CROWN ROOTLESS1 binds DNA with a relaxed specificity and activates OsROP and OsbHLH044 genes involved in crown root formation in rice.

In cereals, the root system is mainly composed of post-embryonic shoot-borne roots, named crown roots. The CROWN ROOTLESS1 (CRL1) transcription factor, belonging to the ASYMMETRIC LEAVES2-LIKE/LATERAL ORGAN BOUNDARIES DOMAIN (ASL/LBD) family, is a key regulator of crown root initiation in rice (Oryza sativa). Here, we show that CRL1 can bind, both in vitro and in vivo, not only the LBD-box, a DNA sequence recognized by several ASL/LBD transcription factors, but also another not previously identified DNA motif that was named CRL1-box. Using rice protoplast transient transactivation assays and a set of previously identified CRL1-regulated genes, we confirm that CRL1 transactivates these genes if they possess at least a CRL1-box or an LBD-box in their promoters. In planta, ChIP-qPCR experiments targeting two of these genes that include both a CRL1- and an LBD-box in their promoter show that CRL1 binds preferentially to the LBD-box in these promoter contexts. CRISPR/Cas9-targeted mutation of these two CRL1-regulated genes, which encode a plant Rho GTPase (OsROP) and a basic helix-loop-helix transcription factor (OsbHLH044), show that both promote crown root development. Finally, we show that OsbHLH044 represses a regulatory module, uncovering how CRL1 regulates specific processes during crown root formation.

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 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.

PUCHI represses early meristem formation in developing lateral roots of Arabidopsis thaliana.

Lateral root organogenesis is a key process in the development of a plant's root system and its adaptation to the environment. During lateral root formation, an early phase of cell proliferation first produces a four-cell-layered primordium, and only from this stage onwards is a root meristem-like structure, expressing root stem cell niche marker genes, being established in the developing organ. Previous studies reported that the gene regulatory network controlling lateral root formation is organized into two subnetworks whose mutual inhibition may contribute to organ patterning. PUCHI encodes an AP2/ERF transcription factor expressed early during lateral root primordium development and required for correct lateral root formation. To dissect the molecular events occurring during this early phase, we generated time-series transcriptomic datasets profiling lateral root development in puchi-1 mutants and wild types. Transcriptomic and reporter analyses revealed that meristem-related genes were expressed ectopically at early stages of lateral root formation in puchi-1 mutants. We conclude that, consistent with the inhibition of genetic modules contributing to lateral root development, PUCHI represses ectopic establishment of meristematic cell identities at early stages of organ development. These findings shed light on gene network properties that orchestrate correct timing and patterning during lateral root formation.

Physiological and genetic control of transpiration efficiency in African rice, Oryza glaberrima Steud.

Improving crop water use efficiency, the amount of carbon assimilated as biomass per unit of water used by a plant, is of major importance as water for agriculture becomes scarcer. In rice, the genetic bases of transpiration efficiency, the derivation of water use efficiency at the whole-plant scale, and its putative component trait transpiration restriction under high evaporative demand remain unknown. These traits were measured in 2019 in a panel of 147 African rice (Oryza glaberrima) genotypes known to be potential sources of tolerance genes to biotic and abiotic stresses. Our results reveal that higher transpiration efficiency is associated with transpiration restriction in African rice. Detailed measurements in a subset of highly contrasted genotypes in terms of biomass accumulation and transpiration confirmed these associations and suggested that root to shoot ratio played an important role in transpiration restriction. Genome wide association studies identified marker-trait associations for transpiration response to evaporative demand, transpiration efficiency, and its residuals, with links to genes involved in water transport and cell wall patterning. Our data suggest that root-shoot partitioning is an important component of transpiration restriction that has a positive effect on transpiration efficiency in African rice. Both traits are heritable and define targets for breeding rice with improved water use strategies.

Inference of the gene regulatory network acting downstream of CROWN ROOTLESS 1 in rice reveals a regulatory cascade linking genes involved in auxin signaling, crown root initiation, and root meristem specification and maintenance.

