Secretory membrane traffic in plant-microbe interactions.

Plant defense responses include the extracellular release of defense-related molecules, such as pathogenesis-related proteins and secondary metabolites, as well as cell wall materials. This primarily depends on the trafficking of secretory vesicles to the plasma membrane, where they discharge their contents into the apoplastic space via soluble N-ethylmaleimide sensitive factor attachment protein receptor-assisted exocytosis. However, some pathogenic and symbiotic microbes have developed strategies to manipulate host plant exocytic pathways. Here, we discuss the mechanisms by which plant exocytic pathways function in immunity and how microbes have evolved to manipulate those pathways.

Comprehensive protein-based artificial microRNA screens for effective gene silencing in plants.

Artificial microRNA (amiRNA) approaches offer a powerful strategy for targeted gene manipulation in any plant species. However, the current unpredictability of amiRNA efficacy has limited broad application of this promising technology. To address this, we developed epitope-tagged protein-based amiRNA (ETPamir) screens, in which target mRNAs encoding epitope-tagged proteins were constitutively or inducibly coexpressed in protoplasts with amiRNA candidates targeting single or multiple genes. This design allowed parallel quantification of target proteins and mRNAs to define amiRNA efficacy and mechanism of action, circumventing unpredictable amiRNA expression/processing and antibody unavailability. Systematic evaluation of 63 amiRNAs in 79 ETPamir screens for 16 target genes revealed a simple, effective solution for selecting optimal amiRNAs from hundreds of computational predictions, reaching ∼100% gene silencing in plant cells and null phenotypes in transgenic plants. Optimal amiRNAs predominantly mediated highly specific translational repression at 5' coding regions with limited mRNA decay or cleavage. Our screens were easily applied to diverse plant species, including Arabidopsis thaliana, tobacco (Nicotiana benthamiana), tomato (Solanum lycopersicum), sunflower (Helianthus annuus), Catharanthus roseus, maize (Zea mays) and rice (Oryza sativa), and effectively validated predicted natural miRNA targets. These screens could improve plant research and crop engineering by making amiRNA a more predictable and manageable genetic and functional genomic technology.

Negative feedback control of jasmonate signaling by an alternative splice variant of JAZ10.

The plant hormone jasmonate (JA) activates gene expression by promoting ubiquitin-dependent degradation of jasmonate ZIM domain (JAZ) transcriptional repressor proteins. A key feature of all JAZ proteins is the highly conserved Jas motif, which mediates both JAZ degradation and JAZ binding to the transcription factor MYC2. Rapid expression of JAZ genes in response to JA is thought to attenuate JA responses, but little is known about the mechanisms by which newly synthesized JAZ proteins exert repression in the presence of the hormone. Here, we show in Arabidopsis (Arabidopsis thaliana) that desensitization to JA is mediated by an alternative splice variant (JAZ10.4) of JAZ10 that lacks the Jas motif. Unbiased protein-protein interaction screens identified three related basic helix-loop-helix transcription factors (MYC2, MYC3, and MYC4) and the corepressor NINJA as JAZ10.4-binding partners. We show that the amino-terminal region of JAZ10.4 contains a cryptic MYC2-binding site that resembles the Jas motif and that the ZIM motif of JAZ10.4 functions as a transferable repressor domain whose activity is associated with the recruitment of NINJA. Functional studies showed that the expression of JAZ10.4 from the native JAZ10 promoter complemented the JA-hypersensitive phenotype of a jaz10 mutant. Moreover, treatment of these complemented lines with JA resulted in the rapid accumulation of JAZ10.4 protein. Our results provide an explanation for how the unique domain architecture of JAZ10.4 links transcription factors to a corepressor complex and suggest how JA-induced transcription and alternative splicing of JAZ10 premessenger RNA creates a regulatory circuit to attenuate JA responses.

MAPK Cascades in Plant Microbiota Structure and Functioning.

Mitogen-activated protein kinase (MAPK) cascades are highly conserved signaling modules that coordinate diverse biological processes such as plant innate immunity and development. Recently, MAPK cascades have emerged as pivotal regulators of the plant holobiont, influencing the assembly of normal plant microbiota, essential for maintaining optimal plant growth and health. In this review, we provide an overview of current knowledge on MAPK cascades, from upstream perception of microbial stimuli to downstream host responses. Synthesizing recent findings, we explore the intricate connections between MAPK signaling and the assembly and functioning of plant microbiota. Additionally, the role of MAPK activation in orchestrating dynamic changes in root exudation to shape microbiota composition is discussed. Finally, our review concludes by emphasizing the necessity for more sophisticated techniques to accurately decipher the role of MAPK signaling in establishing the plant holobiont relationship.

