Relevant Degree Programs
Systemic acquired resistance, SA signaling, ROS signaling, MAPK signaling, signal transduction pathways downstream of plant immune receptors
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Graduate Student Supervision
Doctoral Student Supervision (Jan 2008 - April 2022)
Plants have evolved specific strategies to ward off invading pathogens. The plant immune system consists of two conceptual layers: pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). Plasma membrane-localized pattern-recognition receptors (PRRs) and intracellular nucleotide binding-leucine rich repeat receptors (NLRs) are employed as pathogen receptors for pathogen recognition. Additionally, mitogen-activated protein kinase (MAPK) cascades play critical roles in plant immunity. The cascade formed by the Arabidopsis MEKK1-MKK1/MKK2-MPK4 has dual functions in the regulation of immune responses. It is activated during PTI and contributes to basal resistance against virulent pathogens. The whole cascade is guarded by the NLR protein SUMM2.Although recent studies provided important insights into how disruption of the MEKK1-MKK1/MKK2-MPK4 cascade leads to activation of SUMM2-mediated immunity and how SUMM2-mediated immunity is regulated, the signalling components immediately downstream of SUMM2 are largely unknown. Since several kinases most often are involved in the immune signalling events, the potential role of RLCKs as transducing signalling components downstream of CNL SUMM2 was investigated in Arabidopsis. In this study, reverse genetic analysis was carried out on RLCKs with potentially redundant functions to determine whether they are required for SUMM2-mediated defence responses. Using an egg-cell based CRISPR/Cas9 system, I knocked out the candidate RLCK genes in the mekk1-5 ndr1-1 background and identified a small group of RLCKs (designated as KH9.10) that are required for the autoimmune phenotypes of mekk1-5 ndr1-1. This thesis reports the characterization of RLCKs that belong to subgroup VIII. I provide both genetic and biochemical evidence to support the hypothesis that RLCKs in the KH9.10 group play a key role in SUMM2-mediated immunity. In addition, my study shows that this group of RLCKs is required for PTI responses induced by the elicitor flg22. Overall, this work provides new insights into the role of RLCK subgroup VIII as components in the signal transduction network downstream of SUMM2 in Arabidopsis.
Plant pathogens secrete virulence factors known as effectors to aid in infection and host colonization. Plants possess a multi-tier defense system to detect and halt pathogens. The basidiomycete fungus Ustilago hordei causes covered smut disease of barley. The previously identified effector UhAVR1 from U. hordei causes avirulence in barley cultivars carrying the resistance gene, Ruh1, whereas Ruh1 absence leads to virulence. To date, UhAVR1 is the only proven avirulence effector of smuts. This study aimed to functionally characterize UhAVR1 and to understand the underlying molecular mechanism leading to susceptibility or resistance in barley. Using fungal strains expressing UhAVR1+SP:mCherry, the secretion of UhAVR1 via the Brefeldin A-sensitive ER-Golgi pathway and by the action of its signal peptide was shown. UhAvr1 transcripts were detected only early during infection of barley seedlings confirming in planta induction upon host sensing. This infection-specific induction contributes to susceptibility in cultivar Odessa (ruh1) or to complete immunity in cultivar Hannchen (Ruh1). Transient expression of UhAVR1 in barley, Nicotiana benthamiana, and Arabidopsis thaliana showed it localizes to the cytosol, the site where it likely performs its functions. The delivery of UhAVR1 via foxtail mosaic virus, Pseudomonas bacteria, and Agrobacterium-mediated suppression of cell death inducers in N. benthamiana and barley support a role in the suppression of a conserved component(s) of plant immunity. RNA-seq analysis at 48 hpi of cultivar Odessa infected with U. hordei revealed that UhAvr1 induces plant fatty acids and suppresses plant defense. Whereas in cultivar Hannchen, UhAvr1 induces ETI and down-regulates PTI. Co-immunoprecipitation assays coupled with mass spectrometry from cultivar Odessa and N. benthamiana transiently expressing UhAVR1-SP:GFP revealed candidate host interactors with a chloroplast role. Previous work genetically located Ruh1 on barley chromosome 7H. This information was used to localize Ruh1 to a 10 million base pair region where 21 predicted resistance genes reside. Although identification of Ruh1 among the candidates was unsuccessful, this works paves the way for further studies on host resistance in this pathosystem. Ultimately, generating knowledge on how pathogens cause disease and how plants defend themselves is pivotal in generating control strategies against plant pathogens.
