Three Types of Elicitors Induce Grapevine Resistance against Downy Mildew via Common and Specific Immune Responses.

Three recognized plant defense stimulators (PDS), methyl jasmonate (MeJA), benzothiadiazole (BTH) and phosphonates (PHOS), were sprayed on grapevine Vitis vinifera cuttings and conferred resistance to the biotrophic pathogen Plasmopara viticola. The effects on molecular defense-related genes and polyphenol content (stilbenes and flavanols) were revealed at 6 and 8 days post-elicitation. The transcript accumulation was consistent with the signaling pathway specific to the elicitor, salicylic acid for BTH, and jasmonic acid for MeJA, with some cross-talks. PHOS tended to modulate the defense responses like BTH. Moreover, in response to a downy mildew inoculation, the leaves pre-treated with PHOS and BTH overproduced pterostilbene, and after MeJA treatment, piceids and ε-viniferin, compared to uninoculated elicitor-treated leaves. These results provide evidence of the different modes of action of PDS and their role in sustainable viticulture.

The Biocontrol Root-Oomycete, Pythium Oligandrum, Triggers Grapevine Resistance and Shifts in the Transcriptome of the Trunk Pathogenic Fungus, Phaeomoniella Chlamydospora.

The worldwide increase in grapevine trunk diseases, mainly esca, represents a major threat for vineyard sustainability. Biocontrol of a pioneer fungus of esca, Phaeomoniella chlamydospora, was investigated here by deciphering the tripartite interaction between this trunk-esca pathogen, grapevine and the biocontrol-oomycete, Pythium oligandrum. When P. oligandrum colonizes grapevine roots, it was observed that the wood necroses caused by P. chlamydospora were significantly reduced. Transcriptomic analyses of plant and fungus responses were performed to determine the molecular events occurring, with the aim to relate P.chlamydospora degradation of wood to gene expression modulation. Following P. oligandrum-root colonization, major transcriptomic changes occurred both, in the grapevine-defense system and in the P. chlamydospore-virulence factors. Grapevine-defense was enhanced in response to P. chlamydospora attacks, with P. oligandrum acting as a plant-systemic resistance inducer, promoting jasmonic/ethylene signaling pathways and grapevine priming. P. chlamydospora pathogenicity genes, such as those related to secondary metabolite biosynthesis, carbohydrate-active enzymes and transcription regulators, were also affected in their expression. Shifts in grapevine responses and key-fungal functions were associated with the reduction of P. chlamydospora wood necroses. This study provides evidence of wood fungal pathogen transcriptional changes induced by a root biocontrol agent, P. oligandrum, in which there is no contact between the two microorganisms.

High-throughput gene-expression quantification of grapevine defense responses in the field using microfluidic dynamic arrays.

BACKGROUND: The fight against grapevine diseases due to biotrophic pathogens usually requires the massive use of chemical fungicides with harmful environmental effects. An alternative strategy could be the use of compounds able to stimulate plant immune responses which significantly limit the development of pathogens in laboratory conditions. However, the efficiency of this strategy in natura is still insufficient to be included in pest management programs. To understand and to improve the mode of action of plant defense stimulators in the field, it is essential to develop reliable tools that describe the resistance status of the plant upon treatment. RESULTS: We have developed a pioneering tool ("NeoViGen96" chip) based on a microfluidic dynamic array platform allowing the expression profiling of 85 defense-related grapevine genes in 90 cDNA preparations in a 4 h single run. Two defense inducers, benzothiadiazole (BTH) and fosetyl-aluminum (FOS), have been tested in natura using the "NeoViGen96" chip as well as their efficacy against downy mildew. BTH-induced grapevine resistance is accompanied by the induction of PR protein genes (PR1, PR2 and PR3), genes coding key enzymes in the phenylpropanoid pathway (PAL and STS), a GST gene coding an enzyme involved in the redox status and an ACC gene involved in the ethylene pathway. FOS, a phosphonate known to possess a toxic activity against pathogens and an inducing effect on defense genes provided a better grapevine protection than BTH. Its mode of action was probably strictly due to its fungicide effect at high concentrations because treatment did not induce significant change in the expression level of selected defense-related genes. CONCLUSIONS: The NeoViGen96" chip assesses the effectiveness of plant defense inducers on grapevine in vineyard with an excellent reproducibility. A single run with this system (4 h and 1,500 €), corresponds to 180 qPCR plates with conventional Q-PCR assays (Stragene system, 270 h and 9,000 €) thus a throughput 60-70 times higher and 6 times cheaper. Grapevine responses after BTH elicitation in the vineyard were similar to those obtained in laboratory conditions, whereas our results suggest that the protective effect of FOS against downy mildew in the vineyard was only due to its fungicide activity since no activity on plant defense genes was observed. This tool provides better understanding of how the grapevine replies to elicitation in its natural environment and how the elicitor potential can be used to reduce chemical fungicide inputs.