IMPLANT: a new technique for transgene copy number estimation in plants using a single end-point PCR reaction.

BACKGROUND: Copy number determination is one of the first steps in the characterization of transgenic plant lines. The classical approach to this, Southern blotting, is time-consuming, expensive and requires massive amounts of high-quality genomic DNA. Other PCR-based techniques are either inaccurate, laborious, or expensive. RESULTS: Here, we propose a new technique, IMPLANT (Insertion of competitive PCR calibrator for copy number estimation), a competitive PCR-based technique in which the competitor (based on an endogenous gene) is also incorporated in the T-DNA, which then gets integrated in the genome together with the gene of interest. As the number of integrated competitor molecules directly corresponds to the number of transgene copies, the transgene copy number can be determined by a single PCR reaction. We demonstrate that the results of this technique closely correspond with those obtained by segregation analysis in Arabidopsis and digital PCR In rice, indicating that it is a powerful alternative for other techniques for copy number determination. CONCLUSIONS: We show that this technique is not only reliable, but is also faster, easier, and cheaper as compared with other techniques. Accurate results are obtained in both Arabidopsis and rice, but this technique can be easily extended to other organisms and as such can be widely adopted in the field of biotechnology.

Alternative splicing expands the repertoire of dominant JAZ repressors of jasmonate signaling.

Jasmonates (JAs) are fatty acid-derived signaling compounds that control diverse aspects of plant growth, development and immunity. The F-box protein COI1 functions both as a receptor for jasmonoyl-l-isoleucine (JA-Ile) and as the component of an E3-ubiquitin ligase complex (SCF(COI1) ) that targets JAZ transcriptional regulators for degradation. A key feature of JAZ proteins is the C-terminal Jas motif that mediates the JA-Ile-dependent interaction with COI1. Here, we show that most JAZ genes from evolutionarily diverse plants contain a conserved intron that splits the Jas motif into 20 N-terminal and seven C-terminal (X(5) PY) amino acid submotifs. In most members of the Arabidopsis JAZ family, alternative splicing events involving retention of this intron generate proteins that are truncated before the X(5) PY sequence. In vitro pull-down and yeast two-hybrid assays indicate that these splice variants have reduced capacity to form stable complexes with COI1 in the presence of the bioactive stereoisomer of the hormone (3R,7S)-JA-Ile. cDNA overexpression studies showed that some, but not all, truncated splice variants are dominant repressors of JA signaling. We also show that strong constitutive expression of an intron-containing JAZ10 genomic clone is sufficient to repress JA responses. These findings provide evidence for functional differences between JAZ isoforms, and establish a direct link between the alternative splicing of JAZ pre-mRNA and the dominant repression of JA signal output. We propose that production of dominant JAZ repressors by alternative splicing reduces the negative consequences associated with inappropriate or hyperactivation of the JA response pathway.

Agrobacterium strains and strain improvement: Present and outlook.

Almost 40 years ago the first transgenic plant was generated through Agrobacterium tumefaciens-mediated transformation, which, until now, remains the method of choice for gene delivery into plants. Ever since, optimized Agrobacterium strains have been developed with additional (genetic) modifications that were mostly aimed at enhancing the transformation efficiency, although an optimized strain also exists that reduces unwanted plasmid recombination. As a result, a collection of very useful strains has been created to transform a wide variety of plant species, but has also led to a confusing Agrobacterium strain nomenclature. The latter is often misleading for choosing the best-suited strain for one's transformation purposes. To overcome this issue, we provide a complete overview of the strain classification. We also indicate different strain modifications and their purposes, as well as the obtained results with regard to the transformation process sensu largo. Furthermore, we propose additional improvements of the Agrobacterium-mediated transformation process and consider several worthwhile modifications, for instance, by circumventing a defense response in planta. In this regard, we will discuss pattern-triggered immunity, pathogen-associated molecular pattern detection, hormone homeostasis and signaling, and reactive oxygen species in relationship to Agrobacterium transformation. We will also explore alterations that increase agrobacterial transformation efficiency, reduce plasmid recombination, and improve biocontainment. Finally, we recommend the use of a modular system to best utilize the available knowledge for successful plant transformation.