Plant immunity relies on two groups of pathogen receptors, plasma membrane-localized pattern-recognition receptors (PRRs) and intracellular nucleotide binding-leucine rich repeat receptors (NLRs). Mitogen-activated protein kinase (MAPK) cascades, which generally consist of MAPK kinase kinase (MAPKKK), MAPK kinase (MAPKK) and MAPK, play critical roles in plant immunity. Disruption of the MEKK1-MKK1/MKK2-MPK4 cascade in Arabidopsis results in an autoimmune phenotype in the mekk1, mkk1 mkk2 and mpk4 mutants. A mkk1 mkk2 suppressor screen led to the identification of the NLR SUMM2 (SUPPRESSOR OF MKK1 MKK2 2), suggesting that the MEKK1-MKK1/MKK2-MPK4 cascade is guarded by SUMM2 and that disruption of this cascade can lead to the activation of SUMM2-mediated immunity. This thesis reports the identification and characterization of three additional SUMM proteins, SUMM3, SUMM5 and SUMM6.SUMM3 encodes CALMODULIN-BINDING RECEPTOR-LIKE CYTOPLASMIC KINASE 3 (CRCK3). crck3 mutants suppress the constitutively activated immune responses in mekk1, mkk1 mkk2 and mpk4 mutants. CRCK3 was found to be a substrate of MPK4 and it interacts with SUMM2 in planta, suggesting that SUMM2 utilizes CRCK3 as a “guardee” or “decoy” to monitor the integrity of the MEKK1-MKK1/MKK2-MPK4 cascade. SUMM5 encodes protein phosphatase 5 (PP5), a highly conserved atypical phosphatase with an N-terminal tetratricopeptide domain. SUMM2 was shown to interact with Heat Shock Protein 90 (Hsp90) and PP5 in planta. Accumulation of SUMM2 is reduced in the pp5 knockout mutant, suggesting that PP5 acts as a co-chaperone to regulate the accumulation of SUMM2. SUMM6 encodes the malectin-like domain containing kinase MEDOS1 (MDS1). Mutations in MDS1 and its close homolog MDS2 have additive effects on suppression of the mkk1 mkk2 mutant phenotypes, suggesting that MDS1 and MDS2 have overlapping roles in promoting SUMM2-mediated immunity. MDS1 and CRCK3 were found to associate in planta, indicating that MDS1/MDS2 might promote SUMM2-mediated immunity by targeting CRCK3. Overall, my thesis work provides new insights on how plants sense pathogens using receptor proteins and how they maintain appropriate levels of these receptor proteins to ensure adequate defense response.