Crown roots (CRs) are essential components of the rice root system. Several genes involved in CR initiation or development have been identified but our knowledge about how they organize to form a gene regulatory network (GRN) is still limited. To characterize the regulatory cascades acting during CR formation, we used a systems biology approach to infer the GRN controlling CR formation downstream of CROWN ROOTLESS 1 (CRL1), coding for an ASL (asymmetric leaves-2-like)/LBD (LOB domain) transcription factor necessary for CR initiation. A time-series transcriptomic dataset was generated after synchronized induction of CR formation by dexamethasone-mediated expression of CRL1 expression in a crl1 mutant background. This time series revealed three different genome expression phases during the early steps of CR formation and was further exploited to infer a GRN using a dedicated algorithm. The predicted GRN was confronted with experimental data and 72% of the inferred links were validated. Interestingly, this network revealed a regulatory cascade linking CRL1 to other genes involved in CR initiation, root meristem specification and maintenance, such as QUIESCENT-CENTER-SPECIFIC HOMEOBOX, and in auxin signalling. This predicted regulatory cascade was validated in vivo using transient activation assays. Thus, the CRL1-dependant GRN reflects major gene regulation events at play during CR formation and constitutes a valuable source of discovery to better understand this developmental process.

Genomic, transcriptomic, and proteomic approaches towards understanding the molecular mechanisms of salt tolerance in Frankia strains isolated from Casuarina trees.

BACKGROUND: Soil salinization is a worldwide problem that is intensifying because of the effects of climate change. An effective method for the reclamation of salt-affected soils involves initiating plant succession using fast growing, nitrogen fixing actinorhizal trees such as the Casuarina. The salt tolerance of Casuarina is enhanced by the nitrogen-fixing symbiosis that they form with the actinobacterium Frankia. Identification and molecular characterization of salt-tolerant Casuarina species and associated Frankia is imperative for the successful utilization of Casuarina trees in saline soil reclamation efforts. In this study, salt-tolerant and salt-sensitive Casuarina associated Frankia strains were identified and comparative genomics, transcriptome profiling, and proteomics were employed to elucidate the molecular mechanisms of salt and osmotic stress tolerance. RESULTS: Salt-tolerant Frankia strains (CcI6 and Allo2) that could withstand up to 1000 mM NaCl and a salt-sensitive Frankia strain (CcI3) which could withstand only up to 475 mM NaCl were identified. The remaining isolates had intermediate levels of salt tolerance with MIC values ranging from 650 mM to 750 mM. Comparative genomic analysis showed that all of the Frankia isolates from Casuarina belonged to the same species (Frankia casuarinae). Pangenome analysis revealed a high abundance of singletons among all Casuarina isolates. The two salt-tolerant strains contained 153 shared single copy genes (most of which code for hypothetical proteins) that were not found in the salt-sensitive(CcI3) and moderately salt-tolerant (CeD) strains. RNA-seq analysis of one of the two salt-tolerant strains (Frankia sp. strain CcI6) revealed hundreds of genes differentially expressed under salt and/or osmotic stress. Among the 153 genes, 7 and 7 were responsive to salt and osmotic stress, respectively. Proteomic profiling confirmed the transcriptome results and identified 19 and 8 salt and/or osmotic stress-responsive proteins in the salt-tolerant (CcI6) and the salt-sensitive (CcI3) strains, respectively. CONCLUSION: Genetic differences between salt-tolerant and salt-sensitive Frankia strains isolated from Casuarina were identified. Transcriptome and proteome profiling of a salt-tolerant strain was used to determine molecular differences correlated with differential salt-tolerance and several candidate genes were identified. Mechanisms involving transcriptional and translational regulation, cell envelop remodeling, and previously uncharacterized proteins appear to be important for salt tolerance. Physiological and mutational analyses will further shed light on the molecular mechanism of salt tolerance in Casuarina associated Frankia isolates.

Inhibition of auxin signaling in Frankia species-infected cells in Casuarina glauca nodules leads to increased nodulation.

Actinorhizal symbioses are mutualistic interactions between plants and the soil bacteria Frankia spp. that lead to the formation of nitrogen-fixing root nodules. The plant hormone auxin has been suggested to play a role in the mechanisms that control the establishment of this symbiosis in the actinorhizal tree Casuarina glauca. Here, we analyzed the role of auxin signaling in Frankia spp.-infected cells. Using a dominant-negative version of an endogenous auxin-signaling regulator, INDOLE-3-ACETIC ACID7, we established that inhibition of auxin signaling in these cells led to increased nodulation and, as a consequence, to higher nitrogen fixation per plant even if nitrogen fixation per nodule mass was similar to that in the wild type. Our results suggest that auxin signaling in Frankia spp.-infected cells is involved in the long-distance regulation of nodulation in actinorhizal symbioses.

The Casuarina NIN gene is transcriptionally activated throughout Frankia root infection as well as in response to bacterial diffusible signals.

