Xin Li

Professor

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Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.

Dissecting plant immune responses mediated by the PI4KBs (2024)

The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

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Dissecting signaling pathways mediated by plant TIR-type immune receptors (2024)

Plants utilize sophisticated innate immune systems to fight against pathogens and pests. They have multiple types of immune receptors to detect the presence of pathogens and initiate defense responses. Among them, Toll/interleukin-1 receptor (TIR)-type nucleotide-binding leucine-rich repeat (NLR) immune receptors (TIR-NLRs, TNLs) recognize pathogen effectors intracellularly and activate downstream signaling components, including helper NLRs (hNLRs) consisting of ADR1 (ACTIVATED DISEASE RESISTANCE 1) and NRG1 (N REQUIREMENT GENE 1) families, and two distinct lipase-like protein dimers EDS1 (ENHANCED DISEASE SUSCEPTIBILITY 1)-PAD4 (PHYTOALEXIN DEFICIENT 4) and EDS1-SAG101 (SENESCENCE-ASSOCIATED GENE 101) through enzymatic activities from their TIR domains. However, immune signal transduction from TNL receptors to these downstream modules remains largely unclear. Studies in this dissertation first confirmed EDS1-PAD4-ADR1s and EDS1-SAG101-NRG1s as two distinct modules downstream of TNLs by genetic analysis. Further biochemical experiments indicated that TIR/TNL activation promotes the association between EDS1-PAD4 and one of the ADR1 family members ADR1-L1 (ADR1-Like 1). Enhanced self-association of ADR1-L1 was also detected upon TIR/TNL activation, suggesting a potential EDS1-PAD4-ADR1s protein complex formation for TNL immune signal transduction.Furthermore, an N-terminally truncated hNLR NRG1C was identified with a negative role in plant immunity, which antagonizes immune responses mediated by the TNLs that are dependent on full-length NRG1A/1B. However, NRG1C could not affect the transcript or protein levels of NRG1A/1B, and it did not associate with them either. The interaction of NRG1C with EDS1 and SAG101 suggested that it may interfere with the EDS1-SAG101 complex that signals together with full-length NRG1s to modulate TNL signaling. Furthermore, NRG1Cs are evolutionarily conserved and are functionally exchangeable in Brassicaceae. Finally, the reported NADase and 2’,3’-cAMP/cGMP synthetase activities of plant TIR domains were examined for their importance in TNL-mediated immunity. Through protein structure-based mutagenesis analysis of a typical Arabidopsis TNL SNC1 (SUPPRESSOR OF NPR1-1, CONSTITUTIVE 1), I revealed that the TNL SNC1 requires NADase activity to trigger plant immune responses, while its 2’,3’-cAMP/cGMP synthetase activity is not needed.Altogether, studies in this dissertation contribute to the understanding of TNL signaling pathways in plant immunity. These findings will be helpful for future design of broad-spectrum resistant crops.

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Characterization of two regulators in plant immune receptor-mediated defense (2023)

Plants possess a complex multi-layered immune system that enables them to defend themselves against many potential invaders. However, some virulent pathogens can overcome these defenses to cause plant disease. Plant pathogens are a major obstacle to efficient and sustainable global food production. Understanding the underlying molecular mechanisms behind plant immunity will allow precise science-based engineering and breeding of plant disease resistance. Nucleotide-binding leucine-rich repeat proteins (NLRs) are one type of immune receptor that mediate the recognition of pathogens and the activation of defense responses. Although many regulators contributing to NLR homeostasis and activation have been uncovered, our understanding of plant immune signaling is far from complete. One limitation that has hindered gene discovery is genetic redundancy, which can mask immune phenotypes in single mutant plants. Using ectopic overexpression and CRISPR/Cas9-mediated targeted deletion strategies for reverse genetic screens, these limitations can be overcome. In this study, two novel regulators involved in NLR immunity were characterized. The first gene, called SNIPER7 (SNC1-INFLUENCING PLANT E3 LIGASE REVERSE GENETIC 7), was isolated from a previous E3 ubiquitin ligase overexpression screen. SNIPER7 overexpression results in constitutive activation of NLR-mediated defense. SNIPER7 targets the conserved unfoldase CDC48A (CELL DIVISION CYCLE 48A) for degradation. Since CDC48A is involved in the homeostasis of the NLR SNC1 (SUPPRESSOR OF NPR1-1, CONSTITUTIVE 1), SNIPER7 may contribute to immune receptor accumulation upon pathogen infection. The second gene, called TC1b (TRAF CANDIDATE 1b), was isolated from a new CRISPR/Cas9 screen targeting TRAF (TUMOR NECROSIS FACTOR RECEPTOR ASSOCIATED FACTOR) domain containing genes. TRAF domain genes are hypothesized to play a role in plant NLR immunity since many mammalian TRAF domain proteins are involved in innate immunity. TC1b is required for full SNC1 function, but TC1b does not seem to play a role in SNC1 protein homeostasis nor general NLR downstream signalling. The precise molecular function of TC1b is still unclear. Overall, the study of SNIPER7 and TC1b contributes to knowledge of how NLRs like SNC1 are regulated. These findings may be relevant for the precise engineering of NLRs to mediate disease resistance while avoiding growth/yield trade-off from NLR overexpression.

