Allosteric Modulation of the YAP/TAZ-TEAD Interaction by Palmitoylation and Small-Molecule Inhibitors.

The Hippo signaling pathway is a highly conserved signaling network that plays a central role in regulating cellular growth, proliferation, and organ size. This pathway consists of a kinase cascade that integrates various upstream signals to control the activation or inactivation of YAP/TAZ proteins. Phosphorylated YAP/TAZ is sequestered in the cytoplasm; however, when the Hippo pathway is deactivated, it translocates into the nucleus, where it associates with TEAD transcription factors. This partnership is instrumental in regulating the transcription of progrowth and antiapoptotic genes. Thus, in many cancers, aberrantly hyperactivated YAP/TAZ promotes oncogenesis by contributing to cancer cell proliferation, metastasis, and therapy resistance. Because YAP and TAZ exert their oncogenic effects by binding with TEAD, it is critical to understand this key interaction to develop cancer therapeutics. Previous research has indicated that TEAD undergoes autopalmitoylation at a conserved cysteine, and small molecules that inhibit TEAD palmitoylation disrupt effective YAP/TAZ binding. However, how exactly palmitoylation contributes to YAP/TAZ-TEAD interactions and how the TEAD palmitoylation inhibitors disrupt this interaction remains unknown. Utilizing molecular dynamics simulations, our investigation not only provides detailed atomistic insight into the YAP/TAZ-TEAD dynamics but also unveils that the inhibitor studied influences the binding of YAP and TAZ to TEAD in distinct manners. This discovery has significant implications for the design and deployment of future molecular interventions targeting this interaction.

Structural Insights into the Interaction between the C-Terminal-Deleted BH3-like Motif Peptide of Hepatitis B Virus X Protein and Bcl-xL.

Hepatitis B virus X protein (HBx) plays a crucial role in the development of hepatocellular carcinoma (HCC) associated with hepatitis B virus (HBV) infection. The full-length HBx protein interacts with Bcl-xL and is involved in the HBV replication and cell death processes. The three hydrophobic residues Trp120, Leu123, and Ile127 of the HBx BH3-like motif are essential for the Bcl-xL-binding. On the other hand, various lengths of C-terminal-truncated HBx mutants are frequently detected in HCC tissues, and these mutants, rather than the full-length HBx, appear to be responsible for HCC development. Notably, the region spanning residues 1-120 of HBx [HBx(1 and 120)] has been strongly associated with an increased risk of HCC development. However, the mode of interaction between HBx(1-120) and Bcl-xL remains unclear. HBx(1-120) possesses only Trp120 among the three hydrophobic residues essential for the Bcl-xL-binding. To elucidate this interaction mode, we employed a C-terminal-deleted HBx BH3-like motif peptide composed of residues 101-120. Here, we present the NMR complex structure of Bcl-xL and HBx(101-120). Our results demonstrate that HBx(101-120) binds to Bcl-xL in a weaker manner. Considering the high expression of Bcl-xL in HCC cells, this weak interaction, in conjunction with the overexpression of Bcl-xL in HCC cells, may potentially contribute to HCC development through the interaction between C-terminal-truncated HBx and Bcl-xL.

Identification of potential natural products derived from fungus growing termite, inhibiting Pseudomonas aeruginosa quorum sensing protein LasR using molecular docking and molecular dynamics simulation approach.

Pseudomonas aeruginosa, the most common opportunistic pathogen, is becoming antibiotic-resistant worldwide. The fate of P. aeruginosa, a multidrug-resistant strain, can be determined by multidrug efflux pumps, enzyme synthesis, outer membrane protein depletion, and target alterations. Microbial niches have long used quorum sensing (QS) to synchronize virulence gene expression. Computational methods can aid in the development of novel P. aeruginosa drug-resistant treatments. The tripartite symbiosis in termites that grow fungus may help special microbes find new antimicrobial drugs. To find anti-quorum sensing natural products that could be used as alternative therapies, a library of 376 fungal-growing termite-associated natural products (NPs) was screened for their physicochemical properties, pharmacokinetics, and drug-likeness. Using GOLD, the top 74 NPs were docked to the QS transcriptional regulator LasR protein. The five lead NPs with the highest gold score and drug-like properties were chosen for a 200-ns molecular dynamics simulation to test the competitive activity of different compounds against negative catechin. Fridamycin and Daidzein had stable conformations, with mean RMSDs of 2.48 and 3.67 Å, respectively, which were similar to Catechin's 3.22 Å. Fridamycin and Daidzein had absolute binding energies of -71.186 and -52.013 kcal/mol, respectively, which were higher than the control's -42.75 kcal/mol. All the compounds within the active site of the LasR protein were kept intact by Trp54, Arg55, Asp67, and Ser123. These findings indicate that termite gut and fungus-associated NPs, specifically Fridamycin and Daidzein, are potent QS antagonists that can be used to treat P. aeruginosa's multidrug resistance.Communicated by Ramaswamy H. Sarma.

