Browsing by Author "Dong, Xinnian"
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Item Open Access Balance Between Plant Growth and Defense: Transcriptional and Translational Control of Plant Immune System(2012) Wang, WeiThe activation and maintenance of plant immune responses require a significant amount of energy because they are accompanied by massive transcriptional reprogramming. Spurious activation of plant defense machinery can lead to autoimmune diseases and growth inhibition. So it is important for plants to tightly regulate the immune system to ensure the balance between growth and defense. However, neither the molecular mechanisms nor the design principles of how plants reach this balance are understood.
In this dissertation work, I showed how transcriptional and translational control of plant immune system can help avoid the constant immune surveillance and elicit a proper level of defense responses when necessary. These fine tunings of the immune system ensure the balance between growth and defense.
My research on the transcriptional regulation of plant defense responses led to the surprising discovery that even without pathogen, plant can 'anticipate' potential infection according to a circadian schedule under conditions that favor the initiation of infection. Functional analysis of 22 novel immune components unveiled their transient expression at dawn, when the infection is most likely to happen. This pulse expression pattern was shown to be regulated by the central circadian oscillator, CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) since these 22 genes are no longer induced in the cca1 mutant. Moreover, the temporal control of the transcription level of these 22 immune genes by CCA1 also fine tunes their expression pattern according to the perceptions of different pathogenic signals. At the basal defense level, the expression of these genes can be transiently induced upon perceptions of critical infection stages of the pathogen. When an elevated level of defense response is needed, the high expression levels of these genes are maintained to confer a stronger immunity against pathogen. Since this stronger form of defense may also cause the suicidal death of the plant cells, the interplay between the circadian clock and defense allows a better decision on the proper level of the immunity to minimize the sacrificial death. The circadian clock is also known to regulate the growth-related cellular functions extensively. So the circadian clock can help to balance the energy used in growth and defense through transcriptional regulation on both sides.
Besides the integrated control by the circadian clock, the translational control on a key transcription factor involved in the growth-to-defense transition can also maintain the balance between growth and defense.TBF1 is a major transcription factor that can initiate the growth-to-defense transition through transcriptional repression of growth-associated cellular functions and induction of defense-related machinery. Bioinformatics studies identified 2 upstream open reading frames (uORFs) encoding multiple phenylalanine at 5' of the translation initiation codon of TBF1. Under normal conditions, these 2 uORFs can repress the translation of TBF1 to prevent accidental activation. However, pathogen infection may cause rapid and transient depletion of phenylalanine, a well-known precursor for cell wall components and the SAR signal SA. This depletion signal can be reflected by the increase of uncharged tRNAPhe, which subsequently leads to the phosphorylation of eIF2á and the release of uORFs' repression on TBF1. These findings provided the molecular details of how uORF-based translational control can couple transcriptional reprogramming with metabolic status to coordinately trigger the growth-to-defense transition.
In summary, my dissertation work has identified previously unrecognized regulatory mechanisms by which plant immune responses are balanced with growth. These new findings will further investigations into these novel interfaces between plants and pathogens. Future studies will definitely further improve our understandings of the plant-microbe interactions.
