AP2/ERF transcription factor NbERF-IX-33 is involved in the regulation of phytoalexin production for the resistance of Nicotiana benthamiana to Phytophthora infestans.

Plants recognize molecular patterns unique to a certain group of microbes to induce effective resistance mechanisms. Elicitins are secretory proteins produced by plant pathogenic oomycete genera including Phytophthora and Pythium. Treatment of INF1 (an elicitin produced by P. infestans) induces a series of defense responses in Nicotiana species, including reactive oxygen species (ROS) production, hypersensitive cell death and accumulation of the sesquiterpenoid phytoalexin capsidiol. In this study, we analyzed the expression profiles of N. benthamiana genes after INF1 treatment by RNAseq analysis. Based on their expression patterns, N. benthamiana genes were categorized into 20 clusters and 4,761 (8.3%) out of 57,140 genes were assigned to the clusters for INF1-induced genes. All genes encoding enzymes dedicated for capsidiol production, 5-epi-aristolochene (EA) synthase (NbEAS, 10 copies) and EA dehydrogenase (NbEAH, 6 copies) and some genes for ethylene production, such as 1-aminocyclopropane 1-carboxylate (ACC) synthase (NbACS) and ACC oxidase (NbACO), were significantly upregulated by INF1 treatment. Analysis of NbEAS1 and NbEAS4 promoters revealed that AGACGCC (GCC box-like motif) is the essential cis-element required for INF1-induced expression of NbEAS genes. Given that the GCC box is known to be targeted by ERF (ethylene-responsive factor) transcription factors, we created a complete list of N. benthamiana genes encoding AP2/ERF family transcription factors, and identified 45 out of 337 AP2/ERF genes in the clusters for INF1-induced genes. Among INF1-induced NbERF genes, silencing of NbERF-IX-33 compromised resistance against P. infestans and INF1-induced production of capsidiol. Recombinant NbERF-IX-33 protein can bind to the promoter sequence of NbEAS4, suggesting that NbERF-IX-33 is a transcription factor directly regulating the expression of genes for phytoalexin production.

The Arabidopsis ORA59 coordinates jasmonic acid- and ethylene-responsive gene expression to regulate plant immunity

Jasmonic acid (JA) and ethylene (ET) signaling modulate plant defense against necrotrophic pathogens. These hormone pathways lead to transcriptional reprogramming, which is a major part of plant immunity and requires the roles of transcription factors. ET response factors are responsible for the transcriptional regulation of JA/ET-responsive defense genes, among which ORA59 functions as a key regulator of this process and has been implicated in the JA-ET crosstalk. Here, we identified the ERELEE4 as an ORA59-binding cis-element, in addition to the well-characterized GCC box, demonstrating that ORA59 regulates JA/ET-responsive genes through direct binding to these elements in the gene promoters. Notably, ORA59 exhibited differential preference for the GCC box and ERELEE4, depending on whether ORA59 activation is achieved by JA and ET, respectively. Our results provide insights into how ORA59 can generate specific patterns of gene expression dynamics through JA and ET hormone pathways.

Two Alternative Splicing Variants of AtERF73/HRE1, HRE1α and HRE1β, Have Differential Transactivation Activities in Arabidopsis

AtERF73/HRE1 is an AP2/ERF transcription factor in Arabidopsis and has two distinct alternative splicing variants, HRE1α and HRE1β. In this study, we examined the differences between the molecular functions of HRE1α and HRE1β. We found that HRE1α and HRE1β are both involved in hypoxia response and root development and have transactivation activity. Two conserved motifs in the C-terminal region of HRE1α and HRE1β, EELL and LWSY-like, contributed to their transactivation activity, specifically the four E residues in the EELL motif and the MGLWS amino acid sequence at the end of the LWSY-like motif. The N-terminal region of HRE1β also showed transactivation activity, mediated by the VDDG motif, whereas that of HRE1α did not. The transactivation activity of HRE1β was stronger than that of HRE1α in Arabidopsis protoplasts. Both transcription factors transactivated downstream genes via the GCC box. RNA-sequencing analysis further supported that both HRE1α and HRE1β might regulate gene expression associated with the hypoxia stress response, although they may transactivate different subsets of genes in downstream pathways. Our results, together with previous studies, suggested that HRE1α and HRE1β differentially transactivate downstream genes in hypoxia response and root development in Arabidopsis.