Activated plant defense responses consist of PAMP (pathogen associated molecular pattern)-triggered immunity (PTI) and effector-triggered immunity (ETI) at infected sites and a secondary immune response in distal parts of the host plant, termed systemic acquired resistance (SAR). Salicylic acid (SA) plays critical roles in plant immunity and its level increases upon pathogen infection. Pathogen-induced SA biosynthesis predominantly relies on ICS1 (ISOCHORISMATE SYNTHASE 1), whose induction mainly depends on transcription factors SARD1 (SAR DEFICIENT 1) and CBP60g (CALMODULIN BINDING PROTEIN 60 g). Meanwhile, the expression of SARD1 and CBP60g is also highly induced by pathogens. My Ph.D. research focuses on identification of immune regulators that function upstream and downstream of SARD1 and CBP60g. First, we performed chromatin immunoprecipitation-sequencing experiments to identify candidate targets of SARD1. We found that SARD1 and CBP60g directly control the expression of a large number of key regulators involved in PTI, ETI and SAR. Among them, two genes essential for SAR, ALD1 (AGD2-LIKE DEFENSE RESPONSE PROTEIN 1) and SARD4, are involved in the biosynthesis of pipecolic acid (Pip), a plant secondary metabolite required for SAR. Consistently, the sard1cbp60g double mutant accumulates less Pip than wild type, suggesting that SARD1 and CBP60g regulate Pip biosynthesis in addition to SA. Secondly, we showed that transcription factors TGA1 and TGA4 act upstream of SARD1 and CBP60g and thus regulate the biosynthesis of SA and Pip. Lastly, we revealed a novel mechanism of SA perception by its receptors NPR3 (NPR1-LIKE PROTEIN 3) and NPR4. NPR3/NPR4 interact with transcription factors TGA2, TGA5 and TGA6, and act as transcriptional repressors. SA inhibits the transcriptional repression activities of NPR3/NPR4 and promotes the transcriptional activation activity of NPR1 (NONEXPRESSER OF PR GENES 1); both contribute to SA-induced defense gene expression. We also found that SA induces SARD1 expression, revealing a feedback amplification loop between SA and SARD1, where SARD1 promotes SA biosynthesis via directly activating ICS1 expression and SA induces SARD1 expression by regulating the activities of NPR/TGA complexes. Altogether studies in this dissertation provide new insights on the functions of SARD1 and CBP60g in plant immunity and the mechanism of SA perception and signaling.
In eukaryotes, MAP kinase cascades are important players of signal transduction in many biological processes. In plant immunity, two main MAP kinase pathways, MKK4/MKK5-MPK3/MPK6 and MEKK1-MKK1/MKK2-MPK4, are activated upon elicitor treatments. On the other hand, the YDA-MKK4/MKK5-MPK3/MPK6 cascade is involved in the negative regulation of stomatal differentiation. Plants have evolved different mechanisms to regulate the activity of these cascades. For example, brassinosteroid (BR) signaling has been reported to promote the YDA pathway.This study revealed that the mapkkk3 mapkkk5 double mutant showed reduced phosphorylation of MPK3/MPK6 upon treatment with conserved microbial molecules called PAMPs, suggesting that MAPKKK3/MAPKKK5 function upstream of MKK4/MKK5 to form a cascade in plant immunity. The YDA cascade shares the same MAPKK and MAPK as the MKK4/MKK5-MPK3/MPK6 cascade in plant immunity. Interestingly, loss of YDA or blocking BR signaling leads to increased elicitor-induced activation of MPK3/MPK6. On the other hand, development defects caused by silencing of YDA are suppressed in the mapkkk3 mapkkk5 double mutant. These data suggest that there are antagonistic interactions between the two MAP kinase cascades in stomatal development and plant immunity. Disruption of the MEKK1-MKK1/MKK2-MPK4 cascade leads to immune receptor SUMM2-mediated immune responses. MEKK2, a close paralog of MEKK1, was previously shown to act as a positive regulator of SUMM2-mediated immunity. I discovered that this occurred due to the inhibition of the activity of MPK4 by MEKK2 as MEKK2 is able to inhibit the phosphorylation of MPK4 by MKK2 in vitro. Interestingly, activation of SUMM2-mediated defense responses in the mekk1, mkk1 mkk2 and mpk4 mutant plants leads to increased MEKK2 transcript levels, which contributes to positive feedback regulation of SUMM2-mediated immunity. MEKK2 arose from a recent duplication event resulting in the tandem gene repeat consisting of MEKK1, MEKK2 and MEKK3. My data suggest that MEKK2 underwent dramatic functional divergence from other MAPKKKs to gain the function as a negative regulator of MAP kinases, which is an interesting evolutionary event conferring novel biochemical mechanisms of the paralog. Taken together, the studies in my Ph.D. thesis provide new insights into the regulation of the two main MAP kinase cascades activated during plant immunity.
