Oligomerization-mediated autoinhibition and cofactor binding of a plant NLR.

Nucleotide-binding leucine-rich repeat (NLR) proteins have a pivotal role in plant immunity by recognizing pathogen effectors1,2. Maintaining a balanced immune response is crucial, as excessive NLR expression can lead to unintended autoimmunity3,4. Unlike most NLRs, plant NLR required for cell death 2 (NRC2) belongs to a small NLR group characterized by constitutively high expression without self-activation5. The mechanisms underlying NRC2 autoinhibition and activation are not yet understood. Here we show that Solanum lycopersicum (tomato) NRC2 (SlNRC2) forms dimers and tetramers, and higher-order oligomers at elevated concentrations. Cryo-electron microscopy (cryo-EM) reveals an inactive conformation of SlNRC2 within these oligomers. Dimerization and oligomerization not only stabilize the inactive state but also sequester SlNRC2 from assembling into an active form. Mutations at the dimeric or inter-dimeric interfaces enhance pathogen-induced cell death and immunity in Nicotiana (N.) benthamiana. The cryo-EM structures unexpectedly reveal inositol hexakisphosphate (IP6) or pentakisphosphate (IP5) bound to the inner surface of SlNRC2's C-terminal LRR domain as confirmed by mass spectrometry. Mutations at the IP-binding site impair inositol phosphate binding of SlNRC2 and pathogen-induced SlNRC2-mediated cell death in N. benthamiana. Together, our study unveils a novel negative regulatory mechanism of NLR activation and suggests inositol phosphates as cofactors of NRCs.

NLR signaling in plants: from resistosomes to second messengers.

Nucleotide binding and leucine-rich repeat-containing receptors (NLRs) have a critical role in plant immunity through direct or indirect recognition of pathogen effectors. Recent studies have demonstrated that such recognition induces formation of large protein complexes called resistosomes to mediate NLR immune signaling. Some NLR resistosomes activate Ca2+ influx by acting as Ca2+-permeable channels, whereas others function as active NADases to catalyze the production of nucleotide-derived second messengers. In this review we summarize these studies on pathogen effector-induced assembly of NLR resistosomes and resistosome-mediated production of the second messengers of Ca2+ and nucleotide derivatives. We also discuss downstream events and regulation of resistosome signaling.

Transcriptomics reveals a core transcriptional network of K-type cytoplasmic male sterility microspore abortion in wheat (Triticum aestivum L.).

BACKGROUND: Cytoplasmic male sterility (CMS) plays a crucial role in hybrid production. K-type CMS, a cytoplasmic male sterile line of wheat with the cytoplasms of Aegilops kotschyi, is widely used due to its excellent characteristics of agronomic performance, easy maintenance and easy restoration. However, the mechanism of its pollen abortion is not yet clear. RESULTS: In this study, wheat K-type CMS MS(KOTS)-90-110 (MS line) and it's fertile near-isogenic line MR (KOTS)-90-110 (MR line) were investigated. Cytological analysis indicated that the anthers of MS line microspore nucleus failed to divide normally into two sperm nucleus and lacked starch in mature pollen grains, and the key abortive period was the uninucleate stage to dinuclear stage. Then, we compared the transcriptome of MS line and MR line anthers at these two stages. 11,360 and 5182 differentially expressed genes (DEGs) were identified between the MS and MR lines in the early uninucleate and binucleate stages, respectively. Based on GO enrichment and KEGG pathways analysis, it was evident that significant transcriptomic differences were "plant hormone signal transduction", "MAPK signaling pathway" and "spliceosome". We identified 17 and 10 DEGs associated with the IAA and ABA signal transduction pathways, respectively. DEGs related to IAA signal transduction pathway were downregulated in the early uninucleate stage of MS line. The expression level of DEGs related to ABA pathway was significantly upregulated in MS line at the binucleate stage compared to MR line. The determination of plant hormone content and qRT-PCR further confirmed that hormone imbalance in MS lines. Meanwhile, 1 and 2 DEGs involved in ABA and Ethylene metabolism were also identified in the MAPK cascade pathway, respectively; the significant up regulation of spliceosome related genes in MS line may be another important factor leading to pollen abortion. CONCLUSIONS: We proposed a transcriptome-mediated pollen abortion network for K-type CMS in wheat. The main idea is hormone imbalance may be the primary factor, MAPK cascade pathway and alternative splicing (AS) may also play important regulatory roles in this process. These findings provided intriguing insights for the molecular mechanism of microspore abortion in K-type CMS, and also give useful clues to identify the crucial genes of CMS in wheat.

