Early transcriptomic responses of rice leaves to herbivory by Spodoptera frugiperda.

During herbivory, chewing insects deposit complex oral secretions (OS) onto the plant wound. Understanding how plants respond to the different cues of herbivory remains an active area of research. In this study, we used an herbivory-mimick experiment to investigate the early transcriptional response of rice plants leaves to wounding, OS, and OS microbiota from Spodoptera frugiperda larvae. Wounding induced a massive early response associated to hormones such as jasmonates. This response switched drastically upon OS treatment indicating the activation of OS specific pathways. When comparing native and dysbiotic OS treatments, we observed few gene regulation. This suggests that in addition to wounding the early response in rice is mainly driven by the insect compounds of the OS rather than microbial. However, microbiota affected genes encoding key phytohormone synthesis enzymes, suggesting an additional modulation of plant response by OS microbiota.

A redundant transcription factor network steers spatiotemporal Arabidopsis triterpene synthesis.

Plant specialized metabolites modulate developmental and ecological functions and comprise many therapeutic and other high-value compounds. However, the mechanisms determining their cell-specific expression remain unknown. Here we describe the transcriptional regulatory network that underlies cell-specific biosynthesis of triterpenes in Arabidopsis thaliana root tips. Expression of thalianol and marneral biosynthesis pathway genes depends on the phytohormone jasmonate and is limited to outer tissues. We show that this is promoted by the activity of redundant bHLH-type transcription factors from two distinct clades and coactivated by homeodomain factors. Conversely, the DOF-type transcription factor DAG1 and other regulators prevent expression of the triterpene pathway genes in inner tissues. We thus show how precise expression of triterpene biosynthesis genes is determined by a robust network of transactivators, coactivators and counteracting repressors.

Jasmonate signaling controls negative and positive effectors of salt stress tolerance in rice.

Plant responses to salt exposure involve large reconfigurations of hormonal pathways that orchestrate physiological changes towards tolerance. Jasmonate (JA) hormones are essential to withstand biotic and abiotic assaults, but their roles in salt tolerance remain unclear. Here we describe the dynamics of JA metabolism and signaling in root and leaf tissue of rice, a plant species that is highly exposed and sensitive to salt. Roots activate the JA pathway in an early pulse, while the second leaf displays a biphasic JA response with peaks at 1 h and 3 d post-exposure. Based on higher salt tolerance of a rice JA-deficient mutant (aoc), we examined, through kinetic transcriptome and physiological analysis, the salt-triggered processes that are under JA control. Profound genotype-differential features emerged that could underlie the observed phenotypes. Abscisic acid (ABA) content and ABA-dependent water deprivation responses were impaired in aoc shoots. Moreover, aoc accumulated more Na+ in roots, and less in leaves, with reduced ion translocation correlating with root derepression of the HAK4 Na+ transporter gene. Distinct reactive oxygen species scavengers were also stronger in aoc leaves, along with reduced senescence and chlorophyll catabolism markers. Collectively, our results identify contrasted contributions of JA signaling to different sectors of the salt stress response in rice.

Engineering the plant metabolic system by exploiting metabolic regulation.

Plants are the most sophisticated biofactories and sources of food and biofuels present in nature. By engineering plant metabolism, the production of desired compounds can be increased and the nutritional or commercial value of the plant species can be improved. However, this can be challenging because of the complexity of the regulation of multiple genes and the involvement of different protein interactions. To improve metabolic engineering (ME) capabilities, different tools and strategies for rerouting the metabolic pathways have been developed, including genome editing and transcriptional regulation approaches. In addition, cutting-edge technologies have provided new methods for understanding uncharacterized biosynthetic pathways, protein degradation mechanisms, protein-protein interactions, or allosteric feedback, enabling the design of novel ME approaches.

Rapid evolution of an RNA virus to escape recognition by a rice nucleotide-binding and leucine-rich repeat domain immune receptor.

