Faba bean root exudates alter pea root colonization by the oomycete Aphanomyces euteiches at early stages of infection.

Aphanomyces euteiches is an oomycete pathogen that causes the pea root rot. We investigated the potential role of early belowground defense in pea (susceptible plant) and faba bean (tolerant plant) at three days after inoculation. Pea and faba bean were inoculated with A. euteiches zoospores. Root colonization was examined. Root exudates from pea and faba bean were harvested and their impact on A. euteiches development were assessed by using in vitro assays. A. euteiches root colonization and the influence of the oomycete inoculation on specialized metabolites patterns and arabinogalactan protein (AGP) concentration of root exudates were also determined. In faba bean root, A. euteiches colonization was very low as compared with that of pea. Whereas infected pea root exudates have a positive chemotaxis index (CI) on zoospores, faba bean exudate CI was negative suggesting a repellent effect. While furanoacetylenic compounds were only detected in faba bean exudates, AGP concentration was specifically increased in pea.This work showed that early in the course of infection, host susceptibility to A. euteiches is involved via a plant-species specific root exudation opening new perspectives in pea root rot disease management.

A Sensitive Assay for Rapid Detection and Quantification of Aphanomyces euteiches in Soil.

Aphanomyces euteiches is a widespread oomycete pathogen causing root rot in a wide range of leguminous crops. Losses can reach up to 100% for pea culture and there is currently no registered pesticide for its control. Crop management remains the most efficient tool to control root rot, and avoidance of infested soil seems to be the optimal solution. A test was developed to identify fields suitable for pea crops, consisting of the determination of the inoculum potential of soil using baiting plants. A new rapid, specific, and sensitive molecular method is described allowing the quantification of less than 10 oospores per gram of soil. This challenge is achieved by a real-time polymerase chain reaction procedure targeting internal transcribed spacer 1 from the ribosomal DNA operons. A preliminary study based on typical soils from northwestern France demonstrated that the A. euteiches oospore density in soil is related to the inoculum potential. Furthermore, this method has proved sensitive enough to accurately study the influence of biotic factors that may govern the actual emergence of root rot.

Effect of arabinogalactan proteins from the root caps of pea and Brassica napus on Aphanomyces euteiches zoospore chemotaxis and germination.

Root tips of many plant species release a number of border, or border-like, cells that are thought to play a major role in the protection of root meristem. However, little is currently known on the structure and function of the cell wall components of such root cells. Here, we investigate the sugar composition of the cell wall of the root cap in two species: pea (Pisum sativum), which makes border cells, and Brassica napus, which makes border-like cells. We find that the cell walls are highly enriched in arabinose and galactose, two major residues of arabinogalactan proteins. We confirm the presence of arabinogalactan protein epitopes on root cap cell walls using immunofluorescence microscopy. We then focused on these proteoglycans by analyzing their carbohydrate moieties, linkages, and electrophoretic characteristics. The data reveal (1) significant structural differences between B. napus and pea root cap arabinogalactan proteins and (2) a cross-link between these proteoglycans and pectic polysaccharides. Finally, we assessed the impact of root cap arabinogalactan proteins on the behavior of zoospores of Aphanomyces euteiches, an oomycetous pathogen of pea roots. We find that although the arabinogalactan proteins of both species induce encystment and prevent germination, the effects of both species are similar. However, the arabinogalactan protein fraction from pea attracts zoospores far more effectively than that from B. napus. This suggests that root arabinogalactan proteins are involved in the control of early infection of roots and highlights a novel role for these proteoglycans in root-microbe interactions.

Association between border cell responses and localized root infection by pathogenic Aphanomyces euteiches.

