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

Sensitivity towards DMI fungicides and haplotypic diversity of their CYP51 target in the Mycosphaerella graminicola population of Flanders

Septoria leaf blotch, caused by the fungus Mycosphaerella graminicola, is the most important wheat disease in Northwestern Europe, and is currently controlled by fungicide applications. Since the spread of resistance to methyl benzimidazole carbamates (MBCs) and quinone outside inhibitors (QoIs) in European countries, reliable control is mainly dependent upon sterol 14 alpha-demethylation inhibitors (DMIs). In the last decades however, a slow shift towards reduced sensitivity of M. graminicola to DMIs has been observed. This shift is caused mainly by mutations in the CYP51 gene encoding the 14 alpha-demethylase target protein for these fungicides. In this work, M. graminicola isolates were sampled at fields spread over Flanders, Belgium. In vitro assays were used to analyze the sensitivity of the Flemish M. graminicola population towards different DMIs. Sequencing of the CYP51 gene of these isolates allowed us to identify and map the haplotypes in this population. The results showed that there is a large variability in DMI sensitivity between the isolates, even within one field, which is reflected in a high diversity in CYP51 haplotypes within the M. graminicola population in Flanders. Next to some haplotypes that were not described in literature before, we found that the population is dominated by CYP51 haplotypes which were previously associated with increased resistance towards DMIs

Evolution of GHF5 endoglucanase gene structure in plant-parasitic nematodes: no evidence for an early domain shuffling event

Background Endo-1,4-beta-glucanases or cellulases from the glycosyl hydrolase family 5 (GHF5) have been found in numerous bacteria and fungi, and recently also in higher eukaryotes, particularly in plant-parasitic nematodes (PPN). The origin of these genes has been attributed to horizontal gene transfer from bacteria, although there still is a lot of uncertainty about the origin and structure of the ancestral GHF5 PPN endoglucanase. It is not clear whether this ancestral endoglucanase consisted of the whole gene cassette, containing a catalytic domain and a carbohydrate-binding module (CBM, type 2 in PPN and bacteria) or only of the catalytic domain while the CBM2 was retrieved by domain shuffling later in evolution. Previous studies on the evolution of these genes have focused primarily on data of sedentary nematodes, while in this study, extra data from migratory nematodes were included. Results Two new endoglucanases from the migratory nematodes Pratylenchus coffeae and Ditylenchus africanus were included in this study. The latter one is the first gene isolated from a PPN of a different superfamily (Sphaerularioidea); all previously known nematode endoglucanases belong to the superfamily Tylenchoidea (order Rhabditida). Phylogenetic analyses were conducted with the PPN GHF5 endoglucanases and homologous endoglucanases from bacterial and other eukaryotic lineages such as beetles, fungi and plants. No statistical incongruence between the phylogenetic trees deduced from the catalytic domain and the CBM2 was found, which could suggest that both domains have evolved together. Furthermore, based on gene structure data, we inferred a model for the evolution of the GHF5 endoglucanase gene structure in plant-parasitic nematodes. Our data confirm a close relationship between Pratylenchus spp. and the root knot nematodes, while some Radopholus similis endoglucanases are more similar to cyst nematode genes. Conclusions We conclude that the ancestral PPN GHF5 endoglucanase gene most probably consisted of the whole gene cassette, i.e. the GHF5 catalytic domain and the CBM2, rather than that it evolved by domain shuffling. Our evolutionary model for the gene structure in PPN GHF5 endoglucanases implies the occurrence of an early duplication event, and more recent gene duplications at genus or species level

Biochar-amended potting medium reduces the susceptibility of rice to root-knot nematode infections

Background: Biochar is a solid coproduct of biomass pyrolysis, and soil amended with biochar has been shown to enhance the productivity of various crops and induce systemic plant resistance to fungal pathogens. The aim of this study was to explore the ability of wood biochar to induce resistance to the root-knot nematode (RKN) Meloidogyne graminicola in rice (Oryza sativa cv. Nipponbare) and examine its histochemical and molecular impact on plant defense mechanisms. Results: A 1.2 % concentration of biochar added to the potting medium of rice was found to be the most effective at reducing nematode development in rice roots, whereas direct toxic effects of biochar exudates on nematode viability, infectivity or development were not observed. The increased plant resistance was associated with biochar-primed H2O2 accumulation as well as with the transcriptional enhancement of genes involved in the ethylene (ET) signaling pathway. The increased susceptibility of the Ein2b-RNAi line, which is deficient in ET signaling, further confirmed that biochar-induced priming acts at least partly through ET signaling. Conclusion: These results suggest that biochar amendments protect rice plants challenged by nematodes. This priming effect partially depends on the ET signaling pathway and enhanced H2O2 accumulation

Systemic suppression of the shoot metabolism upon rice root nematode infection

Hirschmanniella oryzae is the most common plant-parasitic nematode in flooded rice cultivation systems. These migratory animals penetrate the plant roots and feed on the root cells, creating large cavities, extensive root necrosis and rotting. The objective of this study was to investigate the systemic response of the rice plant upon root infection by this nematode. RNA sequencing was applied on the above-ground parts of the rice plants at 3 and 7 days post inoculation. The data revealed significant modifications in the primary metabolism of the plant shoot, with a general suppression of for instance chlorophyll biosynthesis, the brassinosteroid pathway, and amino acid production. In the secondary metabolism, we detected a repression of the isoprenoid and shikimate pathways. These molecular changes can have dramatic consequences for the growth and yield of the rice plants, and could potentially change their susceptibility to above-ground pathogens and pests