Jasmonate signaling: a conserved mechanism of hormone sensing.

The lipid-derived hormone jasmonate (JA) regulates diverse aspects of plant immunity and development. Among the central components of the JA signaling cascade are the E3 ubiquitin ligase SCFCOI1 and Jasmonate ZIM-domain (JAZ) proteins that repress transcription of JA-responsive genes. Recent studies provide evidence that amino acid-conjugated forms of JA initiate signal transduction upon formation of a coronatine-insensitive1 (COI1)-JA-JAZ ternary complex in which JAZs are ubiquitinated and subsequently degraded. Coronatine, a virulence factor produced by the plant pathogen Pseudomonas syringae, is a potent agonist of this hormone receptor system. Coronatine-induced targeting of JAZs to COI1 obstructs host immune responses to P. syrinage, providing a striking example of how pathogens exploit hormone signaling pathways in the host to promote disease. These findings, together with homology between COI1 and the auxin receptor, TIR1, extend the paradigm of F-box proteins as intracellular sensors of small molecules, and suggest a common evolutionary origin of the auxin and JA response pathways.

A critical role of two positively charged amino acids in the Jas motif of Arabidopsis JAZ proteins in mediating coronatine- and jasmonoyl isoleucine-dependent interactions with the COI1 F-box protein.

SUMMARY: Coronatine is an important virulence factor produced by several pathovars of the bacterial pathogen Pseudomonas syringae. The structure of coronatine is similar to that of a class of plant hormones called jasmonates (JAs). An important step in JA signaling is the SCF(COI1) E3 ubiquitin ligase-dependent degradation of JAZ repressor proteins. We have recently shown that jasmonoyl isoleucine (JA-Ile) promotes physical interaction between Arabidopsis JAZ1 and COI1 (the F-box component of SCF(COI1)) proteins, and that the JA-Ile-dependent COI1-JAZ1 interaction could be reconstituted in yeast cells (i.e. in the absence of other plant proteins). Here we show that coronatine, but not its two biosynthetic precursors, also promotes interaction between Arabidopsis COI1 and multiple JAZ proteins. The C-terminal Jas motif, but not the N-terminal (NT) domain or central ZIM domain of JAZ proteins, is critical for JA-Ile/coronatine-dependent interaction with COI1. Two positively charged amino acid residues in the Jas domain were identified as essential for coronatine-dependent COI1-JAZ interactions. Mutations of these two residues did not affect the ability of JAZ1 and JAZ9 to interact with the transcription factor AtMYC2. Importantly, transgenic Arabidopsis plants expressing JAZ1 carrying these two mutations exhibited JA-insensitive phenotypes, including male sterility and enhanced resistance to P. syringae infection. These results not only suggest that coronatine and JA-Ile target the physical interaction between COI1 and the Jas domain of JAZ repressors, but also illustrate the critical role of positively charged amino acids in the Jas domain in mediating the JA-Ile/coronatine-dependent JAZ interaction with COI1.

MAPK Assays in Arabidopsis MAMP-PRR Signal Transduction.

Activation of MAPK (Mitogen-Activated Protein Kinase) cascades after MAMP (Microbe-Associated Molecular Pattern) perception through PRR (Pattern Recognition Receptor) is one of the first conserved responses when plants encounter microbial organisms. Phosphorylation of various cellular factors in the MAMP-PRR pathway by MAPK cascades is critical for broad-spectrum plant innate immunity. Measurement of MAPK activation and identification of MAPK phosphorylation targets in the MAMP-PRR signal transduction pathway are essential to understand how plants reprogram their cellular processes to cope with unfavorable microbial attack. Here, we describe detailed protocols of three assays measuring MAPK activity after MAMP perception: (1) immune-blotting analysis with anti-phospho ERK1/2 antibody; (2) in-gel kinase assay using a general substrate myelin basic protein (MBP); (3) an in vitro kinase assay to evaluate phosphorylation of MAPK substrate candidates during MAMP-PRR signaling based on a protoplast expression system.

A critical role for the TIFY motif in repression of jasmonate signaling by a stabilized splice variant of the JASMONATE ZIM-domain protein JAZ10 in Arabidopsis.

