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Plants recognize pathogens and activate immune responses, which usually involve massive transcriptional reprogramming. The evolutionarily conserved kinase, Sucrose non-fermenting-related kinase 1 (SnRK1), functions as a metabolic regulator that is essential for plant growth and stress responses. Here, we identify barley SnRK1 and a WRKY3 transcription factor by screening a cDNA library. SnRK1 interacts with WRKY3 in yeast, as confirmed by pull-down and luciferase complementation assays. Förster resonance energy transfer combined with noninvasive fluorescence lifetime imaging analysis indicates that the interaction occurs in the barley nucleus. Transient expression and virus-induced gene silencing analyses indicate that WRKY3 acts as a repressor of disease resistance to the Bgh fungus. Barley plants overexpressing WRKY3 have enhanced fungal microcolony formation and sporulation. Phosphorylation assays show that SnRK1 phosphorylates WRKY3 mainly at Ser83 and Ser112 to destabilize the repressor, and WRKY3 non-phosphorylation-null mutants at these two sites are more stable than the wild-type protein. SnRK1-overexpressing barley plants display enhanced disease resistance to Bgh . Transient expression of SnRK1 reduces fungal haustorium formation in barley cells, which probably requires SnRK1 nuclear localization and kinase activity. Together, these findings suggest that SnRK1 is directly involved in plant immunity through phosphorylation and destabilization of the WRKY3 repressor, revealing a new regulatory mechanism of immune derepression in plants., (© 2020 The Author(s).)

Background: Bicarbonate-based peritoneal dialysis (PD) fluids enhance the migratory capacity and damage-repair ability of human peritoneal mesothelial cells by upregulating AQP1. However, little is known about the underlying molecular mechanisms., Results: Here we used HEK-293T cells to investigate the effect of pH on AQP1 gene transcription levels. We found that AQP1 mRNA levels increases with pH. Transfection of HEK-293T cells with luciferase reporter vectors containing different regions of the AQP1 promoter identified an upstream region in the AQP1 gene between - 2200 and - 2300 bp as an enhancer required for pH-mediated regulation of AQP1 expression. Site-directed mutagenesis of this specific promoter region revealed a critical region between - 2257 and - 2251 bp, and gene knock-down experiments and ChIP assays suggested that the Spi-B transcription factor SPIB is involved in pH-mediated regulation of AQP1 expression., Conclusions: We identified an upstream region in the AQP1 gene and the transcription factor SPIB that are critically involved in pH-mediated regulation of AQP1 expression. These findings provide the basis for further studies on the pH- and buffer-dependent effects of PD fluids on peritoneal membrane integrity and function.

The glandular secretory trichomes (GSTs) on Artemisia annua leaves have the capacity to secrete and store artemisinin, a compound which is the most effective treatment for uncomplicated malaria. An effective strategy to improve artemisinin content is therefore to increase the density of GSTs in A. annua. However, the formation mechanism of GSTs remains poorly understood. To explore the mechanisms of GST initiation in A. annua, we screened myeloblastosis (MYB) transcription factor genes from a GST transcriptome database and identified a MIXTA transcription factor, AaMIXTA1, which is expressed predominantly in the basal cells of GST in A. annua. Overexpression and repression of AaMIXTA1 resulted in an increase and decrease, respectively, in the number of GSTs as well as the artemisinin content in transgenic plants. Transcriptome analysis and cuticular lipid profiling showed that AaMIXTA1 is likely to be responsible for activating cuticle biosynthesis. In addition, dual-luciferase reporter assays further demonstrated that AaMIXTA1 could directly activate the expression of genes related to cuticle biosynthesis. Taken together, AaMIXTA1 regulated cuticle biosynthesis and prompted GST initiation without any abnormal impact on the morphological structure of the GSTs and so provides a new way to improve artemisinin content in this important medicinal plant., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

The plant Artemisia annua is well known due to the production of artemisinin, a sesquiterpene lactone that is widely used in malaria treatment. Phytohormones play important roles in plant secondary metabolism, such as jasmonic acid (JA), which can induce artemisinin biosynthesis in A. annua. Nevertheless, the JA-inducing mechanism remains poorly understood. The expression of gene AaMYC2 was rapidly induced by JA and AaMYC2 binds the G-box-like motifs within the promoters of gene CYP71AV1 and DBR2, which are key structural genes in the artemisinin biosynthetic pathway. Overexpression of AaMYC2 in A. annua significantly activated the transcript levels of CYP71AV1 and DBR2, which resulted in an increased artemisinin content. By contrast, artemisinin content was reduced in the RNAi transgenic A. annua plants in which the expression of AaMYC2 was suppressed. Meanwhile, the RNAi transgenic A. annua plants showed lower sensitivity to methyl jasmonate treatment than the wild-type plants. These results demonstrate that AaMYC2 is a positive regulator of artemisinin biosynthesis and is of great value in genetic engineering of A. annua for increased artemisinin production., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

