Jasmonate Hypersensitive 3 negatively regulates both jasmonate and ethylene-mediated responses in Arabidopsis.

Jasmonate (JA) is an important hormone involved in regulating diverse responses to environmental factors as well as growth and development, and its signalling is influenced by other hormones such as ethylene (ET). However, our understanding of the regulatory relationship between the JA and ET signalling pathways is limited. In this study, we isolated an Arabidopsis JA-hypersensitive mutant, jah3 (jasmonate hypersensitive3)-1. Map-based cloning revealed that the JAH3 gene corresponds to At4g16535. JAH3 encodes a protein of unknown function whose amino acid sequence has similarity to leukocyte receptor cluster-like protein. The mutation in jah3-1 is caused by a single nucleotide change from A to T at position 220 of 759 bp. Using CRISPR-Cas9, we generated a second allele, jah3-2, that encodes a truncated protein. Both of these loss-of-function alleles resulted in hypersensitivity to JA, ET-induced root growth inhibition, and accelerated dark-induced senescence. Double mutant analyses employing coronatine insensitive 1 (coi1) and ethylene insensitive 3 (ein3) mutants (jah3 coi1 and jah3 ein3) demonstrated that the hypersensitive phenotypes of the jah3 mutants are mediated by JA and ET signalling components COI1 and EIN3. Therefore, we propose that JAH3 is a negative regulator of both JA and ET signalling.

Host-Dependent Producibility of Recombinant Cypridina noctiluca Luciferase With Glycosylation Defects.

Cypridina noctiluca luciferase (CLuc) is a secreted luminescent protein that reacts with its substrate (Cypridina luciferin) to emit light. CLuc is known to be a thermostable protein and has been used for various research applications, including in vivo imaging and high-throughput reporter assays. Previously, we produced a large amount of recombinant CLuc for crystallographic analysis. However, this recombinant protein did not crystallize, probably due to heterogeneous N-glycan modifications. In this study, we produced recombinant CLuc without glycan modifications by introducing mutations at the N-glycan modification residues using mammalian Expi293F cells, silkworms, and tobacco Bright Yellow-2 cells. Interestingly, recombinant CLuc production depended heavily on the expression hosts. Among these selected hosts, we found that Expi293F cells efficiently produced the recombinant mutant CLuc without significant effects on its luciferase activity. We confirmed the lack of N-glycan modifications for this mutant protein by mass spectrometry analysis but found slight O-glycan modifications that we estimated were about 2% of the ion chromatogram peak area for the detected peptide fragments. Moreover, by using CLuc deletion mutants during the investigation of O-glycan modifications, we identified amino acid residues important to the luciferase activity of CLuc. Our results provide invaluable information related to CLuc function and pave the way for its crystallographic analysis.

NINJA-associated ERF19 negatively regulates Arabidopsis pattern-triggered immunity.

Recognition of microbe-associated molecular patterns (MAMPs) derived from invading pathogens by plant pattern recognition receptors (PRRs) initiates a subset of defense responses known as pattern-triggered immunity (PTI). Transcription factors (TFs) orchestrate the onset of PTI through complex signaling networks. Here, we characterized the function of ERF19, a member of the Arabidopsis thaliana ethylene response factor (ERF) family. ERF19 was found to act as a negative regulator of PTI against Botrytis cinerea and Pseudomonas syringae. Notably, overexpression of ERF19 increased plant susceptibility to these pathogens and repressed MAMP-induced PTI outputs. In contrast, expression of the chimeric dominant repressor ERF19-SRDX boosted PTI activation, conferred increased resistance to the fungus B. cinerea, and enhanced elf18-triggered immunity against bacteria. Consistent with a negative role for ERF19 in PTI, MAMP-mediated growth inhibition was weakened or augmented in lines overexpressing ERF19 or expressing ERF19-SRDX, respectively. Using biochemical and genetic approaches, we show that the transcriptional co-repressor Novel INteractor of JAZ (NINJA) associates with and represses the function of ERF19. Our work reveals ERF19 as a novel player in the mitigation of PTI, and highlights a potential role for NINJA in fine-tuning ERF19-mediated regulation of Arabidopsis innate immunity.

The chimeric repressor for the GATA4 transcription factor improves tolerance to nitrogen deficiency in Arabidopsis.

