Nucleotide Binding Site Research Articles

Identification of resistance loci in Chinese and Canadian canola/rapeseed varieties against Leptosphaeria maculans based on genome-wide association studies

BackgroundThe fungal pathogen Leptosphaeria maculans (Lm). causes blackleg disease on canola/rapeseed in many parts of the world. It is important to use resistant cultivars to manage the disease and minimize yield losses. In this study, twenty-two Lm isolates were used to identify resistance genes in a collection of 243 canola/rapeseed (Brassica napus L.) accessions from Canada and China. These Lm isolates carry different compliments of avirulence genes, and the investigation was based on a genome-wide association study (GWAS) and genotype-by-sequencing (GBS).ResultsUsing the CROP-SNP pipeline, a total of 81,471 variants, including 78,632 SNPs and 2839 InDels, were identified. The GWAS was performed using TASSEL 5.0 with GLM + Q model. Thirty-two and 13 SNPs were identified from the Canadian and Chinese accessions, respectively, tightly associated with blackleg resistance with P values < 1 × 10− 4. These SNP loci were distributed on chromosomes A03, A05, A08, A09, C01, C04, C05, and C07, with the majority of them on A08 followed by A09 and A03. The significant SNPs identified on A08 were all located in a 2010-kb region and associated with resistance to 12 of the 22 Lm isolates. Furthermore, 25 resistance gene analogues (RGAs) were identified in these regions, including two nucleotide binding site (NBS) domain proteins, fourteen RLKs, three RLPs and six TM-CCs. These RGAs can be the potential candidate genes for blackleg resistance.ConclusionThis study provides insights into potentially new genomic regions for discovery of additional blackleg resistance genes. The identified regions associated with blackleg resistance in the germplasm collection may also contribute directly to the development of canola varieties with novel resistance genes against blackleg of canola.

ATG4 Mediated Psm ES4326/AvrRpt2-Induced Autophagy Dependent on Salicylic Acid in Arabidopsis Thaliana.

Psm ES4326/AvrRpt2 (AvrRpt2) was widely used as the reaction system of hypersensitive response (HR) in Arabidopsis. The study showed that in npr1 (GFP-ATG8a), AvrRpt2 was more effective at inducing the production of autophagosome and autophagy flux than that in GFP-ATG8a. The mRNA expression of ATG1, ATG6 and ATG8a were more in npr1 during the early HR. Based on transcriptome data analysis, enhanced disease susceptibility 1 (EDS1) was up-regulated in wild-type (WT) but was not induced in atg4a4b (ATG4 deletion mutant) during AvrRpt2 infection. Compared with WT, atg4a4b had higher expression of salicylic acid glucosyltransferase 1 (SGT1) and isochorismate synthase 1 (ICS1); but less salicylic acid (SA) in normal condition and the same level of free SA during AvrRpt2 infection. These results suggested that the consumption of free SA should be occurred in atg4a4b. AvrRpt2 may trigger the activation of Toll/Interleukin-1 receptor (TIR)-nucleotide binding site (NB)-leucine rich repeat (LRR)—TIR-NB-LRR—to induce autophagy via EDS1, which was inhibited by nonexpressor of PR genes 1 (NPR1). Moreover, high expression of NPR3 in atg4a4b may accelerate the degradation of NPR1 during AvrRpt2 infection.

Transcriptome analysis of two lines of Brassica oleracea in response to early infection with Xanthomonas campestris pv. campestris

Black rot, caused by Xanthomonas campestris pv. campestris (Pammel) Dowson (Xcc), is a devastating disease of cruciferous crops worldwide and a threat to the economic production of Brassica oleracea L. in China. Strategies to control this disease are limited. In addition, knowledge of the molecular mechanisms of pathogenicity and host resistance are still only poorly understood. In this study, we performed transcriptomic analyses of the leaves of two contrasting B. oleracea lines (Xcc-resistant line QP07 and Xcc-susceptible line DBP71) to study the early defence response induced by infection with Xcc. We identified 3357 upregulated and 4091 downregulated genes between QP07 and DBP71 at 0, 12, 24, 48 and 96 h post-inoculation (hpi). A functional annotation pathways analysis of the differentially expressed genes indicated that the glucosinolate biosynthetic and catabolic pathways were enhanced, and ROS scavenging, hormonal, receptor-kinase-related genes and nucleotide binding site (NBS)-encoding resistance genes were expressed during the early infection period. In addition, we found that the host plants may respond to Xcc infection by actively regulating photosynthetic energy metabolism. The results provide new insights to help understand the different responses to Xcc in resistant and susceptible host genotypes, and to aid in the development of effective strategies for the prevention of black rot.