Artemisinin is an effective component of drugs against malaria. The regulation of artemisinin biosynthesis is at the forefront of artemisinin research. Previous studies showed that AaWRKY1 can regulate the expression of ADS, which is the first key enzyme in artemisinin biosynthetic pathway. In this study, AaWRKY1 was cloned, and it activated ADSpro and CYPpro in tobacco using dual-LUC assay. To further study the function of AaWRKY1, pCAMBIA2300-AaWRKY1 construct under 35S promoter was generated. Transgenic plants containing AaWRKY1 were obtained, and four independent lines with high expression of AaWRKY1 were analyzed. The expression of ADS and CYP, the key enzymes in artemisinin biosynthetic pathway, was dramatically increased in AaWRKY1-overexpressing A. annua plants. Furthermore, the artemisinin yield increased significantly in AaWRKY1-overexpressing A. annua plants. These results showed that AaWRKY1 increased the content of artemisinin by regulating the expression of both ADS and CYP. It provides a new insight into the mechanism of regulation on artemisinin biosynthesis via transcription factors in the future.

Artemisinin is a sesquiterpenoid especially synthesized in the Chinese herbal plant, Artemisia annua, which is widely used in the treatment of malaria. Artemisinin accumulation can be enhanced by exogenous abscisic acid (ABA) treatment. However, it is not known how ABA signaling regulates artemisinin biosynthesis. A global expression profile and phylogenetic analysis as well as the dual-LUC screening revealed that a basic leucine zipper family transcription factor from A. annua (namely AabZIP1) was involved in ABA signaling to regulate artemisinin biosynthesis. AabZIP1 had a higher expression level in the inflorescences than in other tissues; ABA treatment, drought, and salt stress strongly induced the expression of AabZIP1. Yeast one-hybrid assay and electrophoretic mobility shift assay (EMSA) showed that AabZIP1 bound to the ABA-responsive elements (ABRE) in the promoter regions of the amorpha-4,11-diene synthase (ADS) gene and CYP71AV1, which are two key structural genes of the artemisinin biosynthetic pathway. A mutagenesis assay showed that the C1 domain in the N-terminus of AabZIP1 was important for its transactivation activity. Furthermore, the activation of ADS and CYP71AV1 promoters by AabZIP1 was enhanced by ABA treatment in transient dual-LUC analysis. The AabZIP1 variant with C1 domain deletion lost the ability to activate ADS and CYP71AV1 promoters regardless of ABA treatment. Notably, overexpression of AabZIP1 in A. annua resulted in significantly increased accumulation of artemisinin. Our results indicate that ABA promotes artemisinin biosynthesis, likely through 1 activation of ADS and CYP71AV1 expression by AabZIP in A. annua. Meanwhile, our findings reveal the potential value of AabZIP1 in genetic engineering of artemisinin production., (Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.)

· Six transcription factors of APETALA2/ethylene-response factor (AP2/ERF) family were cloned and analyzed in Artemisia annua. Real-time quantitative polymerase chain reaction (RT-Q-PCR) showed that AaORA exhibited similar expression patterns to those of amorpha-4,11-diene synthase gene (ADS), cytochrome P450-dependent hydroxylase gene (CYP71AV1) and double bond reductase 2 gene (DBR2) in different tissues of A. annua. · AaORA is a trichome-specific transcription factor, which is expressed in both glandular secretory trichomes (GSTs) and nonglandular T-shaped trichomes (TSTs) of A. annua. The result of subcellular localization shows that AaORA is targeted to the nuclei and the cytoplasm. · Overexpression and RNA interference (RNAi) of AaORA in A. annua regulated, positively and significantly, the expression levels of ADS, CYP71AV1, DBR2 and AaERF1. The up-regulated or down-regulated expression levels of these genes resulted in a significant increase or decrease in artemisinin and dihydroartemisinic acid. The results demonstrate that AaORA is a positive regulator in the biosynthesis of artemisinin. · Overexpression of AaORA in Arabidopsis thaliana increased greatly the transcript levels of the defense marker genes PLANT DEFENSIN1.2 (PDF1.2), HEVEIN-LIKE PROTEIN (HEL) and BASIC CHITINASE (B-CHI). After inoculation with Botrytis cinerea, the phenotypes of AaORA overexpression in A. thaliana and AaORA RNAi in A. annua demonstrate that AaORA is a positive regulator of disease resistance to B. cinerea., (© 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.)

