Stefan Jacob

Histidine kinases mediate differentiation, stress response, and pathogenicity in Magnaporthe oryzae

The aim of this study is a functional characterization of 10 putative histidine kinases (HIKs)‐encoding genes in the phytopathogenic fungus Magnaporthe oryzae. Two HIKs were found to be required for pathogenicity in the fungus. It was found that the mutant strains ΔMohik5 and ΔMohik8 show abnormal conidial morphology and furthermore ΔMohik5 is unable to form appressoria. Both HIKs MoHik5p and MoHik8p appear to be essential for pathogenicity since the mutants fail to infect rice plants. MoSln1p and MoHik1p were previously reported to be components of the HOG pathway in M. oryzae. The ΔMosln1 mutant is more susceptible to salt stress compared to ΔMohik1, whereas ΔMohik1 appears to be stronger affected by osmotic or sugar stress. In contrast to yeast, the HOG signaling cascade in phytopathogenic fungi apparently comprises more elements. Furthermore, vegetative growth of the mutants ΔMohik5 and ΔMohik9 was found to be sensitive to hypoxia‐inducing NaNO2‐treatment. Additionally, it was monitored that NaNO2‐treatment resulted in MoHog1p phosphorylation. As a consequence we assume a first simplified model for hypoxia signaling in M. oryzae including the HOG pathway and the HIKs MoHik5p and MoHik9p.

Total Synthesis of (−)-Hymenosetin

The 3-decalinoyltetramic acid (-)-hymenosetin and its N-methyl analogue were prepared in 11 and 8 steps, respectively, from (+)-citronellal using an intramolecular Diels-Alder reaction as the key step. This method represents the first example for the synthesis of a 3-decalinoyltetramic acid with a free NH moiety. The stereochemistry of the title compound, an unnatural diastereomer, and of a decalin building block was studied in detail using circular dichroism spectroscopy in the IR and UV/VIS freqeuncy range. This allowed to determine the absolute configuration of the natural product and to plan the synthetic route.

Cytotoxicity of the Sesquiterpene Lactones Neoambrosin and Damsin from Ambrosia maritima Against Multidrug-Resistant Cancer Cells.

Multidrug resistance is a prevailing phenomenon leading to chemotherapy treatment failure in cancer patients. In the current study two known cytotoxic pseudoguaianolide sesquiterpene lactones; neoambrosin (1) and damsin (2) that circumvent MDR were identified. The two cytotoxic compounds were isolated using column chromatography, characterized using 1D and 2D NMR, MS, and compared with literature values. The isolated compounds were investigated for their cytotoxic potential using resazurin assays and thereafter confirmed with immunoblotting and in silico studies. MDR cells overexpressing ABC transporters (P-glycoprotein, BCRP, ABCB5) did not confer cross-resistance toward (1) and (2), indicating that these compounds are not appropriate substrates for any of the three ABC transporters analyzed. Resistance mechanisms investigated also included; the loss of the functions of the TP53 and the mutated EGFR. The HCT116 p53-/- cells were sensitive to 1 but resistant to 2. It was interesting to note that resistant cells transfected with oncogenic ΔEGFR exhibited hypersensitivity CS toward (1) and (2) (degrees of resistances were 0.18 and 0.15 for (1) and (2), respectively). Immunoblotting and in silico analyses revealed that 1 and 2 silenced c-Src kinase activity. It was hypothesized that inhibition of c-Src kinase activity may explain CS in EGFR-transfected cells. In conclusion, the significant cytotoxicity of 1 and 2 against different drug-resistant tumor cell lines indicate that they may be promising candidates to treat refractory tumors.

Functions of the Magnaporthe oryzae Flb3p and Flb4p transcription factors in the regulation of conidiation

The Magnaporthe oryzae genes FLB3 and FLB4, orthologues of the Aspergillus nidulans regulators of conidiation FlbC and FlbD, were inactivated. These genes encode C2H2 zinc finger and Myb-like transcription factors, respectively, in A. nidulans. Analysis of the resultant mutants demonstrated that FLB4 is essential for spore formation and that strains lacking this gene are fluffy in their colony morphology due to an inability to complete conidiophore formation. Meanwhile, FLB3 is required for normal levels of aerial mycelium formation. We identified genes dependent on both transcription factors using microarray analysis. This analysis revealed that the transcription of several genes encoding proteins implicated in sporulation in Magnaporthe oryzae and other filamentous fungi are affected by FLB3 or FLB4 inactivation. Furthermore, the microarray analysis indicates that Flb3p may effectively reprogramme the cell metabolically by repressing transcription of genes encoding biosynthetic enzymes and inducing transcription of genes encoding catabolic enzymes. Additionally, qRT-PCR was employed and showed that FLB3 and FLB4 transcripts are enriched in synchronously sporulating cultures, as were the transcripts of other genes that are necessary for normal conidiation, consistent with a role for their gene products in this process.

Hog1p activation by marasmic acid through inhibition of the histidine kinase Sln1p.