Item Open Access Circadian Regulation of Plant Physiology: A New Role for Humidity, the Forgotten Zeitgeber(2016) Mwimba, MusokiAir humidity is an environmental cue that influences many physiological responses in terrestrial organisms. In early chronobiology studies, it was thought that humidity could influence the circadian clock. However, no further studies have been performed in pursuit of this idea, nor humidity been investigated as a potential Zeitgeber, despite the fact that daily humidity fluctuation has been widely observed. Here, we report that humidity is a Zeitgeber on par with light and temperature and that it can fundamentally influence plant physiology. Using 10 years of weather records obtained from Harvard Forest and Weather Underground databases, we quantitatively characterized humidity oscillation as a natural phenomenon and found that the circadian oscillation of humidity (i.e., lower during the day and higher at night) is robust and independent of climates or seasons. Moreover, there is a 2-hour overlap of high humidity with light in the morning, suggesting a possible interplay between these two environmental cues and the circadian clock. We then tested and found that humidity can indeed entrain the circadian clock as a bona fide Zeitgeber through regulation of the plant core morning clock gene CCA1 (CIRCADIAN CLOCK ASSOCIATED 1) and the core evening clock gene TOC1 (TIMING OF CAB EXPRESSION 1). However, the core morning clock gene LHY (LATE ELONGATED HYPOCOTYL), partner of CCA1, resists the humidity-entrainment, indicating that the plant circadian clock may have two intertwined loops: one is responsive to both light and humidity via CCA1, whereas the other is responsive only to light mediated by LHY. In the absence of rhythmic light signal, humidity can entrain the clock, whereas in the presence of light in the morning, humidity synergistically reinforces the clock. At the physiological level, the humidity-entrained clock provides additional advantages to plants. It can improve energy storage, reduce the flowering time and improve seed production in a circadian-dependent manner. Additionally, we found that humidity can fundamentally shape plant-microbe interactions because it is a key signal for the initial induction of the bacterial type III secretion system and effector genes required for virulence. Thus, it is necessary for plants to mount an appropriate response to pathogen infections at night when humidity naturally rises. These findings demonstrate how terrestrial organisms could adapt to humidity oscillation, a universal environmental cue, to strategically maximize their own fitness. We propose that humidity-sensing might be an ancient determinant shaping the evolution of terrestrial organisms after their transition away from aquatic environment.
Item Open Access Exploring the Roles of GCN2 and the m6A RNA Modification in Plant Immunity(2019) Motley, JonathanPlants have evolved a robust immune system to fend off pathogens. This response must be tightly regulated, as aberrant activation can have detrimental effects. Much work has been done to understand the transcriptional responses in plant immunity, but less is known about post-transcriptional mechanisms. Here I examine the roles of the general control nonderepressible 2 (GCN2) kinase and the N6-methyladenosine (m6A) RNA modification in regulating plant immunity at the post-transcriptional level.
A previous study revealed that the master immune regulator TBF1 (a transcription factor) is essential for mediating a defense response. TBF1 is under the control of 2 upstream open reading frames (uORFs) which inhibit translation of TBF1’s major open reading frame (mORF). These uORFs’ sequences are enriched with codons for aromatic amino acids, especially phenylalanine. Pathogen treatment resulted in the induction of uncharged tRNAphe and eIF2α phosphorylation. It was proposed that GCN2 would mediate eIF2α phosphorylation and allow readthrough of TBF1’s mORF, analogous to GCN2’s role in promoting translation of GCN4 upon amino acid starvation. To test this, eIF2α phosphorylation was examined in gcn2 mutant plants upon pathogen infection using a phoshpo-specific eIF2α antibody. eIF2α phosphorylation was not observed in gcn2 plants upon pathogen treatment and therefore GCN2 is likely to be the kinase responsible for this induced eIF2α phosphorylation. To test GCN2’s role in positively regulating TBF1, a bacterial infection assay was undertaken with gcn2 plants. Compared to wild-type plants, gcn2 plants did not have any significant difference in bacterial growth and therefore GCN2 is unlikely to play a role in regulating TBF1 or plant immunity.
The m6A modification is the most abundant internal modification present in mRNAs and has been found to regulate several aspects of mRNA metabolism and biological processes. There is less known about m6A in plants, though it has been found to regulate stability of transcripts and is important for development and the salt stress response. It is unknown whether m6A plays a role in plant immunity. To address this, m6A deficient plant lines were used and bacterial infection assays undertaken. These lines displayed significantly higher levels of bacterial growth (susceptibility) and thus m6A plays a positive role in plant immunity. Further assays found that m6A was essential for fully mediating pattern-triggered immunity (PTI) and salicylic acid (SA)-mediated immune responses. m6A-seq was used to map the dynamics of m6A across the transcriptome in response to the immune inducers SA and elf18 (a microbe-associated molecular pattern). Hundreds of pathogen-induced methylation sites were uncovered and gene ontology (GO) analysis revealed a predominance of defense/immune-related transcripts.