Low-Temperature-Mediated Promoter Methylation Relates to the Expression of TaPOR2D, Affecting the Level of Chlorophyll Accumulation in Albino Wheat (Triticum aestivum L.).

Chlorophyll is an indispensable photoreceptor in plant photosynthesis. Its anabolic imbalance is detrimental to individual growth and development. As an essential epigenetic modification, DNA methylation can induce phenotypic variations, such as leaf color transformation, by regulating gene expression. Albino line XN1376B is a natural mutation of winter wheat cultivar XN1376; however, the regulatory mechanism of its albinism is still unclear. In this study, we found that low temperatures induced albinism in XN1376B. The number of chloroplasts decreased as the phenomenon of bleaching intensified and the fence tissue and sponge tissue slowly dissolved. We identified six distinct TaPOR (protochlorophyllide oxidoreductase) genes in the wheat genome, and TaPOR2D was deemed to be related to the phenomenon of albinism based on the expression in different color leaves (green leaves, white leaves and returned green leaves) and the analysis of promoters' cis-acting elements. TaPOR2D was localized to chloroplasts. TaPOR2D overexpression (TaPOR2D-OE) enhanced the chlorophyll significantly in Arabidopsis, especially at two weeks; the amount of chlorophyll was 6.46 mg/L higher than in WT. The methylation rate of the TaPOR2D promoter in low-temperature albino leaves is as high as 93%, whereas there was no methylation in green leaves. Correspondingly, three DNA methyltransferase genes (TaMET1, TaDRM and TaCMT) were up-regulated in white leaves. Our study clarified that the expression of TaPOR2D is associated with its promoter methylation at a low temperature; it affects the level of chlorophyll accumulation, which probably causes the abnormal development of plant chloroplasts in albino wheat XN1376B. The results provide a theoretical basis for in-depth analysis of the regulation of development of plant chloroplasts and color variation in wheat XN1376B leaves.

A superior allele of the wheat gene TaGL3.3-5B, selected in the breeding process, contributes to seed size and weight.

KEY MESSAGE: A superior allele of wheat gene TaGL3.3-5B was identified and could be used in marker-assisted breeding in wheat. Identifying the main genes which mainly regulate the yield-associated traits can significantly increase the wheat production. In this study, gene TaGL3.3 was cloned from common wheat according to the sequence of OsPPKL3. A SNP in the 8th exon of TaGL3.3-5B, T/C in coding sequence (CDS), which resulted in an amino acid change (Val/Ala), was identified between the low 1000-kernel weight (TKW) wheat Chinese Spring and the high TKW wheat Xinong 817 (817). Subsequently, association analysis in the mini-core collection (MCC) and the recombinant inbred lines (RIL) revealed that the allele TaGL3.3-5B-C (from 817) was significantly correlated with higher TKW. The high frequency of TaGL3.3-5B-C in the Chinese modern wheat cultivars indicated that it was selected positively in wheat breeding programs. The overexpression of TaGL3.3-5B-C in Arabidopsis resulted in shorter pods and longer grains than those of wild-type counterparts. Additionally, TaGL3.3 expressed a tissue-specific pattern in wheat as revealed by qRT-PCR. We also found that 817 showed higher expression of TaGL3.3 than that in Chinese Spring (CS) during the seed development. These results demonstrate that TaGL3.3 plays an important role in the formation of seed size and weight. Allele TaGL3.3-5B-C is associated with larger and heavier grains that are beneficial to wheat yield improvement.

The Transcription Factors TaTDRL and TaMYB103 Synergistically Activate the Expression of TAA1a in Wheat, Which Positively Regulates the Development of Microspore in Arabidopsis.