The nucleotide binding domain and Leucine-rich repeat (NLR)-containing proteins in plants and animals mediate pathogen sensing inside host cells and mount innate immune responses against microbial pathogens. The barley (Hordeum vulgare) mildew A (MLA) locus encodes coiled-coil (CC)-type NLRs mediating disease resistance against the powdery mildew pathogen Blumeria graminis. Here, we report direct interactions between MLA and two antagonistically acting transcription factors, MYB6 and WRKY1. The N-terminal CC signaling domain of MLA interacts with MYB6 to stimulate its DNA binding activity. MYB6 functions as a positive regulator of basal and MLA-mediated immunity responses to B. graminis. MYB6 DNA binding is antagonized by direct association with WRKY1 repressor, which in turn also interacts with the MLA CC domain. The activated form of full-length MLA10 receptor is needed to release MYB6 activator from WRKY1 repression and to stimulate MYB6-dependent gene expression. This implies that, while sequestered by the WRKY1 repressor in the presence of the resting immune receptor, MYB6 acts as an immediate and positive postactivation signaling component of the active state of MLA during transcriptional reprogramming for innate immune responses.

Plants are sessile organisms, and they can not move away under abiotic or biotic stresses. Thus plants have evolved a set of genes that response to adverse environment to modulate gene expression. In this study, we characterized and functionally studied an ERF transcription factor from Artemisia annua, AaERF1, which plays an important role in biotic stress responses. The AaERF1 promoter had been cloned and GUS staining results of AaERF1 promoter-GUS transgenic A. annua showed that AaERF1 is expressed ubiquitiously in all organs. Several putative cis-acting elements such as W-box, TGA-box and Py-rich element, which are involved in defense responsiveness, are present in the promoter. The expression of AaERF1 can be induced vigorously by methyl jasmonate as well as by ethephon and wounding, implying that AaERF1 may activate some of the defense genes via the jasmonic acid and ethylene signaling pathways of A. annua. The results of electrophoretic mobility shift assay (EMSA) and yeast one-hybrid experiments showed that AaERF1 was able to bind to the GCC box cis-acting element in vitro and in yeast. Ectopic expression of AaERF1 could enhance the expression levels of the defense marker genes PLANT DEFENSIN1.2 (PDF1.2) and BASIC CHITINASE (ChiB), and increase the resistance to Botrytis cinerea in the 35S::AaERF1 transgenic Arabidopsis. The down-regulated expression level of AaERF1 evidently reduced the resistance to B. cinerea in A. annua. The overall results showed that AaERF1 positively regulated the resistance to B. cinerea in A. annua.

Plant immune responses are triggered by pattern recognition receptors that detect conserved pathogen-associated molecular patterns (PAMPs) or by resistance (R) proteins recognizing isolate-specific pathogen effectors. We show that in barley, intracellular mildew A (MLA) R proteins function in the nucleus to confer resistance against the powdery mildew fungus. Recognition of the fungal avirulence A10 effector by MLA10 induces nuclear associations between receptor and WRKY transcription factors. The identified WRKY proteins act as repressors of PAMP-triggered basal defense. MLA appears to interfere with the WRKY repressor function, thereby de-repressing PAMP-triggered basal defense. Our findings reveal a mechanism by which these polymorphic immune receptors integrate distinct pathogen signals.