Root nodule symbioses (RNS) allow plants to acquire atmospheric nitrogen by establishing an intimate relationship with either rhizobia, the symbionts of legumes or Frankia in the case of actinorhizal plants. In legumes, NIN (Nodule INception) genes encode key transcription factors involved in nodulation. Here we report the characterization of CgNIN, a NIN gene from the actinorhizal tree Casuarina glauca using both phylogenetic analysis and transgenic plants expressing either ProCgNIN::reporter gene fusions or CgNIN RNAi constructs. We have found that CgNIN belongs to the same phylogenetic group as other symbiotic NIN genes and CgNIN is able to complement a legume nin mutant for the early steps of nodule development. CgNIN expression is correlated with infection by Frankia, including preinfection stages in developing root hairs, and is induced by culture supernatants. Knockdown mutants were impaired for nodulation and early root hair deformation responses were severely affected. However, no mycorrhizal phenotype was observed and no induction of CgNIN expression was detected in mycorrhizas. Our results indicate that elements specifically required for nodulation include NIN and possibly related gene networks derived from the nitrate signalling pathways.

Identification of potential transcriptional regulators of actinorhizal symbioses in Casuarina glauca and Alnus glutinosa.

BACKGROUND: Trees belonging to the Casuarinaceae and Betulaceae families play an important ecological role and are useful tools in forestry for degraded land rehabilitation and reforestation. These functions are linked to their capacity to establish symbiotic relationships with a nitrogen-fixing soil bacterium of the genus Frankia. However, the molecular mechanisms controlling the establishment of these symbioses are poorly understood. The aim of this work was to identify potential transcription factors involved in the establishment and functioning of actinorhizal symbioses. RESULTS: We identified 202 putative transcription factors by in silico analysis in 40 families in Casuarina glauca (Casuarinaceae) and 195 in 35 families in Alnus glutinosa (Betulaceae) EST databases. Based on published transcriptome datasets and quantitative PCR analysis, we found that 39% and 26% of these transcription factors were regulated during C. glauca and A. glutinosa-Frankia interactions, respectively. Phylogenetic studies confirmed the presence of common key transcription factors such as NSP, NF-YA and ERN-related proteins involved in nodule formation in legumes, which confirm the existence of a common symbiosis signaling pathway in nitrogen-fixing root nodule symbioses. We also identified an actinorhizal-specific transcription factor belonging to the zinc finger C1-2i subfamily we named CgZF1 in C. glauca and AgZF1 in A. glutinosa. CONCLUSIONS: We identified putative nodulation-associated transcription factors with particular emphasis on members of the GRAS, NF-YA, ERF and C2H2 families. Interestingly, comparison of the non-legume and legume TF with signaling elements from actinorhizal species revealed a new subgroup of nodule-specific C2H2 TF that could be specifically involved in actinorhizal symbioses. In silico identification, transcript analysis, and phylogeny reconstruction of transcription factor families paves the way for the study of specific molecular regulation of symbiosis in response to Frankia infection.

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.

Glutaredoxin regulation of primary root growth is associated with early drought stress tolerance in pearl millet.

Seedling root traits impact plant establishment under challenging environments. Pearl millet is one of the most heat and drought tolerant cereal crops that provides a vital food source across the sub-Saharan Sahel region. Pearl millet's early root system features a single fast-growing primary root which we hypothesize is an adaptation to the Sahelian climate. Using crop modeling, we demonstrate that early drought stress is an important constraint in agrosystems in the Sahel where pearl millet was domesticated. Furthermore, we show that increased pearl millet primary root growth is correlated with increased early water stress tolerance in field conditions. Genetics including genome-wide association study and quantitative trait loci (QTL) approaches identify genomic regions controlling this key root trait. Combining gene expression data, re-sequencing and re-annotation of one of these genomic regions identified a glutaredoxin-encoding gene PgGRXC9 as the candidate stress resilience root growth regulator. Functional characterization of its closest Arabidopsis homolog AtROXY19 revealed a novel role for this glutaredoxin (GRX) gene clade in regulating cell elongation. In summary, our study suggests a conserved function for GRX genes in conferring root cell elongation and enhancing resilience of pearl millet to its Sahelian environment.