Viral diseases are a major limitation for crop production, and their control is crucial for sustainable food supply. We investigated by a combination of functional genetics and experimental evolution the resistance of rice to the rice yellow mottle virus (RYMV), which is among the most devastating rice pathogens in Africa, and the mechanisms underlying the extremely fast adaptation of the virus to its host. We found that the RYMV3 gene that protects rice against the virus codes for a nucleotide-binding and leucine-rich repeat domain immune receptor (NLRs) from the Mla-like clade of NLRs. RYMV3 detects the virus by forming a recognition complex with the viral coat protein (CP). The virus escapes efficiently from detection by mutations in its CP, some of which interfere with the formation of the recognition complex. This study establishes that NLRs also confer in monocotyledonous plants immunity to viruses, and reveals an unexpected functional diversity for NLRs of the Mla clade that were previously only known as fungal disease resistance proteins. In addition, it provides precise insight into the mechanisms by which viruses adapt to plant immunity and gives important knowledge for the development of sustainable resistance against viral diseases of cereals.

Jasmonate: A hormone of primary importance for plant metabolism.

Over the years, jasmonates (JAs) have become recognized as one of the main plant hormones that regulate stress responses by activating defense programs and the production of specialized metabolites. High JA levels have been associated with reduced plant growth, supposedly as a result of the reallocation of carbon sources from primary growth to the biosynthesis of defense compounds. Recent advances suggest however that tight regulatory networks integrate several sensing pathways to steer plant metabolism, and thereby drive the trade-off between growth and defense. In this review, we discuss how JA influences primary metabolism and how it is connected to light-regulated processes, nutrient sensing and energy metabolism. Finally, we speculate that JA, in a conceptual parallelism with adrenaline for humans, overall boosts cellular processes to keep up with an increased metabolic demand during harsh times.

Use of Fluorescent Reporters to Analyse Dynamic and Spatial Responses to Mechanical Wounding.

Mechanical wounding of plant tissues triggers many different responses (Savatin DV, Gramegna G, Modesti V, Front Plant Sci 5:470, 2014). These are primarily mediated by the plant hormone Jasmonic Acid Isoleucine (JA-Ile). Recently, a fluorescent biosensor for JA-Ile showed that sample preparation (i.e., handling of samples) for fluorescent microscopy very often triggers wound response, even without apparent damage to the seedling, affecting downstream analyses (Larrieu A, Champion A, Legrand J, Nat Commun 6:6043, 2015). In this chapter, we describe how to overcome this technical limitation to monitor any fluorescent reporter or dye in response to wounding, using any type of fluorescent or confocal (inverted or upright, laser scanning or spinning disc) microscopes. Pharmacological or wound treatments can easily be performed and responses monitored over long periods of time. We further describe a simple method to extract and analyse quantitative data from confocal images using the open source software Fiji (Fiji Is Just ImageJ (Schindelin J, Arganda-Carreras I, Frise E, Nat Methods 9:676-682, 2012)) and OpenOffice.

CRISPR/Cas9-Mediated Gene Editing of the Jasmonate Biosynthesis OsAOC Gene in Rice.

The function of Jasmonate (JA) is well documented in different plant physiological processes as well as in the interactions with their environment. Mutants impaired in JA production and/or signaling are useful materials to study the function of this phytohormone. Genes involved in the JA biosynthesis pathway in rice have been described, but few mutants deficient in JA production and signaling have been identified. Moreover, these mutants are mostly generated through random mutagenesis approaches, such as irradiation, EMS treatment, or T-DNA insertion, and potentially harbor undesired mutations that could affect other biological processes. The CRISPR/Cas9 system is a precise and efficient genome editing tool that creates DNA modification at specific loci and limit undesired mutations.In this chapter, we describe a procedure to generate new JA-deficient mutant using CRISPR/Cas9 system in rice. The Allene Oxide Cyclase (OsAOC) gene is targeted since it is a single copy gene in the JA biosynthesis pathway in rice. The widely used variety Oryza sativa japonica Kitaake has been chosen due to its short life cycle and its ease of genetic transformation. This protocol describes the selection of the 20-nt target sequence, construction of the binary vector, and strategy for selecting the T-DNA-free mutant.