CUL3BPM E3 ubiquitin ligases regulate MYC2, MYC3, and MYC4 stability and JA responses.

The jasmonate (JA)-pathway regulators MYC2, MYC3, and MYC4 are central nodes in plant signaling networks integrating environmental and developmental signals to fine-tune JA defenses and plant growth. Continuous activation of MYC activity is potentially lethal. Hence, MYCs need to be tightly regulated in order to optimize plant fitness. Among the increasing number of mechanisms regulating MYC activity, protein stability is arising as a major player. However, how the levels of MYC proteins are modulated is still poorly understood. Here, we report that MYC2, MYC3, and MYC4 are targets of BPM (BTB/POZ-MATH) proteins, which act as substrate adaptors of CUL3-based E3 ubiquitin ligases. Reduction of function of CUL3BPM in amiR-bpm lines, bpm235 triple mutants, and cul3ab double mutants enhances MYC2 and MYC3 stability and accumulation and potentiates plant responses to JA such as root-growth inhibition and MYC-regulated gene expression. Moreover, MYC3 polyubiquitination levels are reduced in amiR-bpm lines. BPM3 protein is stabilized by JA, suggesting a negative feedback regulatory mechanism to control MYC activity, avoiding harmful runaway responses. Our results uncover a layer for JA-pathway regulation by CUL3BPM-mediated degradation of MYC transcription factors.

The JA-pathway MYC transcription factors regulate photomorphogenic responses by targeting HY5 gene expression.

Jasmonates are key regulators of the balance between defence and growth in plants. However, the molecular mechanisms by which activation of defence reduces growth are not yet fully understood. Here, we analyze the role of MYC transcription factors (TFs) and jasmonic acid (JA) in photomorphogenic growth. We found that multiple myc mutants share light-associated phenotypes with mutants of the phytochrome B photoreceptor, such as delayed seed germination in the dark and long hypocotyl growth. Overexpression of MYC2 in a phyB background partially suppressed its long hypocotyl phenotype. Transcriptomic analysis of multiple myc mutants confirmed that MYCs are required for full expression of red (R) light-regulated genes, including the master regulator HY5. ChIP-seq analyses revealed that MYC2 and MYC3 bind directly to the promoter of HY5 and that HY5 gene expression and protein levels are compromised in multiple myc mutants. Altogether, our results pinpoint MYCs as photomorphogenic TFs that control phytochrome responses by activating HY5 expression. This has important implications in understanding the trade-off between growth and defence as the same TFs that activate defence responses are photomorphogenic growth regulators.

Jasmonate-Related MYC Transcription Factors Are Functionally Conserved in Marchantia polymorpha.

The lipid-derived phytohormone jasmonoyl-isoleucine regulates plant immunity, growth and development in vascular plants by activating genome-wide transcriptional reprogramming. In Arabidopsis (Arabidopsis thaliana), this process is largely orchestrated by the master regulator MYC2 and related transcription factors (TFs). However, the TFs activating this pathway in basal plant lineages are currently unknown. We report the functional conservation of MYC-related TFs between the eudicot Arabidopsis and the liverwort Marchantia polymorpha, a plant belonging to an early diverging lineage of land plants. Phylogenetic analysis suggests that MYC function first appeared in charophycean algae and therefore predates the evolutionary appearance of any other jasmonate pathway component. M. polymorpha possesses two functionally interchangeable MYC genes, one in females and one in males. Similar to AtMYC2, MpMYCs showed nuclear localization, interaction with JASMONATE-ZIM-DOMAIN PROTEIN repressors, and regulation by light. Phenotypic and molecular characterization of loss- and gain-of-function mutants demonstrated that MpMYCs are necessary and sufficient for activating the jasmonate pathway in M. polymorpha, but unlike their Arabidopsis orthologs, do not regulate fertility. Therefore, despite 450 million years of independent evolution, MYCs are functionally conserved between bryophytes and eudicots. Genetic conservation in an early diverging lineage suggests that MYC function existed in the common ancestor of land plants and evolved from a preexisting MYC function in charophycean algae.