Inositol-requiring enzyme 1alpha is a key regulator of angiogenesis and invasion in malignant glioma.

Inositol-requiring enzyme 1 (IRE1) is a proximal endoplasmic reticulum (ER) stress sensor and a central mediator of the unfolded protein response. In a human glioma model, inhibition of IRE1alpha correlated with down-regulation of prevalent proangiogenic factors such as VEGF-A, IL-1beta, IL-6, and IL-8. Significant up-regulation of antiangiogenic gene transcripts was also apparent. These transcripts encode SPARC, decorin, thrombospondin-1, and other matrix proteins functionally linked to mesenchymal differentiation and glioma invasiveness. In vivo, using both the chick chorio-allantoic membrane assay and a mouse orthotopic brain model, we observed in tumors underexpressing IRE1: (i) reduction of angiogenesis and blood perfusion, (ii) a decreased growth rate, and (iii) extensive invasiveness and blood vessel cooption. This phenotypic change was consistently associated with increased overall survival in glioma-implanted recipient mice. Ectopic expression of IL-6 in IRE1-deficient tumors restored angiogenesis and neutralized vessel cooption but did not reverse the mesenchymal/infiltrative cell phenotype. The ischemia-responsive IRE1 protein is thus identified as a key regulator of tumor neovascularization and invasiveness.

Variability and recombination of clinical human cytomegalovirus strains from transplantation recipients.

BACKGROUND: Human cytomegalovirus (HCMV) is the first cause of viral infection in immunocompromised transplanted patients. OBJECTIVES: Here, five HCMV genes were studied to investigate the existence of recombination events in clinical strains ex vivo. STUDY DESIGN: Sequencing and phylogenetic analysis were conducted on 21 strains from 16 renal and 5 lung transplant recipients. RESULTS: Nucleotidic polymorphism ranged from 6.6% (US3) to 12% (UL40), with a significant proportion of missense mutations (39-69%), some of which could have a functional impact. Analysis of the concatenated sequence (4804 nucleotides for each strain) evidenced two clusters of sequences presenting a reticulate topology suggestive of recombination events (SplitsTree). Phi-test pointed numerous phylogenetically conflicting signals indicating a high statistical probability of recombination. The subsequent bootscan analysis was consistent with these data. CONCLUSIONS: These results reinforce the prominent role of recombination in HCMV evolutionary history and adaptation to its host.

UL40 human cytomegalovirus variability evolution patterns over time in renal transplant recipients.

BACKGROUND: Human cytomegalovirus (HCMV) is the most common opportunistic pathogen infecting immunocompromised patients after transplantation. Although its immunomodulatory capacities and genomic variability participate in immune system evasion, they are poorly studied in clinical strains without culture amplification. One of HCMV immunomodulatory genes, UL40, confers HCMV-infected cells' protection from natural killer-mediated lysis through its encoded nonapeptide presented in the context of human leukocyte antigen-E. METHODS: In three renal transplant recipients with different HCMV serostatus, we aimed to evidence the co-evolution of mixtures of HCMV variants over time with sequencing and cloning of HCMV UL40 gene. RESULTS: Six months after renal transplantation in patient 1Bx, D+/R+, UL40 phylogenetic and bootscan analysis suggested the emergence of a recombination between two previous viral strains. In patient 8Bx, initially D+/R-, distribution of variants in five samples over 43 months was notably stable, with no visible emerging variants despite two renal engraftments and extended episodes of active infection. In patient 9Bx, also D+/R-, phylogenetic tree of viral variants revealed in the first sample a minor clone, confirmed by a specific polymerase chain reaction, related to the three subsequent dominant clones. CONCLUSIONS: In three HCMV-infected renal transplant recipients, we have evidenced different viral evolutive polymorphisms including point mutations, recombination, and occasionally suggesting the intervention of several HCMV strains or a quasispecies-like distribution. This variability could contribute to viral adaptability in pathogenesis.