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Genetic dissection of Sclerotinia sclerotiorum biology using forward genetics (2023)

Diverse plant fungal pathogens that cause worldwide crop losses are understudied due to various technical challenges. With the increasing availability of sequenced whole genomes of these non-model fungi, effective genetic analysis methods are highly desirable. My PhD projects focus on the establishment of a new gene discovery pipeline, which combines a fast forward genetic screening in large scale with high-throughput next-generation sequencing. I applied this pipeline in the notorious soilborne phytopathogen, Sclerotinia sclerotiorum, and identified 32 mutants with various developmental and growth deficiencies. Detailed molecular studies of three melanisation-deficient mutants provide a proof of concept for the effectiveness of our method. Furthermore, using this forward genetic approach, a RAS-GTPase activating protein, SsGAP1, was identified, which plays essential roles in sclerotia formation, compound appressoria production and virulence. To find the responsible RAS-GTPase being activated by SsGAP1 through mutant phenotypes comparison, I knocked down two RAS-GTPases, SsRAS1 and SsRAS2, respectively. My results revealed that both SsRAS1 and SsRAS2 are required for vegetative growth, sclerotia formation, compound appressoria production and virulence. As RNA can move from plant hosts to associating eukaryotic pathogens, trans-species RNAi pathway has been employed for pathogen control through host-induced gene silencing (HIGS). Thereby, I tested whether these RAS signaling genes can be used as HIGS targets to control stem rot caused by S. scleortiorum. Here, when I infiltrated tobacco leaves with Agrobacterium with RNAi constructs targeting SsGAP1 and SsRAS1, I observed reduced virulence. Taken together, my forward genetics gene discovery pipeline in S. sclerotiorum is highly effective in identifying novel HIGS targets to control fungal diseases caused by S. sclerotiorum. As RAS signaling is conserved in eukaryotes, these RAS signaling genes may also have the potential to be used for controlling other fungal diseases in higher plants. Altogether, with the successful identification of these causal genes responsible for the phenotypes of their respective mutants, it is foreseeable that this pipeline can be applied to facilitate efficient in-depth studies of other non-model fungal species and identify more novel HIGS targets against fungal pathogens in the future.

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Negative regulation of FMO1 expression in Arabidopsis thaliana (2023)

The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

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Molecular studies on plant NLR immune receptors (2022)

In both plants and animals, nucleotide-binding leucine-rich repeat (NLR) receptors play crucial roles in the recognition of pathogen-derived molecules and the activation of defense. Sensor NLRs (sNLRs) are polymorphic with the ability to recognize relatively diverse pathogenic effectors. Helper NLRs (hNLRs) with their characteristic RPW8 domain function downstream of diverse sNLRs and are evolutionarily conserved. Proper regulation of immune responses mediated by NLRs is important as over-activation results in growth defects, while under-activation leads to vulnerability to pathogens. Two hNLR families, ADR1s (ACTIVATED DISEASE RESISTANCE 1) and NRG1s (N REQUIREMENT GENE 1), are present in Arabidopsis. Previous studies have shown that the ADR1 family is required for immune response mediated by multiple sNLRs. However, the function of the NRG1 family is unclear. Here, we discovered that the NRG1 family specifically operates downstream of Toll/interleukin-1 (TIR) type sNLRs (TNLs). ADR1s and NRG1s function in two distinct parallel pathways contributing to TNL-specific immunity. Synergistic effects on basal and TNL-mediated defense were detected among ADR1s and NRG1s. In summary, different sensor TNLs differentially use two groups of hNLRs to transduce downstream defense signals. NLRs are controlled at multiple levels to ensure immune responses are effective and timely. Ubiquitin proteasome system (UPS)-mediated NLR turnover plays a pivotal role in the fine-tuned regulation of immune responses. snc1 harbors a gain-of-function mutation in the TNL SNC1 (Suppressor of npr1, Constitutive 1) and exhibits autoimmune phenotype. A snc1-influencing plant E3 ligase reverse (SNIPER) genetic screen identified two snc1-suppressors, SNIPER1 and SNIPER2, both of which completely suppress snc1-mediated autoimmunity upon overexpression. Interestingly, functionally redundant SNIPER1 and SNIPER2 can control the protein levels of diverse sNLRs and the interactions between SNIPER1 and sNLRs appear to be through the common nucleotide-binding (NB) domains of sNLRs. In support, SNIPER1 can ubiquitinate the NB domains of multiple sNLRs in vitro. Our study thus reveals a novel process where global turnover of sNLRs by two master E3 ligases serves to immediately attenuate immune output to effectively avoid autoimmunity. Overall, studies in this dissertation provide new mechanistic insights on NLR activation and the global homeostasis control of NLRs.

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An examination of post-translational regulation of immunity utilising reverse genetic techniques (2021)

The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

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Dissection of molecular events involved in saul1-mediated autoimmunity (2021)

Plant intracellular nucleotide binding and leucine-rich repeat proteins (NB-LRR, NLRs) function as immune receptors to detect microbial pathogens directly or indirectly. Most NLRs guard host proteins that are the direct targets of pathogen effectors. A typical NLR sometimes cooperates with another atypical NLR for effector recognition. Upon effector recognition, plant NLRs oligomerize for defense activation, the mechanism of which is poorly understood. As a positive regulator of plant immunity, E3 ligase SAUL1 is guarded by NLR protein SOC3. The mutant saul1-1 displays seedling lethality from its autoimmunity, which makes it a suitable background for genetic screens. In chapter 2, by using CRISPR/Cas9 gene editing, genetic analysis and biochemical assays, I identified the differential pairings of typical NLR receptor SOC3 with atypical NLR proteins CHS1 or TN2 to guard the homeostasis of the E3 ligase SAUL1. The differential pairing results in autoimmunity with distinct morphological outcomes. SOC3 forms a head-to-head genomic arrangement with CHS1 and TN2, indicative of transcriptional co-regulation. Such cooperative interactions can likely enlarge the recognition spectrum and increase the functional flexibility of NLRs. In chapter 3, SUSA2 and SUSA3 were identified from the saul1-1 suppressor screen. SUSA2 encodes an F-box protein Actin-Related Protein 8 (ARP8) and SUSA3 encodes the chaperone protein HSP90.3. Further characterization showed that susa2-2 only suppresses the autoimmunity mediated by either CHS1-SOC3 or TN2-SOC3 paired NLR proteins, indicating that SUSA2 is specifically involved in NLR protein SOC3-mediated immunity. Moreover, both SUSA proteins are parts of an SCFSUSA2 E3 ligase, forming SCFSUSA2-NLR complex with CHS1-SOC3 or TN2-SOC3 paired NLR proteins. This is the first example of the assembly of plant NLRs into an SCF complex, which likely enables the ubiquitination and degradation/activation of an unknown downstream component to activate defense, a mechanism with remote similarity to mammalian NLR inflammasome activation.Overall, my Ph.D. thesis provides a better understanding of the molecular mechanisms by which NLR immune receptors are activated upon pathogen recognition. Such knowledge will be critical in the future design of pathogen-resistant crops.

