Xin Li

Professor

Relevant Degree Programs

 

Graduate Student Supervision

Doctoral Student Supervision (Jan 2008 - Mar 2019)
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 (2010-2017)
Characterization and cloning of suppressors of chs3-2D in arabidopsis (2015)

No abstract available.

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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