In summary, this dissertation work found that although GCN2 is required for pathogen induced eIF2α phosphorylation, it is dispensable for defense against a bacterial pathogen, and thus is unlikely to be a regulator of TBF1 or the immune response. On the other hand, m6A was established to be broadly essential and dynamic upon immune induction. These findings open the door for future studies to elucidate how m6A machinery is interacting with the defense response and establish a new area for post-transcriptional control of plant immunity.
Item Open Access Global translational reprogramming is a fundamental layer of immune regulation in plants.(Nature, 2017-05-17) Xu, Guoyong; Greene, George H; Yoo, Heejin; Liu, Lijing; Marqués, Jorge; Motley, Jonathan; Dong, XinnianIn the absence of specialized immune cells, the need for plants to reprogram transcription to transition from growth-related activities to defence is well understood. However, little is known about translational changes that occur during immune induction. Using ribosome footprinting, here we perform global translatome profiling on Arabidopsis exposed to the microbe-associated molecular pattern elf18. We find that during this pattern-triggered immunity, translation is tightly regulated and poorly correlated with transcription. Identification of genes with altered translational efficiency leads to the discovery of novel regulators of this immune response. Further investigation of these genes shows that messenger RNA sequence features are major determinants of the observed translational efficiency changes. In the 5' leader sequences of transcripts with increased translational efficiency, we find a highly enriched messenger RNA consensus sequence, R-motif, consisting of mostly purines. We show that R-motif regulates translation in response to pattern-triggered immunity induction through interaction with poly(A)-binding proteins. Therefore, this study provides not only strong evidence, but also a molecular mechanism, for global translational reprogramming during pattern-triggered immunity in plants.Item Open Access H2O2 sulfenylates CHE linking local infection to establishment of systemic acquired resistance.(bioRxiv, 2023-08-01) Cao, Lijun; Yoo, Heejin; Chen, Tianyuan; Mwimba, Musoki; Zhang, Xing; Dong, XinnianIn plants, a local infection can lead to systemic acquired resistance (SAR) through increased production of salicylic acid (SA). For 30 years, the identity of the mobile signal and its direct transduction mechanism for systemic SA synthesis in initiating SAR have been hotly debated. We found that, upon pathogen challenge, the cysteine residue of transcription factor CHE undergoes sulfenylation in systemic tissues, enhancing its binding to the promoter of SA-synthesis gene, ICS1, and increasing SA production. This occurs independently of previously reported pipecolic acid (Pip) signal. Instead, H2O2 produced by NADPH oxidase, RBOHD, is the mobile signal that sulfenylates CHE in a concentration-dependent manner. This modification serves as a molecular switch that activates CHE-mediated SA-increase and subsequent Pip-accumulation in systemic tissues to synergistically induce SAR.Item Open Access Membrane Trafficking in Plant Immunity.(Mol Plant, 2017-07-08) Gu, Yangnan; Zavaliev, Raul; Dong, XinnianPlants employ sophisticated mechanisms to interact with pathogenic as well as beneficial microbes. Of those, membrane trafficking is key in establishing the rapid and precise response. Upon interaction with pathogenic microbes, surface-localized immune receptors undergo endocytosis for signal transduction and activity regulation while cell wall components, antimicrobial compounds, and defense proteins are delivered to pathogen invasion sites through polarized secretion. To sustain mutualistic associations, host cells also reprogram the membrane trafficking system to accommodate invasive structures of symbiotic microbes. Here, we provide analysis of recent advances