Plant immunity is usually governed by 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 and leucine-rich repeats-containing proteins (NLRs) serve as intracellular immune receptors to recognize the presence of relatively diverse pathogen effectors and trigger ETI (effector-triggered immunity). The Arabidopsis thaliana mutant snc2-1D (suppressor of npr1-1, constitutive 2) contains a gain-of-function mutation in a receptor-like protein (RLP) and displays a dwarf morphology. Here I report the characterization of bda4-1D (bian da 4-1D), which was identified as a complete suppressor of snc2-1D dwarf morphology. Positional cloning showed bda4-1D contains a gain-of-function mutation in Non-Expressor of Pathogenesis-Related Proteins 4 (renamed npr4-4D). Functional analysis indicated NPR4, as well as its close homolog NPR3 (Non-Expressor of Pathogenesis-Related Proteins 3), function as transcriptional repressors. They function downstream of SNC2, independent of NPR1 (Non-Expressor of Pathogenesis-Related Proteins 1). In addition, salicylic acid (SA) was shown to inhibit the transcriptional activities of NPR3/4 and promote the expression of key immune regulators. The npr4-4D mutation leads to constitutive repression of SA-induced immune responses, indicating that the mutant protein can no longer respond to SA. On the other hand, the equivalent mutation in NPR1 also abolishes its ability to bind SA and renders reduced SA-induced defence gene expression. My results demonstrated that both NPR1 and NPR3/NPR4 are bona fide SA receptors, but play opposite roles in transcriptional regulation of SA-induced defence gene expression.In the independent eds5-3 snc2-1D npr1-1 suppressor screen, I report the identification and characterization of four more bda mutants, bda3-1D, bda5-1, bda6 and bda7. Cloning of BDA6 and BDA7 showed that they encode FMO1 and ALD1 respectively, which are involved in biosynthesis of N-Hydroxypipecolic Acid (NHP) and pipecolic acid. My results indicate that enzymes involved in Lysine metabolism are also important for signaling in SNC2-mediated immune pathway.Overall, the studies I completed in my Ph.D. thesis expand our knowledge in understanding of the signaling pathways downstream of SNC2 as well as the general regulatory mechanisms of SA receptors in plant innate immunity.
The diamondback moth (DBM), Plutella xylostella, is well known for its extensive adaptation and distribution, high level of genetic variation and polymorphism, and strong resistance to a broad range of synthetic insecticides. Although understanding of the P. xylostella biology and ecology has been considerably improved, knowledge on the genetic basis of these traits remains surprisingly limited. Based on data generated by different sets of molecular markers, we uncovered the history of evolutionary origin and regional dispersal, identified the patterns of genetic diversity and variation, characterized the demographic history, and revealed natural and human-aided factors that are potentially responsible for contemporary distribution of P. xylostella. These findings rewrite our understanding of this exceptional system, revealing that South America might be a potential origin of P. xylostella, and recently colonized across most parts of the world resulting possibly from intensified human activities. With the data from selected continents, we demonstrated signatures of localized selection associated with environmental adaptation and insecticide resistance of P. xylostella. This work brings us to a better understanding of the regional movement and genetic bases on rapid adaptation and development of agrochemical resistance, and provides a solid foundation for better monitoring and management of this worldwide herbivore and forecast of regional pest status of P. xylostella, by taking a cost-effective response to insecticide resistance and better implementation of biological control programs.