A Single JAZ Repressor Controls the Jasmonate Pathway in Marchantia polymorpha.

JAZ proteins are negative regulators of jasmonate responses, acting both as repressors of transcription factors and as co-receptors of JA-Ile. The high redundancy of JAZ genes in angiosperms has hindered the characterization of a complete depletion of JAZ function. Moreover, the recent discovery that dn-OPDA is the jasmonate ligand in Marchantia polymorpha demonstrates that JA-Ile is not the sole COI1/JAZ ligand in land plants and highlights the importance of studying JAZ co-receptors in bryophytes. Here, we have exploited the low gene redundancy of the liverwort M. polymorpha to characterize the single MpJAZ in this early diverging plant lineage. We clarify the phylogenetic history of the TIFY family, demonstrate that MpJAZ is the ortholog of AtJAZ with a conserved function, and characterize its repressor activity of dn-OPDA responses. Our results show that, consistent with previous findings in Arabidopsis, MpJAZ represses jasmonates biosynthesis, senescence, and plant defenses, and promotes cell growth and reproductive fitness, highlighting the power of studies in Marchantia.

Ligand-receptor co-evolution shaped the jasmonate pathway in land plants.

The phytohormone jasmonoyl-isoleucine (JA-Ile) regulates defense, growth and developmental responses in vascular plants. Bryophytes have conserved sequences for all JA-Ile signaling pathway components but lack JA-Ile. We show that, in spite of 450 million years of independent evolution, the JA-Ile receptor COI1 is functionally conserved between the bryophyte Marchantia polymorpha and the eudicot Arabidopsis thaliana but COI1 responds to different ligands in each species. We identified the ligand of Marchantia MpCOI1 as two isomeric forms of the JA-Ile precursor dinor-OPDA (dinor-cis-OPDA and dinor-iso-OPDA). We demonstrate that AtCOI1 functionally complements Mpcoi1 mutation and confers JA-Ile responsiveness and that a single-residue substitution in MpCOI1 is responsible for the evolutionary switch in ligand specificity. Our results identify the ancestral bioactive jasmonate and clarify its biosynthetic pathway, demonstrate the functional conservation of its signaling pathway, and show that JA-Ile and COI1 emergence in vascular plants required co-evolution of hormone biosynthetic complexity and receptor specificity.

JAZ2 controls stomata dynamics during bacterial invasion.

Coronatine (COR) facilitates entry of bacteria into the plant apoplast by stimulating stomata opening. COR-induced signaling events at stomata remain unclear. We found that the COR and jasmonate isoleucine (JA-Ile) co-receptor JAZ2 is constitutively expressed in guard cells and modulates stomatal dynamics during bacterial invasion We analyzed tissue expression patterns of AtJAZ genes and measured stomata opening and pathogen resistance in loss- and gain-of-function mutants. Arabidopsis jaz2 mutants are partially impaired in pathogen-induced stomatal closing and more susceptible to Pseudomonas. Gain-of-function mutations in JAZ2 prevent stomatal reopening by COR and are highly resistant to bacterial penetration. The JAZ2 targets MYC2, MYC3 and MYC4 directly regulate the expression of ANAC19, ANAC55 and ANAC72 to modulate stomata aperture. Due to the antagonistic interactions between the salicylic acid (SA) and JA defense pathways, efforts to increase resistance to biotrophs result in enhanced susceptibility to necrotrophs, and vice versa. Remarkably, dominant jaz2Δjas mutants are resistant to Pseudomonas syringae but retain unaltered resistance against necrotrophs. Our results demonstrate the existence of a COI1-JAZ2-MYC2,3,4-ANAC19,55,72 module responsible for the regulation of stomatal aperture that is hijacked by bacterial COR to promote infection. They also provide novel strategies for crop protection against biotrophs without compromising resistance to necrotrophs.

Rational design of a ligand-based antagonist of jasmonate perception.