Expansion of MIR482/2118 by a class-II transposable element in cotton.

Some plant microRNA (miRNA) families contain multiple members generating identical or highly similar mature miRNA variants. Mechanisms underlying the expansion of miRNA families remain elusive, although tandem and/or segmental duplications have been proposed. In this study of two tetraploid cottons, Gossypium hirsutum and Gossypium barbadense, and their extant diploid progenitors, Gossypium arboreum and Gossypium raimondii, we investigated the gain and loss of members of the miR482/2118 superfamily, which modulates the expression of nucleotide-binding site leucine-rich repeat (NBS-LRR) disease resistance genes. We found significant expansion of MIR482/2118d in G.barbadense, G.hirsutum and G.raimondii, but not in G.arboreum. Several newly expanded MIR482/2118d loci have mutated to produce different miR482/2118 variants with altered target-gene specificity. Based on detailed analysis of sequences flanking these MIR482/2118 loci, we found that this expansion of MIR482/2118d and its derivatives resulted from an initial capture of an MIR482/2118d by a class-II DNA transposable element (TE) in G.raimondii prior to the tetraploidization event, followed by transposition to new genomic locations in G.barbadense, G.hirsutum and G.raimondii. The 'GosTE' involved in the capture and proliferation of MIR482/2118d and its derivatives belongs to the PIF/Harbinger superfamily, generating a 3-bp target site duplication upon insertion at new locations. All orthologous MIR482/2118 loci in the two diploids were retained in the two tetraploids, but mutation(s) in miR482/2118 were observed across all four species as well as in different cultivars of both G.barbadense and G.hirsutum, suggesting a dynamic co-evolution of miR482/2118 and its NBS-LRR targets. Our results provide fresh insights into the mechanisms contributing to MIRNA proliferation and enrich our knowledge on TEs.

Distinct Evolutionary Patterns of NBS-Encoding Genes in Three Soapberry Family (Sapindaceae) Species.

Nucleotide-binding site (NBS)-type disease resistance genes (R genes) play key roles in plant immune responses and have co-evolved with pathogens over the course of plant lifecycles. Comparative genomic studies tracing the dynamic evolution of NBS-encoding genes have been conducted using many important plant lineages. However, studies on Sapindaceae species have not been performed. In this study, a discrepant number of NBS-encoding genes were identified in the genomes of Xanthoceras sorbifolium (180), Dinnocarpus longan (568), and Acer yangbiense (252). These genes were unevenly distributed and usually clustered as tandem arrays on chromosomes, with few existed as singletons. The phylogenetic analysis revealed that NBS-encoding genes formed three monophyletic clades, RPW8-NBS-LRR (RNL), TIR-NBS-LRR (TNL), and CC-NBS-LRR (CNL), which were distinguished by amino acid motifs. The NBS-encoding genes of the X. sorbifolium, D. longan, and A. yangbiense genomes were derived from 181 ancestral genes (three RNL, 23 TNL, and 155 CNL), which exhibited dynamic and distinct evolutionary patterns due to independent gene duplication/loss events. Specifically, X. sorbifolium exhibited a “first expansion and then contraction” evolutionary pattern, while A. yangbiense and D. longan exhibited a “first expansion followed by contraction and further expansion” evolutionary pattern. However, further expansion in D. longan was stronger than in A. yangbiense after divergence, suggesting that D. longan gained more genes in response to various pathogens. Additionally, the ancient and recent expansion of CNL genes generated the dominance of this subclass in terms of gene numbers, while the low copy number status of RNL genes was attributed to their conserved functions.

