Changes in blood perfusion and hypoxia after irradiation of a human squamous cell carcinoma xenograft tumor line

The effect of irradiation depends on the oxygenation status of the tissue, while irradiation itself also changes the oxygenation and perfusion status of tissues. A better understanding of the changes in tumor oxygenation and perfusion over time after irradiation will allow a better planning of fractionated radiotherapy in combination with modifiers of blood flow and oxygenation. Vascular architecture (endothelial marker), perfusion (Hoechst 33342) and oxygenation (pimonidazole) were studied in a human laryngeal squamous cell carcinoma tumor line grown as xenografts in nude mice. The effect of a single dose of 10 Gy X rays on these parameters was evaluated from 2 h to 11 days after irradiation. Shortly after irradiation, there was an 8% increase in perfused blood vessels (from 57% to 65%) followed by a significant decrease, with a minimum value of 42% at 26 h after irradiation, and a subsequent increase to control levels at 7 to 11 days after irradiation. The hypoxic fraction showed a decrease at 7 h after treatment from 13% to 5% with an increase to 19% at 11 days after irradiation. These experiments show that irradiation causes rapid changes in oxygenation and perfusion which may have consequences for the optimal timing of radiotherapy schedules employing multiple fractions per day and the introduction of oxygenation- and perfusion-modifying drugs.     

The Compromised Recognition of Turnip Crinkle Virus1 Subfamily of Microrchidia ATPases Regulates Disease Resistance in Barley to Biotrophic and Necrotrophic Pathogens