Artemisinin, a sesquiterpene lactone obtained from Artemisia annua , is an essential therapeutic against malaria. YABBY family transcription factor AaYABBY5 is an activator of AaCYP71AV1 (cytochrome P450–dependent hydroxylase) and AaDBR2 (double-bond reductase 2); however, the protein–protein interactions of AaYABBY5, as well as the mechanism of its regulation, have not yet been elucidated. AaWRKY9 protein is a positive regulator of artemisinin biosynthesis that activates AaGSW1 (glandular trichome–specific WRKY1) and AaDBR2 (double-bond reductase 2). In this study, YABBY–WRKY interactions are revealed to indirectly regulate artemisinin production. AaYABBY5 significantly increased the activity of the luciferase (LUC) gene fused to the promoter of AaGSW1. Toward the molecular basis of this regulation, AaYABBY5 interaction with AaWRKY9 protein was found. The combined effectors AaYABBY5 + AaWRKY9 showed synergistic effects toward the activities of AaGSW1 and AaDBR2 promoters, respectively. In AaYABBY5 overexpression plants, the expression of GSW1 was found to be significantly increased when compared to that of AaYABBY5 antisense or control plants. In addition, AaGSW1 was identified as an upstream activator of AaYABBY5. Further, it was found that AaJAZ8, a transcriptional repressor of jasmonate signaling, interacted with AaYABBY5 and attenuated its activity. Co-expression of AaYABBY5 and anti- AaJAZ8 in A. annua increased the activity of AaYABBY5 toward artemisinin biosynthesis. This current study provides the first indication of the molecular basis of regulation of artemisinin biosynthesis through YABBY–WRKY interactions, which are regulated through AaJAZ8. This knowledge presents AaYABBY5 overexpression plants as a powerful genetic resource for artemisinin biosynthesis. [ABSTRACT FROM AUTHOR]

Summary: Plant glandular secretory trichomes (GSTs) produce various specialized metabolites. Increasing GST density represents a strategy to enhance the yield of these chemicals; however, the gene regulatory network that controls GST initiation remains unclear.In a previous study of Artemisiaannua L., we found that a HD‐ZIP IV transcription factor, AaHD1, promotes GST initiation by directly regulating AaGSW2. Here, we identified two AaHD1‐interacting transcription factors, namely AaMIXTA‐like 2 (AaMYB16) and AaMYB5.Through the generation and characterization of transgenic plants, we found that AaMYB16 is a positive regulator of GST initiation, whereas AaMYB5 has the opposite effect. Notably, neither of them regulates GST formation independently. Rather, they act competitively, by interacting and modulating AaHD1 promoter binding activity. Additionally, the phytohormone jasmonic acid (JA) was shown to be associated with the AaHD1‐AaMYB16/AaMYB5 regulatory network through transcriptional regulation via a JASMONATE‐ZIM DOMAIN (JAZ) protein repressor.These results bring new insights into the mechanism of GST initiation through regulatory complexes, which appear to have similar functions in a range of vascular plant taxa. [ABSTRACT FROM AUTHOR]

Summary: Artemisinin, a sesquiterpene lactone widely used in malaria treatment, was discovered in the medicinal plant Artemisia annua. The biosynthesis of artemisinin is efficiently regulated by jasmonate (JA) and abscisic acid (ABA) via regulatory factors. However, the mechanisms linking JA and ABA signalling with artemisinin biosynthesis through an associated regulatory network of downstream transcription factors (TFs) remain enigmatic. Here we report AaTCP15, a JA and ABA dual‐responsive teosinte branched1/cycloidea/proliferating (TCP) TF, which is essential for JA and ABA‐induced artemisinin biosynthesis by directly binding to and activating the promoters of DBR2 and ALDH1, two genes encoding enzymes for artemisinin biosynthesis. Furthermore, AaORA, another positive regulator of artemisinin biosynthesis responds to JA and ABA, interacts with and enhances the transactivation activity of AaTCP15 and simultaneously activates AaTCP15 transcripts. Hence, they form an AaORA‐AaTCP15 module to synergistically activate DBR2, a crucial gene for artemisinin biosynthesis. More importantly, AaTCP15 expression is activated by the multiple reported JA and ABA‐responsive TFs that promote artemisinin biosynthesis. Among them, AaGSW1 acts at the nexus of JA and ABA signalling to activate the artemisinin biosynthetic pathway and directly binds to and activates the AaTCP15 promoter apart from the AaORA promoter, which further facilitates formation of the AaGSW1‐AaTCP15/AaORA regulatory module to integrate JA and ABA‐mediated artemisinin biosynthesis. Our results establish a multilayer regulatory network of the AaGSW1‐AaTCP15/AaORA module to regulate artemisinin biosynthesis through JA and ABA signalling, and provide an interesting avenue for future research exploring the special transcriptional regulation module of TCP genes associated with specialized metabolites in plants. [ABSTRACT FROM AUTHOR]