A compendium of genome-wide sequence reads from NBS (nucleotide binding site) domains of resistance genes in the common potato

SolariX is a compendium of DNA sequence tags from the nucleotide binding site (NBS) domain of disease resistance genes of the common potato, Solanum tuberosum Group Tuberosum. The sequences, which we call NBS tags, for nearly all NBS domains from 91 genomes—representing a wide range of historical and contemporary potato cultivars, 24 breeding programs and 200 years—were generated using just 16 amplification primers and high-throughput sequencing. The NBS tags were mapped to 587 NBS domains on the draft potato genome DM, where we detected an average, over all the samples, of 26 nucleotide polymorphisms on each locus. The total number of NBS domains observed, differed between potato cultivars. However, both modern and old cultivars possessed comparable levels of variability, and neither the individual breeder or country nor the generation or time appeared to correlate with the NBS domain frequencies. Our attempts to detect haplotypes (i.e., sets of linked nucleotide polymorphisms) frequently yielded more than the possible 4 alleles per domain indicating potential locus intermixing during the mapping of NBS tags to the DM reference genome. Mapping inaccuracies were likely a consequence of the differences of each cultivar to the reference genome used, coupled with high levels of NBS domain sequence similarity. We illustrate that the SolariX database is useful to search for polymorphism linked with NBS-LRR R gene alleles conferring specific disease resistance and to develop molecular markers for selection.

Identification and Fine Mapping of RppM, a Southern Corn Rust Resistance Gene in Maize.

Southern corn rust (SCR) caused by Puccinia polysora Underw. is a major disease causing severe yield losses during maize production. Here, we identified and mapped the SCR resistance gene RppM from the near-isogenic line Kangxiujing2416 (Jing2416K), which harbors RppM in the genetic background of the susceptible inbred line Jing2416. In this study, the inheritance of SCR resistance was investigated in F2 and F3 populations derived from a cross between Jing2416K and Jing2416. The observed 3:1 segregation ratio of resistant to susceptible plants indicated that the SCR resistance is controlled by a single dominant gene. Using an F2 population, we performed bulked segregant analysis (BSA) sequencing and mapped RppM to a 3.69-Mb region on chromosome arm 10S. To further narrow down the region harboring RppM, we developed 13 insertion/deletion (InDel) markers based on the sequencing data. Finally, RppM was mapped to a region spanning 110-kb using susceptible individuals from a large F2 population. Two genes (Zm00001d023265 and Zm00001d023267) encoding putative CC-NBS-LRR (coiled-coiled, nucleotide-binding site, and leucine-rich repeat) proteins, a common characteristic of R genes, were located in this region (B73 RefGen_v4 reference genome). Sequencing and comparison of the two genes cloned from Jing2416K and Jing2416 revealed sequence variations in their coding regions. The relative expression levels of these two genes in Jing2416K were found to be significantly higher than those in Jing2416. Zm00001d023265 and Zm00001d023267 are thus potential RppM candidates.

Identification of resistance gene analogs involved in Phytophthora capsici recognition in black pepper (Piper nigrum L.)

Black pepper (Piper nigrum L.) is an important spice crop with high economic value. However, its production is severely hampered by the oomycete pathogen, Phytophthora capsici. Integrative disease management strategies have been developed to control the pathogen, but the pathogen is in the phase of evolving its virulence. Absolute resistance against Phytophthora rot was not reported in black pepper germplasm. However, Piper colubrinum, a wild species is reported as resistant. Resistance proteins are involved in continuous surveillance of pathogen entry and activation of plant defense signalling pathways for an effective hypersensitive response to prevent pathogen invasion. In this study, a sequence-based homology approach using the conserved nucleotide-binding site (NBS) of known plant resistance genes was used to isolate Resistance Gene Analogs (RGA) and assess their transcript level during Phytophthora infection. The RGA transcript level was evaluated in resistant wild species (P. colubrinum), two moderately resistant black pepper genotypes (IISR Sakthi and 04-P24-1), and one susceptible genotype (Subhakara). The identified RGAs of black pepper were found to be of non-TIR R gene class with NBS motifs. The expressions of six PnRGAs were assessed employing qRT-PCR at different time points after challenging with highly aggressive Phytophthora isolate. The kinetics of differential expression post-infection with P. capsici indicates the differential timing and magnitude of pathogen recognition in resistant P. colubrinum and moderately resistant black pepper genotypes compared to susceptible genotype. In silico analysis revealed that differentially expressed P. nigrum RGAs function through ADP phosphorylation, which is a key process in pathogen recognition. The identification of P. nigrum RGAs induced by P. capsici should provide valuable information for cloning and characterization of resistance genes.