Line Shape Analysis of 19F NMR-Monitored Chemical Denaturation of a Fold-Switching Protein RfaH Reveals Its Slow Folding Dynamics.

The recent discovery of metamorphic proteins, which can switch between multiple conformations under native conditions, has challenged the well-established one sequence-one structure paradigm of protein folding. This is exemplified in the C-terminal domain of the multidomain transcription factor RfaH, which converts from an α-helical coiled-coil conformation in its autoinhibited state to a ß-barrel conformation upon activation. Here, we use multisite line shape analysis of 19F NMR-monitored equilibrium chemical denaturation measurements of two 19F-labeled variants of full-length RfaH, to show that it folds/unfolds slowly on the NMR time scale, in an apparent all-or-none fashion at physiological pH and room temperature in the 3.3-4.8 M urea concentration range. The significant thermodynamic stability and slow unfolding rate (kinetic stability) are likely responsible for maintaining the closed autoinhibited state of RfaH, preventing spurious interactions with RNA polymerase (RNAP) when not functional. Our results provide a quantitative understanding of the folding-function relationship in the model fold-switching protein RfaH.

The intrinsically disordered transcriptional activation domain of CIITA is functionally tuneable by single substitutions: An exception or a new paradigm?

During protein evolution, some amino acid substitutions modulate protein function ("tuneability"). In most proteins, the tuneable range is wide and can be sampled by a set of protein variants that each contains multiple amino acid substitutions. In other proteins, the full tuneable range can be accessed by a set of variants that each contains a single substitution. Indeed, in some globular proteins, the full tuneable range can be accessed by the set of site-saturating substitutions at an individual "rheostat" position. However, in proteins with intrinsically disordered regions (IDRs), most functional studies-which would also detect tuneability-used multiple substitutions or small deletions. In disordered transcriptional activation domains (ADs), studies with multiple substitutions led to the "acidic exposure" model, which does not anticipate the existence of rheostat positions. In the few studies that did assess effects of single substitutions on AD function, results were mixed: the ADs of two full-length transcription factors did not show tuneability, whereas a fragment of a third AD was tuneable by single substitutions. In this study, we tested tuneability in the AD of full-length human class II transactivator (CIITA). Sequence analyses and experiments showed that CIITA's AD is an IDR. Functional assays of singly-substituted AD variants showed that CIITA's function was highly tuneable, with outcomes not predicted by the acidic exposure model. Four tested positions showed rheostat behavior for transcriptional activation. Thus, tuneability of different IDRs can vary widely. Future studies are needed to illuminate the biophysical features that govern whether an IDR is tuneable by single substitutions.

Electron microscopy mapping of the DNA-binding sites of monomeric, dimeric, and multimeric KSHV RTA protein.

IMPORTANCE: Kaposi's sarcoma-associated herpesvirus (KSHV) is a human herpesvirus associated with several human cancers, typically in patients with compromised immune systems. Herpesviruses establish lifelong infections in hosts in part due to the two phases of infection: the dormant and active phases. Effective antiviral treatments to prevent the production of new viruses are needed to treat KSHV. A detailed microscopy-based investigation of the molecular interactions between viral protein and viral DNA revealed how protein-protein interactions play a role in DNA-binding specificity. This analysis will lead to a more in-depth understanding of KSHV DNA replication and serve as the basis for anti-viral therapies that disrupt and prevent the protein-DNA interactions, thereby decreasing spread to new hosts.

Synergistic Binding of ATP and Nucleic Acids Necessitates UPF1's ATPase Functional Cycle.

