The role of posttranslational hypusination of the eukaryotic translation initiation factor 5A in Zea mays and Fusarium graminearum

The posttranslational modification of the eukaryotic translation initiation factor 5A (eIF5A) is indispensable for its function. This modification requeries two enzymes, deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH) to produce the unique amino acid hypusine. The genes encoding for the biosynthetic enzymes DHS, DOHH and the substrate eIF5A are indispensable in plants and fungi. Therefore, a way to study the eIF5A-hypusine synthesis and its implications is by altering the expression levels of the modifying genes DHS or DOHH. In the first part of this study, the importance of DHS expression levels was investigated in Zea mays. In previous studies, silencing or overexpressing ZmDHS1 maize lines were generated. Here, these transgenic maize lines were cultivated until T3 generation and verified. The ZmDHS1 gene expression results provided a strong evidence of activation of the silencing and overexpressing DHS constructs in the transgenic maize plants. In addition, in silico analysis revealed the presence of two DHS genes in Z. mays. Expression analysis of the DHS genes indicated a transcriptional co-expression of ZmDHS1 and ZmDHS2 genes, but no effect on the expression of ZmDOHH or the three ZmeIF5A genes in the silencing and overexpressing lines. Aditionally, differences in expression of ZmDHS1 produced diverse effects in germination, growth and development in the silencing and overexpressing maize lines. Nevertheless, no evident physiological phenotype could be attributed to silencing or overexpression of ZmDHS1. Exceptionally, the DHS-overexpressing lines exhibit resistance towards the fungal pathogens Bipolaris sorokiniana, Cochliobolus heterostrophus, and Colletotrichum graminicola, while the DHS-silencing lines showed similar susceptibility as the control plants. Expression analysis of key genes involved in plant defence response revealed that more than a hormonal defence response, the resistance of overexpressing lines could be attributed to the thickening of a physical barrier. In the second part of this study, a comparative analysis of differentially expressed genes was produced using two mutants and the wild type strain of Fusarium graminearum. The two mutants were generated in previous studies, a DHS overexpressing mutant (DHSoex) with a hypervirulent phenotype and a DOHH overexpressing mutant (DOHHoex) with an avirulent phenotype. By using different microscopic techniques, infection structures of the WT and overexpressing mutants growing on wheat glumes were analyzed. While DHSoex produced similar infection structures (IC) to the wild type, the DOHHoex did not produce infection structures, only epiphytically growing runner hyphae (RH). A combination of laser microdissection and LD_PCR was used to create cDNA libraries from low amount of mRNA extracted from RH or IC of the wild type strain and the two overexpressing mutants. Using Ilumina Next Generation Sequencing we determined the differentially transcribed genes of all samples. The results revealed a global viewpoint of F. graminearum gene expression during early infection of wheat. Here, we analysed not only the total expression but also a number of regulated genes involved in several functions such as transcription factors (TF), transporters (TP), glycoside hydrolases (GH), secondary metabolite (SM), ROS-related proteins, effectors, fungal cell wall remodeling enzymes (FCWRE), protein kinases (PK) and plant cell wall degrading enzymes (PCWDE). During the analyses, several specific genes emerged as being responsible for expansion on the plant surface but not for penetration or pathogenicity. Among the SM gene clusters, while the aurofusarin gene cluster is necessary during F. graminearum expansion on plant surfaces where competitors grow, it is not needed during penetration. On the contrary, the gene clusters such as C31, C37, C40 and C11 were involved during penetration more than during expansion of the fungus. The comparison of F. graminearum gene expression in vitro and in planta of wild type and the overexpressing DHSoex and DOHHoex mutants allowed to restrict the large number of genes expressed during infection and revealed novel virulence factors involved specifically during penetration of the host plant. Finally, the transcriptome analysis confirmed the influence of diverse genes affecting the morphological differentiation and pathogenicity in F. graminearum due to the alteration of hypusine-eIF5A biosynthesis.