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Investigating plant-nepovirus interactions: non-canonical translation of tomato ringspot virus RNAs and plant antiviral defence responses associated with symptom recovery (2020)

Tomato ringspot virus (ToRSV, genus Nepovirus) was used as a model to study two essential steps of the infection cycle: the translation of uncapped viral RNAs and the induction of host antiviral defences. Using reporter transcripts containing the 5’ and 3’ untranslated regions (UTRs) of ToRSV RNAs and a portion of the viral coding region, I show that translation initiates at the first AUG of the viral RNA (nt 77) in spite of its suboptimal context. A predicted downstream stem-loop structure (5’ SL) may enhance initiation at this AUG. I show that both viral UTRs are required for efficient translation. A region of 386 nt in the viral 3’ UTR was necessary and sufficient for translation in conjunction with the viral 5’ UTR. In this region, a predicted stem-loop (SL 3’) may be involved in a long-distance RNA-RNA interaction with the 5’ SL as suggested by the complementarity of the loop sequences. The viral 3’ UTR inhibited the cap-dependent translation of control transcripts, suggesting that it contains a cis-acting translation element that binds one or several translation factors. These results highlight a unique non-canonical translation initiation mechanism that shares similarities with that described for Blackcurrant reversion virus (another nepovirus), although the cis-acting elements required for translation differed between the two viruses. Symptom recovery (an asymptomatic phase of infection that follows an initial symptomatic phase) has been attributed to antiviral RNA silencing. Using Nicotiana benthamiana plants infected with two ToRSV isolates (Rasp1 and GYV), I show that symptom recovery is dependent on environmental conditions and the isolate. The recovery from GYV- or Rasp1-infection shared similar hallmarks of RNA silencing but GYV-infected plants recovered at a wider range of temperatures (21–27°C). ARGONAUTE (AGO) proteins are key components of the RNA silencing pathway. Silencing of AGO1, which prevents the recovery of Rasp1-infected plants, did not prevent recovery from GYV infection. I show differential accumulation of AGO2 depending on the temperature and the isolate. However, although silencing or mutation of AGO2 increased virus accumulation in infected plants, it did not prevent symptom recovery. Taken together, these results highlight the complexity of plant-virus interactions.

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Regulation of plant immunity: lessons from SNIPER4 and CDK8 (2019)

Plants employ sophisticated innate immune systems to ward off the invasion of pathogens. Upon perception of pathogens, plants transduce signals to downstream components and activate defense responses. To investigate the regulation of immune responses and the mechanisms of immune signaling, my Ph.D. projects focus on the characterization of immune regulators isolated from two genetic screens conducted in autoimmune mutants snc1 and camta1/2/3.SNIPER4, identified from the snc1-influencing plant E3 ligase reverse genetic screen (SNIPER), encodes an F-box protein being part of an SCF E3 complex. Two redundant tumor necrosis factor receptor associated factors (TRAF) proteins MUSE13 and MUSE14 serve as adaptors in the SCFCPR1 E3 complex to facilitate the degradation of NLRs including SNC1 and RPS2. Accumulation of MUSE13 and MUSE14 is decreased by overexpression of SNIPER4, but is increased when dominant-negative (DN)-SNIPER4 is overexpressed. In addition, SNIPER4 interacts with MUSE13 and MUSE14. Collectively, my data suggest that SNIPER4 fine-tunes the output of NLR proteins by modulating the turnover of MUSE13 and MUSE14 of the SCFCPR1 E3 complex.Three calmodulin binding transcription activators (CAMTAs), including CAMTA1, CAMTA2 and CAMTA3, play redundant roles in plant immunity. However, their major function in immune responses remains ambiguous. By conducting the Suppressor of camta1/2/3 (SUCA) screen, I found that loss-of function ICS1, a gene indispensable for SA biosynthesis, almost fully suppresses the drastic autoimmunity of camta1/2/3 triple mutant, suggesting that the major role of CAMTAs is to inhibit SA biosynthesis. In support of this, SA levels are decreased in the gain-of-function camta3-3D mutant. Transcriptional analysis revealed that expression of SA-related genes, including ICS1, EDS5 and PBS3, is compromised in camta3-3D plants. On the other hand, mutations in Cyclin-Dependent Kinase 8 (CDK8), isolated from the SUCA screen, compromise SA accumulation and systemic required resistance (SAR). cdk8 mutants exhibit reduced transcript levels of ICS and EDS5. Taken together, my results indicate that CAMTAs and CDK8 function oppositely in transcriptional regulation of SA biosynthesis. Overall, my thesis work adds to the current literature on the regulation of plant immunity. Such knowledge will assist the future development of sustainable methods for controlling diseases of our crop plants.