UPF1 is a core protein in the nonsense mRNA degradation (NMD) surveillance pathway that degrades aberrant mRNA. UPF1 has both ATPase and RNA helicase activities, but it exhibits mutually exclusive binding of ATP and RNA. This suggests intricate allosteric coupling between ATP and RNA binding that remains unresolved. In this study, we used molecular dynamics simulations and dynamic network analyses to probe the dynamics and free energy landscapes covering UPF1 crystal structures resolved in the Apo state, the ATP bound state, and the ATP-RNA bound (catalytic transition) state. Free energy calculations show that in the presence of ATP and RNA, the transition from the Apo state to the ATP bound state is an uphill process but becomes a downhill process when transitioning to the catalytic transition state. Allostery potential analyses reveal that the Apo and catalytic transition states are mutually allosterically activated toward each other, reflecting the intrinsic ATPase function of UPF1. The Apo state is also allosterically activated toward the ATP bound state. However, binding ATP alone leads to an allosterically trapped state that is difficult to revert to either the Apo or the catalytic transition state. The high allostery potential of Apo UPF1 toward different states results in a "first come, first served" mechanism that requires the synergistic binding of ATP and RNA to drive the ATPase cycle. Our results reconcile UPF1's ATPase and RNA helicase activities within an allostery framework and may apply to other SF1 helicases, as we demonstrate that UPF1's allostery signaling pathways prefer the RecA1 domain over the equally fold-conserved RecA2 domain, and this preference coincides with higher sequence conservation in the RecA1 domain across typical human SF1 helicases.

Biochemical analysis of protein-protein interfaces underlying the regulation of bacterial secretion systems.

Bacterial pathogens such as Pseudomonas aeruginosa use complex regulatory networks to tailor gene expression patterns to meet complex environmental challenges. P. aeruginosa is capable of causing both acute and chronic persistent infections, each type being characterized by distinct symptoms brought about by distinct sets of virulence mechanisms. The GacS/GacA phosphorelay system sits at the heart of a complex regulatory network that reciprocally governs the expression of virulence factors associated with either acute or chronic infections. A second non-enzymatic signaling cascade involving four proteins, ExsA, ExsC, ExsD, and ExsE is a key player in regulating the expression of the type three secretion system, an essential facilitator of acute infections. Both signaling pathways involve a remarkable array of non-canonical interactions that we sought to characterize. In the following section, we will outline several strategies, we adapted to map protein-protein interfaces and quantify the strength of biomolecular interactions by pairing complex mutational analyses with FRET binding assays and Bacterial-Two-Hybrid assays with appropriate functional assays. In the process, protocols were developed for disrupting large hydrophobic interfaces, deleting entire domains within a protein, and for mapping protein-protein interfaces formed primarily through backbone interactions.

Computational investigations on the potential role of hygrophorones as quorum sensing inhibitors against LasR protein of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a gram negative, rod shape bacterium that infects people with compromised immune systems, such as those suffering from AIDS, organ transplantation and cancer. This bacterium is responsible for diseases like cystic fibrosis, chronic lung infection, and ulcerative keratitis. It is diagnosed in most of the patients who were on prolonged ventilation with long term critical care stay. P. aeruginosa develops rapid antimicrobial resistance that is challenging for the treatment and eventually it causes high mortality rate. Thus, the search for potential novel inhibitors that can inhibit the pathogenic activity of P. aeruginosa is of utmost importance. In P. aeruginosa, an important protein, LasR that participates in the gene regulations and expressions has been proposed to be a suitable drug target. Here, we identify a set of hygrophorone molecules as effective inhibitors for this LasR protein based on molecular docking and simulations studies. At first, large number of hygrophorone series of small molecules were screened against the LasR protein and their binding affinities were assessed based on the docking scores. Top scored molecules were selected for calculating various pharmacophore properties, and finally, their potential in inhibiting the LasR protein was delineated by atomistic molecular dynamics simulations and molecular mechanics Poisson-Boltzmann surface area-based calculations. Both docking and simulations studies reveal that a subset of hygrophorone molecules have a good binding affinity for LasR protein and form stable LasR-inhibitor complexes. The present study illustrates that the hygrophorones can be effective inhibitors for the LasR protein and will spur further in vitro studies that would aid to the ongoing search for new antibiotics.Communicated by Ramaswamy H. Sarma.

In silico study reveals unconventional interactions between MDC1 of DDR and Beclin-1 of autophagy.

