The small-molecule compound fipexide (FPX) has been shown to promote callus formation in several plants, but its effects on forage crops remain unexplored, and its molecular mechanism is not yet fully understood. In this study, we evaluated FPX-induced callus formation from seeds for up to four weeks in four elite cultivars of Medicago sativa, finding it to be faster than the classical 2,4-D/6-BA treatment for the first two weeks. Notably, the cellular organization of FPX-induced calli differed from those induced by 2,4-D/6-BA by showing almost no conducting tissues. Comparative transcriptome analysis revealed dynamic gene expression changes during the early and late stages of callus induction, such as multicellular organism development and response to auxin. Interestingly, in both M. sativa and Arabidopsis, FPX regulates a group of small auxin upregulated RNA (SAUR) family genes, which are known to fine-tune growth in response to internal and external signals. This suggests a potential evolutionary conserved molecular mechanism underlying FPX-induced callus formation across plant species.

Fusarium acuminatum is a major pathogenic fungus causing root rot in alfalfa (Medicago sativa). DNA polymerase theta is known to play a crucial role in repairing DNA double-strand breaks. However, its biological function in F. acuminatum remains unknown. In this study, the POLQ gene was deleted by homologous recombination using Agrobacterium tumefaciens-mediated transformation. Compared to the wild type (with the POLQ gene), the mutants (without the POLQ gene) showed significant phenotypic changes: they produced brown-yellow pigments instead of pink, slowed mycelial growth, and exhibited changes in macroconidia size and shape. The virulence of the mutants was greatly reduced, inducing only mild symptoms in alfalfa. In addition, FITC-WGA staining showed impaired spore germination and hyphal growth. These results suggest that POLQ is a key gene regulating growth and development of F. acuminatum, indicating that DNA repair may play an essential role in the pathogenicity of the pathogen in alfalfa. The POLQ gene could thus be a promising target for limiting F. acuminatum infections in alfalfa.