Glandular secreting trichomes (GSTs) synthesize and secrete large quantities of secondary metabolites, some of which have well-established commercial value. An example is the anti-malarial compound artemisinin, which is synthesized in the GSTs of Artemisia annua. Accordingly, there is considerable interest in understanding the processes that regulate GST density as a strategy to increase artemisinin production. In this study, we identified a GST-specific WRKY transcription factor from A. annua , AaGSW2, which is positively regulated by the direct binding of the homeodomain proteins AaHD1 and AaHD8 to the L1-box of the AaGSW2 promoter. Overexpression of AaGSW2 in A. annua significantly increased GST density, while AaGSW2 knockdown lines showed impaired GST initiation. Ectopic expression of AaGSW2 homologs from two mint cultivars, Mentha spicata and Mentha haplocalyx , in A. annua also induced GST formation. These results reveal a molecular mechanism involving homeodomain and WRKY proteins that controls glandular trichome initiation, at least part of which is shared by A. annua and mint. [ABSTRACT FROM AUTHOR]

Artemisinin-based therapies are the only effective treatment for malaria, which reached to 219 million cases and killed 435,000 people in 2017. To meet the growing demand for artemisinin and make it accessible to the poorest, genetic engineering of Artemisia annua becomes one of the most promising approaches to improve artemisinin yield. In this work, AabZIP9 transcription factor has been identified and characterized. The expression profile of AabZIP9 revealed that it was clustered with the artemisinin specific biosynthetic pathway genes ADS , CYP71AV1 , DBR2 , and ALDH1. Furthermore, the transiently dual-LUC analysis showed that the activation of ADS promoter was enhanced by AabZIP9. Meanwhile, yeast one-hybrid assay showed that AabZIP9 was able to bind to the "ACGT" cis -element present in both ADS and CYP71AV1 promoters. AabZIP9 gene was driven by the constitutive CaMV35S promoter and the glandular trichome specific CYP71AV1 promoter and stably transformed into A. annua plants. The transcript level of AabZIP9 was increased in both of the 35S and CYP71AV1 driven transgenic plants compared with the wild type or GUS control plants. All the transgenic A. annua plants overexpressing AabZIP9 showed elevated transcript level of ADS , but the transcription levels of CYP71AV1 , DBR2 , and ALDH1 have no significant change in both types of transgenic plants. The significantly upregulated ADS promoted the accumulation of artemisinin, dihydroartemisinic acid, and artemisinic acid biosynthesis in the transgenic A. annua plants. These results suggest that AabZIP9 can positively regulate the biosynthesis of artemisinin. [ABSTRACT FROM AUTHOR]

Artemisinin is an effective antimalarial sesquiterpene lactone synthesized in Artemisia annua. Various transcription factors have been previously reported that can influence the biosynthesis of artemisinin; however, the effect of YABBY family transcription factors on artemisinin biosynthesis was unknown. In the present study, we cloned and characterized AaYABBY5: a homolog of MsYABBY5 in Mentha spicata which is involved in modulating the monoterpenes, as a positive regulator of artemisinin biosynthesis in A. annua. AaYABBY5 was found localized to the nucleus, and its expression was found to be induced by exogenous methyl jasmonic acid (MeJA) treatment. In the dual-luciferase reporter assay, it was found that AaYABBY5 significantly increased the activities of promoters of amorpha-4,11-diene synthase (ADS), cytochrome P450 monooxygenase (CYP71AV1), double-bond reductase 2 (DBR2), and aldehyde dehydrogenase 1 (ALDH1) genes. Yeast one hybrid assay showed that AaYABBY5 directly bonds to the promoters of CYP71AV1 and DBR2 genes. Quantitative real-time polymerase chain reaction (qPCR) of AaYABBY5 overexpression and AaYABBY5 antisense plants revealed a significant increase in the expression of ADS , CYP71AV1 , DBR2 , and ALDH1 in AaYABBY5 overexpression plants and a significant decrease in the expression of these genes in AaYABBY5 antisense A. annua , respectively. Furthermore, the results of high-performance liquid chromatography (HPLC) showed that the artemisinin and its precursor dihydroartemisinic acid were significantly increased in the AaYABBY5 overexpression plants while AaYABBY5 downregulation resulted in a significant decrease in the concentration of artemisinin. Taken together, these results explicitly represent that AaYABBY5 is a positive regulator of artemisinin biosynthesis in A. annua. [ABSTRACT FROM AUTHOR]