Master's Student Supervision (2010 - 2021)
Plants are constantly exposed to different pathogens in their environment and they have evolved complex defense strategies to avoid potential disease. The two general layers of plant defense are pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI). PTI is activated upon recognition of PAMPs by plant’s pattern recognition receptors (PRRs) localized on plasma membrane, while ETI is triggered when the presence of bacterial effector proteins is detected by intracellular plant nucleotide-binding leucine-rich repeat (NLR) proteins. Salicylic acid (SA) is a major regulator of plant immunity. A new model of SA signaling in Arabidopsis thaliana was recently established. Non-expressor of Pathogenesis-Related (PR) genes 1 (NPR1), NPR3 and NPR4 are SA receptors that have opposite roles in transcriptional regulation of defense-related genes. NPR1 functions as a transcriptional co-activator, while NPR3 and NPR4 are redundant transcriptional co-repressors of defense-related genes. Binding of SA activates NPR1, while it blocks transcriptional repression activity of NPR3 and NPR4. Although general function of these proteins in plant defense is understood, their requirement for different regulatory pathways in plant immunity is not entirely explored. In this thesis I analyze how NPR1 and NPR3/4 are involved in regulation of PTI response, and ETI response mediated by different NLR proteins. Infection assays with different strains of Pseudomonas syringae pv. tomato DC3000 revealed that PTI is regulated through both NPR1- and NPR3/4-dependent signaling, and that basal levels of SA contribute to PTI and AvrRPS4- or AvrRpt2-triggered ETI. The knock-out mutant npr1-1 and a dominant gain-of-function mutant allele npr4-4D fully suppress expression of PR1 and resistance to H.a. Noco2, but partially suppress dwarf morphology and constitutive PR2 expression of autoimmune snc1 mutant. Similarly, npr1-8 and npr4-4D partially suppress autoimmune phenotype of mkk1/2 mutants. Results of ion-leakage assay suggest that SA serves as a negative regulator of AvrRpt2-triggered hypersensitive response through NPR1 and NPR4-mediated signaling. My results support the new model of SA-dependent signaling, confirming that NPR1 and NPR3/4 function in parallel pathways. Although SA is the major regulator of plant defense, not all aspects of immune response rely entirely on SA receptors NPR1 and NPR3/4, confirming existence of SA-independent signaling.
As an early defense response, MAP kinase cascade activation plays important roles in transduction and amplification of signals upon pathogen perception in plants. The Arabidopsis MEKK1-MKK1/MKK2-MPK4 kinase cascade was previously shown to negatively regulate plant immunity. In this study, two suppressors of the mkk1 mkk2 double mutant – summ4-1D and summ4-2D have been identified and characterized. summ4-1D and summ4-2D contain mutations in the promoter region of MKK6, which leads to elevated expression of MKK6, causing suppression of the mkk1 mkk2 autoimmune phenotypes. However, the autoimmune phenotypes of mekk1 and mpk4 cannot be suppressed by summ4-1D. MKK6 interacts with MEKK1 and MPK4, and MPK4 activation is blocked in mkk1 mkk2, but is recovered in the summ4-1D mkk1 mkk2 triple mutant background. These results suggest that MKK6 functions in parallel with MKK1 and MKK2 to negatively regulate plant immunity.
Recognition of pathogens through pathogen-associated molecular patterns (PAMPs) or effectors in plants activates a variety of defense responses including MAPKs signaling pathways and defense related genes expression. ANPs (Arabidopsis Nucleus- and Phragmoplast- localized kinase 1 related protein kinases), including ANP1, ANP2, ANP3 are three MAP kinase kinase kinases that form a MAP kinase cascade with downstream MKK6 and MPK4 to regulate cytokinesis process. In this study, we showed that the anp2 anp3 double mutants exhibit constitutive expression of PR (Pathogenesis-Related) genes and enhanced resistance against oomycete pathogen H. a. Noco2, suggesting that ANP2 and ANP3 negatively regulate plant immunity. In addition, loss function of MKK6 causes high levels of PR gene expression, indicating that MKK6 is involved in negative regulation of defense responses. Since MPK4 was previously shown to function as a negative regulator of plant immunity, we tested whether MPK4 functions downstream of ANP2/ANP3 and MKK6 in plant immunity by introducing CA-MPK4 transgene, which expresses a constitutively active (CA) variant of MPK4, to anp2 anp3 and mkk6. Constitutive expression of PR genes and enhanced resistance to H.a. Noco2 in anp2 anp3 and mkk6 were partially suppressed by expressing CA MPK4, suggesting that the ANP2/ANP3, MKK6 and MPK4 function in a MAPK cascade to negatively regulate defense responses. To find out components that function downstream of ANP2/ANP3- MKK6-MPK4 cascade in plant immunity, two mutants summ2-8 (SUPPRESSOR OF MKK1 MKK2 2) and pad4-1 (PHYTOALEXIN DEFICIENT 4) were crossed into anp2 anp3 respectively. The constitutive defense responses in anp2 anp3 were fully suppressed by pad4-1, but not affected by the summ2-8 mutation, suggesting that PAD4 functions downstream of ANP2/ANP3 and that immune responses mediated by certain TIR-NB-LRR R proteins might be activated in the anp2 anp3 mutant.