Variability of UL18, UL40, UL111a and US3 immunomodulatory genes among human cytomegalovirus clinical isolates from renal transplant recipients.

BACKGROUND: Variability of human cytomegalovirus (HCMV) genes counteracting immune responses is poorly investigated in non-cultured clinical strains. OBJECTIVES: In HCMV-infected renal graft recipients, we aimed to (i) investigate the variability of four HCMV immunomodulatory genes, without any culture-related viral selection, (ii) provide evolutionary sequence data, and (iii) study co-existing HCMV variants and their evolution. STUDY DESIGN: UL18, UL40, UL111a and US3 were sequenced in 31 blood samples from 17 patients (8 with sequential samples). Cloning of UL40 PCR products was performed in one donor-positive/recipient-positive (D+/R+) patient's samples. RESULTS: Each patient harboured a unique strain (combination of four genes), however single identical genes were demonstrated among various patients, suggesting recombination events. Sequencing showed in D+/R- recipients, either complete gene stability (four patients) or significant variability (one patient); in three D+/R+ patients, multiple gene variations, possibly linked to super- or co-infections. Cloning evidenced different variants at each time point with an increasing variability over time, illustrating possibly viral reactivations and the subsequent evolution of the variants mixture. CONCLUSION: A noticeable HCMV natural polymorphism was shown, with different evolutive patterns. Moreover, we described the co-evolution of variants mixtures in one patient. Consequences on HCMV infection and graft function deserve further studying.

Resistance mutations in subtype C HIV type 1 isolates from Indian patients of Mumbai receiving NRTIs plus NNRTIs and experiencing a treatment failure: resistance to AR.

We present here the first data available on resistance to nucleoside reverse transcriptase inhibitors (NRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs) in India. In these subtype C isolates, we have observed most of the mutations noted in reverse transcriptase (RT) for subtype B with some additional substitutions (at positions 98, 203, 208, and 221) that will warrant attention in the algorithms used.

Photosynthetic and other phosphoenolpyruvate carboxylase isoforms in the single-cell, facultative C(4) system of Hydrilla verticillata.

The submersed monocot Hydrilla verticillata (L.f.) Royle is a facultative C(4) plant. It typically exhibits C(3) photosynthetic characteristics, but exposure to low [CO(2)] induces a C(4) system in which the C(4) and Calvin cycles co-exist in the same cell and the initial fixation in the light is catalyzed by phosphoenolpyruvate carboxylase (PEPC). Three full-length cDNAs encoding PEPC were isolated from H. verticillata, two from leaves and one from root. The sequences were 95% to 99% identical and shared a 75% to 85% similarity with other plant PEPCs. Transcript studies revealed that one isoform, Hvpepc4, was exclusively expressed in leaves during C(4) induction. This and enzyme kinetic data were consistent with it being the C(4) photosynthesis isoform. However, the C(4) signature serine of terrestrial plant C(4) isoforms was absent in this and the other H. verticillata sequences. Instead, alanine, typical of C(3) sequences, was present. Western analyses of C(3) and C(4) leaf extracts after anion-exchange chromatography showed similar dominant PEPC-specific bands at 110 kD. In phylogenetic analyses, the sequences grouped with C(3), non-graminaceous C(4), and Crassulacean acid metabolism PEPCs but not with the graminaceous C(4), and formed a clade with a gymnosperm, which is consistent with H. verticillata PEPC predating that of other C(4) angiosperms.