Structural insights into Arabidopsis ethylene response factor 96 with an extended N-terminal binding to GCC box

The phytohormone ethylene is widely involved in many developmental processes and is a crucial regulator of defense responses against biotic and abiotic stresses in plants. Ethylene-responsive element binding protein, a member of the APETALA2/ethylene response factor (AP2/ERF) superfamily, is a transcription factor that regulates stress-responsive genes by recognizing a specific cis-acting element of target DNA. A previous study showed only the NMR structure of the AP2/ERF domain of AtERF100 in complex with a GCC box DNA motif. In this report, we determined the crystal structure of AtERF96 in complex with a GCC box at atomic resolution. We analyzed the binding residues of the conserved AP2/ERF domain in the DNA recognition sequence. In addition to the AP2/ERF domain, an N-terminal α-helix of AtERF96 participates in DNA interaction in the flanking region. We also demonstrated the structure of AtERF96 EDLL motif, a unique conserved motif in the group IX of AP2/ERF family, might involve in the transactivation of defense-related genes. Our study establishes the structural basis of the AtERF96 transcription factor in complex with the GCC box, as well as the DNA binding mechanisms of the N-terminal α-helix and AP2/ERF domain.

An AC-Rich Bean Element Serves as an Ethylene-Responsive Element in Arabidopsis

Ethylene-responsive elements (EREs), such as the GCC box, are critical for ethylene-regulated transcription in plants. Our previous work identified a 19-bp AC-rich element (ACE) in the promoter of bean (Phaseolus vulgaris) metal response element-binding transcription factor 1 (PvMTF-1). Ethylene response factor 15 (PvERF15) directly binds ACE to enhance PvMTF-1 expression. As a novel ERF-binding element, ACE exhibits a significant difference from the GCC box. Here, we demonstrated that ACE serves as an ERE in Arabidopsis. It conferred the minimal promoter to respond to the ethylene stress and inhibition of ethylene. Moreover, the cis-acting element ACE could specifically bind the nuclear proteins in vitro. We further revealed that the first 9-bp sequence of ACE (ACEcore) is importantly required by the binding of nuclear proteins. In addition, PvERF15 and PvMTF-1 were strongly induced by ethylene in bean seedlings. Since PvERF15 activates PvMTF-1 via ACE, ACE is involved in ethylene-induced PvMTF-1 expression. Taken together, our findings provide genetic and biochemical evidence for a new ERE.

Tinkering Cis Motifs Jigsaw Puzzle Led to Root-Specific Drought-Inducible Novel Synthetic Promoters

Following an in-depth transcriptomics-based approach, we first screened out and analyzed (in silico) cis motifs in a group of 63 drought-inducible genes (in soybean). Six novel synthetic promoters (SynP14-SynP19) were designed by concatenating 11 cis motifs, ABF, ABRE, ABRE-Like, CBF, E2F-VARIANT, G-box, GCC-Box, MYB1, MYB4, RAV1-A, and RAV1-B (in multiple copies and various combination) with a minimal 35s core promoter and a 222 bp synthetic intron sequence. In order to validate their drought-inducibility and root-specificity, the designed synthetic assemblies were transformed in soybean hairy roots to drive GUS gene using pCAMBIA3301. Through GUS histochemical assay (after a 72 h 6% PEG6000 treatment), we noticed higher glucuronidase activity in transgenic hairy roots harboring SynP15, SynP16, and SynP18. Further screening through GUS fluorometric assay flaunted SynP16 as the most appropriate combination of efficient drought-responsive cis motifs. Afterwards, we stably transformed SynP15, SynP16, and SynP18 in Arabidopsis and carried out GUS staining as well as fluorometric assays of the transgenic plants treated with simulated drought stress. Consistently, SynP16 retained higher transcriptional activity in Arabidopsis roots in response to drought. Thus the root-specific drought-inducible synthetic promoters designed using stimulus-specific cis motifs in a definite fashion could be exploited in developing drought tolerance in soybean and other crops as well. Moreover, the rationale of design extends our knowledge of trial-and-error based cis engineering to construct synthetic promoters for transcriptional upgradation against other stresses.

