Selection of miRNA reference genes for plant defence studies in rice (Oryza sativa)

RT-qPCR is a widely used method to investigate the expression levels of genes under certain conditions. A key step, however, to have reliable results is the normalization of expression. For every experimental condition, suitable reference genes must be chosen. These reference genes must not be affected by differences in experimental conditions. MicroRNAs are regulatory RNA molecules, able to direct the expression levels of protein coding genes. In plants, their attributed functions range from roles in development to immunity. In this work, microRNAs (miRNAs) are evaluated for their suitability as reference genes in rice after infection with root-knot nematode Meloidogyne graminicola or after priming with beta-amino butyric acid. The evaluation was based on their amplification efficiency and their stability estimates according to geNorm, NormFinder and BestKeeper. All tested miRNAs, excluding one, were considered acceptable for normalization. Furthermore, miRNAs were validated using miRNA sequencing data. The set of microRNAs miR390-5p and miR7694-3p was found to be the most stable combination under the tested conditions. Another miRNA set consisting of miR7694-3p, miR1868 and miR1849 also shows potential to be used for miRNA expression normalization under experimental conditions beyond the scope of this study. This work is the first report on reference miRNAs in rice for the purpose of plant defence studies

Genome-wide shifts in histone modifications at early stage of rice infection with Meloidogyne graminicola

Epigenetic processes play a crucial role in the regulation of plant stress responses, but their role in plant-pathogen interactions remains poorly understood. Although histone-modifying enzymes have been observed to be deregulated in galls induced by root-knot nematodes (RKN, Meloidogyne graminicola) in rice, their influence on plant defence and their genome-wide impact has not been comprehensively investigated. First, the role of histone modifications in plant-nematode interactions was confirmed by pharmacological inhibition of histone-modifying enzymes, which all significantly affected rice susceptibility to RKN. For a more specific view, three histone marks, H3K9ac, H3K9me2, and H3K27me3, were subsequently studied by chromatin-immunoprecipitation-sequencing on RKN-induced galls at 3 days postinoculation. While levels of H3K9ac and H3K27me3 were strongly enriched, H3K9me2 was generally depleted in galls versus control root tips. Differential histone peaks were generally associated with plant defence-related genes. Transcriptome analysis using RNA-Seq and RT-qPCR-based validation revealed that genes marked with H3K9ac or H3K9me2 showed the expected activation or repression gene expression pattern, but this was not the case for H3K27me3 marks. Our results indicate that histone modifications respond dynamically to RKN infection, and that posttranslational modifications mainly at H3K9 specifically target plant defence-related genes

Genome‐wide DNA hypomethylation shapes nematode pattern‐triggered immunity in plants

A role for DNA hypomethylation has recently been suggested in the interaction between bacteria and plants; it is unclear whether this phenomenon reflects a conserved response. Treatment of plants of monocot rice and dicot tomato with nematode-associated molecular patterns from different nematode species or bacterial pathogen-associated molecular pattern flg22 revealed global DNA hypomethylation. A similar hypomethylation response was observed during early gall induction by Meloidogyne graminicola in rice. Evidence for the causal impact of hypomethylation on immunity was revealed by a significantly reduced plant susceptibility upon treatment with DNA methylation inhibitor 5-azacytidine. Whole-genome bisulphite sequencing of young galls revealed massive hypomethylation in the CHH context, while not for CG or CHG nucleotide contexts. Further, CHH hypomethylated regions were predominantly associated with gene promoter regions, which was not correlated with activated gene expression at the same time point but, rather, was correlated with a delayed transcriptional gene activation. Finally, the relevance of CHH hypomethylation in plant defence was confirmed in rice mutants of the RNA-directed DNA methylation pathway and DECREASED DNA METHYLATION 1. We demonstrated that DNA hypomethylation is associated with reduced susceptibility in rice towards root-parasitic nematodes and is likely to be part of the basal pattern-triggered immunity response in plants

Molecular insights into the compatible and incompatible interactions between sugar beet and the beet cyst nematode

Background: Sugar beet (Beta vulgarissubsp.vulgaris) is an economically important crop that provides nearly one third of the global sugar production. The beet cyst nematode (BCN),Heterodera schachtii, causes major yield losses in sugar beet and other crops worldwide. The most effective and economic approach to control this nematode is growing tolerant or resistant cultivars. To identify candidate genes involved in susceptibility and resistance, the transcriptome of sugar beet and BCN in compatible and incompatible interactions at two time points was studied using mRNA-seq. Results: In the susceptible cultivar, most defense-related genes were induced at 4 dai while suppressed at 10 dai but in the resistant cultivar Nemakill, induction of genes involved in the plant defense response was observed at both time points. In the compatible interaction, alterations in phytohormone-related genes were detected. The effect of exogenous application of Methyl Jasmonate and ET-generator ethephon on susceptible plants was therefore investigated and the results revealed significant reduction in plant susceptibility. Genes putatively involved in the resistance of Nemakill were identified, such as genes involved in phenylpropanoid pathway and genes encoding CYSTM domain-containing proteins, F-box proteins, chitinase, galactono-1,4-lactone dehydrogenase and CASP-like protein. Also, the transcriptome of the BCN was analyzed in infected root samples and several novel potential nematode effector genes were found. Conclusions: Our data provides detailed insights into the plant and nematode transcriptional changes occurring during compatible and incompatible interactions between sugar beet and BCN. Many important genes playing potential roles in susceptibility or resistance of sugar beet against BCN, as well as some BCN effectors with a potential role asavrproteins were identified. In addition, our findings indicate the effective role of jasmonate and ethylene in enhancing sugar beet defense response against BCN. This research provides new molecular insights into the plant-nematode interactions that can be used to design novel management strategies against BCN

