Primers used in this study.

<div><p>Anaplerosis refers to enzymatic reactions or pathways replenishing metabolic intermediates in the tricarboxylic acid (TCA) cycle. Pyruvate carboxylase (PYC) plays an important anaplerotic role by catalyzing pyruvate carboxylation, forming oxaloacetate. Although PYC orthologs are well conserved in prokaryotes and eukaryotes, their pathobiological functions in filamentous pathogenic fungi have yet to be fully understood. Here, we delve into the molecular functions of the ortholog gene <i>PYC1</i> in <i>Fusarium graminearum</i> and <i>F</i>. <i>oxysporum</i>, prominent fungal plant pathogens with distinct pathosystems, demonstrating variations in carbon metabolism for pathogenesis. Surprisingly, the <i>PYC1</i> deletion mutant of <i>F</i>. <i>oxysporum</i> exhibited pleiotropic defects in hyphal growth, conidiation, and virulence, unlike <i>F</i>. <i>graminearum</i>, where <i>PYC1</i> deletion did not significantly impact virulence. To further explore the species-specific effects of <i>PYC1</i> deletion on pathogenicity, we conducted comprehensive metabolic profiling. Despite shared metabolic changes, distinct reprogramming in central carbon and nitrogen metabolism was identified. Specifically, alpha-ketoglutarate, a key link between the TCA cycle and amino acid metabolism, showed significant down-regulation exclusively in the <i>PYC1</i> deletion mutant of <i>F</i>. <i>oxysporum</i>. The metabolic response associated with pathogenicity was notably characterized by S-methyl-5-thioadenosine and S-adenosyl-L-methionine. This research sheds light on how <i>PYC1</i>-mediated anaplerosis affects fungal metabolism and reveals species-specific variations, exemplified in <i>F</i>. <i>graminearum</i> and <i>F</i>. <i>oxysporum</i>.</p></div>