Abstract BACKGROUND The histidine kinase (HK) MoHik1p within the high‐osmolarity glycerol (HOG) pathway is known to be the target of the fungicide fludioxonil. Treatment of the fungus with fludioxonil causes an uncontrolled hyperactivation of the pathway and cell death. In this study, we used a target‐based in vivo test system with mutant strains of the rice blast fungus Magnaporthe oryzae to search for new fungicidal compounds having various target locations within the HOG pathway. Mutants with inactivated HOG signalling are resistant to fungicides having the target located in the HOG pathway. RESULTS The HK MoSln1p was identified as being involved in the new antifungal mode of action of marasmic acid, as single inactivation of the genes MoSLN1, MoSSK1, MoSSK2, MoPBS2 and MoHOG1 resulted in mutant strains resistant against the sesquiterpenoid, whereas the wild‐type strain and the ΔMohik1 mutant were susceptible. Western blot analysis of phosphorylated MoHog1p confirmed the hypothesis that marasmic acid interferes with the HOG pathway, as a strong phosphorylation of MoHog1p was detectable after sesquiterpenoid treatment in the wild‐type strain but not in the ΔMosln1 mutant. CONCLUSION This study provides evidence for marasmic acid activating the HOG pathway via the HK MoSln1p, and we propose that the sesquiterpenoid has a new mode of action in M. oryzae that differs from that of known HOG inhibitors, e.g. fludioxonil.

Identification of factors involved in dimorphism and pathogenicity of Zymoseptoria tritici.

A forward genetics approach was applied in order to investigate the molecular basis of morphological transition in the wheat pathogenic fungus Zymoseptoria tritici. Z. tritici is a dimorphic plant pathogen displaying environmentally regulated morphogenetic transition between yeast-like and hyphal growth. Considering the infection mode of Z. tritici, the switching to hyphal growth is essential for pathogenicity allowing the fungus the host invasion through natural openings like stomata. We exploited a previously developed Agrobacterium tumefaciens-mediated transformation (ATMT) to generate a mutant library by insertional mutagenesis including more than 10,000 random mutants. To identify genes involved in dimorphic switch, a plate-based screening system was established. With this approach eleven dimorphic switch deficient random mutants were recovered, ten of which exhibited a yeast-like mode of growth and one mutant predominantly growing filamentously, producing high amount of mycelium under different incubation conditions. Using genome walking approach previously established, the T-DNA integration sites were recovered and the disrupted genomic loci of corresponding mutants were identified and validated within reverse genetics approach. As prove of concept, two of the random mutants obtained were selected for further investigation using targeted gene inactivation. Both genes deduced were found to encode known factors, previously characterized in other fungi: Ssk1p being constituent of HOG pathway and Ade5,7p involved in de novo purine biosynthesis. The targeted mutant strains defective in these genes exhibit a drastically impaired virulence within infection assays on whole wheat plants. Moreover exploiting further physiological assays the predicted function for both gene products could be confirmed in concordance with conserved biological role of homologous proteins previously described in other fungal organisms.

Multistep phosphorelay in fungi: the enigma of multiple signals and a limited number of signaling pathways

Current knowledge of fungal ecology depends on the understanding of the perception and processing of biotic and abiotic factors facilitating environmental interactions. Ongoing research regarding fungal signaling mechanisms has not only increased our appreciation for multistep phosphorelay signaling systems as an asset, but also sheds light on the role that these upgrades of the simple two-component signaling systems play in fungal biology, stress response, differentiation processes, metabolism, and pathogenicity. In contrast to two-component signaling systems, multistep phosphorelay systems comprise a phosphotransfer protein enabling modular phosphate transfer from the sensor hybrid histidine kinase to the response regulator protein. Histidine kinases were found to perceive a lot of environmental signals and integrate them into a number of signaling pathways. The major question is how multistep phosphorelay signal transduction takes place in detail, since most fungi possess only one single histidine-containing phosphotransfer protein enabling phosphate transfer. One single phosphotransfer protein routes various signals in a specific way and coordinates the phosphates precisely to different target locations within the cell. This mini review opens the door and presents three hypotheses as the basis for further investigations to unravel the complex signaling mechanisms in fungal multistep phosphorelay systems. Scaffold proteins, frequency-based signaling, and alternative splicing are discussed as putative candidates to implement fungal signaling in multistep phosphorelay systems having only one phosphotransfer protein for coordinated phosphate transfer.

Unravelling the biosynthesis of pyriculol in the rice blast fungus Magnaporthe oryzae

Pyriculol was isolated from the rice blast fungus Magnaporthe oryzae and found to induce lesion formation on rice leaves. These findings suggest that it could be involved in virulence. The gene MoPKS19 was identified to encode a polyketide synthase essential for the production of the polyketide pyriculol in the rice blast fungus M. oryzae. The transcript abundance of MoPKS19 correlates with the biosynthesis rate of pyriculol in a time-dependent manner. Furthermore, gene inactivation of MoPKS19 resulted in a mutant unable to produce pyriculol, pyriculariol and their dihydro derivatives. Inactivation of a putative oxidase-encoding gene MoC19OXR1, which was found to be located in the genome close to MoPKS19, resulted in a mutant exclusively producing dihydropyriculol and dihydropyriculariol. By contrast, overexpression of MoC19OXR1 resulted in a mutant strain only producing pyriculol. The MoPKS19 cluster, furthermore, comprises two transcription factors MoC19TRF1 and MoC19TRF2, which were both found individually to act as negative regulators repressing gene expression of MoPKS19. Additionally, extracts of ΔMopks19 and ΔMoC19oxr1 made from axenic cultures failed to induce lesions on rice leaves compared to extracts of the wild-type strain. Consequently, pyriculol and its isomer pyriculariol appear to be the only lesion-inducing secondary metabolites produced by M. oryzae wild-type (MoWT) under these culture conditions. Interestingly, the mutants unable to produce pyriculol and pyriculariol were as pathogenic as MoWT, demonstrating that pyriculol is not required for infection.