Pearl millet is a staple food for over 90 million people living in regions of Africa and India that typically experience high temperatures and little rainfall. It was domesticated about 4,500 years ago in the Sahel region of West Africa and is one of the most heat and drought tolerant cereal crops worldwide. In most plants, organs known as roots absorb water and essential nutrients from the soil. Young pearl millet plants develop a fast-growing primary root, but it is unclear how this unique feature helps the crop to grow in hot and dry conditions. Using weather data collected from the Sahel over a 20-year period, Fuente, Grondin et al. predicted by modelling that early drought stress is the major factor limiting pearl millet growth and yield in this region. Field experiments found that plants with primary roots that grow faster within soil were better at tolerating early drought than those with slower growing roots. Further work using genetic approaches revealed that a gene known as PgGRXC9 promotes the growth of the primary root. To better understand how this gene works, the team examined a very similar gene in a well-studied model plant known as Arabidopsis. This suggested that PgGRXC9 helps the primary root to grow by stimulating cell elongation within the root. Since it is well adapted to dry conditions, pearl millet is expected to play an important role in helping agriculture adjust to climate change. The findings of Fuente, Grondin et al. may be used by plant breeders to create more resilient and productive varieties of pearl millet.

Identification of the Hevea brasiliensis AP2/ERF superfamily by RNA sequencing.

BACKGROUND: Rubber tree (Hevea brasiliensis) laticifers are the source of natural rubber. Rubber production depends on endogenous and exogenous ethylene (ethephon). AP2/ERF transcription factors, and especially Ethylene-Response Factors, play a crucial role in plant development and response to biotic and abiotic stresses. This study set out to sequence transcript expressed in various tissues using next-generation sequencing and to identify AP2/ERF superfamily in the rubber tree. RESULTS: The 454 sequencing technique was used to produce five tissue-type transcript libraries (leaf, bark, latex, embryogenic tissues and root). Reads from all libraries were pooled and reassembled to improve mRNA lengths and produce a global library. One hundred and seventy-three AP2/ERF contigs were identified by in silico analysis based on the amino acid sequence of the conserved AP2 domain from the global library. The 142 contigs with the full AP2 domain were classified into three main families (20 AP2 members, 115 ERF members divided into 11 groups, and 4 RAV members) and 3 soloist members. Fifty-nine AP2/ERF transcripts were found in latex. Alongside the microRNA172 already described in plants, eleven additional microRNAs were predicted to inhibit Hevea AP2/ERF transcripts. CONCLUSIONS: Hevea has a similar number of AP2/ERF genes to that of other dicot species. We adapted the alignment and classification methods to data from next-generation sequencing techniques to provide reliable information. We observed several specific features for the ERF family. Three HbSoloist members form a group in Hevea. Several AP2/ERF genes highly expressed in latex suggest they have a specific function in Hevea. The analysis of AP2/ERF transcripts in Hevea presented here provides the basis for studying the molecular regulation of latex production in response to abiotic stresses and latex cell differentiation.

Genetic analysis of the rice jasmonate receptors reveals specialized functions for OsCOI2.

COI1-mediated perception of jasmonate is critical for plant development and responses to environmental stresses. Monocots such as rice have two groups of COI genes due to gene duplication: OsCOI1a and OsCOI1b that are functionally equivalent to the dicotyledons COI1 and OsCOI2 whose function remains unclear. In order to assess the function of OsCOI2 and its functional redundancy with COI1 genes, we developed a series of rice mutants in the 3 genes OsCOI1a, OsCOI1b and OsCOI2 by CRISPR Cas9-mediated editing and characterized their phenotype and responses to jasmonate. Characterization of OsCOI2 uncovered its important roles in root, leaf and flower development. In particular, we show that crown root growth inhibition by jasmonate relies on OsCOI2 and not on OsCOI1a nor on OsCOI1b, revealing a major function for the non-canonical OsCOI2 in jasmonate-dependent control of rice root growth. Collectively, these results point to a specialized function of OsCOI2 in the regulation of plant development in rice and indicate that sub-functionalisation of jasmonate receptors has occurred in the monocot phylum.

Regulatory mechanisms underlying oil palm fruit mesocarp maturation, ripening, and functional specialization in lipid and carotenoid metabolism.

Fruit provide essential nutrients and vitamins for the human diet. Not only is the lipid-rich fleshy mesocarp tissue of the oil palm (Elaeis guineensis) fruit the main source of edible oil for the world, but it is also the richest dietary source of provitamin A. This study examines the transcriptional basis of these two outstanding metabolic characters in the oil palm mesocarp. Morphological, cellular, biochemical, and hormonal features defined key phases of mesocarp development. A 454 pyrosequencing-derived transcriptome was then assembled for the developmental phases preceding and during maturation and ripening, when high rates of lipid and carotenoid biosynthesis occur. A total of 2,629 contigs with differential representation revealed coordination of metabolic and regulatory components. Further analysis focused on the fatty acid and triacylglycerol assembly pathways and during carotenogenesis. Notably, a contig similar to the Arabidopsis (Arabidopsis thaliana) seed oil transcription factor WRINKLED1 was identified with a transcript profile coordinated with those of several fatty acid biosynthetic genes and the high rates of lipid accumulation, suggesting some common regulatory features between seeds and fruits. We also focused on transcriptional regulatory networks of the fruit, in particular those related to ethylene transcriptional and GLOBOSA/PISTILLATA-like proteins in the mesocarp and a central role for ethylene-coordinated transcriptional regulation of type VII ethylene response factors during ripening. Our results suggest that divergence has occurred in the regulatory components in this monocot fruit compared with those identified in the dicot tomato (Solanum lycopersicum) fleshy fruit model.