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Diverse roles of the ubiquitin proteasome system in regulation of plant immunity (2018)

Plants rely on a variety of immune signaling pathways to recognize pathogens and defend against pathogen attacks. In order to effectively recognize pathogens without causing autoimmunity, homeostasis of the proteins in these pathways must be maintained at optimal levels. The ubiquitin proteasome system (UPS) is an important mechanism by which the turnover of proteins, including immune system components, is achieved. This thesis describes novel roles for three UPS-related proteins in the regulation of plant immunity. The conserved E3 ligase C-TERMINUS OF HSP70 INTERACTING PROTEIN (AtCHIP) is a positive regulator of immunity in Arabidopsis, as overexpression of AtCHIP causes enhanced disease resistance. Loss of AtCHIP function causes enhanced susceptibility to virulent pathogens, but does not affect resistance mediated by Nucleotide-binding domain and leucine-rich repeat (NLR) receptors. The stability of the chaperone HEAT SHOCK PROTEIN 90.3 (HSP90.3) is also normal in atchip plants.Previous studies have shown that UPS also functions as a negative regulator of immunity, by preventing excessive accumulation of NLR proteins such as SUPPRESSOR OF NPR1, CONSTITUTIVE 1 (SNC1). CELL DIVISION CYCLE 48A (AtCDC48A) was identified as a negative regulator of immunity in a forward genetic screen for enhancers of autoimmunity caused by a gain-of-function allele of snc1. atcdc48A-4 plants exhibit dwarf morphology and enhanced disease resistance to the oomycete pathogen Hyaloperonospora arabidopsidis (H.a.) Noco2. SNC1 protein level is increased in atcdc48A plants, and AtCDC48A interacts with the E4 enzyme MUTANT, SNC1 ENHANCING 3 (MUSE3) in co-immunoprecipitation experiments, supporting a role for AtCDC48A in NLR turnover. Loss of PROTEASOME REGULATOR 1 (PTRE1), a positive regulator of protein turnover through the UPS, also causes enhanced disease resistance and increases SNC1 protein stability, further supporting the importance of proteasomal degradation in NLR homeostasis. Thus, the UPS plays both positive and negative roles in immune regulation.

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Identification and Analysis of E3 Ligases and Helper NLRs in Plant Immunity (2016)

Plant immunity is usually initiated with two types of immune receptors: 1) pattern recognition receptors (PRRs) recognize the conserved molecular features of pathogens (pathogen-associated molecular patterns, PAMPs) and trigger PTI (PAMP-triggered immunity) and; 2) nucleotide-binding/leucine-rich repeats (NLRs) serve as intracellular immune receptors with the ability to recognize the presence of relatively diverse pathogen effectors and trigger ETI (effector-triggered immunity). The Arabidopsis thaliana mutant snc1 contains a gain-of-function mutation in a Toll/interleukin-1 (TIR)-type NLR (TNL) gene and displays a dwarf morphology. Here, I report on the results of a snc1-influencing plant E3 ligase reverse genetic (SNIPER) screen that looked for snc1 plants with altered dwarfism in the presence of overexpressed E3 ligases. Six SNIPER genes were identified with four snc1-suppressors and two snc1-enhancers. SNIPER1/2/3 were selected for further characterization. The analysis of SNIPER1/2 is incomplete, thus is not included in this thesis. Chapter 3 describes SNIPER3, previously known as SAUL1 (Senescence-Associated E3 Ubiquitin Ligase 1) or PUB44 (Plant U-box 44), which encodes a U-box-type E3 ligase. Our data suggests that SAUL1 plays a dual role in plant immunity: on one hand, SAUL1 positively regulates basal resistance; on the other hand, SAUL1 suppresses a typical TNL immune receptor SUSA1 (Suppressor of saul1) to prevent its autoimmunity. ADR1, ADR1-L1 and ADR1-L2 are three homologous coiled-coil (CC)-type NLRs (CNLs), which were previously shown to work as helper NLRs. Chapter 4 further explores the specificity of the genetic requirement of ADR1s for typical TNLs, SNC1 and CHS2 (CHILLING SENSITIVE 2). Among the three ADR1 members, ADR1 is the leading contributor while ADR1-L1 is the least. Moreover, loss-of-function mutation of ADR1-L1 leads to over compensation of the transcript expression level of ADR1 and ADR1-L2 and results in the enhancement of snc1-mediated immunity. Overall, the studies I completed as part of my Ph.D. thesis expand our knowledge of the roles of E3 ligases and ADR1s in plant defense and help us to better understand the sophisticated regulatory mechanisms of plant innate immunity.

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Regulating the plant innate immune system: the roles of three Arabidopsis MUSE proteins (2016)