JASMONATE ZIM-domain (JAZ) proteins act as repressors of jasmonate (JA) signaling. Perception of bioactive JAs by the F-box protein CORONATINE INSENSITIVE1 (COI1) causes degradation of JAZs via the ubiquitin-proteasome pathway, which in turn activates the expression of genes involved in plant growth, development, and defense. JAZ proteins contain two highly conserved sequence regions: the Jas domain that interacts with COI1 to destabilize the repressor and the ZIM domain of unknown function. Here, we show that the conserved TIFY motif (TIFF/YXG) within the ZIM domain mediates homo- and heteromeric interactions between most Arabidopsis thaliana JAZs. We have also identified an alternatively spliced form (JAZ10.4) of JAZ10 that lacks the Jas domain and, as a consequence, is highly resistant to JA-induced degradation. Strong JA-insensitive phenotypes conferred by overexpression of JAZ10.4 were suppressed by mutations in the TIFY motif that block JAZ10.4-JAZ interactions. We conclude that JAZ10.4 functions to attenuate signal output in the presence of JA and further suggest that the dominant-negative action of this splice variant involves protein-protein interaction through the ZIM/TIFY domain. The ability of JAZ10.4 to interact with MYC2 is consistent with a model in which a JAZ10.4-containing protein complex directly represses the activity of transcription factors that promote expression of JA response genes.

Top hits in contemporary JAZ: an update on jasmonate signaling.

The phytohormone jasmonate (JA) regulates a wide range of growth, developmental, and defense-related processes during the plant life cycle. Identification of the JAZ family of proteins that repress JA responses has facilitated rapid progress in understanding how this lipid-derived hormone controls gene expression. Recent analysis of JAZ proteins has provided insight into the nature of the JA receptor, the chemical specificity of signal perception, and cross-talk between JA and other hormone response pathways. Functional diversification of JAZ proteins by alternative splicing, together with the ability of JAZ proteins to homo- and heterodimerize, provide mechanisms to enhance combinatorial diversity and versatility in gene regulation by JA.

Regulation and function of Arabidopsis JASMONATE ZIM-domain genes in response to wounding and herbivory.

Jasmonate (JA) and its amino acid conjugate, jasmonoyl-isoleucine (JA-Ile), play important roles in regulating plant defense responses to insect herbivores. Recent studies indicate that JA-Ile promotes the degradation of JASMONATE ZIM-domain (JAZ) transcriptional repressors through the activity of the E(3) ubiquitin-ligase SCF(COI1). Here, we investigated the regulation and function of JAZ genes during the interaction of Arabidopsis (Arabidopsis thaliana) with the generalist herbivore Spodoptera exigua. Most members of the JAZ gene family were highly expressed in response to S. exigua feeding and mechanical wounding. JAZ transcript levels increased within 5 min of mechanical tissue damage, coincident with a large (approximately 25-fold) rise in JA and JA-Ile levels. Wound-induced expression of JAZ and other CORONATINE-INSENSITIVE1 (COI1)-dependent genes was not impaired in the jar1-1 mutant that is partially deficient in the conversion of JA to JA-Ile. Experiments performed with the protein synthesis inhibitor cycloheximide provided evidence that JAZs, MYC2, and genes encoding several JA biosynthetic enzymes are primary response genes whose expression is derepressed upon COI1-dependent turnover of a labile repressor protein(s). We also show that overexpression of a modified form of JAZ1 (JAZ1Delta3A) that is stable in the presence of JA compromises host resistance to feeding by S. exigua larvae. These findings establish a role for JAZ proteins in the regulation of plant anti-insect defense, and support the hypothesis that JA-Ile and perhaps other JA derivatives activate COI1-dependent wound responses in Arabidopsis. Our results also indicate that the timing of JA-induced transcription in response to wounding is more rapid than previously realized.

Identification of a peroxisomal acyl-activating enzyme involved in the biosynthesis of jasmonic acid in Arabidopsis.