(+)-7-iso-Jasmonoyl-L-isoleucine (JA-Ile) regulates developmental and stress responses in plants. Its perception involves the formation of a ternary complex with the F-box COI1 and a member of the JAZ family of co-repressors and leads to JAZ degradation. Coronatine (COR) is a bacterial phytotoxin that functionally mimics JA-Ile and interacts with the COI1-JAZ co-receptor with higher affinity than JA-Ile. On the basis of the co-receptor structure, we designed ligand derivatives that spatially impede the interaction of the co-receptor proteins and, therefore, should act as competitive antagonists. One derivative, coronatine-O-methyloxime (COR-MO), has strong activity in preventing the COI1-JAZ interaction, JAZ degradation and the effects of JA-Ile or COR on several JA-mediated responses in Arabidopsis thaliana. Moreover, it potentiates plant resistance, preventing the effect of bacterially produced COR during Pseudomonas syringae infections in different plant species. In addition to the utility of COR-MO for plant biology research, our results underscore its biotechnological potential for safer and sustainable agriculture.

Light regulates motility, attachment and virulence in the plant pathogen Pseudomonas syringae pv tomato DC3000.

Pseudomonas syringae pv tomato DC3000 (Pto) is the causal agent of the bacterial speck of tomato, which leads to significant economic losses in this crop. Pto inhabits the tomato phyllosphere, where the pathogen is highly exposed to light, among other environmental factors. Light represents a stressful condition and acts as a source of information associated with different plant defence levels. Here, we analysed the presence of both blue and red light photoreceptors in a group of Pseudomonas. In addition, we studied the effect of white, blue and red light on Pto features related to epiphytic fitness. While white and blue light inhibit motility, bacterial attachment to plant leaves is promoted. Moreover, these phenotypes are altered in a blue-light receptor mutant. These light-controlled changes during the epiphytic stage cause a reduction in virulence, highlighting the relevance of motility during the entry process to the plant apoplast. This study demonstrated the key role of light perception in the Pto phenotype switching and its effect on virulence.

Distinct and conserved transcriptomic changes during nematode-induced giant cell development in tomato compared with Arabidopsis: a functional role for gene repression.

Root-knot nematodes (RKNs) induce giant cells (GCs) from root vascular cells inside the galls. Accompanying molecular changes as a function of infection time and across different species, and their functional impact, are still poorly understood. Thus, the transcriptomes of tomato galls and laser capture microdissected (LCM) GCs over the course of parasitism were compared with those of Arabidopsis, and functional analysis of a repressed gene was performed. Microarray hybridization with RNA from galls and LCM GCs, infection-reproduction tests and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) transcriptional profiles in susceptible and resistant (Mi-1) lines were performed in tomato. Tomato GC-induced genes include some possibly contributing to the epigenetic control of GC identity. GC-repressed genes are conserved between tomato and Arabidopsis, notably those involved in lignin deposition. However, genes related to the regulation of gene expression diverge, suggesting that diverse transcriptional regulators mediate common responses leading to GC formation in different plant species. TPX1, a cell wall peroxidase specifically involved in lignification, was strongly repressed in GCs/galls, but induced in a nearly isogenic Mi-1 resistant line on nematode infection. TPX1 overexpression in susceptible plants hindered nematode reproduction and GC expansion. Time-course and cross-species comparisons of gall and GC transcriptomes provide novel insights pointing to the relevance of gene repression during RKN establishment.

Identification of water stress genes in Pinus pinaster Ait. by controlled progressive stress and suppression-subtractive hybridization.

Climate change is a major challenge particularly for forest tree species, which will have to face the severe alterations of environmental conditions with their current genetic pool. Thus, an understanding of their adaptive responses is of the utmost interest. In this work we have selected Pinus pinaster as a model species. This pine is one of the most important conifers (for which molecular tools and knowledge are far more scarce than for angiosperms) in the Mediterranean Basin, which is characterised in all foreseen scenarios as one of the regions most drastically affected by climate change, mainly because of increasing temperature and, particularly, by increasing drought. We have induced a controlled, increasing water stress by adding PEG to a hydroponic culture. We have generated a subtractive library, with the aim of identifying the genes induced by this stress and have searched for the most reliable expressional candidate genes, based on their overexpression during water stress, as revealed by microarray analysis and confirmed by RT-PCR. We have selected a set of 67 candidate genes belonging to different functional groups that will be useful molecular tools for further studies on drought stress responses, adaptation, and population genomics in conifers, as well as in breeding programs.

