The aim of this study is a functional characterization of 10 putative histidine kinases (HIKs)‐encoding genes in the phytopathogenic fungusMagnaporthe oryzae. TwoHIKs were found to be required for pathogenicity in the fungus. It was found that the mutant strainsΔMohik5andΔMohik8show abnormal conidial morphology and furthermoreΔMohik5is unable to form appressoria. BothHIKs 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 theHOGpathway inM. oryzae. TheΔMosln1mutant is more susceptible to salt stress compared toΔMohik1, whereasΔMohik1appears to be stronger affected by osmotic or sugar stress. In contrast to yeast, theHOGsignaling cascade in phytopathogenic fungi apparently comprises more elements. Furthermore, vegetative growth of the mutantsΔMohik5andΔMohik9was 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 inM. oryzaeincluding theHOGpathway and theHIKs MoHik5p and MoHik9p.

This study comprises a first functional analysis of an YPD1-homologue in filamentous phytopathogenic fungi and its role in the HOG signalling pathway. We generated a gene deletion mutant of the gene MoYPD1 in Magnaporthe oryzae and characterized the resulting mutant strain. We have shown that MoYpd1p is a component of the phosphorelay system acting in the HOG pathway due to its Y2H protein interaction with the HKs MoHik1p and MoSln1p as well as with the response regulator MoSsk1p. Fungicidal activity of fludioxonil was reported to be based on the inhibition of MoHik1p resulting in hyperactivation of the HOG signalling pathway and lethality. Western analysis proved that both, osmotic stress and fludioxonil application resulted in the phosphorylation of the MoHog1p in a MoYpd1p-dependent manner. We therefore consider MoYpd1p to be essential for the regulation capability of the HOG pathway and the fungicide action of fludioxonil, but dispensible for viability. The results indicate that MoYpd1p functions as signal transfer protein between MoSln1p, MoHik1p, and MoSsk1p. Manipulations of the HOG signalling pathway affects the infection-related morphogenesis in M. oryzae, since the mutant strain ΔMoypd1 has a white and fluffy phenotype on complete media, is not able to form spores in various conditions and fails to colonize rice plants.

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.

Multidrug resistance (MDR) is a prevailing phenomenon leading to chemotherapy treatment failure in cancer patients. In the current study two known cytotoxic pseudoguaianolide sesquiterpene lactones; neombrosin (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 towards neoambrosin (1) and damsin (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 tumor suppressor TP53 and the mutated epidermal growth factor receptor (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 (collateral sensitivity) towards neoambrosin (1) and damsin (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 collateral sensitivity 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.