MORC1 and MORC2, two of the seven members of the Arabidopsis (Arabidopsis thaliana) Compromised Recognition of Turnip Crinkle Virus1 subfamily of microrchidia Gyrase, Heat Shock Protein90, Histidine Kinase, MutL (GHKL) ATPases, were previously shown to be required in multiple layers of plant immunity. Here, we show that the barley (Hordeum vulgare) MORCs also are involved in disease resistance. Genome-wide analyses identified five MORCs that are 37% to 48% identical on the protein level to AtMORC1. Unexpectedly, and in clear contrast to Arabidopsis, RNA interference-mediated knockdown of MORC in barley resulted in enhanced basal resistance and effector-triggered, powdery mildew resistance locus A12-mediated resistance against the biotrophic powdery mildew fungus (Blumeria graminis f. sp. hordei), while MORC overexpression decreased resistance. Moreover, barley knockdown mutants also showed higher resistance to Fusarium graminearum. Barley MORCs, like their Arabidopsis homologs, contain the highly conserved GHKL ATPase and S5 domains, which identify them as members of the MORC superfamily. Like AtMORC1, barley MORC1 (HvMORC1) binds DNA and has Mn²⁺-dependent endonuclease activities, suggesting that the contrasting function of MORC1 homologs in barley versus Arabidopsis is not due to differences in their enzyme activities. In contrast to AtMORCs, which are involved in silencing of transposons that are largely restricted to pericentromeric regions, barley MORC mutants did not show a loss-of-transposon silencing regardless of their genomic location. Reciprocal overexpression of MORC1 homologs in barley and Arabidopsis showed that AtMORC1 and HvMORC1 could not restore each other’s function. Together, these results suggest that MORC proteins function as modulators of immunity, which can act negatively (barley) or positively (Arabidopsis) dependent on the species.The evolution of a complex defense system has been the consequence of plants being constantly exposed to pathogenic microbes and pests. One of the first lines of active defense is based on a perception of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors located in the plant cell membrane. The defense response to PAMP recognition is called PAMP-triggered immunity (PTI). While PTI is sufficient to stop colonization by many microbes, some microorganisms overcome this immune response by releasing effectors (formerly called virulence factors). In a coevolutionary process, some plants have evolved resistance (R) proteins for direct or indirect recognition of microbial effectors (avirulence [Avr] factors) leading to effector-triggered immunity (ETI). ETI is frequently characterized by a rapid and locally restricted programmed cell death response (also known as hypersensitive reaction [HR]), which helps to limit pathogen proliferation and disease symptoms. On the contrary, the absence of an Avr-R protein interaction results in virulence of the pathogen. In addition, ETI is counteracted by some microbes by the release of additional virulence factors that block or overcome effector recognition and ensure pathogenicity. The mutual evolution of host and microbe leading to elicitation or suppression of ETI is summarized by the “zigzag” model proposed by Jones and Dangl (2006). PTI and ETI are associated with activation of various defense responses both at infection sites and in distal tissue, including production and accumulation of reactive oxygen species, salicylic acid, and pathogenesis-related proteins. Systemic activation of such responses, triggered in the uninfected tissue, leads to long-lasting, broad-based resistance to subsequent pathogen infections, termed systemic acquired resistance.A genetic screen in Arabidopsis (Arabidopsis thaliana) searching for mutants with compromised resistance mediated by the R protein HR to Turnip Crinkle Virus (HRT) against Turnip Crinkle Virus (TCV) led to the discovery of the Compromised Recognition of TCV1 (CRT1) subfamily of the microrchidia (MORC) subclade of the GHKL (for Gyrase, Heat Shock Protein90, Histidine Kinase, MutL) ATPase superfamily (Watson et al., 1998; Iyer et al., 2008; Kang et al., 2008). Genome analysis of Arabidopsis revealed that MORC1 (formerly named CRT1 in Kang et al., 2008, 2010, 2012) has two close (>70% sequence similarity on amino acid [aa] level) and four distant (<50% aa similarity) homologs. A double knockout mutant, morc1-2 morc2-1, lacking MORC1 and its closest homolog MORC2 also displayed compromised ETI to avirulent Pseudomonas syringae, suppressed basal resistance, systemic acquired resistance, and/or PTI to TCV and virulent P. syringae and compromised nonhost resistance to Phytophthora infestans (Kang et al., 2012). Arabidopsis MORC1 physically interacts with at least eleven R proteins belonging to three different structural classes (Martin et al., 2003), including HRT, the R protein involved in recognition of TCV. This interaction is a dynamic process, as MORC1 bound inactive R proteins, while little or no interaction was observed when the R proteins were activated (Kang et al., 2010). Taken together, these results argued that MORC1 protein family members in Arabidopsis are key components in multiple layers of resistance against a variety of pathogens. Recently, it was shown that a small fraction of AtMORC1 translocates to the plant nucleus after ETI and PTI activation (Kang et al., 2012). Because Arabidopsis MORC1 possesses DNA/RNA-binding capacity and endonuclease activity in vitro, these findings suggest a potential role in DNA recombination and repair (Kang et al., 2012). In addition, three recent independent studies identified Arabidopsis MORC1 and its homolog MORC6 (also named Defective in Meristem Silencing11) as novel factors involved in gene silencing and/or chromatin superstructure remodeling in response to epigenetic signals (Lorković et al., 2012; Moissiard et al., 2012; Brabbs et al., 2013).Given that the CRT1 subfamily of MORC ATPases is involved in multiple layers of disease resistance against various pathogens, these genes may have relevance for agronomic applications. To assess whether MORCs are involved in crop plant resistance and thus could be exploited in breeding strategies, MORC1 homologous genes were identified in the model cereal crop barley (Hordeum vulgare). We show here that all five barley MORCs, discovered in the not yet fully annotated barley genome, are involved in resistance to agronomically important diseases. Unexpectedly, however, and in clear contrast to Arabidopsis, barley plants silenced for MORC genes were more resistant, while overexpression compromised resistance to infections by both biotrophic and necrotrophic fungal pathogens. Moreover, reciprocal overexpression in Arabidopsis and barley showed that AtMORC1 and HvMORC1 homologs are not functionally interchangeable.     