DNA damage response (DDR) and autophagy are concerned with maintaining cellular homeostasis and dysregulation of these two pathways lead to pathologic conditions including tumorigenesis. Autophagy is activated as a protective mechanism during DDR which is indicative of their functional cooperativity but the molecular mechanism leading to the convergence of these two pathways during genotoxic stress remains elusive. In this study, through in silico analysis, we have shown an interaction between the Mediator of DNA damage checkpoint 1 (MDC1), an important DDR-associated protein, and Beclin-1, an autophagy inducer. MDC1 is an adaptor or scaffold protein known to regulate DDR, apoptosis, and cell cycle progression. While, Beclin-1 is involved in autophagosome nucleation and exhibits affinity for binding to Fork-head-associated domain (FHA) containing proteins. The FHA domain is commonly conserved in DDR-related proteins including MDC1. Through molecular docking, we have predicted the modeled complex between the MDC1 FHA domain and the Beclin-1 Coiled coil domain (CCD). The docking complex was modeled using ClusPro2.0, based on the crystal structure for the dimerized MDC1 FHA domain and Beclin-1 CCD. The complex stability and binding affinities were assessed using a Ramachandran plot, MD simulation, MM/GBSA, and PRODIGY webserver. Finally, the hot-spot residues at the interface were determined using computational alanine scanning by the DrugScorePPI webserver. Our analysis unveils significant interaction between MDC1 and Beclin-1, involving hydrogen bonds, non-bonded contacts, and salt bridges and indicates MDC1 possibly recruits Beclin-1 to the DSBs, as a consequence of which Beclin-1 is able to modulate DDR.

Self-Effected Allosteric Coupling and Cooperativity in Hypoxic Response Regulation with Disordered Proteins.

Hypersensitive regulation of cellular hypoxic response relies on cooperative displacement of one disordered protein (HIF-1α) by another disordered protein (CITED2) from the target in a negative feedback loop. Considering the weak intramolecule coupling in disordered proteins, the molecular mechanism of high cooperativity in the molecular displacement event remains elusive. Herein, we show that disordered proteins utilize a "self-effected allostery" mechanism to achieve high binding cooperativity. Different from the conventional allostery mechanisms shown by many structured or disordered proteins, this mechanism utilizes one part of the disordered protein as the effector to trigger the allosteric coupling and enhance the binding of the remaining part of the same disordered protein, contributing to high cooperativity of the displacement event. The conserved charge motif of CITED2 is the key determinant of the molecular displacement event by serving as the effector of allosteric coupling. Such self-effected allostery provides an efficient strategy to achieve high cooperativity in the molecular events involving disordered proteins.

Phenol sensing in nature is modulated via a conformational switch governed by dynamic allostery.

The NtrC family of proteins senses external stimuli and accordingly stimulates stress and virulence pathways via activation of associated σ54-dependent RNA polymerases. However, the structural determinants that mediate this activation are not well understood. Here, we establish using computational, structural, biochemical, and biophysical studies that MopR, an NtrC protein, harbors a dynamic bidirectional electrostatic network that connects the phenol pocket to two distal regions, namely the "G-hinge" and the "allosteric linker." While the G-hinge influences the entry of phenol into the pocket, the allosteric linker passes the signal to the downstream ATPase domain. We show that phenol binding induces a rewiring of the electrostatic connections by eliciting dynamic allostery and demonstrates that perturbation of the core relay residues results in a complete loss of ATPase stimulation. Furthermore, we found a mutation of the G-hinge, ∼20 Å from the phenol pocket, promotes altered flexibility by shifting the pattern of conformational states accessed, leading to a protein with 7-fold enhanced phenol binding ability and enhanced transcriptional activation. Finally, we conducted a global analysis that illustrates that dynamic allostery-driven conserved community networks are universal and evolutionarily conserved across species. Taken together, these results provide insights into the mechanisms of dynamic allostery-mediated conformational changes in NtrC sensor proteins.

Cyclin-dependent kinase-mediated phosphorylation and the negative regulatory domain of transcription factor B-Myb modulate its DNA binding.

B-Myb is a highly conserved member of the vertebrate Myb family of transcription factors that plays a critical role in cell-cycle progression and proliferation. Myb proteins activate Myb-dependent promoters by interacting specifically with Myb-binding site (MBS) sequences using their DNA-binding domain (DBD). Transactivation of MBS promoters by B-Myb is repressed by its negative regulatory domain (NRD), and phosphorylation of the NRD by Cdk2-CyclinA relieves the repression to activate B-Myb-dependent promoters. However, the structural mechanisms underlying autoinhibition and activation of B-Myb-mediated transcription have been poorly characterized. Here, we determined that a region in the B-Myb NRD (residues 510-600) directly associates with the DBD and inhibits binding of the DBD to the MBS DNA sequence. We demonstrate using biophysical assays that phosphorylation of the NRD at T515, T518, and T520 is sufficient to disrupt the interaction between the NRD and the DBD, which results in increased affinity for MBS DNA and increased B-Myb-dependent promoter activation in cell assays. Our biochemical characterization of B-Myb autoregulation and the activating effects of phosphorylation provide insight into how B-Myb functions as a site-specific transcription factor.

