Two <i>FgLEU2</i> Genes with Different Roles in Leucine Biosynthesis and Infection-Related Morphogenesis in <i>Fusarium graminearum</i>

Abstract

<div><p>3-isopropylmalate dehydrogenase (IPMD) encoded by <i>LEU2</i> is a key enzyme in leucine (Leu) biosynthetic pathway. Analysis of the genome sequence of <i>Fusarium graminearum</i> revealed two paralogous <i>LEU2</i> genes (designated as <i>FgLEU2A</i> and <i>FgLEU2B</i>) in this fungus and the deduced amino acid sequences of FgLeu2A and FgLeu2B share 45% identity. Targeted disruption of individual <i>FgLEU2A/B</i> gene in <i>F</i>. <i>graminearum</i> assigned a more crucial role of FgLeu2A in Leu biosynthesis as disruption of <i>FgLEU2A</i> resulted in mutant (ΔFgLeu2A-10) that was Leu-auxotrophic and could not grow in minimal medium limited for amino acids, whereas <i>FgLEU2B</i> deletion mutant ΔFgLeu2B-2 was morphologically indistinguishable from the wild type strain PH-1. The growth defects of ΔFgLeu2A-10 could be overcome by exogenous addition of Leu at 0.25 mM. Double deletion of <i>FgLEU2A</i> and <i>FgLEU2B</i> (ΔFgLeu2AB-8) caused a more severe Leu-auxotrophic phenotype as the concentration of Leu exogenously added to medium to rescue the growth defect of ΔFgLeu2AB-8 should be raised to 1.25 mM, indicating a less important but nonnegligible role of FgLeu2B in Leu biosynthesis. Disturb of Leu biosynthesis caused by <i>FgLEU2A</i> deletion leads to slower growth rate, reduced aerial hyphal formation and red pigmentation on PDA plates and completely blocked conidial production and germination. All of the defects above could be overcome by Leu addition or complementation of the full-length <i>FgLEU2A</i> gene. ΔFgLeu2A-10 also showed significantly increased sensitivity to osmotic and oxidative stresses. Pathogenicity assay results showed that virulence of mutants lacking <i>FgLEU2A</i> were dramatically impaired on wheat heads and non-host cherry tomatoes. Additionally, a low level of deoxynivalenol (DON) production of ΔFgLeu2A-10 and ΔFgLeu2AB-8 in wheat kernels was also detected. Taken together, results of this study indicated a crucial role of FgLeu2A and a less important role of FgLeu2B in Leu biosynthesis and fungal infection-related morphogenesis in <i>F</i>. <i>graminearum</i> and FgLeu2A may serve as a potential target for novel antifungal development.</p></div