be performed out respectively to evaluate the effect of IBH1 knock out on morphological and yield traits and resistance of the edited lines to environmental stressors.
Conference(s)
LEVERAGING GENETIC INNOVATION FOR FUTURE-PROOFING CROPS – From conventional breeding to multi-omics and AI in sustainable agriculture - SIGA 2025