NINJA connects the co-repressor TOPLESS to jasmonate signalling.

Jasmonoyl-isoleucine (JA-Ile) is a plant hormone that regulates a broad array of plant defence and developmental processes. JA-Ile-responsive gene expression is regulated by the transcriptional activator MYC2 that interacts physically with the jasmonate ZIM-domain (JAZ) repressor proteins. On perception of JA-Ile, JAZ proteins are degraded and JA-Ile-dependent gene expression is activated. The molecular mechanisms by which JAZ proteins repress gene expression remain unknown. Here we show that the Arabidopsis JAZ proteins recruit the Groucho/Tup1-type co-repressor TOPLESS (TPL) and TPL-related proteins (TPRs) through a previously uncharacterized adaptor protein, designated Novel Interactor of JAZ (NINJA). NINJA acts as a transcriptional repressor whose activity is mediated by a functional TPL-binding EAR repression motif. Accordingly, both NINJA and TPL proteins function as negative regulators of jasmonate responses. Our results point to TPL proteins as general co-repressors that affect multiple signalling pathways through the interaction with specific adaptor proteins. This new insight reveals how stress-related and growth-related signalling cascades use common molecular mechanisms to regulate gene expression in plants.

Early transcriptomic events in microdissected Arabidopsis nematode-induced giant cells.

Root-knot nematodes differentiate highly specialized feeding cells in roots (giant cells, GCs), through poorly characterized mechanisms that include extensive transcriptional changes. While global transcriptome analyses have used galls, which are complex root structures that include GCs and surrounding tissues, no global gene expression changes specific to GCs have been described. We report on the differential transcriptome of GCs versus root vascular cells, induced in Arabidopsis by Meloidogyne javanica at a very early stage of their development, 3 days after infection (d.p.i.). Laser microdissection was used to capture GCs and root vascular cells for microarray analysis, which was validated through qPCR and by a promoter-GUS fusion study. Results show that by 3 d.p.i., GCs exhibit major gene repression. Although some genes showed similar regulation in both galls and GCs, the majority had different expression patterns, confirming the molecular distinctiveness of the GCs within the gall. Most of the differentially regulated genes in GCs have no previously assigned function. Comparisons with other transcriptome analyses revealed similarities between GCs and cell suspensions differentiating into xylem cells. This suggests a molecular link between GCs and developing vascular cells, which represent putative GC stem cells. Gene expression in GCs at 3 d.p.i. was also found to be similar to crown galls induced by Agrobacterium tumefaciens, a specialized root biotroph.

Isolation of RNA from laser-capture-microdissected giant cells at early differentiation stages suitable for differential transcriptome analysis.

Plant organ gene expression profile analyses are complicated by the various cell types, and therefore transcription patterns, present in each organ. For example, each gall formed in roots following root knot nematode infection contains between four and eight specialized feeding cells (giant cells, GCs) embedded within hypertrophied root tissues. A recent goal in plant science has been the isolation of nematode feeding cell mRNAs for subsequent gene expression analysis. By adapting current protocols for different plant species and cells, we have developed a simple and rapid method for obtaining GCs from frozen tissue sections of tomato with good morphology and preserved RNA. The tissue sections obtained were suitable for the laser capture microdissection of GCs 6-7 days post-infection, and even of very early developing GCs (48-72 h post-infection), by fixation of tissue with ethanol-acetic acid, infiltration with sucrose and freezing in isopentane with optimal cutting temperature medium. This process was also successful for obtaining control vascular cells from uninfected roots for direct comparison with GCs. A minimum of about 300 GCs and 600 control vascular cells was required for efficient linear RNA amplification through in vitro transcription. Laser capture microdissection-derived RNA, after two rounds of amplification, was successfully used for microarray hybridization and validated with several differentially expressed genes by quantitative polymerase chain reaction. Consistent with our results, 117 homologous genes were found to be co-regulated in a previous microarray analysis of Arabidopsis galls at the same developmental stage. Therefore, we conclude that our method allows the isolation of a sufficient quantity of RNA with a high quality/integrity, appropriate for differential transcriptome analysis.