Pollen fertility plays an important role in the application of heterosis in wheat (Triticum aestivum L.). However, the key genes and mechanisms underlying pollen abortion in K-type male sterility remain unclear. TAA1a is an essential gene for pollen development in wheat. Here, we explored the mechanism involved in its transcriptional regulation during pollen development, focusing on a 1315-bp promoter region. Several cis-acting elements were identified in the TAA1a promoter, including binding motifs for Arabidopsis thaliana AtAMS and AtMYB103 (CANNTG and CCAACC, respectively). Evolutionary analysis indicated that TaTDRL and TaMYB103 were the T. aestivum homologs of AtAMS and AtMYB103, respectively, and encoded nucleus-localized transcription factors containing 557 and 352 amino acids, respectively. TaTDRL and TaMYB103 were specifically expressed in wheat anthers, and their expression levels were highest in the early uninucleate stage; this expression pattern was consistent with that of TAA1a. Meanwhile, we found that TaTDRL and TaMYB03 directly interacted, as evidenced by yeast two-hybrid and bimolecular fluorescence complementation assays, while yeast one-hybrid and dual-luciferase assays revealed that both TaTDRL and TaMYB103 could bind the TAA1a promoter and synergistically increase its transcriptional activity. Furthermore, TaTDRL-EAR and TaMYB103-EAR transgenic Arabidopsis plants displayed abnormal microspore morphology, reduced pollen viability, and lowered seed setting rates. Additionally, the expression of AtMS2, a TAA1a homolog, was significantly lower in the two repressor lines than in the corresponding overexpression lines or WT plants. In summary, we identified a potential transcriptional regulatory mechanism associated with wheat pollen development.

Cyclin-Dependent Kinase Inhibitor Gene TaICK1 acts as a Potential Contributor to Wheat Male Sterility induced by a Chemical Hybridizing Agent.

Heterosis has been widely accepted as an effective strategy to increase yields in plant breeding. Notably, the chemical hybridization agent SQ-1 induces male sterility in wheat, representing a critical potential tool in hybrid seed production. However, the mechanisms underlying the male sterility induced by SQ-1 still remain poorly understood. In this study, a cyclin-dependent kinase inhibitor gene, TaICK1, which encodes a 229 amino acid protein, was identified as a potential contributor to male sterility in common wheat. The expression of TaICK1 was upregulated during the development of anthers in Xinong1376 wheat treated with SQ-1. Meanwhile, the seed setting rate was found to be significantly decreased in TaICK1 transgenic rice. Furthermore, we identified two cyclin proteins, TaCYCD2;1 and TaCYCD6;1, as interactors through yeast two-hybrid screening using TaICK1 as the bait, which were validated using bimolecular fluorescence complementation. Subcellular localization revealed that the proteins encoded by TaICK1, TaCYCD2;1, and TaCYCD6;1 were localized in the cell nucleus. The expression levels of TaCYCD2;1 and TaCYCD6;1 were lower in Xinong1376 treated with SQ-1. A further analysis demonstrated that the expression levels of OsCYCD2;1 and OsCYCD6;1 were lower in transgenic TaICK1 rice lines as well. Taken together, these results suggest that the upregulation of TaICK1, induced by SQ-1, may subsequently suppress the expression of TaCYCD2;1 and TaCYCD6;1 in anthers, resulting in male sterility. This study provides new insights into the understanding of SQ-1-induced wheat male sterility, as well as the developmental mechanisms of anthers.

Isolation and Identification of a TaTDR-Like Wheat Gene Encoding a bHLH Domain Protein, Which Negatively Regulates Chlorophyll Biosynthesis in Arabidopsis.

Chlorophyll biosynthesis plays a vital role in chloroplast development and photosynthesis in plants. In this study, we identified an orthologue of the rice gene TDR (Oryza sativa L., Tapetum Degeneration Retardation) in wheat (Triticum aestivum L.) called TaTDR-Like (TaTDRL) by sequence comparison. TaTDRL encodes a putative 557 amino acid protein with a basic helix-loop-helix (bHLH) conserved domain at the C-terminal (295-344 aa). The TaTDRL protein localised to the nucleus and displayed transcriptional activation activity in a yeast hybrid system. TaTDRL was expressed in the leaf tissue and expression was induced by dark treatment. Here, we revealed the potential function of TaTDRL gene in wheat by utilizing transgenic Arabidopsis plants TaTDRL overexpressing (TaTDRL-OE) and TaTDRL-EAR (EAR-motif, a repression domain of only 12 amino acids). Compared with wild-type plants (WT), both TaTDRL-OE and TaTDRL-EAR were characterized by a deficiency of chlorophyll. Moreover, the expression level of the chlorophyll-related gene AtPORC (NADPH:protochlorophyllide oxidoreductase C) in TaTDRL-OE and TaTDRL-EAR was lower than that of WT. We found that TaTDRL physically interacts with wheat Phytochrome Interacting Factor 1 (PIF1) and Arabadopsis PIF1, suggesting that TaTDRL regulates light signaling during dark or light treatment. In summary, TaTDRL may respond to dark or light treatment and negatively regulate chlorophyll biosynthesis by interacting with AtPIF1 in transgenic Arabidopsis.

