Dynamic transcriptome and GWAS uncover a hydroxyproline-rich glycoprotein that suppresses Agrobacterium-mediated...

Genetic transformation is a crucial tool for investigating gene function and advancing molecular breeding in crops, with Agrobacterium tumefaciens-mediated transformation being the primary method for plant genetic modification. However, this approach exhibits significant genotypic dependence in maize. Therefore, to overcome these limitations, we combined dynamic transcriptome analysis and genome-wide association study (GWAS) to identify the key genes controlling Agrobacterium infection frequency (AIF) in immature maize embryos. Transcriptome analysis of Agrobacterium-infected embryos uncovered 8483 and 1580 genotype-specific response genes in the maize line 18-599R with low AIF and A188 with high AIF, respectively. A weighted gene co-expression network analysis (WGCNA) revealed five and seven stage-specific co-expression modules in each corresponding line. Based on a self-developed AIF quantitation method, the GWAS revealed 30 AIF-associated single-nucleotide polymorphisms and 315 candidate genes under multiple environments. Integration of GWAS and WGCNA further identified 12 key genes associated with high AIF in A188. ZmHRGP, encoding a hydroxyproline-rich glycoprotein, was functionally validated as a key factor of AIF in immature embryos. Knockout of ZmHRGP enabled us to establish a high-efficiency genetic transformation system for the 18-599R line, with the transformation frequency being approximately 80%. Moreover, the transient reduction of ZmHRGP expression significantly enhanced the AIF of maize calluses and leaves. Collectively, these findings advance our understanding of plant factors controlling Agrobacterium infection and contribute to developing more efficient Agrobacterium-mediated transformation systems in crops.