Identification of NBS-LRR Resistance Gene Analogues (RGA) from Rose (IIHRR13-4) Resistant to Powdery Mildew (Podosphaera pannosa (Wallr.:Fr.) de Bary)

Resistance is the best strategy to manage powdery mildew (Podosphaera pannosa (Wallr.:Fr.) de Bary) of rose. Identification of resistant genes (R genes) from plant species will help in breeding programs. Nucleotide Binding Site - Leucine Rich Repeats (NBS- LRR) is a major class of R gene family in plants. This study reports the identification and molecular characterization of resistance gene analogues from roses maintained at ICAR- Indian Institute of Horticultural Research (IIHR). The powdery mildew resistant line IIHRR13-4 was compared with the susceptible commercial cultivar, konfetti. PCR based approaches with degenerative primers based on different conserved motifs of NBS-LRR were employed to isolate resistance gene analogues (RGAs) from rose. Eleven RGAs (IIHRR13-4R1, IIHRR13-4R2, IIHRR13-4R3, IIHRR13-4R4, IIHRR13-4R5, IIHRR13- 4R6, IIHRR13-4R7, IIHRR13-4R8 IIHRR13-4R9 and IIHRR13-4R10) were identified from powdery mildew resistant germplasm line, IIHRR13-4, based on the sequence and similarity to RGAs from rosaceae family and other crops. The major similarity to rose RGAs reported are from Fragaria vesca, Rosa hybrid cultivar, Prunus and Rosa chinensis. RGAs isolated from IIHRR13-4 belonged to Toll Interleukin Receptor (TIR)-NBS-LRR and Non-TIR-NBS-LRR RGAs (Lecine Zipper (LZ) type). Different motifs of RGAs identified were P-loop, RNBS A, kinase 2, kinase 3a, RNBS-D and GLPL of NBS domain. This study reports the existence of resistance at genetic level in powdery mildew resistant genotype IIHRR13-4. These RGAs will be useful for mapping and characterization of R genes in IIHRR13-4 and breeding for improved powdery mildew resistance in roses.

Identification of NBS-LRR Resistance Gene Analogues (RGA) from Rose (IIHRR13-4) Resistant to Powdery Mildew (Podosphaera pannosa (Wallr.:Fr.) de Bary)

Resistance is the best strategy to manage powdery mildew (Podosphaera pannosa (Wallr.:Fr.) de Bary) of rose. Identification of resistant genes (R genes) from plant species will help in breeding programs. Nucleotide Binding Site - Leucine Rich Repeats (NBS- LRR) is a major class of R gene family in plants. This study reports the identification and molecular characterization of resistance gene analogues from roses maintained at ICAR- Indian Institute of Horticultural Research (IIHR). The powdery mildew resistant line IIHRR13-4 was compared with the susceptible commercial cultivar, konfetti. PCR based approaches with degenerative primers based on different conserved motifs of NBS-LRR were employed to isolate resistance gene analogues (RGAs) from rose. Eleven RGAs (IIHRR13-4R1, IIHRR13-4R2, IIHRR13-4R3, IIHRR13-4R4, IIHRR13-4R5, IIHRR13- 4R6, IIHRR13-4R7, IIHRR13-4R8 IIHRR13-4R9 and IIHRR13-4R10) were identified from powdery mildew resistant germplasm line, IIHRR13-4, based on the sequence and similarity to RGAs from rosaceae family and other crops. The major similarity to rose RGAs reported are from Fragaria vesca, Rosa hybrid cultivar, Prunus and Rosa chinensis. RGAs isolated from IIHRR13-4 belonged to Toll Interleukin Receptor (TIR)-NBS-LRR and Non-TIR-NBS-LRR RGAs (Lecine Zipper (LZ) type). Different motifs of RGAs identified were P-loop, RNBS A, kinase 2, kinase 3a, RNBS-D and GLPL of NBS domain. This study reports the existence of resistance at genetic level in powdery mildew resistant genotype IIHRR13-4. These RGAs will be useful for mapping and characterization of R genes in IIHRR13-4 and breeding for improved powdery mildew resistance in roses.