Identification of the WUSCHEL-Related Homeobox (WOX) Gene Family, and Interaction and Functional Analysis of TaWOX9 and TaWUS in Wheat.

The WUSCHEL-related homeobox (WOX) is a family of plant-specific transcription factors, with important functions, such as regulating the dynamic balance of division and differentiation of plant stem cells and plant organ development. We identified 14 distinct TaWOX genes in the wheat (Triticum aestivum L.) genome, based on a genome-wide scan approach. All of the genes under evaluation had positional homoeologs on subgenomes A, B and D except TaWUS and TaWOX14. Both TaWOX14a and TaWOX14d had a paralogous copy on the same genome due to tandem duplication events. A phylogenetic analysis revealed that TaWOX genes could be divided into three groups. We performed functional characterization of TaWOX genes based on the evolutionary relationships among the WOX gene families of wheat, rice (Oryza sativa L.), and Arabidopsis. An overexpression analysis of TaWUS in Arabidopsis revealed that it affected the development of outer floral whorl organs. The overexpression analysis of TaWOX9 in Arabidopsis revealed that it promoted the root development. In addition, we identified some interaction between the TaWUS and TaWOX9 proteins by screening wheat cDNA expression libraries, which informed directions for further research to determine the functions of TaWUS and TaWOX9. This study represents the first comprehensive data on members of the WOX gene family in wheat.

Comparative proteomic analysis of multi-ovary wheat under heterogeneous cytoplasm suppression.

BACKGROUND: DUOII is a multi-ovary wheat (Triticum aestivum L.) line with two or three pistils and three stamens in each floret. The multi-ovary trait of DUOII is controlled by a dominant gene, whose expression can be suppressed by the heterogeneous cytoplasm of TeZhiI (TZI), a line with the nucleus of common wheat and the cytoplasm of Aegilops. Crosses between female DUOII plants and male TZI plants resulted in multi-ovary F1s; whereas, the reciprocal crosses resulted in mono-ovary F1s. Although the multi-ovary trait is inherited as single trait controlled by a dominant allele in lines with a Triticum cytoplasm, the mechanism by which the special heterogeneous cytoplasm suppresses the expression of multi-ovary is not well understood. RESULTS: Observing the developmental process, we found that the critical stage of additional pistil primordium development was when the young spikes were 2-6 mm long. Then, we compared the quantitative proteomic profiles of 2-6 mm long young spikes obtained from the reciprocal crosses between DUOII and TZI. A total of 90 differentially expressed proteins were identified and analyzed based on their biological functions. These proteins had obvious functional pathways mainly implicated in chloroplast metabolism, nuclear and cell division, plant respiration, protein metabolism, and flower development. Importantly, we identified two key proteins, Flowering Locus K Homology Domain and PEPPER, which are known to play an essential role in the specification of pistil organ identity. By drawing relationships between the 90 differentially expressed proteins, we found that these proteins revealed a complex network which is associated with multi-ovary gene expression under heterogeneous cytoplasmic suppression. CONCLUSIONS: Our proteomic analysis has identified certain differentially expressed proteins in 2-6 mm long young spikes, which was the critical stage of additional primordium development. This paper provided a universal proteomic profiling involved in the cytoplasmic suppression of wheat floral meristems; and our findings have laid a solid foundation for further mechanistic studies on the underlying mechanisms that control the heterogeneous cytoplasm-induced suppression of the nuclear multi-ovary gene in wheat.

Identification and Functional Analysis of the CLAVATA3/EMBRYO SURROUNDING REGION (CLE) Gene Family in Wheat.