Colonization of rice leaf blades by an African strain of Xanthomonas oryzae pv. oryzae depends on a new TAL effector that induces the rice nodulin-3 Os11N3 gene.

African strains of Xanthomonas oryzae pv. oryzae contain fewer TAL effectors than Asian strains, and their contribution to pathogenicity is unknown. Systematic mutagenesis of tal genes was used to decipher the contribution of each of the eight TAL effector paralogs to pathogenicity of African X. oryzae pv. oryzae BAI3. A strain mutated in talC was severely affected in the production of disease symptoms. Analysis of growth in planta upon leaf-clip inoculation showed that mutant bacteria multiplied only at the site of inoculation at the apex of the leaf, suggesting a requirement for talC during colonization of vascular tissues. Such tissue-specific effect of a tal mutant is a novel phenotype, which has not yet been characterized in other xanthomonads. Microarray experiments comparing the host response of rice leaves challenged with BAI3(R) vs. BAI3(R)ΔtalC were performed to identify genes targeted by TalC. A total of 120 upregulated and 21 downregulated genes were identified, among them Os11N3, which is a member of the MtN3/saliva family. Based on semiquantitative reverse transcription-polymerase chain reaction and ß-glucuronidase reporter assays, we show that Os11N3 is directly upregulated by TalC and identify a TalC DNA target box within the Os11N3 upstream sequence.

Two GCC boxes and AP2/ERF-domain transcription factor ORA59 in jasmonate/ethylene-mediated activation of the PDF1.2 promoter in Arabidopsis.

Plant defense against microbial pathogens depends on the action of several endogenously produced hormones, including jasmonic acid (JA) and ethylene (ET). In defense against necrotrophic pathogens, the JA and ET signaling pathways synergize to activate a specific set of defense genes including PLANT DEFENSIN1.2 (PDF1.2). The APETALA2/Ethylene Response Factor (AP2/ERF)-domain transcription factor ORA59 acts as the integrator of the JA and ET signaling pathways and is the key regulator of JA- and ET-responsive PDF1.2 expression. The present study was aimed at the identification of elements in the PDF1.2 promoter conferring the synergistic response to JA/ET and interacting with ORA59. We show that the PDF1.2 promoter was activated synergistically by JA and the ET-releasing agent ethephon due to the activity of two GCC boxes. ORA59 bound in vitro to these GCC boxes and trans-activated the PDF1.2 promoter in transient assays via these two boxes. Using the chromatin immunoprecipitation technique we were able to show that ORA59 bound the PDF1.2 promoter in vivo. Finally, we show that a tetramer of a single GCC box conferred JA/ethephon-responsive expression, demonstrating that the JA and ET signaling pathways converge to a single type of GCC box. Therefore ORA59 and two functionally equivalent GCC box binding sites form the module that enables the PDF1.2 gene to respond synergistically to simultaneous activation of the JA and ET signaling pathways.

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.

Establishment of Actinorhizal Symbiosis in Response to Ethylene, Salicylic Acid, and Jasmonate.

Phytohormones play a crucial role in regulating plant developmental processes. Among them, ethylene and jasmonate are known to be involved in plant defense responses to a wide range of biotic stresses as their levels increase with pathogen infection. In addition, these two phytohormones have been shown to inhibit plant nodulation in legumes. Here, exogenous salicylic acid (SA), jasmonate acid (JA), and ethephon (ET) were applied to the root system of Casuarina glauca plants before Frankia inoculation, in order to analyze their effects on the establishment of actinorhizal symbiosis. This protocol further describes how to identify putative ortholog genes involved in ethylene and jasmonate biosynthesis and/or signaling pathways in plant, using the Arabidopsis Information Resource (TAIR), Legume Information System (LIS), and Genevestigator databases. The expression of these genes in response to the bacterium Frankia was analyzed using the gene atlas for Casuarina-Frankia symbiosis (SESAM web site).

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.