Jasmonic acid (JA) is a lipid-derived signal that regulates a wide variety of developmental and defense-related processes in higher plants. JA is synthesized from linolenic acid via an enzymatic pathway that initiates in the plastid and terminates in peroxisomes. The C18 JA precursor 12-oxo-phytodienoic acid (OPDA) is converted in the peroxisome to 3-oxo-2-(2'-[Z]-pentenyl)cyclopentane-1-octanoic acid (OPC-8:0), which subsequently undergoes three rounds of beta-oxidation to yield JA. Although most JA biosynthetic enzymes have been identified, several key steps in the pathway remain to be elucidated. To address this knowledge gap, we employed co-expression analysis to identify genes that are coordinately regulated with known JA biosynthetic components in Arabidopsis. Among the candidate genes uncovered by this approach was a 4-coumarate-CoA ligase-like member of the acyl-activating enzyme (AAE) gene family, which we have named OPC-8:0 CoA Ligase1 (OPCL1). In response to wounding, opcl1 null mutants exhibited reduced levels of JA and hyperaccumulation of OPC-8:0. Recombinant OPCL1 was active against both OPDA and OPC-8:0, as well as medium-to-long straight-chain fatty acids. Subcellular localization studies with green fluorescent protein-tagged OPCL1 showed that the protein is targeted to peroxisomes. These findings establish a physiological role for OPCL1 in the activation of JA biosynthetic precursors in leaf peroxisomes, and further indicate that OPC-8:0 is a physiological substrate for the activation step. The results also demonstrate the utility of co-expression analysis for identification of factors that contribute to jasmonate homeostasis.

Heterologous expression and molecular and cellular characterization of CaPUB1 encoding a hot pepper U-Box E3 ubiquitin ligase homolog.

The U-box motif is a conserved domain found in the diverse isoforms of E3 ubiquitin ligase in eukaryotes. From water-stressed hot pepper (Capsicum annuum L. cv Pukang) plants, we isolated C. annuum putative U-box protein 1 (CaPUB1), which encodes a protein containing a single U-box motif in its N-terminal region. In vitro ubiquitination and site-directed mutagenesis assays revealed that CaPUB1 possessed E3 ubiquitin ligase activity and that the U-box motif was indeed essential for its enzyme activity. RNA gel-blot analysis showed that CaPUB1 mRNA was induced rapidly by a broad spectrum of abiotic stresses, including drought, high salinity, cold temperature, and mechanical wounding, but not in response to ethylene, abscisic acid, or a bacterial pathogen, suggesting its role in the early events in the abiotic-related defense response. Because transgenic work was extremely difficult in hot pepper, in this study we overexpressed CaPUB1 in Arabidopsis (Arabidopsis thaliana) to provide cellular information on the function of this gene in the development and plant responses to abiotic stresses. Transgenic Arabidopsis plants that constitutively expressed the CaPUB1 gene under the control of the cauliflower mosaic virus 35S promoter had markedly longer hypocotyls and roots and grew more rapidly than the wild type, leading to an early bolting phenotype. Microscopic analysis showed that 35S::CaPUB1 roots had increased numbers of small-sized cells, resulting in disordered, highly populated cell layers in the cortex, endodermis, and stele. In addition, CaPUB1-overexpressing plants displayed increased sensitivity to water stress and mild salinity. These results indicate that CaPUB1 is functional in Arabidopsis cells, thereby effectively altering cell and tissue growth and also the response to abiotic stresses. Comparative proteomic analysis showed that the level of RPN6 protein, a non-ATPase subunit of the 26S proteasome complex, was significantly reduced in 35SCaPUB1 seedlings as compared to the wild type. Pull-down and ubiquitination assays demonstrated that RPN6 interacted physically with CaPUB1 and was ubiquitinated in a CaPUB1-dependent manner in vitro. Although the physiological function of CaPUB1 is not yet clear, there are several possibilities for its involvement in a subset of physiological responses to counteract dehydration and high-salinity stresses in transgenic Arabidopsis seedlings.

Generation and analysis of end sequence database for T-DNA tagging lines in rice.

We analyzed 6749 lines tagged by the gene trap vector pGA2707. This resulted in the isolation of 3793 genomic sequences flanking the T-DNA. Among the insertions, 1846 T-DNAs were integrated into genic regions, and 1864 were located in intergenic regions. Frequencies were also higher at the beginning and end of the coding regions and upstream near the ATG start codon. The overall GC content at the insertion sites was close to that measured from the entire rice (Oryza sativa) genome. Functional classification of these 1846 tagged genes showed a distribution similar to that observed for all the genes in the rice chromosomes. This indicates that T-DNA insertion is not biased toward a particular class of genes. There were 764, 327, and 346 T-DNA insertions in chromosomes 1, 4 and 10, respectively. Insertions were not evenly distributed; frequencies were higher at the ends of the chromosomes and lower near the centromere. At certain sites, the frequency was higher than in the surrounding regions. This sequence database will be valuable in identifying knockout mutants for elucidating gene function in rice. This resource is available to the scientific community at http://www.postech.ac.kr/life/pfg/risd.