Introduction to Plant Immune Responses
Immunity is a double-edged sword: without it, the organism will succumb to infection, whereas too much of it could subject the host to autoimmune disease. The situation is even more perplexing in plants as they do not have specialized immune cells. Immune responses have to be balanced with other biological functions in each cell to ultimately protect the whole organism. The main research goal of my laboratory is to understand the mechanisms and the dynamic regulation of plant immune responses. Specifically, we use Arabidopsis thaliana as a reference organism and apply modern genetic, genomic, and proteomic approaches to our studies at molecular, cellular and organismal levels. Compared to the sophisticated immune system in jawed vertebrates, the immune system of plants appears far less complex. However, using strategies that are distinct from animals, plants are capable of mounting highly specific defense responses with restricted self-reactivity. Memory of pathogen exposure can last for weeks, if not longer. How do plants achieve these immune properties? At the cell surface, microbe-associated molecular patterns (MAMPs) are detected by pattern recognizing receptors (PRRs) leading to pattern-triggered immunity (PTI). Adapted pathogens can normally overcome PTI by directly delivering effectors into plant cells. Even though these effectors are highly polymorphic among pathogen strains, they target only a few critical host proteins to suppress PTI and enhance virulence. Perturbations of the host target caused by some effectors can be detected by the plant intracellular nucleotide-binding and leucine-rich repeat (NB-LRR) immune receptors resulting in effector-triggered immunity (ETI). ETI is the major immune mechanism in plants, which is characterized by programmed cell death (PCD) at the site of attempted infection and the release immune signals such as salicylic acid (SA). Each plant genome encodes hundreds of NB-LRRs to “guard” key cellular targets (i.e., self) and to provide immunity against a wide range of effector specificities. Activation of local ETI can often lead to systemic acquired resistance (SAR) which is a long-lasting broad-spectrum resistance. SA is a necessary and sufficient signal for SAR. An increase in SA either through endogenous synthesis or by exogenous application can lead to major transcription reprogramming and secretion of a large array of antimicrobial PR proteins.
Elucidating the molecular function of the master immune regulator NPR1
The main research interest of my laboratory has been focused on SAR. Through a genetic screen, we identified the npr1 (nonexpresser of PR genes 1) mutant that failed to respond to SA induction and showed compromised SAR. All the evidence showed that NPR1 is a master regulator in plants, playing a role similar to NF-kB/IkB in the mammalian immune system. Since the cloning of NPR1 in 1997, we made several key discoveries in NPR1 function. We first found that nuclear translocation is a key regulatory step for this protein. In the absence of pathogen challenge, NPR1 is retained in the cytoplasm as an oligomer through redox-sensitive intermolecular disulfide bonds. Upon induction, the NPR1 monomer is released to enter the nucleus, a process that is mediated by thioredoxins. Discovery of this novel regulatory mechanism provided new insights into how pathogen-induced cellular redox changes lead to induction of immune responses in plants. In the nucleus, NPR1 serves as a cofactor to transcription factors to induce PR genes encoding antimicrobial proteins and ER-resident genes to ensure proper secretion of the PR proteins.
The transcriptional activity and protein stability of NPR1 are controlled by various posttranslational modifications (PTMs). The ultimate modification seems to be sumoylation which is required for NPR1 transcriptional activity as well as its association with its paralogs, NPR3 and NPR4, whose role as CUL3 E3 ubiquitin ligase adaptors depends on their direct associations with SA. Our future work will be focused on NPR1 nuclear function and regulation. We will characterize the NPR1 nuclear complex to identify enzymes that are involved in NPR1 PTMs and determine how the stability and activity of this complex is dynamically regulated to control the transcriptional cascade during SAR. This project is currently supported by grants from NIH and hhmi.
Understanding the central role of the circadian clock in anticipation of infection and in gating plant immune responses
In a functional genomic study of ETI-output genes, we were surprised to find that they are not only induced in an NB-LRR-dependent manner upon pathogen challenge, but also controlled by the circadian clock in the absence of infection. This observation initiated our new line of inquiry on the interplay between plant immunity and the circadian clock. We found that these clock-regulated ETI genes are normally pulse-expressed in the morning when environmental conditions are the most favorable for infection by many pathogens. More recently, we discovered that in addition to ETI-genes, the SA-biosynthetic gene, ICS1, which encodes isochorismate synthase, is also directly regulated by a circadian clock component, CHE. In a che mutant, the daily oscillation in SA levels, which normally peak before dawn, is compromised. Additionally, CHE is required for both SA- and pathogen-induced SA synthesis in systemic tissue, suggesting a mechanism for systemic SA synthesis. We are currently studying the oscillatory regulation of CHE and its role in the positive feedback-loop of SA synthesis. The involvement of CHE in both SA- and pathogen-induced SA synthesis suggests that the role of the circadian clock in immunity goes beyond anticipating infection; it may also play a role in response to infection. Indeed, we found that SA-treatment of plants reinforces the circadian clock through NPR1-mediated induction of LHY in the morning, and TOC1 in the evening. Since LHY and TOC1 are respectively positive and negative regulators of defense, reinforcement of the clock leads to gating of immune responses towards the morning (i.e., higher response when induced in the morning than in the evening). We then demonstrated experimentally that gating of SA-sensitivity towards the morning allows plants to minimize immune interference with normal cellular functions scheduled at other times of the day, because treating plants with SA in darkness caused a severe loss in fresh-weight due to interference with water-transport activity under such conditions. Our study showed that controlling the time of chemical deployment is critical for maximizing its efficacy and minimizing side effects. We plan to further explore how the circadian clock serves as a central regulator of plant immune responses by integrating both environmental cues and internal metabolic activities to optimize plant fitness. This project is currently supported by grants from NIH and hhmi.