Artemisinin is a sesquiterpene lactone produced by the Chinese traditional herb Artemisia annua and is used for the treatment of malaria. It is known that salicylic acid (SA) can enhance artemisinin content but the mechanism by which it does so is not known. In this study, we systematically investigated a basic leucine zipper family transcription factor, AaTGA6, involved in SA signaling to regulate artemisinin biosynthesis. We found specific in vivo and in vitro binding of the AaTGA6 protein to a 'TGACG' element in the AaERF1 promoter. Moreover, we demonstrated that AaNPR1 can interact with AaTGA6 and enhance its DNA-binding activity to its cognate promoter element 'TGACG' in the promoter of AaERF1 , thus enhancing artemisinin biosynthesis. The artemisinin contents in AaTGA6-overexpressing and RNAi transgenic plants were increased by 90–120% and decreased by 20–60%, respectively, indicating that AaTGA6 plays a positive role in artemisinin biosynthesis. Importantly, heterodimerization with AaTGA3 significantly inhibits the DNA-binding activity of AaTGA6 and plays a negative role in target gene activation. In conclusion, we demonstrate that binding of AaTGA6 to the promoter of the artemisinin-regulatory gene AaERF1 is enhanced by AaNPR1 and inhibited by AaTGA3. Based on these findings, AaTGA6 has potential value in the genetic engineering of artemisinin production. [ABSTRACT FROM AUTHOR]

Artemisinin, the frontline drug against malaria, is a sesquiterpenoid extracted from Artemisia annua. Light has been proposed to play an important role in the activation of artemisinin biosynthesis. Here, we report the basic leucine zipper transcription factor (TF) AaHY5 as a key regulator of light-induced biosynthesis of artemisinin. We show that AaHY5 transcription overlaps with that of artemisinin biosynthesis genes in response to light and in A. annua tissues. Analysis of AaHY5 overexpression and RNAi-suppression lines suggests that AaHY5 is a positive regulator of the expression of artemisinin biosynthesis genes and accumulation of artemisinin. We show that AaHY5 complements the hy5 mutant in Arabidopsis thaliana. Our data further suggest that AaHY5 interacts with AaCOP1, the ubiquitin E3 ligase CONSTITUTIVE PHOTOMORPHOGENIC1 in A. annua. In yeast one-hybrid and transient expression assays, we demonstrate that AaHY5 acts via the TF GLANDULAR TRICHOME-SPECIFIC WRKY 1 (AaGSW1) in artemisinin regulation. In summary, we present a novel regulator of artemisinin gene expression and propose a model in which AaHY5 indirectly controls artemisinin production in response to changing light conditions. [ABSTRACT FROM AUTHOR]

Six transcription factors of APETALA2/ethylene-response factor ( AP2/ ERF) family were cloned and analyzed in Artemisia annua. Real-time quantitative polymerase chain reaction ( RT- Q- PCR) showed that Aa ORA exhibited similar expression patterns to those of amorpha-4,11-diene synthase gene ( ADS), cytochrome P450-dependent hydroxylase gene ( CYP71AV1) and double bond reductase 2 gene ( DBR2) in different tissues of A. annua., Aa ORA is a trichome-specific transcription factor, which is expressed in both glandular secretory trichomes ( GSTs) and nonglandular T-shaped trichomes ( TSTs) of A. annua. The result of subcellular localization shows that Aa ORA is targeted to the nuclei and the cytoplasm., Overexpression and RNA interference ( RNAi) of Aa ORA in A. annua regulated, positively and significantly, the expression levels of ADS, CYP71 AV1, DBR2 and Aa ERF1. The up-regulated or down-regulated expression levels of these genes resulted in a significant increase or decrease in artemisinin and dihydroartemisinic acid. The results demonstrate that Aa ORA is a positive regulator in the biosynthesis of artemisinin., Overexpression of Aa ORA in Arabidopsis thaliana increased greatly the transcript levels of the defense marker genes PLANT DEFENSIN1.2 ( PDF1.2), HEVEIN-LIKE PROTEIN ( HEL) and BASIC CHITINASE ( B-CHI). After inoculation with Botrytis cinerea, the phenotypes of Aa ORA overexpression in A. thaliana and Aa ORA RNAi in A. annua demonstrate that Aa ORA is a positive regulator of disease resistance to B. cinerea. [ABSTRACT FROM AUTHOR]