Nitrogen limits crop yield, but application of nitrogen fertilizer can cause environmental problems and much fertilizer is lost without being absorbed by plants. Increasing nitrogen use efficiency in plants may help overcome these problems and is, therefore, an important and active subject of agricultural research. Here, we report that the expression of the chimeric repressor for the GATA4 transcription factor (35S:GATA4-SRDX) improved tolerance to nitrogen deficiency in Arabidopsis thaliana. 35S:GATA4-SRDX seedlings were significantly larger than wild type under nitrogen-sufficient and -deficient conditions (10 and 0.5 mM NH4NO3, respectively). 35S:GATA4-SRDX plants exhibited shorter primary roots, fewer lateral roots, and higher root hair density compared with wild type. The expression levels of NITRATE TRANSPORTER 2.1, ASPARAGINE SYNTHETASE and NITRATE REDUCTASE 1 were significantly higher in roots of 35S:GATA4-SRDX plants than in wild type under nitrogen-sufficient conditions. Under nitrogen-deficient conditions, the expression of genes for cytosolic glutamine synthetases was upregulated in shoots of 35S:GATA4-SRDX plants compared with wild type. This upregulation of nitrogen transporter and nitrogen assimilation-related genes might confer tolerance to nitrogen deficiency in 35S:GATA4-SRDX plants.

WUSCHEL-RELATED HOMEOBOX 2 is a transcriptional repressor involved in lateral organ formation and separation in Arabidopsis.

In this study, we characterized the function of WUSCHEL-RELATED HOMEOBOX 2 (WOX2) using overexpression, CRES-T, and VP16 fusion techniques. Although the function of WOX2 has been described mainly in embryogenesis, it was unclear whether it also plays a role in the post-embryogenic developmental stage. We found that WOX2 has transcriptional repression activity and that either overexpression of WOX2 or expression of its chimeric repressor causes severe growth defects and other morphological phenotypes by impairing plant organ formation and separation. By contrast, VP16-fused WOX2-expressing plants did not display such severe phenotypic defects. In addition, some of them displayed phenotypic defects such as fusion of organs and induction of undifferentiated cells in the boundary regions of organs where GUS staining was clearly observed in the proWOX2:GUS transgenic plants. We suggest that WOX2 is involved in regulation of lateral organ formation and separation during the post-embryogenic development processes.

The ERF transcription factor EPI1 is a negative regulator of dark-induced and jasmonate-stimulated senescence in Arabidopsis.

Identification of the factors involved in the regulation of senescence and the analysis of their function are important for both a biological understanding of the senescence mechanism and the improvement of agricultural productivity. In this study, we identified an ERF gene termed "ERF gene conferring Postharvest longevity Improvement 1" (EPI1) as a possible regulator of senescence in Arabidopsis. We found that EPI1 possesses transcriptional repression activity and that the transgenic plants overexpressing EPI1 and expressing its chimeric repressor, EPI1-SRDX, commonly suppressed the darkness-induced senescence in their excised aerial parts. These transgenic plants additionally maintained a high level of chlorophyll, even after the methyl jasmonate (MeJA) treatment, which stimulated senescence in the dark. In addition, we found that senescence-induced and -reduced genes are down- and upregulated, respectively, in the MeJA-treated transgenic plants under darkness. Our results suggest that EPI1 functions as a negative regulator of the dark-induced and JA-stimulated senescence.

RPT2a, a 26S proteasome AAA-ATPase, is directly involved in Arabidopsis CC-NBS-LRR protein uni-1D-induced signaling pathways.

Arabidopsis semi-dominant uni-1D shows both constitutive defense responses and diverse morphological defects. In particular, uni-1D homozygote (uni-1D) mutants exhibit severe phenotypes including not only highly up-regulated pathogenesis related-1(PR-1) gene expression, but also lethality in the early stage of true leaf formation after germination. The gene responsible for the mutant encodes a coiled-coil-nucleotide-binding site-leucine-rich repeat (CC-NBS-LRR)-type R protein that functions in the recognition of pathogen and the triggering of defense responses. However, the molecular basis of how uni-1D can induce these phenotypes was unknown. In this study, we isolated the regulatory particle triple-ATPase (RPT) subunits 2a and 2b, base components of the 19S regulatory particle in the 26S proteasome, as uni-1D-interacting proteins using yeast two-hybrid screening. Genetic studies showed that crossing with the rpt2a mutant reduces the level of uni-1D-induced PR-1 gene expression and suppresses the lethality of uni-1D, by leading to restoration of lost expression of the WUSCHEL gene, which functions to maintain meristem activity, in the shoot apical mersitem of uni-1D. These results suggest that RPT2a is a major interacting partner of uni-1D/UNI, and that the interaction between uni-1D and RPT2a is responsible for activating both morphology and defense signals.

New perspectives on plant defense responses through modulation of developmental pathways.