CLAVATA3/EMBRYO SURROUNDING REGION (CLE) peptides are post-translationally cleaved and modified peptides from their corresponding pre-propeptides. Although they are only 12 to 13 amino acids in length, they are important ligands involved in regulating cell proliferation and differentiation in plant shoots, roots, vasculature, and other tissues. They function by interacting with their corresponding receptors. CLE peptides have been studied in many plants, but not in wheat. We identified 104 TaCLE genes in the wheat genome based on a genome-wide scan approach. Most of these genes have homologous copies distributed on sub-genomes A, B, and D. A few genes are derived from tandem duplication and segmental duplication events. Phylogenetic analysis revealed that TaCLE genes can be divided into five different groups. We obtained functional characterization of the peptides based on the evolutionary relationships among the CLE peptide families of wheat, rice, and Arabidopsis, and expression pattern analysis. Using chemically synthesized peptides (TaCLE3p and TaCLE34p), we found that TaCLE3 and TaCLE34 play important roles in regulating wheat and Arabidopsis root development, and wheat stem development. Overexpression analysis of TaCLE3 in Arabidopsis revealed that TaCLE3 not only affects the development of roots and stems, but also affects the development of leaves and fruits. These data represent the first comprehensive information on TaCLE family members.

Cytological and Proteomic Analysis of Wheat Pollen Abortion Induced by Chemical Hybridization Agent.

In plants, pollen grain transfers the haploid male genetic material from anther to stigma, both between flowers (cross-pollination) and within the same flower (self-pollination). In order to better understand chemical hybridizing agent (CHA) SQ-1-induced pollen abortion in wheat, comparative cytological and proteomic analyses were conducted. Results indicated that pollen grains underwent serious structural injury, including cell division abnormality, nutritional deficiencies, pollen wall defect and pollen grain malformations in the CHA-SQ-1-treated plants, resulting in pollen abortion and male sterility. A total of 61 proteins showed statistically significant differences in abundance, among which 18 proteins were highly abundant and 43 proteins were less abundant in CHA-SQ-1 treated plants. 60 proteins were successfully identified using MALDI-TOF/TOF mass spectrometry. These proteins were found to be involved in pollen maturation and showed a change in the abundance of a battery of proteins involved in multiple biological processes, including pollen development, carbohydrate and energy metabolism, stress response, protein metabolism. Interactions between these proteins were predicted using bioinformatics analysis. Gene ontology and pathway analyses revealed that the majority of the identified proteins were involved in carbohydrate and energy metabolism. Accordingly, a protein-protein interaction network involving in pollen abortion was proposed. These results provide information for the molecular events underlying CHA-SQ-1-induced pollen abortion and may serve as an additional guide for practical hybrid breeding.

Chemical hybridizing agent SQ-1-induced male sterility in Triticum aestivum L.: a comparative analysis of the anther proteome.

BACKGROUND: Heterosis is widely used to increase the yield of many crops. However, as wheat is a self-pollinating crop, hybrid breeding is not so successful in this organism. Even though male sterility induced by chemical hybridizing agents is an important aspect of crossbreeding, the mechanisms by which these agents induce male sterility in wheat is not well understood. RESULTS: We performed proteomic analyses using the wheat Triticum aestivum L.to identify those proteins involved in physiological male sterility (PHYMS) induced by the chemical hybridizing agent CHA SQ-1. A total of 103 differentially expressed proteins were found by 2D-PAGE and subsequently identified by MALDI-TOF/TOF MS/MS. In general, these proteins had obvious functional tendencies implicated in carbohydrate metabolism, oxidative stress and resistance, protein metabolism, photosynthesis, and cytoskeleton and cell structure. In combination with phenotypic, tissue section, and bioinformatics analyses, the identified differentially expressed proteins revealed a complex network behind the regulation of PHYMS and pollen development. Accordingly, we constructed a protein network of male sterility in wheat, drawing relationships between the 103 differentially expressed proteins and their annotated biological pathways. To further validate our proposed protein network, we determined relevant physiological values and performed real-time PCR assays. CONCLUSIONS: Our proteomics based approach has enabled us to identify certain tendencies in PHYMS anthers. Anomalies in carbohydrate metabolism and oxidative stress, together with premature tapetum degradation, may be the cause behind carbohydrate starvation and male sterility in CHA SQ-1 treated plants. Here, we provide important insight into the mechanisms underlying CHA SQ-1-induced male sterility. Our findings have practical implications for the application of hybrid breeding in wheat.

Structure of the Shq1-Cbf5-Nop10-Gar1 complex and implications for H/ACA RNP biogenesis and dyskeratosis congenita.