The primary layer of plant immunity is pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). In PTI, when pattern recognition receptors (PRRs) recognize PAMPs, a highly regulated, rapid downstream signaling response such as activation of mitogen-activated protein (MAP) kinases, production of ROS and defense gene expression is initiated. My study involves the identification of MAP kinases activated in PTI and regulation of PTI responses by E3 ubiquitin ligases. In Arabidopsis thaliana, MPK3, MPK4, and MPK6 are activated upon PAMP treatment. However, previous studies suggest that there are more MAP kinases activated upon PAMP treatment. To identify the additional MAP kinases phosphorylated upon PAMP perception, transgenic plants expressing candidate MPKs with a ZZ-FLAG double tag (approx. 17kD) were generated in Col-0 background. Western blot analysis identified three MAP kinases, MPK1, MPK11 and MPK13 that are phosphorylated upon PAMP treatment. To identify E3 ligases involved in PTI, E3 ligases whose transcripts are up-regulated upon PAMP treatment were selected for our study. Transgenic lines overexpressing candidate E3 ligases were assayed for deficiencies in PTI. Overexpression of U6, one of the selected E3 ligases, lead to severe reduction of flg22-induced reactive oxygen species (ROS) production and increased susceptibility to Pseudomonas syringae pv. tomato (P.s.t.) DC3000 hrcC-. Furthermore, when U6 was overexpressed in BIK1 (a positive regulator of PTI)-HA transgenic plants, there was a decrease in BIK1-HA protein expression, leading to the hypothesis that BIK1 may be a potential target of U6. Overall, my thesis contributes to a better understanding of the signaling and negative regulation of PTI. Advancing our knowledge in plant immunity leads to the potential of its use in agriculture and plant protection.
Plants are sessile organisms that are surrounded by pathogens. To stay healthy, they need a complex and sensitive immune system. Specific pattern-recognition receptors (PRRs) localized on the plasma membrane can recognize conserved motifs from pathogens and transduce the signal into the cell to initiate defence responses. The receptor-like kinase BAK1-INTERACTING RECEPTOR-LIKE KINASE 1 (BIR1), functions as a negative regulator of plant immunity. bir1-1 exhibits spontaneous cell death and constitutive defence responses that are dependent on SUPPRESSOR OF BIR1,1 (SOBIR1) and PHYTOALEXIN DEFICIENT 4 (PAD4). Here I present the evidence that ER-quality control, a collective mechanism ensuring that only native proteins are produced by the secretary pathway, plays important roles in regulating defence responses in bir1-1. Five components in ER-quality control pathways, including CRT3, UGGT, STT3a, ERdj3b and SDF2, are all required for the immune responses in bir1-1. Western blot analysis showed that mutations in CRT3, ERdj3b and UGGT lead to reduced accumulation of SOBIR1 protein. The data suggest that ER-quality control plays an important role in the accumulation of SOBIR1 and is required for the defence responses in bir1-1.