The plant innate immune system is highly effective in impeding infection by a broad spectrum of microbial pathogens. Strict regulation of immune signaling in plants is required to both facilitate rapid defense response induction upon pathogen detection and prevent the precocious activation of immunity, the latter of which has associated fitness costs. Despite their significance, the regulatory mechanisms governing plant immunity have only been partially characterized. Previously, members of the Li research group employed a forward genetic screen to identify positive regulators of innate immunity. This suppressor screen was performed using the unique Arabidopsis autoimmune mutant snc1 (suppressor of npr1, constitutive 1), which contains a gain-of-function mutation in an NLR (NOD-LIKE RECEPTOR) protein. The identified MOS (MODIFIER OF SNC1) genes highlighted the importance of diverse biological processes in the regulation of disease resistance. More recently, a snc1 enhancer screen was conducted to identify negative regulators of plant immune signaling. This thesis describes the cloning and characterization of three mutants isolated from this MUSE (MUTANT, SNC1-ENHANCING) screen.The muse9 mutant carries a molecular lesion in the gene encoding the chromatin remodeler SPLAYED (SYD). Molecular analyses showed that SYD negatively regulates SNC1 expression and thus functions antagonistically to MOS1 and MOS9, both of which were previously shown to positively regulate SNC1 transcription. This study emphasizes the importance of finely-tuned transcriptional control in NLR-mediated immunity. The muse4 mutant contains a partial loss-of-function mutation in NRPC7, which encodes an RNA polymerase III (Pol III) subunit. This is the first reported viable Pol III mutant. Using RT-PCR, it was established that the mutation in NRPC7 affects the expression of a diverse suite of genes and results in distortions in alternative splicing. The mutation responsible for the muse7 phenotypes is in a gene that encodes a protein of unknown function. MUSE7 negatively regulates SNC1 at the protein level, although no interactions were detected between MUSE7 and other known regulators of NLR protein turnover. This suggests that MUSE7 either regulates protein synthesis or is involved in an alternate degradation pathway. Taken together, these characterizations underscore the complexity inherent in the molecular mechanisms that control plant immune signaling.

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Regulation of immune receptor homeostasis: lessons from four MUSE proteins (2016)

Plants are sessile organisms and rely on their innate immune systems to defend against pathogens. Nucleotide-binding (NB) and leucine-rich repeat (LRR) domain-containing proteins (NLRs) serve as immune receptors in both plants and animals. It remains poorly understood how NLRs are activated and very few components downstream of NLR activation have been identified. Autoimmune mutants provide a powerful tool to study NLR-mediated immune pathways through genetic screens. To search for negative regulators of plant immunity, we performed a MUSE (MUTANT, SNC1-ENHANCING) screen in Arabidopsis thaliana, where 15 novel muse mutants were identified. My Ph.D. dissertation consists of the cloning and characterization of four muse mutants: muse10, muse12, muse13 and muse14.muse10 and muse12 are alleles of Arabidopsis thaliana hsp90.3 and hsp90.2, respectively. HSP90s are conserved molecular chaperones that play diverse roles. Previous studies have shown that HSP90s are positive regulators in NLR protein assembly. We found that they also exert negative roles in NLR protein turnover. Specific mutations in HSP90.3 lead to increased protein levels of multiples NLRs. Using an immunoprecipitation assay we also demonstrated that SNC1 is a client of HSP90.3. MUSE13 and MUSE14 are both TRAF domain-containing proteins. TRAF domains are conserved in eukaryotes, and TRAF domain proteins are predominantly involved in protein ubiquitination or protein processing. MUSE13 and MUSE14 function redundantly in the negative regulation of protein turnover of two NLRs. We found that MUSE13 forms a protein complex with SCFCPR1 and NLRs, which suggests the existence of a plant type TRAFasome that modulates NLR homeostasis. Overall, my study on these MUSE proteins provides insights into the regulation of plant NLR turnover, and broadens our knowledge of the tight control of the plant immune system.

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The roles of MUSE1 and MUSE15 in plant innate immunity (2016)

Nucleotide-binding leucine-rich repeat (NLR) immune receptors play crucial roles in pathogen recognition and defense activation in animals and plants. The immune responses mediated by NLR proteins are tightly regulated in plants so that the host effectively responds to pathogen attack without experiencing autoimmunity. However, the mechanisms underlying this regulation are not fully understood. To better understand this process, a mutant snc1-enhancing (MUSE) forward genetic screen was performed in model plant Arabidopsis thaliana. This thesis describes the identification and characterization of two genes encoding negative regulators of defense responses, MUSE1 and MUSE15, respectively. MUSE1 encodes a previously uncharacterized RING domain protein exhibiting E3 ubiquitin ligase activity. It has a close paralog in the Arabidopsis genome, which is MUSE2. Albeit both muse1 and muse2 single mutants are wild type (WT)-like, the muse1 muse2 double knockout mutant displays severe autoimmunity, suggesting their overlapping functions in regulating defense. Through epistatic analysis, it was found that the autoimmunity of muse1 muse2 is fully dependent on SNC1, suggesting that MUSE1 and MUSE2 are specifically involved in the regulation of SNC1-mediated immunity. Genetic and biochemical analyses excluded SNC1, bHLH84 and MOS10 from being potential ubiquitination substrates of MUSE1 and MUSE2, and offered clues to the identity of the substrates of MUSE1 and MUSE2. These findings add to the growing list of characterized E3 ubiquitin ligases involved in the stringent regulation of NLR-mediated immunity.MUSE15 encodes ADR1-L1, which belongs to the ADR1 helper NLR family. Previous studies have demonstrated that the ADR1 family is required for defense mediated by multiple sensor NLRs. Unexpectedly, loss of ADR1-L1 enhances immunity-related phenotypes in multiple autoimmune mutants including snc1, cpr1, bal and lsd1. This immunity-enhancing effect is not mediated by increased SNC1 protein stability, nor is it fully dependent on the accumulation of defense hormone salicylic acid (SA). Transcriptional analysis revealed an up-regulation of ADR1 and ADR1-L2 in the adr1-L1 background, which may over-compensate the loss of ADR1-L1, leading to stronger defense responses. The complex regulation within the ADR1 family extends our knowledge on the interplay among helper NLRs.