Piriformospora indica-a mutualistic basidiomycete with an exceptionally large plant host range

Piriformospora indica is a basidiomycete of the order Sebacinales, representing a model for the study of mutualistic symbiosis and, beyond that, the plant immune system. The fungus colonizes the roots of a wide range of vascular plants, increasing their growth, seed yield and adaptation to abiotic and biotic stresses. The fungal colonization of roots begins with a biotrophic growth phase, in which living cells are colonized, and continues with a cell death-dependent phase, in which root cells are actively killed by the fungus. The complexity of sebacinalean symbiosis is further enhanced by the presence of endocellular bacteria which may represent significant determinants for a successful outcome of the symbioses. Molecular ecological analyses have revealed an exceptional relevance of sebacinoid fungi in natural ecosystems worldwide. This natural competence could be rooted in their phenotypic adaptability, which, for instance, allows P. indica to grow readily on various synthetic media and to colonize distinct hosts. In molecular and genetic studies, P. indica's mutualistic colonization strategy has been partly unravelled, showing that the jasmonate pathway is exploited for immune suppression and successful development in roots. Research on P. indica supports efforts to make the bioprotective potential of the fungus accessible for agricultural plant production. The decoding of P. indica's genome has revealed its potential for application as bioagent and for targeted improvement of crop plants in biotechnology-based approaches.     

Late effects of radiation on the central nervous system: role of vascular endothelial damage and glial stem cell survival

Selective irradiation of the vasculature of the rat spinal cord was used in this study, which was designed specifically to address the question as to whether it is the endothelial cell or the glial progenitor cell that is the target responsible for late white matter necrosis in the CNS. Selective irradiation of the vascular endothelium was achieved by the intraperitoneal (ip) administration of a boron compound known as BSH (Na(2)B(12)H(11)SH), followed by local irradiation with thermal neutrons. The blood-brain barrier is known to exclude BSH from the CNS parenchyma. Thirty minutes after the ip injection of BSH, the boron concentration in blood was 100 microg (10)B/ g, while that in the CNS parenchyma was below the detection limit of the boron analysis system, <1 microg (10)B/g. An ex vivo clonogenic assay of the O2A (oligodendrocyte-type 2 astrocyte) glial progenitor cell survival was performed 1 week after irradiation and at various times during the latent period before white matter necrosis in the spinal cord resulted in myelopathy. One week after 4.5 Gy of thermal neutron irradiation alone (approximately one-third of the dose required to produce a 50% incidence of radiation myelopathy), the average glial progenitor cell surviving fraction was 0.03. The surviving fraction of glial progenitor cells after a thermal neutron irradiation with BSH for a comparable effect was 0.46. The high level of glial progenitor cell survival after irradiation in the presence of BSH clearly reflects the lower dose delivered to the parenchyma due to the complete exclusion of BSH by the blood-brain barrier. The intermediate response of glial progenitor cells after irradiation with thermal neutrons in the presence of a boron compound known as BPA (p-dihydroxyboryl-phenylalanine), again for a dose that represents one-third the ED(50) for radiation-induced myelopathy, reflects the differential partition of boron-10 between blood and CNS parenchyma for this compound, which crosses the blood-brain barrier, at the time of irradiation. The large differences in glial progenitor survival seen 1 week after irradiation were also maintained during the 4-5-month latent period before the development of radiation myelopathy, due to selective white matter necrosis, after irradiation with doses that would produce a high incidence of radiation myelopathy. Glial progenitor survival was similar to control values at 100 days after irradiation with a dose of thermal neutrons in the presence of BSH, significantly greater than the ED(100), shortly before the normal time of onset of myelopathy. In contrast, glial progenitor survival was less than 1% of control levels after irradiation with 15 Gy of thermal neutrons alone. This dose of thermal neutrons represents the approximate ED(90-100) for myelopathy. The response to irradiation with an equivalent dose of X rays (ED(90): 23 Gy) was intermediate between these extremes as it was to thermal neutrons in the presence of BPA at a slightly lower dose equivalent to the approximate ED(60) for radiation myelopathy. The conclusions from these studies, performed at dose levels approximately iso-effective for radiation-induced myelopathy as a consequence of white matter necrosis, were that the large differences observed in glial progenitor survival were directly related to the dose distribution in the parenchyma. These observations clearly indicate the relative importance of the dose to the vascular endothelium as the primary event leading to white matter necrosis.     

