Molecular Biology and Fungal Genetics

In general, molecular biology is a fundamental field in experimental biology and an important support for almost all research areas at the IBWF. With this, the “Molecular Biology and Fungal Genetics” group operates interdisciplinary in many research and industrial projects along with the other groups, such as Enzymes & Materials Research (particularly heterologous expression systems), Plant Protection & Biocontrol or Natural Products & Analytics.

Furthermore, the research profile of the Jacob group is about molecular mechanisms of cellular signaling associated with plant pathogenic fungi. In particular, we do research on the filamentous fungus Magnaporthe oryzae, which causes rice blast disease.

Magnaporthe oryzae represents from the economic and the scientific view one of the most important plant pathogens all over the world. M. oryzae is a suitable organism to work with in our labs since it is facultative pathogen and hemi-biotrophic, we have detailed knowledge of its infection related morphogenesis and a rich toolbox of methods for directed genetic manipulations.

In a multidisciplinary approach of molecular biotechnology, we investigate the its signaling system for osmoregulation, which is named the high osmolarity glycerol (HOG) signaling pathway, and study the various aspects of how the rice blast fungus adapts to its environment and cause disease in plants.

With this, we have three main research areas with the following research questions:

  • Alternative splicing in fungal development and disease

The locus MGG_07173 occurs only once in the genome of M. oryzae and encodes the phosphotransfer protein MoYpd1p, which plays an important role in the high osmolarity glycerol (HOG) signaling pathway for osmoregulation. Originating from this locus, at least three MoYPD1 isoforms are produced in a signal-specific manner. The transcript levels of these MoYPD1-isoforms were individually affected by external stress. Salt (KCI) stress raised MoYPD1_T0 and MoYPD1_T2 abundance, whereas osmotic stress by sorbitol elevates MoYPD1_T1 levels. In line with this, signal-specific nuclear translocation of green fluorescent protein-fused MoYpd1p isoforms in response to stress was observed. Mutant strains that produce only one of the MoYpd1p isoforms are less virulent, suggesting a combination thereof is required to invade the host successfully. In summary, we research signal-specific production of MoYpd1p isoforms that individually increase signal diversity and orchestrate virulence in M. oryzae.

  • Molecular programming of eukaryotic MAPK signaling

The MoA of the successful phenylpyrrole fungicide fludioxonil as well as the fundamental molecular mechanisms of how exactly multiple different signals are transmitted and encrypted from environment to the target site in the cell are still enigmatic, particularly because of the limited number of signaling proteins and a multitude of transitions inside the living cell. Protein phosphorylation is one of the hallmarks enabling transmission and encryption of signals whereby it is often induced by mitogen activated protein kinases (MAPK). The MAPK MoHog1p is a key regulator in the HOG pathway, well conserved in eukaryotes and classified as a p38-type MAPK. Signal transduction at this MAPK is achieved by dual phosphorylation of a conserved TxY-motif, but it is not known how various stimuli are individually encrypted and transmitted. We aspire to assemble a dynamic multidimensional model of the individual time- and stimulus-dependent intensity of T, Y and T/Y phosphorylation to unravel the molecular MAPK programming in vivo. The multiple dimensions comprise (1) the intensity of phosphorylation at T, Y and T/Y, (2) variation of stimuli, (3) different concentrations of stimuli, (4) time course and (5) different (transcriptional) responses. We recently were able to pinpoint key differences in the degree of the intensity of phosphorylated T, Y and T/Y over time after activation of the signaling pathway upon salt stress and found this pattern individually different as compared to the pattern upon fludioxonil fungicide stress. Consequently, we set up the hypothesis, that temporal dynamic site-specific phosphorylation at the single amino acids of the TxY-motif is the molecular mechanism to encrypt and encode multiple signals at MAPKs.

  • Rapid evolution of eukaryotic signaling networks

The question of how the remarkably diverse array of eukaryotic signaling networks has evolved is of enormous scientific relevance. Evolutionary adaptation of living organisms is commonly thought to be the result of processes that acted over long periods of time. Here, we are motivated by observations that microorganisms are able to rapidly adapt to new environments and establish stable phenotypes by natural selection, even within few generations. We found the filamentous rice blast fungus Magnaporthe oryzae to rapidly rewire signal transduction required for osmoregulation in several independent “loss of function” (lof)-mutants of the High Osmolarity Glycerol (HOG)-pathway upon exposure to salt stress. Adaptation resulted in stable mutants being restored in osmoregulation arising as individuals outgrowing from salt-sensitive lof-mutants. The major compatible solute produced upon salt stress by these rapidly “adapted” strains was found to be glycerol whereas it is arabitol in the wildtype strain. These findings lead to the hypothesis that stable adaptation-events under continuously environmental evolutionary pressure enable Magnaporthe oryzae to rapidly restore or modify entire signaling networks. To address this hypothesis, we aim to identify the molecular or biochemical mechanisms of this rapid evolutionary adaption and characterize associated factors and signalling pathways which enable or prevent adaption. We combine expertise of theoretical approaches to integrate sequencing data from genomics and transcriptomics with modern quantitative (phospho)-proteomics techniques. Furthermore, reversed molecular genetics will be used to validate the candidate genes or even other factors (e.g. phosphorylation patterns) found to be putatively promote or constrain rapid evolutionary adaptation.