BACKGROUND AND AIMS: The oomycete Aphanomyces euteiches causes up to 80 % crop loss in pea (Pisum sativum). Aphanomyces euteiches invades the root system leading to a complete arrest of root growth and ultimately to plant death. To date, disease control measures are limited to crop rotation and no resistant pea lines are available. The present study aims to get a deeper understanding of the early oomycete-plant interaction at the tissue and cellular levels. METHODS: Here, the process of root infection by A. euteiches on pea is investigated using flow cytometry and microscopic techniques. Dynamic changes in secondary metabolism are analysed with high-performance liquid chromatography with diode-array detection. KEY RESULTS: Root infection is initiated in the elongation zone but not in the root cap and border cells. Border-cell production is significantly enhanced in response to root inoculation with changes in their size and morphology. The stimulatory effect of A. euteiches on border-cell production is dependent on the number of oospores inoculated. Interestingly, border cells respond to pathogen challenge by increasing the synthesis of the phytoalexin pisatin. CONCLUSIONS: Distinctive responses to A. euteiches inoculation occur at the root tissue level. The findings suggest that root border cells in pea are involved in local defence of the root tip against A. euteiches. Root border cells constitute a convenient quantitative model to measure the molecular and cellular basis of plant-microbe interactions.

The inflammation-induced down-regulation of plasma Fetuin-A (alpha2HS-Glycoprotein) in liver results from the loss of interaction between long C/EBP isoforms at two neighbouring binding sites.

Fetuin-A is an hepatic protein whose mRNA transiently falls during the inflammatory acute phase via unknown transcriptional mechanisms. Various FETUA promoter/cat constructs transiently transfected in the Hep3B hepatoma cell line allowed us to identify four NF-1 and C/EBP binding sites (N, C) arranged in a 5'-N2-C2-N1-C1-3' order and required for basal promoter activity. Mutant constructs demonstrated that C1 and C2 but not N1 nor N2 are required for the cytokine-driven down-regulation of the promoter. A variable spacing between C2 and N1 showed that the alignment of the (C1+N1) and (C2+N2) areas is critical for the promoter activity in quiescent but not cytokine-stimulated cells. Co-transfection of a plasmid only producing either a long or short C/EBPbeta isoform prevented FETUA regulation by cytokines. Electromobility shift assays with liver nuclear extracts showed that during the acute phase the complexes formed over N1 and N2 are not modified whereas short C/EBPalpha and -beta isoforms replace the long isoforms bound to C1 and C2 in the quiescent liver. Therefore the basal promoter activity requires an interaction between the long C/EBP isoforms bound to C1 and C2 whereas the inflammation-induced down-regulation results from the loss of interaction between the cytokine-induced, short C/EBP isoforms.

Amino acid control of the human glyceraldehyde 3-phosphate dehydrogenase gene transcription in hepatocyte.

Glutamine (Gln) is the most potent of the amino acids (AAs) that regulate liver anabolism, and its effect is similar to that of insulin in peripheral tissues. However, the influence of AAs on regulation of metabolic enzyme-encoding genes is not known at the molecular level in liver. We now report that Gln and some essential AAs activate the human GAPDH gene that codes for GAPDH, a central enzyme of glycolysis and a target for insulin regulation. In HepG2 cells, Gln upregulated the GAPDH mRNA level, and this effect was additive to that of insulin. Transient transfection of GAPDH promoter/cat constructs demonstrated that a gene-specific and insulin-independent transcriptional step is involved in the Gln responsiveness of GAPDH. Transfected HepG2 cells challenged with various AAs, Gln metabolites or inhibitors of Gln metabolism showed that the Gln-induced effect is similar to that of some essential AAs and that Gln metabolism is a necessary step for GAPDH activation. Deletion mutants and site-directed mutagenesis of the GAPDH promoter indicated that the Gln responsiveness is mediated by a sequence that is distinct from insulin-responsive elements and from positively acting elements previously described in this promoter. This motif located at -126/-118 clearly differs from AA-responsive elements recently identified in other genes. Electromobility shift assay and supershifts showed that the transcription factors bound to the Gln-responsive element in the GAPDH promoter are C/EBPalpha and -delta. This finding is consistent with the role of C/EBP family members in controlling the hepatic expression of genes involved in nutrient metabolism.