Shq1 is a conserved protein required for the biogenesis of eukaryotic H/ACA ribonucleoproteins (RNPs), including human telomerase. We report the structure of the Shq1-specific domain alone and in complex with H/ACA RNP proteins Cbf5, Nop10 and Gar1. The Shq1-specific domain adopts a novel helical fold and primarily contacts the PUA domain and the otherwise disordered C-terminal extension (CTE) of Cbf5. The structure shows that dyskeratosis congenita mutations found in the CTE of human Cbf5 likely interfere with Shq1 binding. However, most mutations in the PUA domain are not located at the Shq1-binding surface and also have little effect on the yeast Cbf5-Shq1 interaction. Shq1 binds Cbf5 independently of the H/ACA RNP proteins Nop10, Gar1 and Nhp2 and the assembly factor Naf1, but shares an overlapping binding surface with H/ACA RNA. Shq1 point mutations that disrupt Cbf5 interaction suppress yeast growth particularly at elevated temperatures. Our results suggest that Shq1 functions as an assembly chaperone that protects the Cbf5 protein complexes from non-specific RNA binding and aggregation before assembly of H/ACA RNA.

Comparative studies of mitochondrial proteomics reveal an intimate protein network of male sterility in wheat (Triticum aestivum L.).

Plant male sterility has often been associated with mitochondrial dysfunction; however, the mechanism in wheat (Triticum aestivum L.) has not been elucidated. This study set out to probe the mechanism of physiological male sterility (PHYMS) induced by the chemical hybridizing agent (CHA)-SQ-1, and cytoplasmic male sterility (CMS) of wheat at the proteomic level. A total of 71 differentially expressed mitochondrial proteins were found to be involved in pollen abortion and further identified by MALDI-TOF/TOF MS (matrix-assisted laser desorption/ionization-time of fight/time of flight mass spectrometry). These proteins were implicated in different cellular responses and metabolic processes, with obvious functional tendencies toward the tricarboxylic acid cycle, the mitochondrial electron transport chain, protein synthesis and degradation, oxidation stress, the cell division cycle, and epigenetics. Interactions between identified proteins were demonstrated by bioinformatics analysis, enabling a more complete insight into biological pathways involved in anther abortion and pollen defects. Accordingly, a mitochondria-mediated male sterility protein network in wheat is proposed; this network was further confirmed by physiological data, RT-PCR (real-time PCR), and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling) assay. The results provide intriguing insights into the metabolic pathway of anther abortion induced by CHA-SQ-1 and also give useful clues to identify the crucial proteins of PHYMS and CMS in wheat.

Kinetic and thermodynamic characterization of the reaction pathway of box H/ACA RNA-guided pseudouridine formation.

The box H/ACA RNA-guided pseudouridine synthase is a complicated ribonucleoprotein enzyme that recruits substrate via both the guide RNA and the catalytic subunit Cbf5. Structural studies have revealed multiple conformations of the enzyme, but a quantitative description of the reaction pathway is still lacking. Using fluorescence correlation spectroscopy, we here measured the equilibrium dissociation constants and kinetic association and dissociation rates of substrate and product complexes mimicking various reaction intermediate states. These data support a sequential model for substrate loading and product release regulated by the thumb loop of Cbf5. The uridine substrate is first bound primarily through interaction with the guide RNA and then loaded into the active site while progressively interacted with the thumb. After modification, the subtle chemical structure change from uridine to pseudouridine at the target site triggers the release of the thumb, resulting in an intermediate complex with the product bound mainly by the guide RNA. By dissecting the role of Gar1 in individual steps of substrate turnover, we show that Gar1 plays a major role in catalysis and also accelerates product release about 2-fold. Our biophysical results integrate with previous structural knowledge into a coherent reaction pathway of H/ACA RNA-guided pseudouridylation.

TIR-catalyzed nucleotide signaling molecules in plant defense.

Toll and interleukin-1 receptor (TIR) domain is a conserved immune module in prokaryotes and eukaryotes. Signaling regulated by TIR-only proteins or TIR domain-containing intracellular immune receptors is critical for plant immunity. Recent studies demonstrated that TIR domains function as enzymes encoding a variety of activities, which manifest different mechanisms for regulation of plant immunity. These enzymatic activities catalyze metabolism of NAD+, ATP and other nucleic acids, generating structurally diversified nucleotide metabolites. Signaling roles have been revealed for some TIR enzymatic products that can act as second messengers to induce plant immunity. Herein, we summarize our current knowledge about catalytic production of these nucleotide metabolites and their roles in plant immune signaling. We also highlight outstanding questions that are likely to be the focus of future investigations about TIR-produced signaling molecules.