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Dissecting negative regulation of plant immunity through studying muse (mutant, snc1-enhancing) mutants (2014)

Plants respond in various ways to defend themselves against pathogen infections. Resistance (R) protein-mediated defense is one of the most effective mechanisms, through which plants can detect the activity of secreted pathogen-derived molecules (effectors) that promote infection. In Arabidopsis, snc1 encodes a TIR-NB-LRR R-like protein that carries a unique gain-of-function mutation, leading to constitutive activation of defense. Mutant snc1 has been used as a successful tool to dissect R protein-mediated immunity. Over a dozen MOS (Modifier of SNC1) proteins have been identified as positive regulators of plant immunity, indicating a complicated signaling network involved in R protein activation. To study negative regulation of snc1-mediated resistance, genetic screens were performed in mos snc1 backgrounds. Mutants restoring snc1-mediated autoimmunity phenotypes were isolated and named as muse (mutants, snc1-enhancing) mutants. The sensitized mos snc1 background enables us to find mutants that may have subtle defense phenotypes by themselves.This PhD thesis reports the identification and characterization of two muse mutants, muse3 and muse5, both enhancing snc1-associated autoimmune phenotypes in the mos4 snc1 background. MUSE3 is an Arabidopsis ortholog of yeast E4 ubiquitin conjugating factor required for polyubiquitin chain assembly. From the genetic and biochemical data, we found that MUSE3 functions downstream of the E3 complex SCFCPR1 to facilitate the degradation of at least two R proteins, SNC1 and RPS2. My study is the first report on E4 function in plants and adds another key step in R protein turnover pathway.MUSE5 encodes an ortholog of yeast PAM16, part of the mitochondrial inner membrane protein import motor and therefore, is renamed AtPAM16. In yeast pam16 mutants, preprotein import into the matrix is defective. Knocking out AtPAM16 leads to elevated ROS production and enhanced PR gene expression, suggesting that a negative regulator of plant immunity may not be properly imported into mitochondria in Atpam16. This unknown negative regulator is probably involved in preventing ROS accumulation and autoimmunity in mitochondria. This study highlights the significance of negative regulation of plant immunity in mitochondria.In summary, my PhD research contributes to better understanding of the negative regulatory mechanisms plants utilize to defend themselves against pathogen attack.

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Genetic and molecular analysis of resistance protein mediated plant immunity (2014)

Upon recognition of pathogen effectors, plant resistance proteins trigger strong defence responses that restrict the growth and spread of pathogens. Most of these immune receptors are nucleotide-binding (NB) and leucine-rich repeat (LRR) domain-containing proteins (NLRs). SNC1 (Suppressor of npr1, constitutive 1) is an Arabidopsis Toll/interleukin-1 Receptor (TIR)-type NLR that was originally identified through a gain-of-function autoimmune mutant, snc1, from a forward genetic screen. My Ph.D. thesis is comprised of three projects, all taking advantage of the unique autoimmune phenotypes of snc1 to enable efficient genetic screening.Firstly, MOS12 was identified from the snc1 suppressor screen, which encodes a protein homologous to cyclin L of mouse and human. MOS12 is required for both SNC1- and RPS4- mediated defense response through its involvement in alternative splicing of SNC1 and RPS4. MOS12 associates with the MAC in planta and MAC components also contribute to proper splicing of SNC1 and RPS4. This study provides the emerging regulatory details of alternative splicing of certain NLR genes. Secondly, the transcription factor bHLH84 was isolated from a reverse genetic screen by its ability to confer enhanced immunity when overexpressed. bHLH84 and its homologs function redundantly in SNC1- and RPS4-mediated defense response. While bHLH84 does not seem to regulate the expression of NLR-encoding genes directly, it associates with SNC1 and RPS4 to fulfil their function in plant immunity. Being a transcription activator, bHLH84 associates with nuclear NLR proteins, probably in parallel with NLR-associated transcription repressors, enabling potentially fast and robust transcriptional reprogramming upon pathogen recognition.Lastly, the identification and functional study of MUSE6 revealed the crucial impact of N-terminal acetylation on the turnover of SNC1. Biochemical analysis uncovered that SNC1 undergoes alternative translation initiation, which provides respective substrates for NatA and NatB. SNC1 peptides translated from the first Met are targeted by NatA, and the acetylation serves as a degron, while SNC1 peptides initiated from the second Met are targeted by NatB, which stabilizes the protein. Different acetylation events on SNC1 are speculated to provide more flexibility to maintain its homeostasis. Overall, my Ph.D thesis research contributes to the better understanding of NLR protein regulation and activation.

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Dissecting plant innate immunity using SNC1 - a sensitive immune receptor (2013)

No abstract available.

Towards Comprehensive Understanding of PLEIOTROPIC REGULATORY LOCUS 1 Associated Resistance Signalling (2012)

Plants employ a multi-layered protection system to recognize pathogen presence and act uponintrusion. The conserved MOS4-associated complex (MAC) participates in the triggered signaltransduction relay and contributes to the build-up of sound resistance. PLEIOTROPICREGULATORY LOCUS 1 (PRL1), a MAC component with predicted structural function, isneeded for a healthy immune response. Loss of this WD40 protein results in substantially higherpathogen colonization in Arabidopsis mutants. To dissect signalling steps downstream of theMAC, a mutant allele of PRL1 was chosen as the basis for a genetic suppressor screen. Fromthis screen, both dominant and recessive mutants with defects in candidate genes wereisolated, and two suppressors were cloned using map-based cloning techniques.Characterization of the first dominant mutant revealed a gain-of-function mutation in PRL2, thehomolog of PRL1. Although similar in sequence, the expression of PRL2 is greatly reduced inwild-type plants and functional analysis had not been attempted. Using the dominant prl2-1dallele and complementary mutants, full functional equivalence between the related proteins wasestablished by means of defence –testing assays and evaluation of morphological criteria. Thisinvestigation revealed unequal genetic redundancy between the homologs; PRL2 has retainedresidual but relevant expression levels compared to the higher expressed PRL1. PRL2 alsodisplays modified expression patterns, potentially indicative of developing tissue specificity.The haplo-insufficient SUPPRESSOR OF prl1, 2 (SOP2) gene is an intriguing discovery inPRL1 signal relay. Devoid of known sequence motifs, SOP2 encodes a novel nuclear proteinwith homologs limited to the plant kingdom. Several lines of evidence support a dosagedependentmechanism, mediated by SOP2, which is prone to interference by a spoiler protein.Both the obtained dominant-negative sop2-1D allele and a recessive sop2 mutation fullysuppress prl1-related phenotypes, however neither one causes impaired resistance in singlemutant analysis. Although specifics of SOP2 functionality in the context of plant resistancesignalling remain to be fully resolved, clues from epistasis analysis point towards a PRL1centered relationship and do not support SOP2 as a target of the MAC