Ascorbate oxidation activates systemic defence against root-knot nematode Meloidogyne graminicola in rice

Ascorbic acid (AA) is the major antioxidant buffer produced in the shoot tissue of plants. Previous studies on root-knot nematode (RKN; Meloidogyne graminicola)-infected rice (Oryza sativa) plants showed differential expression of AA-recycling genes, although their functional role was unknown. Our results confirmed increased dehydroascorbate (DHA) levels in nematode-induced root galls, while AA mutants were significantly more susceptible to nematode infection. External applications of ascorbate oxidase (AO), DHA, or reduced AA, revealed systemic effects of ascorbate oxidation on rice defence versus RKN, associated with a primed accumulation of H2O2 upon nematode infection. To confirm and further investigate these systemic effects, a transcriptome analysis was done on roots of foliar AO-treated plants, revealing activation of the ethylene (ET) response and jasmonic acid (JA) biosynthesis pathways in roots, which was confirmed by hormone measurements. Activation of these pathways by methyl-JA, or ethephon treatment can complement the susceptibility phenotype of the rice Vitamin C (vtc1) mutant. Experiments on the jasmonate signalling (jar1) mutant or using chemical JA/ET inhibitors confirm that the effects of ascorbate oxidation are dependent on both the JA and ET pathways. Collectively, our data reveal a novel pathway in which ascorbate oxidation induces systemic defence against RKNs

Exceedance of critical loads and of critical limits impacts tree nutrition across Europe

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Elucidation of the Mode of Action of a New Antibacterial Compound Active against Staphylococcus aureus and Pseudomonas aeruginosa.

Nosocomial and community-acquired infections caused by multidrug resistant bacteria represent a major human health problem. Thus, there is an urgent need for the development of antibiotics with new modes of action. In this study, we investigated the antibacterial characteristics and mode of action of a new antimicrobial compound, SPI031 (N-alkylated 3, 6-dihalogenocarbazol 1-(sec-butylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol), which was previously identified in our group. This compound exhibits broad-spectrum antibacterial activity, including activity against the human pathogens Staphylococcus aureus and Pseudomonas aeruginosa. We found that SPI031 has rapid bactericidal activity (7-log reduction within 30 min at 4x MIC) and that the frequency of resistance development against SPI031 is low. To elucidate the mode of action of SPI031, we performed a macromolecular synthesis assay, which showed that SPI031 causes non-specific inhibition of macromolecular biosynthesis pathways. Liposome leakage and membrane permeability studies revealed that SPI031 rapidly exerts membrane damage, which is likely the primary cause of its antibacterial activity. These findings were supported by a mutational analysis of SPI031-resistant mutants, a transcriptome analysis and the identification of transposon mutants with altered sensitivity to the compound. In conclusion, our results show that SPI031 exerts its antimicrobial activity by causing membrane damage, making it an interesting starting point for the development of new antibacterial therapies

Epilepsy in Dcx Knockout Mice Associated with Discrete Lamination Defects and Enhanced Excitability in the Hippocampus

Patients with Doublecortin (DCX) mutations have severe cortical malformations associated with mental retardation and epilepsy. Dcx knockout (KO) mice show no major isocortical abnormalities, but have discrete hippocampal defects. We questioned the functional consequences of these defects and report here that Dcx KO mice are hyperactive and exhibit spontaneous convulsive seizures. Changes in neuropeptide Y and calbindin expression, consistent with seizure occurrence, were detected in a large proportion of KO animals, and convulsants, including kainate and pentylenetetrazole, also induced seizures more readily in KO mice. We show that the dysplastic CA3 region in KO hippocampal slices generates sharp wave-like activities and possesses a lower threshold for epileptiform events. Video-EEG monitoring also demonstrated that spontaneous seizures were initiated in the hippocampus. Similarly, seizures in human patients mutated for DCX can show a primary involvement of the temporal lobe. In conclusion, seizures in Dcx KO mice are likely to be due to abnormal synaptic transmission involving heterotopic cells in the hippocampus and these mice may therefore provide a useful model to further study how lamination defects underlie the genesis of epileptiform activities