in understanding the roles of secretory and endocytic membrane trafficking pathways in the plant immune activation. We also discuss strategies deployed by adapted microbes to manipulate these pathways to subvert or inhibit plant defense.Item Embargo Next-Generation Mapping of the Salicylic Acid Signaling Hub and Transcriptional Cascade(2024) Powers, Jordan JohnFor over 60 years, salicylic acid (SA) has been known as a key signaling molecule for local and systemic immunity. SA causes drastic cellular reprogramming in a phenomenon known as Systemic Acquired Resistance (SAR). During SAR, thousands of genes transcriptional respond. This transcriptional response is regulated by the cofactor NONEXPRESSER OF PR1 (NPR1), despite NPR1 not having a DNA binding domain. With no DNA binding domain, how this cofactor functions in this transcriptional reprogramming became a question at the forefront of the field. Despite decades of investigation, we have only superficially understood its function, largely due to the reliance on marker genes and low-throughput, molecular biology approaches. While higher throughput methodologies have been employed in previous attempts to identify NPR1 partners and target genes, all efforts yielded futile results. However, with the development of new targeted proteomic techniques and higher signal chromatin profiling techniques, we are poised to adapt these technologies to identify NPR1 proximal proteins and direct targets. Utilizing TurboID, we identified 234 NPR1 proximal partner proteins, including most known NPR1 interactors, validating our methodology. These proximal proteins are enriched with chromatin remodelers, splicing machinery, the Mediator complex, and transcriptional regulators. Strikingly these proximal partners can play either a positive or negative role in establishing SAR. Beyond NPR1 partners, we identified NPR1 target genes upon SA treatment using greenCUT&RUN. Despite a proposed transcription factor switching model, our time course greenCUT&RUN showed NPR1 binds consistently to the same loci after SA treatment through TGA transcription factors and induces a transcriptional cascade comprised of WRKY, NAC, ERF, and MYB transcription factors. This induction requires the condensate formation of NPR1 to stabilize the NPR1 TGA interaction. Through this induced cascade, WRKYs continue propagating this SA transcriptional reprogramming and activating many signaling kinases and defense genes. Beyond NPR1, many conical ideas about transcription induction seem to be amiss in SA-induced genes, including increased chromatin accessibility and histone acetylation, leading rise to the new yet old question; how does NPR1, and the subsequent SA-induce transcription factors, cause this transcriptional reprogramming?
Item Open Access Pervasive downstream RNA hairpins dynamically dictate start-codon selection(NATURE, 2023) Xiang, Yezi; Huang, Wenze; Tan, Lianmei; Chen, Tianyuan; He, Yang; Irving, Patrick S; Weeks, Kevin M; Zhang, Qiangfeng Cliff; Dong, XinnianItem Open Access Post-translational regulation of plant immunity.(Curr Opin Plant Biol, 2017-05-21) Withers, John; Dong, XinnianPlants have evolved multi-layered molecular defense strategies to protect against pathogens. Plant immune signaling largely relies on post-translational modifications (PTMs) to induce rapid alterations of signaling pathways to achieve a response that is appropriate to the type of pathogen and infection pressure. In host cells, dynamic PTMs have emerged as powerful regulatory mechanisms that cells use to adjust their immune response. PTM is also a virulence strategy used by pathogens to subvert host immunity through the activities of effector proteins secreted into the host cell. Recent studies focusing on deciphering post-translational mechanisms underlying plant immunity have offered an in-depth view of how PTMs facilitate efficient immune responses and have provided a more dynamic and holistic view of plant immunity.Item Open Access Protective plant immune responses are elicited by bacterial outer membrane vesicles.