Studying the interplay between DNA repair response, cell cycle control, and plant immunity
Through genetic screens for components involved in defense gene induction, we unexpectedly identified multiple components of the DNA damage response machinery such as BRCA2 and RAD51. In humans, BRCA2 is a mediator of RAD51 in pairing of homologous DNA; and mutations in BRCA2 cause a predisposition to breast/ovarian cancers. However, it is puzzling how a defect in DNA homologous recombination (HR), a mechanism required for all cells, leads to malignancy in specific tissues. Our work in Arabidopsis suggests that components of the HR pathway may play a noncanonical role in defense gene transcription in plants. Treating plants with the DNA damage agent bleomycin primes the defense genes for induction by SA, whereas both brca2 and rad51 mutants are hyper-susceptible to not only genotoxic substances, but also pathogen infection. Strikingly, chromatin immunoprecipitation demonstrated that RAD51 is specifically recruited to the promoters of defense genes during SAR in an SA- and BRCA2-dependent manner. Since both biotic and abiotic stresses can trigger the release of reactive oxygen species (ROS), we are currently testing two hypotheses: (1) DNA damage repair proteins are recruited to the defense gene promoters to induce chromatin remodeling as well as to safeguard against transcription-associated DNA instability. (2) Pathogen-induced DNA recombination facilitates generation of new NB-LRR genes through DNA rearrangement in the gene clusters, serving as a long-term survival strategy. Apart from engaging HR components, our genetic study also showed an involvement of cell cycle regulators in ETI. We recently found that induction of ETI can lead to an oligomer-to-monomer conformational change of CPR5 in the nuclear pore complex. This not only permeabilizes the nuclear pore resulting in spurious activation of multiple signaling pathways, but also releases the cyclin-dependent kinase inhibitors (CKIs) and activates TF E2F, leading to ETI and PCD. For the future, we are interested in (1) identifying the signaling pathway that causes the CPR5 oligomer-to-monomer switch upon NB-LRR activation; and (2) understanding the noncanonical role of CKI and E2F in conferring ETI and PCD. This project is currently supported by hhmi.
Understanding translational regulation in plant immune induction and applying the knowledge to genetically enhance disease resistance in crops
Most studies of plant immunity have been focused on transcriptional regulation. Little is known about whether defense-related mRNAs are selectively translated upon pathogen challenge. 5’-RACE performed on the mRNA of TBF1, a key immune TF, led us to the surprising discovery of two upstream open reading frames (uORFs). These uORFs normally block ribosomes from reaching the major ORF. Upon immune induction, this inhibitory effect is rapidly and transiently alleviated, leading to TBF1 translation. This suggests that de-repressing translation of pre-existing immune TF mRNA may be a strategy for rapid response to pathogen challenge. We recently performed a global translatome profiling using ribosome footprinting (RF) on PTI-induced plants and found that changes in translation did not correlate well with transcription. Genes with altered translational efficiency (TE) during PTI belong to diverse functional groups and contain novel regulators of this immune response. Moreover, a purine-rich mRNA consensus (“R-motif”) was found to be significantly enriched in the 5’ leader regions of transcripts and shown to be both necessary and sufficient for the increased TE during PTI. Currently, we are (1) performing in-depth studies of mRNA features that are responsible for immune-induced translation, with the focus on uORFs; (2) examining other factors affecting translation including mRNA modifications and tRNA populations; (3) identifying trans-acting regulatory components of uORFs and defense-induced translation using both molecular and genetic approaches. Understanding how defense protein production is regulated will allow us to develop better strategies of engineering disease resistance in crops because ectopic expression of defense proteins in plants often results in compromised growth due to their toxicity. Applying pathogen-inducible translational control to engineering disease resistance may be a revolutionary step towards new environmentally friendly agricultural practices, which reduce not only the use of pesticides but also the selective pressure for resistant pathogens. This project is currently supported by grants from NSF and hhmi.
Overall vision for the future
Our studies have shown that plant immune mechanisms are intrinsically linked to other key cellular functions. In our future research, we aim to understand the tripartite interplay between immunity, the cellular redox rhythm, and the circadian clock with the focus on: (1) the master immune regulator NPR1, which is a redox sensor and an intrinsic regulator of the circadian clock; (2) the circadian clock which “schedules” (anticipation) and “oversees” (gating) metabolic activities, including defense, in coordination with environmental changes; and (3) translational programming triggered by pathogen-induced perturbation in metabolic activities, about which little is known. Understanding mechanisms and homeostasis of these processes will significantly enrich our knowledge about immunity in general and lead to development of strategies to control infection while minimizing costs to hosts.
To see an overview of our current research view our Power Point
Areas of Interest
- plant-microbe interactions
- immune signal transduction
- interplay between immunity and other cellular processes
- environmental influence on plant immunity
- regulation of defense gene expression
- control of defense protein production