Interplay between carotenoids, abscisic acid and jasmonate guides the compatible rice-Meloidogyne graminicola interaction

In this study, we have characterized the role of carotenoids and chlorophyll in the compatible interaction between the sedentary root knot nematode (RKN) Meloidogyne graminicola and the monocot model plant rice (Oryza sativa). Previous transcriptome data showed a differential expression of carotenoid and chlorophyll biosynthesis genes in nematode-induced giant cells and gall tissue. Metabolite measurement showed that galls indeed accumulate chlorophyll a, b and carotenoids, as well as the hormone abscisic acid (ABA). When ABA was externally applied on rice plants, or when ABA-biosynthesis was inhibited, a significant increase in gall formation and nematode development was found, showing the complex role of ABA in this interaction. ABA application suppressed jasmonic acid (JA) levels in the plants, while ABA-biosynthesis inhibition lead to increased JA levels confirming an antagonism between ABA and JA in rice roots. In addition, combined applications of ABA and JA showed that the ABA-effect can overcome JA-induced defense. Based on these observations, we hypothesized that the accumulation of chlorophyll and carotenoid precursors would be beneficial to nematode infection. Indeed, when chemically blocking the carotenoid biosynthesis pathway at different steps, which leads to differential accumulation of carotenoids and chlorophyll in the plants, a positive and clear link between accumulation of carotenoids and chlorophyll and rice susceptibility to RKN was detected

A phytochemical perspective on plant defense against nematodes

Given the large yield losses attributed to plant-parasitic nematodes and the limited availability of sustainable control strategies, new plant-parasitic nematode control strategies are urgently needed. To defend themselves against nematode attack, plants possess sophisticated multi-layered immune systems. One element of plant immunity against nematodes is the production of small molecules with anti-nematode activity, either constitutively or after nematode infection. This review provides an overview of such metabolites that have been identified to date and groups them by chemical class (e.g., terpenoids, flavonoids, glucosinolates, etc.). Furthermore, this review discusses strategies that have been used to identify such metabolites and highlights the ways in which studying anti-nematode metabolites might be of use to agriculture and crop protection. Particular attention is given to emerging, high-throughput approaches for the identification of anti-nematode metabolites, in particular the use of untargeted metabolomics techniques based on nuclear magnetic resonance (NMR) and mass spectrometry (MS)

Species relationships in the genus Vasconcellea (Caricaceae) based on molecular and morphological evidence

Validity of the taxa currently recognized in the genus Vasconcellea was analyzed by investigating morphological and molecular data from 105 specimens of this genus and six specimens of the related genus Carica. Taxon identification of these specimens was compared with clustering in two phenetic dendrograms generated with 36 morphological characters and 254 amplified fragment length polymorphic (AFLP) markers. Moreover, cytoplasmic haplotypes were assessed using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) of one mitochondrial and two chloroplast DNA regions. Results show that the morphological data set, containing mainly vegetative characteristics, merely reveals external resemblance between specimens, which is not directly associated with genetic relationships and taxon validity. Phenotypic plasticity and intercompatibility between several species are likely to confuse morphological delimitation of the taxa. Based on the results of our study, several specimens that could not be identified with the currently used identification key (1) could be attributed to a known taxon, which should be extended to include a higher range of morphological variability or (2) could be hypothesized to be of hybrid origin. Because of the high intraspecific variation within V. microcarpa and V. X heilbornii, revision of these taxa is recommended

Ascorbate oxidase induces systemic resistance in sugar beet against cyst nematode Heterodera schachtii

Ascorbate oxidase (AO) is an enzyme involved in catalyzing the oxidation of apoplastic ascorbic acid (AA) to dehydroascorbic acid (DHA). In this research, the potential of AO spraying to induce systemic resistance was demonstrated in the interaction between sugar beet root and cyst nematode Heterodera schachtii and the mechanism was elucidated. Plant bioassays showed that roots of AO-sprayed plants were infested by a significantly lower number of females and cysts when compared with mock-sprayed control plants. Hormone measurements showed an elevated level of jasmonic acid (JA) salicylic acid (SA) and ethylene (ET) in the roots of AO-sprayed plants, with a dynamic temporal pattern of activation. Experiments with chemical inhibitors showed that AO-induced systemic resistance is partially dependent on the JA, ET and SA pathways. Biochemical analyses revealed a primed accumulation of hydrogen peroxide (H2O2), and phenylalanine ammonia lyase (PAL) activity in the roots of AO-sprayed plants upon infection by cyst nematodes. In conclusion, our data shows that AO works as an effective systemic defense priming agent in sugar beet against cyst nematode infection, through activation of multiple basal plant defense pathways

Interactions between the oomycete Pythium arrhenomanes and the rice root-knot nematode Meloidogyne graminicola in aerobic Asian rice varieties

Background: Aerobic rice fields are frequently infested by pathogenic oomycetes (Pythium spp.) and the rice root-knot nematode Meloidogyne graminicola. Here, the interaction between Pythium arrhenomanes and Meloidogyne graminicola was studied in rice roots of two aerobic rice varieties. In different experimental set-ups and infection regimes, plant growth, rice yield, Pythium colonization, as well as establishment, development and reproduction of M. graminicola were studied. Results: In this study, it is shown that the presence of P. arrhenomanes delays the establishment, development and reproduction of M. graminicola compared to single nematode infected plants. The delay in establishment and development of M. graminicola becomes stronger with higher P. arrhenomanes infection pressure. Conclusions: Our data indicate that P. arrhenomanes antagonizes M. graminicola in the rice root and that the plant benefits from this antagonism as shown by the yield data, especially when either of the pathogens is present in high levels