High-Precision Radiosurgical Dose Delivery by Interlaced Microbeam Arrays of High-Flux Low-Energy Synchrotron X-Rays

Microbeam Radiation Therapy (MRT) is a preclinical form of radiosurgery dedicated to brain tumor treatment. It uses micrometer-wide synchrotron-generated X-ray beams on the basis of spatial beam fractionation. Due to the radioresistance of normal brain vasculature to MRT, a continuous blood supply can be maintained which would in part explain the surprising tolerance of normal tissues to very high radiation doses (hundreds of Gy). Based on this well described normal tissue sparing effect of microplanar beams, we developed a new irradiation geometry which allows the delivery of a high uniform dose deposition at a given brain target whereas surrounding normal tissues are irradiated by well tolerated parallel microbeams only. Normal rat brains were exposed to 4 focally interlaced arrays of 10 microplanar beams (52 µm wide, spaced 200 µm on-center, 50 to 350 keV in energy range), targeted from 4 different ports, with a peak entrance dose of 200Gy each, to deliver an homogenous dose to a target volume of 7 mm3 in the caudate nucleus. Magnetic resonance imaging follow-up of rats showed a highly localized increase in blood vessel permeability, starting 1 week after irradiation. Contrast agent diffusion was confined to the target volume and was still observed 1 month after irradiation, along with histopathological changes, including damaged blood vessels. No changes in vessel permeability were detected in the normal brain tissue surrounding the target. The interlacing radiation-induced reduction of spontaneous seizures of epileptic rats illustrated the potential pre-clinical applications of this new irradiation geometry. Finally, Monte Carlo simulations performed on a human-sized head phantom suggested that synchrotron photons can be used for human radiosurgical applications. Our data show that interlaced microbeam irradiation allows a high homogeneous dose deposition in a brain target and leads to a confined tissue necrosis while sparing surrounding tissues. The use of synchrotron-generated X-rays enables delivery of high doses for destruction of small focal regions in human brains, with sharper dose fall-offs than those described in any other conventional radiation therapy

Etude des interactions riz-Magnaporthe grisea (Caractérisation et clonage du gène de résistance Pi33)

MONTPELLIER-SupAgro La Gaillarde (341722306) / SudocSudocFranceF

Translocation of Magnaporthe oryzae effectors into rice cells and their subsequent cell-to-cell movement

Citation: Khang, C. & Berruyer, R. (2010). Translocation of Magnaporthe oryzae Effectors into Rice Cells and Their Subsequent Cell-to-Cell Movement. The Plant Cell, 22(4), 1388-1403. https://doi.org/10.1105/tpc.109.069666Knowledge remains limited about how fungal pathogens that colonize living plant cells translocate effector proteins inside host cells to regulate cellular processes and neutralize defense responses. To cause the globally important rice blast disease, specialized invasive hyphae (IH) invade successive living rice (Oryza sativa) cells while enclosed in host-derived extrainvasive hyphal membrane. Using live-cell imaging, we identified a highly localized structure, the biotrophic interfacial complex (BIC), which accumulates fluorescently labeled effectors secreted by IH. In each newly entered rice cell, effectors were first secreted into BICs at the tips of the initially filamentous hyphae in the cell. These tip BICs were left behind beside the first-differentiated bulbous IH cells as the fungus continued to colonize the host cell. Fluorescence recovery after photobleaching experiments showed that the effector protein PWL2 (for prevents pathogenicity toward weeping lovegrass [Eragrostis curvula]) continued to accumulate in BICs after IH were growing elsewhere. PWL2 and BAS1 (for biotrophy-associated secreted protein 1), BIC-localized secreted proteins, were translocated into the rice cytoplasm. By contrast, BAS4, which uniformly outlines the IH, was not translocated into the host cytoplasm. Fluorescent PWL2 and BAS1 proteins that reached the rice cytoplasm moved into uninvaded neighbors, presumably preparing host cells before invasion. We report robust assays for elucidating the molecular mechanisms that underpin effector secretion into BICs, translocation to the rice cytoplasm, and cell-to-cell movement in rice

Cloning of Pi33, the rice resistance gene corresponding to the cloned avirulence gene ACE1 of Magnaporthe grisea