(Cell reports, 2021-01) McMillan, Hannah M; Zebell, Sophia G; Ristaino, Jean B; Dong, Xinnian; Kuehn, Meta JBacterial outer membrane vesicles (OMVs) perform a variety of functions in bacterial survival and virulence. In mammalian systems, OMVs activate immune responses and are exploited as vaccines. However, little work has focused on the interactions of OMVs with plant hosts. Here, we report that OMVs from Pseudomonas syringae and P. fluorescens activate plant immune responses that protect against bacterial and oomycete pathogens. OMV-mediated immunomodulatory activity from these species displayed different sensitivity to biochemical stressors, reflecting differences in OMV content. Importantly, OMV-mediated plant responses are distinct from those triggered by conserved bacterial epitopes or effector molecules alone. Our study shows that OMV-induced protective immune responses are independent of the T3SS and protein, but that OMV-mediated seedling growth inhibition largely depends on proteinaceous components. OMVs provide a unique opportunity to understand the interplay between virulence and host response strategies and add a new dimension to consider in host-microbe interactions.Item Open Access Quantification of the humidity effect on HR by Ion leakage assay.(Bio-protocol, 2019-04-05) Mwimba, Musoki; Dong, XinnianWe describe a protocol to measure the contribution of humidity on cell death during the effector-triggered immunity (ETI), the plant immune response triggered by the recognition of pathogen effectors by plant resistance genes. This protocol quantifies tissue cell death by measuring ion leakage due to loss of membrane integrity during the hypersensitive response (HR), the ETI-associated cell death. The method is simple and short enough to handle many biological replicates, which improves the power of test of statistical significance. The protocol is easily applicable to other environmental cues, such as light and temperature, or treatment with chemicals.Item Open Access Regulation of Cell Death During Arabidopsis Effector Triggered Immunity(2019) Zebell, SophiaIn the plant innate immune system, diverse signals from a wide range of pathogens converge on the same output, effector triggered immunity (ETI) and the associated programmed cell death (PCD). Past genetic studies have succeeded in uncovering the role of R-genes in recognizing the presence of pathogen effectors, and in identifying a number of downstream executors of the immune response. However, the gap between effector recognition and phenotype regulation remains poorly understood, with each signaling component only contributing a minor quantitative effect to the phenotype of ETI-PCD. In this dissertation, my goal is to fill in a portion of that gap.
I demonstrate that there is a prolonged nuclear increase of calcium ions during ETI, and that that nuclear calcium signal is essential for PCD. I also utilize cpr5, a point mutant identified for its constitutive defense response and programmed cell death lesions, to identify a new role for cell cycle regulators in regulating ETI-PCD. I show that phosphorylation of the cell cycle regulator Retinoblastoma-Related 1 (RBR1) is responsive to ETI. The RBR1 target transcription factors E2Fa, E2Fb, and E2Fc have an additive role regulating ETI, and a triple e2fabc mutant is susceptible to pathogens. Using a reverse genetics approach in e2fabc, I identify repression of nonphotochemical quenching in the chloroplasts as a key step in ETI-PCD regulation.
Together, these studies emphasize the role of organelles in PCD regulation, with the nucleus serving as a hub of second messenger signaling and transcription and the chloroplasts responding to ETI by remodeling to serve a new role as a platform for ROS production. In addition, they define a new pathway of ETI regulation that contributes quantitatively to ETI-PCD.