Genome-Wide Identification and Expression Analysis of TUA and TUB Genes in Wheat (Triticum aestivum L.) during Its Development.

Microtubules play a fundamental role in plant development, morphogenesis, and cytokinesis; they are assembled from heterodimers containing an α-tubulin (TUA) and a ß-tubulin (TUB) protein. However, little research has been conducted on the TUA and TUB gene families in hexaploid wheat (Triticum aestivum L.). In this study, we identified 15 TaTUA and 28 TaTUB genes in wheat. Phylogenetic analysis showed that 15 TaTUA genes were divided into two major subfamilies, and 28 TaTUB genes were divided into five major subfamilies. Mostly, there were similar motif compositions and exon-intron structures among the same subfamilies. Segmental duplication of genes (WGD/segmental) is the main process of TaTUA and TaTUB gene family expansion in wheat. It was found that TaTUA and TaTUB genes presented specific temporal and spatial characteristics based on the expression profiles of 17 tissues during wheat development using publicly available RNA-seq data. It was worth noting, via qRT-PCR, that two TaTUA and five TaTUB genes were highly expressed in fertile anthers compared to male sterility. These were quite different between physiological male sterile lines and S-type cytoplasmic male sterile lines at different stages of pollen development. This study offers fundamental information on the TUA and TUB gene families during wheat development and provides new insights for exploring the molecular mechanism of wheat male sterility.

TIR-catalyzed ADP-ribosylation reactions produce signaling molecules for plant immunity.

Plant pathogen-activated immune signaling by nucleotide-binding leucine-rich repeat (NLR) receptors with an N-terminal Toll/interleukin-1 receptor (TIR) domain converges on Enhanced Disease Susceptibility 1 (EDS1) and its direct partners, Phytoalexin Deficient 4 (PAD4) or Senescence-Associated Gene 101 (SAG101). TIR-encoded nicotinamide adenine dinucleotide hydrolase (NADase) produces signaling molecules to promote exclusive EDS1-PAD4 and EDS1-SAG101 interactions with helper NLR subclasses. In this work, we show that TIR-containing proteins catalyze adenosine diphosphate (ADP)-ribosylation of adenosine triphosphate (ATP) and ADP ribose (ADPR) through ADPR polymerase-like and NADase activity, forming ADP-ribosylated ATP (ADPr-ATP) and ADPr-ADPR (di-ADPR), respectively. Specific binding of ADPr-ATP or di-ADPR allosterically promotes EDS1-SAG101 interaction with helper NLR N requirement gene 1A (NRG1A) in vitro and in planta. Our data reveal an enzymatic activity of TIRs that enables specific activation of the EDS1-SAG101-NRG1 immunity branch.

Identification and receptor mechanism of TIR-catalyzed small molecules in plant immunity.

Plant nucleotide-binding leucine-rich repeat-containing (NLR) receptors with an N-terminal Toll/interleukin-1 receptor (TIR) domain sense pathogen effectors to enable TIR-encoded nicotinamide adenine dinucleotide hydrolase (NADase) activity for immune signaling. TIR-NLR signaling requires the helper NLRs N requirement gene 1 (NRG1), Activated Disease Resistance 1 (ADR1), and Enhanced Disease Susceptibility 1 (EDS1), which forms a heterodimer with each of its paralogs Phytoalexin Deficient 4 (PAD4) and Senescence-Associated Gene 101 (SAG101). Here, we show that TIR-containing proteins catalyze the production of 2'-(5''-phosphoribosyl)-5'-adenosine monophosphate (pRib-AMP) and diphosphate (pRib-ADP) in vitro and in planta. Biochemical and structural data demonstrate that EDS1-PAD4 is a receptor complex for pRib-AMP and pRib-ADP, which allosterically promote EDS1-PAD4 interaction with ADR1-L1 but not NRG1A. Our study identifies TIR-catalyzed pRib-AMP and pRib-ADP as a missing link in TIR signaling through EDS1-PAD4 and as likely second messengers for plant immunity.