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Understanding the Immune Mechanisms and Regulation in Plants

Research Summary

Xinnian Dong is interested in elucidating the mechanisms and regulation of immunity in plants.

Immunity is a double-edged sword: without it, an organism will succumb to infection; too much of it is detrimental to the growth and survival of the organism. The situation is even more delicate in plants, which lack specialized immune cells. To protect the whole plant, immune responses have to be balanced with other cellular functions. The main goal of my research is to understand the mechanisms of plant immune responses and the dynamics of their regulation, using modern genetic, genomic, molecular, and systems approaches.

Compared to the sophisticated immune system of jawed vertebrates, the plant immune system appears to be less complex. However, using distinctly different defense strategies, plants are capable of mounting both signal specific and broad-spectrum defense responses. Moreover, plants exposed to pathogens can develop an immune memory that lasts for weeks or even in the subsequent generation.

How do plants achieve these immune properties? At the cell surface, microbe-associated molecular patterns (MAMPs) are detected by pattern recognition receptors (PRRs) leading to MAMP-triggered immunity (MTI). Adapted pathogens can normally overcome MTI by delivering effectors directly into plant cells. Even though these effectors are highly polymorphic among pathogen strains, they target a few critical host proteins to suppress MTI and enhance virulence. Perturbations of the host targets caused by some effectors can be detected by the intracellular nucleotide-binding leucine-rich repeat (NB-LRR) immune receptors, resulting in effector-triggered immunity (ETI). ETI, a major immune mechanism in plants, is characterized by programmed cell death (PCD) at the site of attempted infection. Each plant genome encodes hundreds of such NB-LRR proteins to "guard" these 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), a long-lasting, broad-spectrum resistance. The plant hormone salicylic acid (SA) is a necessary and sufficient signal for the establishment of SAR. An increase in plant SA levels, either by de novo biosynthesis or by exogenous application, can trigger major transcriptional reprogramming and secretion of a large array of antimicrobial pathogenesis-related (PR) proteins.

Understanding the Function of the Master Immune Regulator, NPR1, in SA-Mediated SAR
The research of my laboratory has been focused on SAR. Through a genetic screen, we identified the mutant npr1 (nonexpresser of PR genes 1), which fails to respond to SA and is compromised in SAR. Like NF-kB/IkB in the mammalian immune system, NPR1 is a master regulator of the immune response in plants. We 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. SA treatment causes changes in cellular redox state and release of NPR1 monomers, which are able to enter the nucleus. In the nucleus, NPR1 is sumoylated to turn on its activity as a transcription cofactor and, at the same time, enhance its turnover by the proteasome to render the immune induction transient. We recently found that NPR1 stability is controlled by its paralogs NPR3 and NPR4 whose interactions with NPR1 as CUL3 E3 ubiquitin ligase adaptors are determined by their direct binding to SA. This SA concentration-dependent interaction between NPR1 and NPR3/NPR4 plays a critical role in controlling cell death (infected cells) and survival (uninfected peripheral cells).

Our future work on NPR1 will be focused on its nuclear function and regulation. We will characterize the NPR1 nuclear complex, and determine how this protein serves as a cofactor for multiple transcription factors (TFs) and controls gene expression in distinct immune responses. The significance of our study on SA is not limited to the plant field. SA and its derivatives, such as aspirin, are among the oldest and the most fascinating drugs known to mankind. The effects of salicylates include not only fever reduction and pain relief, but also prevention of cardiovascular diseases, significant reduction in deaths by certain types of cancer as well as treatment of type II diabetes. However, the underlying mechanisms of these medicinal uses cannot all be explained by the currently known aspirin targets COX2 and IkB kinase because animals with mutations in these genes still have intact responses. Our system allows dissection of its drug mechanisms, which are more difficult to probe in animal models, not to mention in humans.

DNA Repair Machinery Synergistically Stimulates Plant Immunity
To identify additional signaling components in SAR, we performed two rounds of suppressor screens. The first one identified sni1 as a suppressor of npr1. In the subsequent screen for suppressors of sni1, we unexpectedly found multiple components of the DNA damage repair machinery, such as ATR, RAD17, BRCA2, and RAD51. In humans, mutations in these genes are either lethal or lead to a predisposition to cancer. For example, BRCA2 is a mediator of RAD51 in pairing of homologous DNA. Mutations in BRCA2 result in a predisposition to breast and ovarian cancers. It is puzzling how a defect in a mechanism as general as DNA homologous recombination can lead to a specific type of cancer. In Arabidopsis, treatment with the DNA damage agent bleomycin primes the defense genes for induction by SA. The atr, rad17, brca2 and rad51 mutants are all hypersusceptible to pathogen infection. Strikingly, chromatin immunoprecipitation demonstrated that RAD51 is specifically recruited to the promoters of defense genes during SAR in an SA- and RAD17/BRCA2-dependent manner.