Salt tolerance of barley induced by the root endophyte Piriformospora indica is associated with a strong increase in antioxidants

The root endophytic basidiomycete Piriformospora indica has been shown to increase resistance against biotic stress and tolerance to abiotic stress in many plants. Biochemical mechanisms underlying P. indica-mediated salt tolerance were studied in barley (Hordeum vulgare) with special focus on antioxidants. Physiological markers for salt stress, such as metabolic activity, fatty acid composition, lipid peroxidation, ascorbate concentration and activities of catalase, ascorbate peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase and glutathione reductase enzymes were assessed. Root colonization by P. indica increased plant growth and attenuated the NaCl-induced lipid peroxidation, metabolic heat efflux and fatty acid desaturation in leaves of the salt-sensitive barley cultivar Ingrid. The endophyte significantly elevated the amount of ascorbic acid and increased the activities of antioxidant enzymes in barley roots under salt stress conditions. Likewise, a sustained up-regulation of the antioxidative system was demonstrated in NaCl-treated roots of the salt-tolerant barley cultivar California Mariout, irrespective of plant colonization by P. indica. These findings suggest that antioxidants might play a role in both inherited and endophyte-mediated plant tolerance to salinity.     

Nitric oxide generation in Vicia faba phloem cells reveals them to be sensitive detectors as well as possible systemic transducers of stress signals

Vascular tissue was recently shown to be capable of producing nitric oxide (NO), but the production sites and sources were not precisely determined. Here, NO synthesis was analysed in the phloem of Vicia faba in response to stress- and pathogen defence-related compounds. The chemical stimuli were added to shallow paradermal cortical cuts in the main veins of leaves attached to intact plants. NO production in the bare-lying phloem area was visualized by real-time confocal laser scanning microscopy using the NO-specific fluorochrome 4,5-diaminofluorescein diacetate (DAF-2 DA). Abundant NO generation in companion cells was induced by 500 microm salicylic acid (SA) and 10 microm hydrogen peroxide (H(2)O(2)), but the fungal elicitor chitooctaose was much less effective. Phloem NO production was found to be dependent on Ca(2+) and mitochondrial electron transport and pharmacological approaches found evidence for activity of a plant NO synthase but not a nitrate reductase. DAF fluorescence increased most strongly in companion cells and was occasionally observed in phloem parenchyma cells. Significantly, accumulation of NO in sieve elements could be demonstrated. These findings suggest that the phloem perceives and produces stress-related signals and that one mechanism of distal signalling involves the production and transport of NO in the phloem.     

Systemic resistance in Arabidopsis conferred by the mycorrhizal fungus Piriformospora indica requires jasmonic acid signaling and the cytoplasmic function of NPR1

We analyzed the requirement of specific defense pathways for powdery mildew (Golovinomyces orontii) resistance induced by the basidiomycete Piriformospora indica in Arabidopsis. Piriformospora indica root colonization reduced G. orontii conidia in wild-type (Col-0), npr1-3 (nonexpressor of PR genes 1-3) and NahG plants, but not in the npr1-1 null mutant. Therefore, cytoplasmic but not nuclear localization of NPR1 is required for P. indica-induced resistance. Two jasmonate signaling mutants were non-responsive to P. indica, and jasmonic acid-responsive vegetative storage protein expression was primed and thus elevated in response to powdery mildew, suggesting that P. indica confers resistance reminiscent of induced systemic resistance (ISR).