Invasion mechanisms of pathogens and counteracting defense mechanisms of plants are highly diverse and perpetually evolving. While most classical studies of plant defense have focused only on defense-specific factor-mediated responses, recent work is beginning to shed light on the involvement of non-stress signal components, especially growth and developmental processes. This shift in focus links plant resistance more closely with growth and development. In this review, we summarize our current understanding of how pathogens manipulate host developmental processes and, conversely, of how plants deploy their developmental processes for self-protection. We conclude by introducing our recent work on UNI, a novel R protein in Arabidopsis which mediates cross-talk between developmental processes and defense responses.

Finding a missing link in MAP kinase cascade.

Mitogen-activated protein kinase (MAPK) cascade is one of the major signaling systems in eukaryotes. External signals are tranduced through three protein kinases, which successively relay phosphorylation to finally activate target genes/proteins. However, few information on targets of MAPK have so far been available. In this study, we identified a novel transcription factor, NtWIF, which is directly phosphorylated by a wound-induced protein kinase (WIPK), a typical MAPK from tobacco plants. Phosphorylated NtWIF recognizes the auxin responsive element (ARE), and transcriptionally activates ARE-driven Luciferase-reporter genes. Transgenic tobacco plants, in which NtWIF was overexpressed or suppressed, showed distinct features not only in pathogen resistance, but also in seed development and root growth. Micro-array assay using transgenic lines identified 178 differentially expressed genes, among which nearly half was related to defense and development. Screening of the available promoter regions revealed that multiple genes encoding such as pathogenesis-related protein Q (PR-Q), beta-1,3-glucanase, aminocyclopropane carboxylic acid synthase 2, P-450 and WIPK itself possess the ARE motif. Upon coexpression in cultured cells, NtWIF transcriptionally activated the Luciferase-reporter gene driven by intact promoters of PR-Q and WIPK. Since ARE is commonly found in auxin-responsive genes, NtWIF possibly targets diverse genes for defense and development by sharing processes that involve auxins. Transcriptional activation of WIPK by NtWIF suggests that WIPK is produced through a feed-back controlling system. It was thus concluded that NtWIF is a missing link between WIPK and its down-stream proteins, and that WIPK cascade is auto-regulated through a self-amplifying circuit.

Transactivation of wound-responsive genes containing the core sequence of the auxin-responsive element by a wound-induced protein kinase-activated transcription factor in tobacco plants.

Mitogen-activated protein kinases (MAPKs) constitute one of the most critical signaling components in plants. A typical example is wound-induced protein kinase (WIPK), which functions during pathogen responses in tobacco plants (Nicotiana tabacum). Searching for direct down-stream components, we previously isolated a novel transcription factor, which was activated upon phosphorylation by WIPK and designated as N. tabacum WIPK-interacting factor (NtWIF). Overexpression of NtWIF in tobacco plants enhanced the hypersensitive response (HR) upon tobacco mosaic virus infection and cryptogein treatment, while its silencing by RNAi suppressed such HR. NtWIF contains a specific motif similar to the B3 DNA binding domain, which recognizes the core TGTCTC motif called the auxin-responsive element (ARE). Using synthetic ARE sequences, NtWIF was also shown to recognize the ARE motifs and to transactivate the Luciferase (Luc)-reporter gene driven by such AREs in tobacco BY2 cultured cells. Subsequent microarray screening of NtWIF overexpressing tobacco identified 49 stress-responsive genes, and in silico analyses of available promoter regions of these genes revealed beta-1,3-glucanase, ACS2, P-450, and WIPK itself to contain the ARE core motif consisted of either TGTCTC or TGTCCT. Gel shift assay showed NtWIF to efficiently bind to both sequences. Assays with 1.5-kb PR-Q and 1.2 kb WIPK promoter regions, each fused to the Luc-reporter gene, indicated NtWIF to exhibit a clear transactivation activity, which was increased up to 3-fold upon phosphorylation by WIPK. These results revealed that NtWIF directly regulates multiple stress-responsive genes containing the ARE motif in their promoters, thereby partly filling up the last step of the MAPK cascade.

Expression of a WIPK-activated transcription factor results in increase of endogenous salicylic acid and pathogen resistance in tobacco plants.

NtWIF is a transcription factor activated upon phosphorylation by wound-induced protein kinase (WIPK) in tobacco plants. Transgenic tobacco plants overexpressing NtWIF exhibited constitutive accumulation of transcripts for pathogenesis-related genes, PR-1a and PR-2. Salicylic acid levels were 50-fold higher than those in wild-type plants. The levels of jasmonic acid and IAA did not significantly differ, while an increase of ABA upon wounding was delayed by 3 h in the transgenics. When challenged with tobacco mosaic virus, lesions developed faster and were smaller in the transgenic plants. The results suggest that NtWIF is likely to influence salicylic acid biosynthesis, being located downstream of WIPK.