Respiratory Syncytial Virus NS1 Protein Targets the Transactivator Binding Domain of MED25.

Human RSV is the leading cause of infantile bronchiolitis in the world and one of the major causes of childhood deaths in resource-poor settings. It is a major unmet target for vaccines and anti-viral drugs. Respiratory syncytial virus has evolved a unique strategy to evade host immune response by coding for two non-structural proteins NS1 and NS2. Recently it was shown that in infected cells, nuclear NS1 could be involved in transcription regulation of host genes linked to innate immune response, via interactions with chromatin and the Mediator complex. Here we identified the MED25 Mediator subunit as an NS1 interactor in a yeast two-hybrid screen. We demonstrate that NS1 directly interacts with MED25 in vitro and in cellula, and that this interaction involves the MED25 transactivator binding ACID domain on the one hand, and the C-terminal α3 helix of NS1, with an additional contribution of the globular domain of NS1, on the other hand. By NMR we show that the NS1 α3 sequence primarily binds to the MED25 ACID H2 face, similarly to the α-helical transactivation domains (TADs) of transcription regulators such as Herpex simplex VP16 and ATF6α, a master regulator of ER stress response activated upon viral infection. Moreover, we found out that the NS1 could compete with ATF6α TAD for binding to MED25. These findings point to a mechanism of NS1 interfering with innate immune response by impairing recruitment by cellular TADs of the Mediator via MED25 and hence transcription of specific genes by RNA polymerase II.

Coevolution-derived native and non-native contacts determine the emergence of a novel fold in a universally conserved family of transcription factors.

The NusG protein family is structurally and functionally conserved in all domains of life. Its members directly bind RNA polymerases and regulate transcription processivity and termination. RfaH, a divergent sub-family in its evolutionary history, is known for displaying distinct features than those in NusG proteins, which allows them to regulate the expression of virulence factors in enterobacteria in a DNA sequence-dependent manner. A striking feature is its structural interconversion between an active fold, which is the canonical NusG three-dimensional structure, and an autoinhibited fold, which is distinctively novel. How this novel fold is encoded within RfaH sequence to encode a metamorphic protein remains elusive. In this work, we used publicly available genomic RfaH protein sequences to construct a complete multiple sequence alignment, which was further augmented with metagenomic sequences and curated by predicting their secondary structure propensities using JPred. Coevolving pairs of residues were calculated from these sequences using plmDCA and GREMLIN, which allowed us to detect the enrichment of key metamorphic contacts after sequence filtering. Finally, we combined our coevolutionary predictions with molecular dynamics to demonstrate that these interactions are sufficient to predict the structures of both native folds, where coevolutionary-derived non-native contacts may play a key role in achieving the compact RfaH novel fold. All in all, emergent coevolutionary signals found within RfaH sequences encode the autoinhibited and active folds of this protein, shedding light on the key interactions responsible for the action of this metamorphic protein.

Dynamics of the protein search for targets on DNA in quorum-sensing cells.

Quorum sensing is a bacterial cell-cell communication process that regulates gene expression. The search and binding of the autoinducer molecule (AHL)-bound LuxR-type proteins to specific sites on DNA in quorum-sensing cells in Gram-negative bacteria is a complex process and has been theoretically investigated based on a discrete-state stochastic approach. It is shown that several factors such as the rate of formation of the AHL-bound LuxR protein within the cells and its dissociation to freely diffusing AHL, the diffusion of the latter in and out of the cells, positive feedback loops, and the cell population density play important roles in the protein target search and can control the gene regulation processes. Physical-chemical arguments to explain these observations are presented. Our calculations of the dynamic properties are also supplemented by Monte Carlo computer simulations. Our theoretical model provides physical insights into the complex mechanisms of protein target search in quorum-sensing cells.

Global Fold Switching of the RafH Protein: Diverse Structures with a Conserved Pathway.