Item Open Access Salicylic acid biosynthesis is enhanced and contributes to increased biotrophic pathogen resistance in Arabidopsis hybrids.(Nat Commun, 2015-06-12) Yang, Li; Li, Bosheng; Zheng, Xiao-yu; Li, Jigang; Yang, Mei; Dong, Xinnian; He, Guangming; An, Chengcai; Deng, Xing WangHeterosis, the phenotypic superiority of a hybrid over its parents, has been demonstrated for many traits in Arabidopsis thaliana, but its effect on defence remains largely unexplored. Here, we show that hybrids between some A. thaliana accessions show increased resistance to the biotrophic bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. Comparisons of transcriptomes between these hybrids and their parents after inoculation reveal that several key salicylic acid (SA) biosynthesis genes are significantly upregulated in hybrids. Moreover, SA levels are higher in hybrids than in either parent. Increased resistance to Pst DC3000 is significantly compromised in hybrids of pad4 mutants in which the SA biosynthesis pathway is blocked. Finally, increased histone H3 acetylation of key SA biosynthesis genes correlates with their upregulation in infected hybrids. Our data demonstrate that enhanced activation of SA biosynthesis in A. thaliana hybrids may contribute to their increased resistance to a biotrophic bacterial pathogen.Item Open Access Salicylic acid receptors activate jasmonic acid signalling through a non-canonical pathway to promote effector-triggered immunity.(Nature communications, 2016-10-11) Liu, Lijing; Sonbol, Fathi-Mohamed; Huot, Bethany; Gu, Yangnan; Withers, John; Mwimba, Musoki; Yao, Jian; He, Sheng Yang; Dong, XinnianIt is an apparent conundrum how plants evolved effector-triggered immunity (ETI), involving programmed cell death (PCD), as a major defence mechanism against biotrophic pathogens, because ETI-associated PCD could leave them vulnerable to necrotrophic pathogens that thrive on dead host cells. Interestingly, during ETI, the normally antagonistic defence hormones, salicylic acid (SA) and jasmonic acid (JA) associated with defence against biotrophs and necrotrophs respectively, both accumulate to high levels. In this study, we made the surprising finding that JA is a positive regulator of RPS2-mediated ETI. Early induction of JA-responsive genes and de novo JA synthesis following SA accumulation is activated through the SA receptors NPR3 and NPR4, instead of the JA receptor COI1. We provide evidence that NPR3 and NPR4 may mediate this effect by promoting degradation of the JA transcriptional repressor JAZs. This unique interplay between SA and JA offers a possible explanation of how plants can mount defence against a biotrophic pathogen without becoming vulnerable to necrotrophic pathogens.Item Open Access Selective mRNA Translation during the Plant Immune Response(2020) Greene, George HThe ability to rapidly alter gene expression is essential to surviving fluctuating environments. The disjunction between mRNA abundance and mRNA translation is of particular interest in stress responses that elicit large scale gene expression reprogramming. Targeted changes in the translation efficiency of select mRNA, within the standing pool of total mRNA, allow for rapid alterations in the proteome upon environmental cue. Here, I discover large changes in the translational landscape that are independent from the underlying transcriptional dynamics during the early stages of PAMP-Triggered Immunity (PTI) and a coordinated translational response during the later stage Effector-Triggered Immunity (ETI). Certain mRNAs borne features, such as mRNA methylation and uAUGs, which initiate uORFs, that are known to affect mRNA stability and translation activity, yet little is known about how these elements affect mRNA translation during infection stress. Through observations of translation dynamics, I investigated the impact of known and novel mRNA features on local immunity. In addition, I discovered additional facets of the plant immune response including a novel mRNA sequence consensus, which I call the R-motif that induces mRNA translation upon pathogen detection.
Item Open Access The molecular interplay between the circadian clock and the plant immune signal, salicylic acid(2014) Zhou, MianPlants have evolved the circadian clock to anticipate environmental changes and coordinate internal biological processes. Recent studies unveiled the circadian regulation on plant immune responses as well as a reciprocal effect of immune activation on the clock activity. However, it is still largely unknown how the circadian clock interacts with specific immune signals. Plant hormone salicylic acid (SA) is a key immune signal. Its accumulation is sufficient to trigger immune responses and establish broad-spectrum resistance, known as systemic acquired resistance (SAR). My dissertation work studied whether SA could interact with the circadian clock and what potential mechanisms and the biological significance are.
I first found that SA could reinforce the circadian clock through the modulation of redox state in an NONEXPRESSER OF PR 1 (NPR1)-dependent manner. The basal redox state manifested by the NADPH abundance is shown to display a circadian rhythm. Perturbation in this cellular redox rhythm caused by the immune signal SA is sensed by the master immune regulator NPR1. NPR1 then triggers defense genes expression to generate SAR as well as transcriptionally activates several clock genes to reinforce the circadian clock. Since the basal redox state, which reflects the cellular metabolic activities, is under the circadian control, the reinforced circadian clock may negate the SA-triggered redox perturbation to restore the normal redox rhythm. One of NPR1-regulated clock components is TIMMING OF CAB2 EXPRESSION 1 (TOC1). SA/NPR1-mediated increase in TOC1 expression alone could lead to dampening of SAR through direct transcriptional repression on defense genes. Since maintenance of the immune responses is an energy-costly process, the strength and duration of SAR, a preventative defense strategy, need to be fine-tuned to reduce unnecessary energy expenditure. Therefore, both SA-dependent circadian clock reinforcement and the specific clock component TOC1 induction help to ensure a proper immune induction and a balanced energy allocation between defense and normal metabolic activities.