Genome-wide identification of small RNA targets based on target enrichment and microarray hybridizations.

MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are two classes of abundant 21-24 nucleotide small RNAs (smRNAs) that control gene expression in plants, mainly by guiding cleavage and degradation of target transcripts. Target identification based on predictive algorithms for base-paired complementarity requires further experimental validation and often fails to recognize miRNA::target pairs that escape from stringent complementarity rules. Here, we report on a microarray-based methodology to identify target mRNAs of miRNAs and siRNAs at a genomic scale. This strategy takes advantage of the RNA ligase-mediated amplification of 5' cDNA ends (RLM-RACE) to isolate miRNA or siRNA cleavage products from biological samples. Cleaved transcripts are then subjected to T7 RNA polymerase-mediated amplification and microarray hybridizations. The use of suitable hybridization controls is what makes our strategy outperform previous analyses. We applied this method and identified more than 100 putative novel miRNA or siRNA target mRNAs that had not been previously predicted by computational or microarray-based methods. Our data expand the regulatory role of endogenous smRNAs to a wide range of cellular processes, with prevalence in the regulation of cellular solute homeostasis. The methodology described here is straightforward, avoids extensive computational analysis and allows simultaneous analyses of several biological replicates, thus reducing the biological variability inherent in genomic analysis. The application of this simple methodology offers a framework for systematic analysis of smRNA-guided cleaved transcriptomes in different plant tissues, genotypes or stress conditions, and should contribute to understanding of the physiological role of smRNAs in plants.

A protein phosphatase 2A catalytic subunit is a negative regulator of abscisic acid signalling.

The key regulatory role of abscisic acid (ABA) in many physiological processes in plants is well established. However, compared with other plant hormones, the molecular mechanisms underlying ABA signalling are poorly characterized. In this work, a specific catalytic subunit of protein phosphatase 2A (PP2Ac-2) has been identified as a component of the signalling pathway that represses responses to ABA. A loss-of-function pp2ac-2 mutant is hypersensitive to ABA. Moreover, pp2ac-2 plants have altered responses in developmental and environmental processes that are mediated by ABA, such as primary and lateral root development, seed germination and responses to drought and high salt and sugar stresses. Conversely, transgenic plants overexpressing PP2Ac-2 are less sensitive to ABA than wild type, a phenotype that is manifested in all the above-mentioned physiological processes. DNA microarray hybridization experiments reveal that PP2Ac-2 is negatively involved in ABA responses through regulation of ABA-dependent gene expression. Moreover, the results obtained indicate that ABA antagonistically regulates PP2Ac-2 expression and PP2Ac-2 activity thus allowing plant sensitivity to the hormone to be reset after induction. Phenotypic, genetic and gene expression data strongly suggest that PP2Ac-2 is a negative regulator of the ABA pathway. Activity of protein phosphatase 2A thus emerges as a key element in the control of ABA signalling.

Identification of IgE-binding epitopes of the major peach allergen Pru p 3.