It is generally believed that a protein's sequence uniquely determines its structure, the basis for a protein to perform biological functions. However, as a representative metamorphic protein, RfaH can be encoded by a single amino acid sequence into two distinct native state structures. Its C-terminal domain (CTD) either takes an all-α-helical configuration to pack tightly with its N-terminal domain (NTD), or the CTD disassociates from the NTD, transforms into an all-ß-barrel fold, and further attaches to the ribosome, leaving the NTD exposed to bind RNA polymerases. Therefore, the RfaH protein couples transcription and translation processes. Although previous studies have provided a preliminary understanding of its function, the full course of the conformational change of RfaH-CTD at the atomic level is elusive. We used teDA2, a feature space-based enhanced sampling protocol, to explore the transformation of RfaH-CTD. We found that it undergoes a large-scale structural rearrangement, with characteristic spectra as the fingerprint, and a global unfolding transition with a tighter and energetically moderate molten globule-like nucleus formed in between. The formation of this nucleus limits the possible intermediate conformations, facilitates the formation of secondary and tertiary structures, and thus ensures the efficiency of transformation. The key features along the transition path disclosed from this work are likely associated with the evolution of RfaH, such that encoding a single sequence into multiple folds with distinct biological functions is energetically unhindered.

OFF-switching property of quorum sensor LuxR via As(III)-induced insoluble form.

Whole-cell sensors for arsenite detection have been developed exclusively based on the natural arsenite (As(III)) sensory protein ArsR for arsenic metabolism. This study reports that the quorum-sensing LuxR/Plux system from Vibrio fischeri, which is completely unrelated to arsenic metabolism, responds to As(III) in a dose-dependent manner. Due to as many as 9 cysteine residues, which has a high binding affinity with As(III), LuxR underwent As(III)-induced insoluble form, thereby reducing its effective cellular concentration. Accordingly, the expression level of green fluorescent protein under the control of Plux gradually decreased with increasing As(III) concentration in the medium. This is a novel As(III)-detection system that has never been proposed before, with a unique ON-to-OFF transfer function.

Non-TZF Transcriptional Activator AtC3H12 Negatively Affects Seed Germination and Seedling Development in Arabidopsis.

CCCH zinc finger proteins are a large protein family and are classified as either tandem CCCH zinc finger (TZF) or non-TZF proteins. The roles of TZF genes in several plants have been well determined, whereas the functions of many non-TZF genes in plants remain uncharacterized. Herein, we describe biological and molecular functions of AtC3H12, an Arabidopsis non-TZF protein containing three CCCH zinc finger motifs. AtC3H12 has orthologs in several plant species but has no paralog in Arabidopsis. AtC3H12-overexpressing transgenic plants (OXs) germinated slower than wild-type (WT) plants, whereas atc3h12 mutants germinated faster than WT plants. The fresh weight (FW) and primary root lengths of AtC3H12 OX seedlings were lighter and shorter than those of WT seedlings, respectively. In contrast, FW and primary root lengths of atc3h12 seedlings were heavier and longer than those of WT seedlings, respectively. AtC3H12 was localized in the nucleus and displayed transactivation activity in both yeast and Arabidopsis. We found that the 97-197 aa region of AtC3H12 is an important part for its transactivation activity. Detection of expression levels and analysis of Arabidopsis transgenic plants harboring a PAtC3H12::GUS construct showed that AtC3H12 expression increases as the Arabidopsis seedlings develop. Taken together, our results demonstrate that AtC3H12 negatively affects seed germination and seedling development as a nuclear transcriptional activator in Arabidopsis. To our knowledge, this is the first report to show that non-TZF proteins negatively affect plant development as nuclear transcriptional activators.

A NAC Transcription Factor TuNAC69 Contributes to ANK-NLR-WRKY NLR-Mediated Stripe Rust Resistance in the Diploid Wheat Triticum urartu.

Stripe rust is one of the most devastating diseases in wheat. Nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domain receptors (NLRs) recognize pathogenic effectors and trigger plant immunity. We previously identified a unique NLR protein YrU1 in the diploid wheat Triticum urartu, which contains an N-terminal ANK domain and a C-terminal WRKY domain and confers disease resistance to stripe rust fungus Puccinia striiformis f. sp. Tritici (Pst). However, how YrU1 functions in disease resistance is not clear. In this study, through the RNA-seq analysis, we found that the expression of a NAC member TuNAC69 was significantly up-regulated after inoculation with Pst in the presence of YrU1. TuNAC69 was mainly localized in the nucleus and showed transcriptional activation in yeast. Knockdown TuNAC69 in diploid wheat Triticum urartu PI428309 that contains YrU1 by virus-induced gene silencing reduced the resistance to stripe rust. In addition, overexpression of TuNAC69 in Arabidopsis enhanced the resistance to powdery mildew Golovinomyces cichoracearum. In summary, our study indicates that TuNAC69 participates in the immune response mediated by NLR protein YrU1, and likely plays an important role in disease resistance to other pathogens.