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Characterization of the mos4-associated complex in plant defense signaling (2010)

The plant immune system is governed in part by Resistance (R) proteins that recognize pathogenic microorganisms and initiate enduring defense responses. While the terminal outputs of R protein activation are fairly well understood, information about signaling components involved in plant immunity is scarce. We previously showed that MODIFIER OF SNC1, 4 (MOS4) associates with the transcription factor AtCDC5 and the WD-40 protein PRL1 in vivo, forming the MOS4-Associated Complex (MAC). The MAC is required for plant defense responses, including those activated in the autoimmune mutant snc1, in which the R protein SNC1 is constitutively active. To identify additional MAC proteins, hemagglutinin-tagged MOS4 was purified by affinity chromatography and over 20 associated proteins were subsequently identified by mass spectrometry. In addition to MOS4, AtCDC5, and PRL1, we identified two homologous U-box proteins as well as several nucleic-acid binding proteins and snRNP subunits predicted to be integral components of the spliceosome. This thesis describes the characterization of selected MAC proteins in plant defense as well as EDS17, a gene unrelated to the MAC but that is likewise required for innate immunity in Arabidopsis. MAC3A and MAC3B encode highly similar E3 ubiquitin ligases with homology to the yeast and human protein Prp19. Through the analysis of loss-of-function mutants, we found that these loci are genetically redundant and are required for innate immunity in plants. MAC5A and MAC5B encode highly homologous putative RNA-binding proteins similar to the human protein RBM22. Analysis of these loci by reverse genetics revealed that they are partially redundant in a dosage-dependent manner and that both genes are essential for viability in Arabidopsis. Importantly, the loss of either MAC3A/3B or MAC5A suppresses snc1-associated autoimmune responses, indicating that these loci function in the snc1 pathway similar to MOS4. Even though the MAC is closely associated with the spliceosome, we could not detect obvious splicing defects in MAC mutants, indicating that this protein complex is probably not required for general splicing in plants. Together, our data suggest that the MAC likely functions as a transcriptional regulator to fine-tune plant immune responses.

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Master's Student Supervision

Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.

Genetic Dissection of Sclerotial Formation in Sclerotinia Sclerotiorum (2024)

The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

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Identification and characterization of suppressors of anp2/anp3 (2024)

A diverse array of defense mechanisms and signaling pathways are activated following the detection of pathogens by the plant immune system. Immunity is kept under tight regulation in order to respond appropriately to threats while avoiding costly autoimmune activity in the absence of pathogens. ANP2 (Arabidopsis NPK1-related Protein kinase 2) and ANP3 (Arabidopsis NPK1-related Protein kinase 3) operate as part of a mitogen-activated protein kinase (MAPK) cascade that modulates downstream responses to pathogen attack. Loss of ANP2 and ANP3 leads to autoimmunity and dwarfism. In this study we investigate a large number of suppressor of anp2/anp3 (san) mutants in the anp2/anp3 double mutant background. We show that these san mutants suppress the morphological deficiencies exhibited by the anp2/anp3 mutant. In addition, the san mutants show broad suppression of the autoimmunity present in the anp2/anp3 double mutant, suggesting that these mutations are located within genes that encode immune-relevant proteins, likely downstream components of the same signaling pathway that ANP2 and ANP3 operate in. We mapped these mutations and identified three genomic regions containing candidate genes of interest.

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Dissecting plant immune signaling (2021)

Pathogen infection in plants can result in plant disease. In crop plants, this can cause large losses in yield and profit, as well as endanger food security. The plant immune system that has evolved to fight off these infections differs from that of mammals greatly, but also involves tight regulation and complex signaling networks, to prevent under- or over-active immune expression. The many layers of plant immune signaling are still not completely understood. Here, two aspects of immune signaling are considered. Firstly, the theoretical explanations and practical considerations for the clustering of NLRs in genomes, both in pairs and in larger clusters were examined. I put forward that the frequency with which functionally linked NLRs are found to also have close genomic proximity is not coincident, and thus should be taken into consideration when examining the functions of NLRs located adjacent to other NLRs, and broader recognition capabilities considered in these cases. Secondly, mutants found through a suppressor screen of the autoimmune triple mutant camta1/2/3 were studied in order to better understand downstream signaling and immune crosstalk in Arabidopsis thaliana. Complementation tests revealed that most of the mutants identified carry mutations in known genes. However, a handful of mutants were identified that will pave the way to future discoveries of novel mechanisms of plant immune regulation. This suggests that new mutants could be found through continued screening, as we are moving closer to screen saturation.