Besides the SA effects on the circadian clock, the circadian clock is found to reciprocally regulate SA biosynthesis. The clock gene, CCA1 HIKING EXPEDITION (CHE), and the major SA synthesis gene, ISOCHORISMATE SYNTHASE 1 (ICS1), show in-phase oscillatory rhythms, indicating that CHE may contribute to generation of the circadian rhythm of the basal SA level. I found that CHE, as a transcription factor, directly binds to the promoter of ICS1 to positively regulate its expression. After pathogen infection, CHE promotes endogenous SA biosynthesis and acts as a positive regulator of SAR. The function of the clock component CHE in activating ICS1 not only reveals a novel transcriptional regulatory mechanism of SA accumulation but also provides a new molecular link between the circadian clock and plant immunity.
In summary, my dissertation studies identified previously unknown molecular mechanisms of how the circadian clock mediates SA biosynthesis and SA-triggered immune responses. The interplay between the circadian clock and SA achieves a balance between activation of immune responses and maintenance of normal metabolic activities. Further studies may explore how other plant immune signals affect the circadian clock as well as how different clock components coordinately regulate the plant immunity. These future directions will broaden our understanding about the clock-immunity crosstalk.
Item Open Access The Transcriptional Regulation of the Central Plant Defense Signal, Salicylic Acid(2014) Zheng, XiaoyuSalicylic acid (SA) is a central plant defense signal. It is not only required for closing the stomata upon infection to prevent pathogens from entering into the plant apoplast, but also mediates defense responses activated by pathogen-originated microbe-associated molecular patterns (MAMPs) and effectors in the infected tissues. In addition, SA is a necessary and sufficient signal for systemic acquired resistance (SAR). In Arabidopsis thaliana, SA level increases in response to pathogen attack, which is essential for activating defense responses. This SA accumulation involves transcriptional activation of several genes including ICS1 (ISOCHORISMATE SYNTHASE 1), EDS5 (ENHANCED DISEASE SUSCEPTIBILITY 5), EDS1 (ENHANCED DISEASE SUSCEPTIBILITY 1), PAD4 (PHYTOALEXIN-DEFICIENT 4) and PBS3 (avrPphB SUSCEPTIBLE 3). However, it is not well understood how pathogenic signals induce these SA accumulation genes. Interestingly, our time-course transcriptome analysis showed that these five genes share a similar pathogen-induced expression pattern, suggesting the existence of common transcription factors (TFs). Through yeast-one-hybrid screening, a TF NTL9 was identified for its interactions with the promoters of the SA accumulation genes. Preferentially expressed in guard cells, NTL9 activates the expression of SA accumulation genes in guard cells. The ntl9 mutant is defective in pathogen-induced stomatal closure mediated by a well-characterized MAMP, flg22. Consistent with the stomatal closure defect, the ntl9 mutant exhibits elevated susceptibility to surface-inoculated pathogens. The stomatal closure defect of the ntl9 mutant can be rescued by exogenous application of SA, demonstrating that NTL9 acts upstream of SA in stomatal closure response. These results suggest that NTL9-mediated activation of SA accumulation genes is essential for MAMP-triggered stomatal closure.