National audienceFew reports on the distribution of resistance genes to plant pathogens within a species are available. Because of its genetic structuration, Oryza sativa can be an interesting model for such a study. We were interested in the interaction between the resistance gene to blast Pi33 and its corresponding avirulence gene ACE1. Although Pi33 was not cloned, its allele(s) conferring resistance can be identified by inoculation of pairs of isogenic strains of Magnaporthe grisea differing only for ACE1. One-hundred-eighty-three varieties of O. sativa were inoculated with isogenic strains. Twenty-three varieties of the Indica subspecies (mainly modem semi-dwarf varieties) carrying a resistance allele of Pi33 were identified representing 21,6 percent of the Indica varieties tested. None of the 45 Japonica varieties tested carried the resistance allele. In order to identify the original donors of Pi33, the genealogies of the varieties carrying Pi33 were examined. In addition, 28 parental lines from IR64 were tested to s ek the donor of resistance for this variety. Two possible origins of resistance could be identified: Tsai Yuan Chung and the wild rice O. rufipogon. These two candidates were confirmed as possible sources of resistance by genotyping and sequencing. It appears that both resistance sources were commonly used in breeding programmes leading to modem semi-dwarf Indica varieties. The presence of Pi33 in O. rufipogon led us to the hypothesis that Pi33 existed before domestication. This hypothesis was confirmed by the detection of Pi33 in O. latifolia (CCDD genome), O. barthii (AA) and diverse accessions of O. rufipogon (AA)

Blast Interfacial Complex, a novel in planta structure that accumulates effector proteins of rice blast fungus Magnaporthe oryzae

International audienceThe hemibiotrophic fungus Magnaporthe oryzae causes the devastating rice blast disease using specialized intracellular invasive hyphae (IH) that successively invade living plant cells. IH differentiate from thin filamentous primary hyphae that form immediately af ter the appressorial penetration peg breaches the exterior plant surface. At this point, IH become enclosed in a generally tight-fitting, plant-derived Extra-Invasive Hyphal Membrane (EIHM). Blast effector proteins are secreted from IH to manipulate host responses, but the route of their delive ry across the EIHM to reach the host cytoplasm is not understood. Here we show that fusion proteins between blast effectors, AVR-Pita and PWL, and enhanced green fluorescent protein (EGFP) are secreted by the fungus into a previously unrecognized structure, the Blast Interfacial Complex (BIC). Live cell imaging showed that fusion proteins first appeared in primary BICs at the growing tips of primary hyphae, in the previously reported EIHM caps. At the point where primary hyphae differentiated into bulbous IH, the fluorescent BICs moved to the sides of enlarging IH and remained fluorescent at these same locations as long as IH grew within the cell. When the fungus moved into neighboring cells, fluorescence accumulated in secondary BICs at the tips of filamentous IH that grew immediately after crossing the plan t cell wall. Again, the fluorescent tip structures moved to the side when the thin hyphae thickened into IH. BICs reside between the fungal cell wall and the EIHM in dynamic association with plant cytoplasm as IH begin to grow. Correlative light and electron microscopy showed that BICs contained complex lamellar membranes and vesicles. BIC localization requires the N-terminal signal peptide of these effector proteins. We hypothesize that BI Cs are involved in delivering fungal effectors proteins inside plant cells

Mesure de la biomasse fongique in planta d'Alternaria dauci et description du cycle infectieux en fonction du niveau de résistance de la carotte.

National audienc

Aldaulactone – an original phytotoxic secondary metabolite involved in the aggressiveness of Alternaria dauci on carrot

Qualitative plant resistance mechanisms and pathogen virulence have been extensively studied since the formulation of the gene-for-gene hypothesis. The mechanisms involved in the quantitative traits of aggressiveness and plant partial resistance are less well-known. Nevertheless, they are prevalent in most plant-necrotrophic pathogen interactions, including the Daucus carota–Alternaria dauci interaction. Phytotoxic metabolite production by the pathogen plays a key role in aggressiveness in these interactions. The aim of the present study was to explore the link between A. dauci aggressiveness and toxin production. We challenged carrot embryogenic cell cultures from a susceptible genotype (H1) and two partially resistant genotypes (I2 and K3) with exudates from A. dauci strains with various aggressiveness levels. Interestingly, A. dauci-resistant carrot genotypes were only affected by exudates from the most aggressive strain in our study (ITA002). Our results highlight a positive link between A. dauci aggressiveness and the fungal exudate cell toxicity. We hypothesize that the fungal exudate toxicity was linked with the amount of toxic compounds produced by the fungus. Interestingly, organic exudate production by the fungus was correlated with aggressiveness. Hence, we further analyzed the fungal organic extract using HPLC, and correlations between the observed peak intensities and fungal aggressiveness were measured. One observed peak was closely correlated with fungal aggressiveness. We succeeded in purifying this peak and NMR analysis revealed that the purified compound was a novel 10-membered benzenediol lactone, a polyketid that we named ‘aldaulactone’.  We used a new automated image analysis method and found that aldaulactone was toxic to in vitro cultured plant cells at those concentrations. The effects of both aldaulactone and fungal organic extracts were weaker on I2-resistant carrot cells compared to H1 carrot cells. Taken together, our results suggest that: (i) aldaulactone is a new phytotoxin, (ii) there is a relationship between the amount of aldaulactone produced and fungal aggressiveness, and (iii) carrot resistance to A. dauci involves mechanisms of resistance to aldaulactone