BACKGROUND: Lipid transfer proteins (LTPs) are clinically relevant plant food panallergens and have been proposed as ideal tools to study true food allergy. Pru p 3, the major peach allergen in the Mediterranean area, is among the best-characterized allergenic members of the LTP family. Its diagnostic value for Rosaceae fruit allergy has been demonstrated both in vivo and in vitro. OBJECTIVE: We sought to locate major IgE-binding epitopes of Pru p 3. METHODS: A serum pool and individual sera from patients with peach allergy and positive skin prick test results to Pru p 3 were used. Three-dimensional modeling was achieved by using experimentally available structures of Pru p 3 homologues as templates. Theoretical prediction of potential IgE-binding regions was performed by selecting specific residues on the molecular surface displaying prominent electrostatic potential features. Point mutants of Pru p 3 were constructed by standard polymerase chain reaction procedures with the appropriate primers. Mutants were expressed in P pastoris by means of the pPIC 9 vector and purified from the corresponding supernatants by gel-filtration chromatography followed by RP-HPLC. IgE binding by Pru p 3 mutants was tested by immunodetection and quantified by ELISA and ELISA inhibition assays. Synthetic peptides (10 mer; 5 amino acids overlapping) covering the full Pru p 3 sequence were used to detect IgE epitopes by (125)I-anti-IgE immunodetection. RESULTS: Pru p 3 showed a 3-dimensional structure comprising 4 alpha-helixes and a nonstructured C-terminal coil (residues 73 to 91). Regions around amino acids in positions 23 to 36, 39 to 44, and 80 to 91, particularly residues R39, T40, and R44, K80 and K91, were predicted as potential antibody recognition sites according to their relevant surface and electrostatic properties. Point mutants K80A and K91A were found to have an IgE-binding capacity similar to that of recombinant Pru p 3, but the triple mutant R39A/T40A/R44A showed a substantial decrease (approximately 5 times) of IgE binding. IgE immunodetection of synthetic peptides led to the identification of Pru p 3 sequence regions 11 to 25, 31 to 45, and 71 to 80 as major IgE epitopes. CONCLUSIONS: Main IgE-binding regions of the Pru p 3 amino acid sequence were identified. The three major ones comprised the end of an alpha-helix and some residues of the following interhelix loop. These data can help to search for Pru p 3 hypoallergenic forms.

Recombinant Pru p 3 and natural Pru p 3, a major peach allergen, show equivalent immunologic reactivity: a new tool for the diagnosis of fruit allergy.

BACKGROUND: The peach lipid transfer protein Pru p 3 has been identified as a major allergen from this fruit. Homologous cross-reactive allergens have been found in several plant foods and pollens. Recombinant Pru p 3 has been recently produced in the yeast Pichia pastoris. OBJECTIVE: We sought to evaluate the potential role of recombinant Pru p 3 as a novel tool for the diagnosis of fruit allergy. METHODS: Circular dichroism analysis was used to compare the protein folding of natural Pru p 3 and recombinant Pru p 3. IgE binding by both molecular forms was quantified by means of ELISA and ELISA inhibition assays, and their biologic activity was estimated by using basophil activation, histamine release, and sulphidoleukotriene production tests. Individual sera or blood samples from patients with peach allergy (up to 17) were used in the assays. RESULTS: A nearly identical circular dichroism spectra was shown by using natural Pru p 3 and recombinant Pru p 3, indicating that both protein forms are similarly folded. No difference was detected in the IgE-binding capacity of the 2 mo-lecular versions. Basophil activation and induction of sulphidoleukotriene production were positive in 9 of 10 patients, and histamine release was induced in at least half of the patients, with similar effects of the natural and recombinant forms in the 3 assays. CONCLUSION: Recombinant Pru p 3 shows a strong immunologic activity equivalent to that of its natural counterpart, and therefore it can be a useful tool for diagnosis (and future immunotherapy) of fruit allergy.

Immunoassay to quantify the major peach allergen Pru p 3 in foodstuffs. Differential allergen release and stability under physiological conditions.

Pru p 3 is a lipid transfer protein (LTP) that has been identified as the major peach (Prunus persica) allergen. However, little is known about the amount present in both raw and processed foodstuffs. Moreover, the in vivo release upon consumption of peach-containing foods remains unclear. We have developed a sensitive monoclonal antibody-based ELISA for Pru p 3. The method has been applied to measure the allergen levels in foodstuffs and the allergen release under different physiological conditions. A significant variability in all raw peaches and peach-containing foods tested has been detected. The allergen was extracted more efficiently at a low pH, and it was highly resistant to pepsin. This ELISA will be very useful in controlling the allergen concentration in diagnostics, in evaluating threshold levels in provocation tests, and in detecting hidden allergens in processed foods and cosmetics.