Identification and Distribution of NBS-Encoding Resistance Genes of Dactylis glomerata L. and Its Expression Under Abiotic and Biotic Stress.

Orchardgrass (Dactylis glomerata L.) is drought resistant and tolerant to barren landscapes, making it one of the most important forages for animal husbandry, as well as ecological restoration of rocky landscapes that are undergoing desertification. However, orchardgrass is susceptible to rust, which can significantly reduce its yield and quality. Therefore, understanding the genes that underlie resistance against rust in orchardgrass is critical. The evolution, cloning of plant disease resistance genes, and the analysis of pathogenic bacteria induced expression patterns are important contents in the study of interaction between microorganisms and plants. Genes with nucleotide binding site (NBS) structure are disease-resistant genes ubiquitous in plants and play an important role in plant attacks against various pathogens. Using sequence analysis and re-annotation, we identified 413 NBS resistance genes in orchardgrass. Similar to previous studies, NBS resistance genes containing TIR (toll/interleukin-1 receptor) domain were not found in orchardgrass. The NBS resistance genes can be divided into four types: NBS (up to 264 homologous genes, accounting for 64% of the total number of NBS genes in orchardgrass), NBS-LRR, CC-NBS, and CC-NBS-LRR (minimum of 26 homologous genes, only 6% of the total number of NBS genes in orchardgrass). These 413 NBS resistance genes were unevenly distributed across seven chromosomes where chromosome 5 had up to 99 NBS resistance genes. There were 224 (54%) NBS resistance genes expressed in different tissues (roots, stems, leaves, flowers, and spikes), and we did not detect expression for 45 genes (11%). The remaining 145 (35%) were expressed in some tissues. And we found that 11 NBS resistance genes were differentially expressed under waterlogging stress, 5 NBS resistance genes were differentially expressed under waterlogging and drought stress, and 1 NBS resistance was is differentially expressed under waterlogging and heat stress. Most importantly, we found that 65 NBS resistance genes were significantly expressed in different control groups. On the 7th day of inoculation, 23 NBS resistance genes were differentially expressed in high resistance materials alone, of which 7 NBS resistance genes regulate the "plant-pathogen interaction" pathway by encoding RPM1. At the same time, 2 NBS resistance genes that were differentially expressed in the high resistance material after inoculation were also differentially expressed in abiotic stress. In summary, the NBS resistance gene plays a crucial role in the resistance of orchardgrass to rust.

QTL mapping of web blotch resistance in peanut by high-throughput genome-wide sequencing

BackgroundWeb blotch is one of the most important foliar diseases worldwide in peanut (Arachis hypogaea L.). The identification of quantitative trait loci (QTLs) for peanut web blotch resistance represents the basis for gene mining and the application of molecular breeding technologies.ResultsIn this study, a peanut recombinant inbred line (RIL) population was used to map QTLs for web blotch resistance based on high-throughput genome-wide sequencing. Frequency distributions of disease grade and disease index in five environments indicated wide phenotypic variations in response to web blotch among RILs. A high-density genetic map was constructed, containing 3634 bin markers distributed on 20 peanut linkage groups (LGs) with an average genetic distance of 0.5 cM. In total, eight QTLs were detected for peanut web blotch resistance in at least two environments, explaining from 2.8 to 15.1% of phenotypic variance. Two major QTLs qWBRA04 and qWBRA14 were detected in all five environments and were linked to 40 candidate genes encoding nucleotide-binding site leucine-rich repeat (NBS-LRR) or other proteins related to disease resistances.ConclusionsThe results of this study provide a basis for breeding peanut cultivars with web blotch resistance.