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HSP90 contributes to chs3-2D-mediated autoimmunity (2021)

Plants employ multi-layered immune systems against infections by various pathogens. Different receptors in plants are able to detect the invasion activities caused by pathogens, transduce signals to downstream components, and activate defense responses. Among them, nucleotide-binding domain leucine-rich repeat containing proteins (NLRs) are the major intracellular immune receptor proteins. CHILLING SENSITIVE 3 (CHS3) is one of the NLRs with an additional Lin-11, Isl-1 and Mec-3 (LIM) domain at its C terminus. The gain-of-function mutant, chs3-2D, exhibiting severe dwarfism and constitutively activated defense responses, was selected as a genetic background in this study for a forward genetic screen. An allele of hsp90.2 was isolated as a partial suppressor of chs3-2D, which suggests that HSP90.2 is a positive regulator of CHS3-mediated defense signaling. In addition, HSP90s are also either fully or partially required for the autoimmunity of Dominant Negative (DN) SNIPER1 and gain-of-function ADR1 mutants, suggesting a broader role of HSP90 in NLR-mediated defense. Overall, my thesis work dissecting the CHS3-mediated autoimmunity may assist the future development of crop disease control in a more sustainable way.

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Characterization and cloning of suppressors of chs3-2D in arabidopsis (2015)

Plant innate immunity depends on the function of a large number of intracellular immune receptor proteins, the majority of which are structurally similar to mammalian NUCLEOTIDE-BINDING OLIGOMERIZATION DOMAIN (NOD)-LIKE RECEPTOR (NLR) proteins. CHILLING SENSITIVE 3 (CHS3) encodes an atypical Toll/Interleukin 1 Receptor (TIR)-type NLR protein with an additional Lin-11, Isl-1 and Mec-3 (LIM) domain at its C terminus. The gain-of-function mutant allele chs3-2D exhibits severe dwarfism and constitutively activated defense responses, including enhanced resistance to virulent pathogens, high PATHOGENESIS-RELATED (PR) gene expression, and salicylic acid accumulation. To search for novel regulators involved in the CHS3-mediated immune signaling pathway, we conducted suppressor screens in the chs3-2D and chs3-2D pad4-1 genetic backgrounds. Alleles of sag101 and eds1-90 were isolated as complete suppressors of chs3-2D, and alleles of sgt1b were isolated as partial suppressors of chs3-2D pad4-1. This suggests that SAG101, EDS1-90, and SGT1b are all positive regulators of CHS3-mediated defense signaling. Additionally, the TIR-type NLR-encoding CSA1 locus located in the Arabidopsis genome adjacent to CHS3 was found to be required for CHS3-mediated signaling. CSA1 is located 3.9kb upstream of CHS3 and is transcribed in the opposite direction. Altogether, these data illustrate the distinct genetic requirements for CHS3-mediated defense signaling.

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Using chemical genetics to discover regulators in plant immunity (2013)

Plants fend off pathogens by using Resistance (R) protein receptors to recognize pathogenic effectors. Classical genetic methods have been used to study R protein activation. However, the usage of classical genetics is limited by redundancy and lethality. To overcome these problems, I performed a high-throughput chemical genetics screen. I screened a chemical library containing 3,600 small-molecule compounds with known functions in animal systems. I identified one compound, 353D7, an NMDA (N-methyl-D-aspartate) subtype glutamate receptor antagonist in animals, that was able to completely suppress the autoimmune phenotypes of chs3-2D (chilling sensitive 3, 2D), including the arrested growth morphology and PR (pathogenesis related) gene expression. I also demonstrated that 353D7 suppressed the morphology of chs3-2D in a dosage dependent manner. In addition, 353D7 could partially suppress flg22‒induced reactive oxygen species production. In Arabidopsis, there are twenty glutamate-like receptors (GLR) ‒encoding genes that share high homology with the animal ionotropic glutamate receptors (iGluRs). Activation of NMDA receptors triggers ion fluxes including Ca²⁺ influx across the plasma membrane (PM). Cytosolic Ca²⁺ is one of the most ubiquitous secondary messengers mediating numerous biological processes including plant defense. We hypothesize that 353D7 probably perturbs plant GLR‒mediated Ca²⁺ influx. This work will potentially help us discover the longsought Ca²⁺ channels on the plasma membrane necessary for plant defense.

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Characterization and cloning of defence-related suppressors of mos4-1 snc1 in Arabidopsis thaliana (2011)

In response to pathogen infection, plants typically use RESISTANCE (R) proteins to recognize and induce a strong defence response. SNC1 belongs to a class of R-proteins, and the gain of function snc1 mutant has constitutively active immune responses. MOS4 has been identified through a snc1 suppressor screen. MOS4 interacts in a complex called the mos4-associated complex (MAC) which is homologous to splicing-related complexes in yeast and humans. However, no splicing defects have been observed in any MAC single mutants. Two MAC proteins, AtCDC5 (a transcription factor) and MAC3A/3B (an E3 ubiquitin ligase) could be responsible for defence signalling downstream of the MAC. Since mos4-1 has the same defence phenotype as both atcdc5 and mac3a/3b, mutations to any of these genes probably has the same effect on perturbing the MAC. We performed a mos4-1 snc1 suppressor screen to identify signalling components downstream of the MAC.The suppressor screen identified 31 dwarf mutants that all had either high PR2 defence gene expression or resistance to a virulent pathogen, H.a. Noco2, suggesting that the mutations affect defence signalling. Three mutants, 60B-1, 83-2 and 39-1, were characterized in greater detail and each of their respective mutations were mapped. 60B-1 carries a mutation to a known negative regulator of defence signalling, BON1. 83-2 carries a further gain of function mutation to snc1, however, unlike snc1 which causes snc1 protein accumulation, snc1 protein accumulation in 83-2 does not appear to be affected, suggesting that the protein is converted into a more active form. The molecular lesion in 39-1 was mapped near the southern telomere of chromosome 1, its exact location awaits discovery.We have shown that a mos4 snc1 suppressor screen can successfully identify both recessive negative regulators of defence and dominant positive regulators of defence.

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