Pathogen Inducible cis-acting Elements of Synthetic Promoters in Plants-review

Synthetic pathogen inducible promoters are used for the improvement and application of transgenic techniques in research and to increase agriculture production. The promoter contains specific cis-regulatory elements (W box, GCC box, Box S and D box) which induce anti pathogen molecular cascades. Insertion of dimerized form of cis acting elements at upstream region of promoter in promoter probe vector drives the expression of resistance gene or reporter gene. The expression indicates that synthetic promoters are responded to fungal elicitors. Expression of resistance restrict to infection sites which boost disease resistance in plants.

Abscisic acid is essential for rewiring of jasmonic acid-dependent defenses during herbivory

Jasmonic acid (JA) is an important plant hormone in the regulation of defenses against chewing herbivores and necrotrophic pathogens. In Arabidopsis thaliana, the JA response pathway consists of two antagonistic branches that are regulated by MYC- and ERF-type transcription factors, respectively. The role of abscisic acid (ABA) and ethylene (ET) in the molecular regulation of the MYC/ERF antagonism during plant-insect interactions is still unclear. Here, we show that production of ABA induced in response to leaf-chewing Pieris rapae caterpillars is required for both the activation of the MYC-branch and the suppression of the ERF-branch during herbivory. Exogenous application of ABA suppressed ectopic ERF-mediated PDF1.2 expression in 35S::ORA59 plants. Moreover, the GCC-box promoter motif, which is required for JA/ET-induced activation of the ERF-branch genes ORA59 and PDF1.2, was targeted by ABA. Application of gaseous ET counteracted activation of the MYC-branch and repression of the ERF-branch by P. rapae, but infection with the ET-inducing necrotrophic pathogen Botrytis cinerea did not. Accordingly, P. rapae performed equally well on B. cinerea-infected and control plants, whereas activation of the MYC-branch resulted in reduced caterpillar performance. Together, these data indicate that upon feeding by P. rapae, ABA is essential for activating the MYC-branch and suppressing the ERF-branch of the JA pathway, which maximizes defense against caterpillars.

The Roles of Arabidopsis C1-2i Subclass of C2H2-type Zinc-Finger Transcription Factors

The Cys2His2 (C2H2)-type zinc-finger protein (ZFP) family, which includes 176 members in Arabidopsis thaliana, is one of the largest families of putative transcription factors in plants. Of the Arabidopsis ZFP members, only 33 members are conserved in other eukaryotes, with 143 considered to be plant specific. C2H2-type ZFPs have been extensively studied and have been shown to play important roles in plant development and environmental stress responses by transcriptional regulation. The ethylene-responsive element binding-factor-associated amphiphilic repression (EAR) domain (GCC box) has been found to have a critical role in the tolerance response to abiotic stress. Many of the plant ZFPs containing the EAR domain, such as AZF1/2/3, ZAT7, ZAT10, and ZAT12, have been shown to function as transcriptional repressors. In this review, we mainly focus on the C1-2i subclass of C2H2 ZFPs and summarize the latest research into their roles in various stress responses. The role of C2H2-type ZFPs in response to the abiotic and biotic stress signaling network is not well explained, and amongst them, C1-2i is one of the better-characterized classifications in response to environmental stresses. These studies of the C1-2i subclass ought to furnish the basis for future studies to discover the pathways and receptors concerned in stress defense. Research has implied possible protein-protein interactions between members of C1-2i under various stresses, for which we have proposed a hypothetical model.