Optimization of Ustilago nuda Inoculum Concentration for Screening Un8-Mediated Loose Smut Resistance in Barley Reveals a Resistance Reaction that Disrupts Seed Germination and Suggests a Role for Abscisic Acid in Disease Development.

Barley loose smut has been effectively controlled for decades through resistance conferred by the Un8 gene. However, evaluation of loose smut reaction using floret inoculation at the standard inoculum concentration is associated with the production of small, discolored seeds in Un8 carriers and susceptible genotypes. Interestingly, Un8 carriers also displayed significantly poorer germination than susceptible genotypes and produce short-lived seedlings following inoculation. To understand these observations, a Un8 carrier (TR11698) and susceptible non-Un8 carrier (CDC Austenson) were assessed for seed traits, Ustilago nuda biomass in the seed, infection rate, and phytohormone profile across a range of lower inoculum concentrations. At lower inoculum concentrations, seed appearance and weight improved in both genotypes, and infection rate increased in CDC Austenson. Pathogen load in the seed was similar in both genotypes and was positively correlated with the CDC Austenson infection rate. No infection was ever observed in TR11698. Significantly, germination rate improved in CDC Austenson, whereas the very low germination rate and short-lived seedlings remained associated with TR11698. It appears that poor seed appearance in both genotypes and low germination rate in the susceptible genotype can be improved by lowering the inoculum concentration. However, the very low germination rates and seedling death associated with the Un8 carrier TR11698 are indicative of Un8-mediated resistance to loose smut. Finally, profiling of 38 phytohormones revealed that larger seeds observed at some inoculum concentrations compared with mock inoculation had higher abscisic acid concentrations. This could represent a pathogen survival strategy by ensuring better growth of the host.

Study on the interaction between methyl jasmonate and the coiled-coil domain of rice blast resistance protein Pi36 by spectroscopic methods.

Interaction between the coiled-coil domain of rice blast resistance protein Pi36 and methyl-jasmonate (MeJA) was studied by fluorescence and UV-vis spectroscopic techniques. The quenching mechanism of fluorescence of MeJA by this domain was discussed to be a static quenching procedure. Fluorescence quenching was explored to measure the number of binding sites n and apparent binding constants K. The thermodynamics parameters ΔH, ΔG, ΔS were also calculated. The results indicate the binding reaction was not entropy-driven but enthalpy-driven, and hydrophobic binding played major role in the interaction. The binding sites of MeJA with the coiled-coil structural domain of rice blast resistance protein Pi36 were found to approach the microenvironment of both Tyr and Trp by the synchronous fluorescence spectrometry. The distance r between donor (the coiled-coil domain of rice blast resistance protein Pi36) and acceptor (MeJA) was obtained according to Förster theory of non-radioactive energy transfer.

The combination of 5-fluorouracil plus p53 pathway restoration is associated with depletion of p53-deficient or mutant p53-expressing putative colon cancer stem cells.

The cancer stem cell hypothesis suggests that rare populations of tumor-initiating cells may be resistant to therapy, lead to tumor relapse and contribute to poor prognosis for cancer patients. We previously demonstrated the feasibility of p53 pathway restoration in p53-deficient tumor cell populations using small molecules including ellipticine or its derivatives. We now establish a single cell p53-regulated green fluorescent protein (EGFP)-reporter system in human DLD1 colon tumor cells expressing mutant p53 protein. We use these p53-EGFP reporter DLD1 cells to investigate the status of p53 transcriptional activity in putative colon cancer stem cell populations following exposure to p53 pathway-restoring drugs and/or classical chemotherapy. We demonstrate induction of p53-specific EGFP reporter fluorescence following overexpression of p53 family member p73 by an Adenovirus vector. We further show that p53-reporter activity is induced in DLD1 putative cancer stem cell side-populations analyzed by their Hoechst dye efflux properties following treatment with the p53 pathway restoring drug ellipticine. Combination of ellipticine with the cytotoxic agent 5-fluorouracil resulted in increased cytotoxicity as compared to either agent alone and this was associated with depletion of putative cancer stem cell populations as compared with 5-FU alone treatment. Our results support the feasibility of therapeutic targeting of mutant p53 in putative cancer stem cells as well as the potential to enhance cytotoxic chemotherapy.