The synergism between the plant DNA damage response and the plant immune response is surprising, but the implications are compelling. DNA damage response is the most fundamental and evolutionarily ancient stress response upon which other defense mechanisms could be built. It is well known that both biotic and abiotic stresses can trigger the release of reactive oxygen species (ROS). ROS, while serving as signals, can also cause collateral damage to the host genome. Based on this knowledge, we would like to test the following hypotheses: (1) DNA damage repair proteins are recruited to the defense gene promoters to induce chromatin remodeling and to safeguard against transcription-associated DNA instability. (2) Pathogen-induced DNA recombination facilitates generation of new NB-LRR genes through DNA rearrangement in the NB-LRR gene clusters, serving as a long-term survival strategy.

Cell Cycle Regulators Are Required for Effector-Triggered PCD and Immunity
It has been a mystery how ETI triggers PCD in plants because plant genomes do not contain close homologs of caspases involved in pyroptosis during animal immune response. Through a series of genetic screens and characterization of suppressors of a mutant with spontaneous PCD, cpr5, we found that the CKI-RB-E2F cell cycle signaling pathway plays a key role in conferring PCD in plants during ETI mediated by both TIR-NB-LRR and CC-NB-LRR, two major classes of immune receptors in plants. Upon NB-LRR activation, CKIs are specifically released from association with the nuclear membrane protein CPR5, triggering E2F-mediated defense gene expression through hyperphosphorylation of RB. Because CKIs are normally found to inhibit the E2F TF activity during cell cycle, the CKI-RB-E2F signaling cascade discovered for ETI is a noncanonical pathway.

Currently, we are characterizing the CPR5 protein and searching for the signal that connects NB-LRR to CPR5 on the nuclear membrane. We are also looking for the downstream kinase that hyperphosphorylates RB upon ETI induction, and for the partner TF(s) of E2F in ETI-mediated gene expression.

The Interplay between the Circadian Clock and Plant Immune Responses
In a functional genomic study, my lab found that genes conferring ETI against the obligate biotrophic pathogen Hyaloperonospora are driven by the circadian clock and expressed in the morning. We showed experimentally for the first time that this regulation by the circadian clock allows anticipation of infection at a time of the day (i.e., morning) when conditions (e.g., humidity, temperature, and pathogen spore dissemination) are the most favorable for many pathogens. In a collaborative study with Dr. Steve Kay’s lab, we discovered that besides ETI, the expression of the immune signal SA is regulated by the clock component, CHE, using a yeast one-hybrid screen. CHE is one of the transcriptional activators of the SA synthesis gene isochorismate synthase (ICS1). In the che mutant, the daily oscillation in SA level, which peaks before dawn, is significantly compromised. Moreover, the che mutant is also defective in SAR due to a failure to induce expression of ICS1 in systemic tissue. In a subsequent investigation of whether the plant immune response in turn affects the circadian clock, we were once again surprised to find that perturbation of the redox rhythm by SA treatment does not change the clock but rather reinforces it. Mathematical modeling and experiments showed that this is accomplished by NPR1-mediated induction of both an evening clock gene and two morning clock genes. This balanced network architecture ensures better time keeping when cellular redox rhythm is disturbed by pathogens, to minimize effects on fitness.

The interplay between cellular redox rhythm and the circadian clock are also being actively studied in animals due to its significance to human health and medicine. With the substantial collection of genetic materials generated through our previous studies of redox regulation of plant responses and the circadian clock, we are in a unique position to elucidate the complex relationship between these oscillatory signals and their influences on immunity.

Translational Regulation during Plant Immune Responses
In our study of TBF1, a TF important for the switch between growth and defense in plants, we found that its translation is tightly controlled by two upstream open reading frames (uORFs). Because translation of eukaryotic mRNAs begins at the AUG closest to the 5’ end, translation of these uORFs would cause dissociation of the ribosome from the mRNA before it reaches the downstream main open reading frame (mORF), TBF1. However, upon immune induction by the MAMP signal, elf18, or by the bacterial pathogen Pseudomonas syringae pv. maculicola ES4326 carrying the effector gene AvrRpt2 (Psm ES4326/AvrRpt2), the inhibitory effects of uORF1 and uORF2 are rapidly and transiently alleviated, leading to TBF1 protein production. Recently reported ribosome footprint-sequencing (Ribo-Seq) results showed that uORFs are much more widely present in both mammalian (>40%) and plant (>22% according to ours and others’ data) mRNAs.

Currently, there are only a limited number of mechanistic studies reported about uORF-mediated translation regulation. In yeast, the translation of GCN4 downstream of 4 uORFs is induced by amino acid starvation through accumulation of uncharged tRNA which activates GCN2 kinase to phosphorylate and inactivate the translation initiation factor eIF2a. Our current data show that immune-related translation induction of TBF1 is through a novel mechanism. We have initiated a major effort of studying this translational regulatory mechanism using both genetic and genomic approaches (e.g., Ribo-Seq and RNA-Seq). For agricultural application, we are developing new tools to regulate both transcription and translation of defense genes in crops to minimize fitness costs commonly associated with enhanced immunity.

This work is partially supported by grants from the National Institutes of Health and the National Science Foundation.