Abstract
3-isopropylmalate dehydrogenase (IPMD) encoded by LEU2 is a key enzyme in leucine (Leu) biosynthetic pathway. Analysis of the genome sequence of Fusarium graminearum revealed two paralogous LEU2 genes (designated as FgLEU2A and FgLEU2B) in this fungus and the deduced amino acid sequences of FgLeu2A and FgLeu2B share 45% identity. Targeted disruption of individual FgLEU2A/B gene in F. graminearum assigned a more crucial role of FgLeu2A in Leu biosynthesis as disruption of FgLEU2A resulted in mutant (ΔFgLeu2A-10) that was Leu-auxotrophic and could not grow in minimal medium limited for amino acids, whereas FgLEU2B 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 FgLEU2A and FgLEU2B (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 FgLEU2A 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 FgLEU2A gene. ΔFgLeu2A-10 also showed significantly increased sensitivity to osmotic and oxidative stresses. Pathogenicity assay results showed that virulence of mutants lacking FgLEU2A 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 F. graminearum and FgLeu2A may serve as a potential target for novel antifungal development.
Highlights
Branched-chain amino acids (BCAAs) including leucine (Leu), valine (Val) and isoleucine (Ile) can be synthesized in bacteria, fungi and high plants, but not mammals
Leu is synthesized as a branch that leads from 2-ketoisovalerate (2-KIV), the intermediate precursor of Val, to Leu and its biosynthesis contains four enzymatic reactions catalyzed by isopropylmalate synthase (IPMS, encoded by LEU4), isopropylmalate isomerase (IPMI, encoded by LEU1), isopropylmalate dehydrogenase (IPMD, encoded by LEU2) and branchedchain aminotransferase (BCAT, encoded by BCA1/2) [1]
We found two paralogous LEU2 genes in the genome of F. graminearum, using targeted-gene disruption strategy we have constructed single and double deletion mutants of FgLEU2A/B gene in F. graminearum and investigated the different roles of each gene in Leu biosynthesis and in other important cellular processes
Summary
Branched-chain amino acids (BCAAs) including leucine (Leu), valine (Val) and isoleucine (Ile) can be synthesized in bacteria, fungi and high plants, but not mammals. The third Leu biosynthetic specific enzyme IPMD catalyzes the conversion of 3-isopropylmalate (3-IPPM) to 2-ketoisocaproate (2-KIC) and its encoding gene LEU2 is one of the best-studied BCAA biosynthetic genes because of its wide use as selected marker for gene-transformation experiments in Saccharomyces cerevisiae and several other yeast species [5,6]. Very few documents characterized functions of LEU2 in filamentous fungi except for a relatively ancient and primary research in Aspergillus niger showed that this fungus elaborates two isoenzymes of IPMD and these two encoding genes were differentially expressed [8]. The detailed function of these two paralogous LEU2 genes in Leu biosynthesis or other cellular processes was not investigated in A. niger or other filamentous fungi
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