While plants induce SA to activate defense responses, pathogens can also produce virulence factors to counteract the effects of SA. Coronatine is one such virulence factor produced by Pseudomonas syringae. Coronatine is known to promote opening of stomata for bacterial entry, bacterial growth in the apoplast, systemic susceptibility and development of disease symptoms such as chlorosis. In the process of examining the mechanisms underlying coronatine-mediated virulence, three homologous TFs, ANAC019, ANAC055 and ANAC072, were found to be activated by coronatine directly through the TF, MYC2. Genetic characterization of these three TF mutants revealed that these TFs mediate multiple virulence effects of coronatine by inhibiting SA accumulation. To exert this inhibitory effect, these TFs repress ICS1 and activate BSMT1, genes involved in SA biosynthesis and inactivation modification, respectively. Thus, a signaling cascade downstream of coronatine was illustrated to dampen SA-mediated defense responses through differential transcriptional regulation of genes related to SA level.
Taken together, my dissertation studies revealed novel transcriptional regulation of SA production and demonstrated that this transcriptional regulation is a vital point not only for plant defense activation but also for pathogen manipulation to counteract defense responses. Further studies on the interplay of this transcriptional regulation by different TFs would broaden our understanding about the dynamics of plant-pathogen interaction.
Item Embargo Translation regulation during pattern-triggered immunity(2023) Xiang, YeziTranslational reprogramming allows organisms to adapt to changing conditions. In regulating translation, upstream start codons (uAUGs), prevalently present in mRNAs, play crucial roles by providing alternative translation start sites. However, what determines this selective translation initiation between conditions remains a fundamental question. By integrating transcriptome-wide translational and structural analyses during Arabidopsis pattern-triggered immunity, I found that transcripts with immune-induced translation are enriched with upstream open reading frames (uORFs). Under normal conditions, these uORFs are selectively translated due to highly structured regions immediately downstream of uAUGs by slowing and engaging the scanning preinitiation complex. Deep learning modelling provides unbiased support for recognizable double-stranded RNA structures downstream of uAUGs (“uAUG-ds”) being responsible for the selective translation of uAUGs. I showed that uAUG-ds regulation is generalizable in human cells. Moreover, I found that uAUG-ds-mediated start codon selection is dynamically regulated. Upon immune challenge in plants, induced Ded1p/DDX3X-homologous RNA helicases resolve these structures, allowing ribosomes to bypass uAUGs to translate downstream defensce proteins. This study demonstrates that mRNA structures, rather than primary sequences, dynamically regulate start codon selection. The prevalence of this RNA structural feature and the conservation of RNA helicases across kingdoms suggest the generality of mRNA structural remodelling in mediating translational reprogramming.
Item Open Access Unfolded protein response genes regulated by CED-1 are required for Caenorhabditis elegans innate immunity.(2008) Haskins, Kylie AnneThe first line of defense against pathogens is the phylogenetically ancient innate immune system. This system consists of physical barriers and conserved signaling pathways are activated upon infection to produce effector molecules that mount a microbicidal response. Recently, C. elegans has been established as a model organism for the study of innate immunity due to C. elegans genetic tractability and origins predating the evolution of adaptive immunity. Conserved defense pathways essential for mammalian innate immunity have been identified in C. elegans. However, most receptors critical for the activation of the defense signaling pathways in C. elegans remain unknown. The goal of this work was to study CED-1 and its potential role as a cell-surface signaling receptor essential for C. elegans immune response. In this study, we performed a full-genome microarray analysis and discovered that CED-1 functions to activate the expression of pqn/abu unfolded protein response (UPR) genes. The unfolded protein response has been implicated in the normal physiology of immune defense and in several disorders including diabetes, cancer, and neurodegenerative disease. Here we show that ced-1 and pqn/abu genes are required for the survival of C. elegans exposed to live S. enterica. We also show that the overexpression of pqn/abu genes confers protection to pathogen-mediated killing. Taken together, these results indicate that the apoptotic receptor CED-1 and a network of PQN/ABU proteins involved in a non-canonical UPR response are required for proper defense to pathogen infection in Caenorhabditis elegans.