The genome sequence of star fruit (Averrhoa carambola)

Oxalidaceae is one of the most important plant families in horticulture, and its key commercially relevant genus, Averrhoa, has diverse growth habits and fruit types. Here, we describe the assembly of a high-quality chromosome-scale genome sequence for Averrhoa carambola (star fruit). Ks distribution analysis showed that A. carambola underwent a whole-genome triplication event, i.e., the gamma event shared by most eudicots. Comparisons between A. carambola and other angiosperms also permitted the generation of Oxalidaceae gene annotations. We identified unique gene families and analyzed gene family expansion and contraction. This analysis revealed significant changes in MADS-box gene family content, which might be related to the cauliflory of A. carambola. In addition, we identified and analyzed a total of 204 nucleotide-binding site, leucine-rich repeat receptor (NLR) genes and 58 WRKY genes in the genome, which may be related to the defense response. Our results provide insights into the origin, evolution and diversification of star fruit.

D-Alanine–d-alanine ligase as a model for the activation of ATP-grasp enzymes by monovalent cations

The ATP-grasp superfamily of enzymes shares an atypical nucleotide-binding site known as the ATP-grasp fold. These enzymes are involved in many biological pathways in all domains of life. One ATP-grasp enzyme, d-alanine-d-alanine ligase (Ddl), catalyzes ATP-dependent formation of the d-alanyl-d-alanine dipeptide essential for bacterial cell wall biosynthesis and is therefore an important antibiotic drug target. Ddl is activated by the monovalent cation (MVC) K+, but despite its clinical relevance and decades of research, how this activation occurs has not been elucidated. We demonstrate here that activating MVCs bind adjacent to the active site of Ddl from Thermus thermophilus and used a combined biochemical and structural approach to characterize MVC activation. We found that TtDdl is a type II MVC-activated enzyme, retaining activity in the absence of MVCs. However, the efficiency of TtDdl increased ∼20-fold in the presence of activating MVCs, and it was maximally activated by K+ and Rb+ ions. A strict dependence on ionic radius of the MVC was observed, with Li+ and Na+ providing little to no TtDdl activation. To understand the mechanism of MVC activation, we solved crystal structures of TtDdl representing distinct catalytic stages in complex with K+, Rb+, or Cs+ Comparison of these structures with apo TtDdl revealed no evident conformational change on MVC binding. Of note, the identified MVC binding site is structurally conserved within the ATP-grasp superfamily. We propose that MVCs activate Ddl by altering the charge distribution of its active site. These findings provide insight into the catalytic mechanism of ATP-grasp enzymes.

Genetic Diversity and Evolutionary Analyses Reveal the Powdery Mildew Resistance Gene Pm21 Undergoing Diversifying Selection.

Wheat powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) is a devastating disease that threatens wheat production and yield worldwide. The powdery mildew resistance gene Pm21, originating from wheat wild relative Dasypyrum villosum, encodes a coiled-coil, nucleotide-binding site, leucine-rich repeat (CC-NBS-LRR) protein and confers broad-spectrum resistance to wheat powdery mildew. In the present study, we isolated 73 Pm21 alleles from different powdery mildew-resistant D. villosum accessions, among which, 38 alleles were non-redundant. Sequence analysis identified seven minor insertion-deletion (InDel) polymorphisms and 400 single nucleotide polymorphisms (SNPs) among the 38 non-redundant Pm21 alleles. The nucleotide diversity of the LRR domain was significantly higher than those of the CC and NB-ARC domains. Further evolutionary analysis indicated that the solvent-exposed LRR residues of Pm21 alleles had undergone diversifying selection (dN/dS = 3.19734). In addition, eight LRR motifs and four amino acid sites in the LRR domain were also experienced positive selection, indicating that these motifs and sites play critical roles in resistance specificity. The phylogenetic tree showed that 38 Pm21 alleles were divided into seven classes. Classes A (including original Pm21), B and C were the major classes, including 26 alleles (68.4%). We also identified three non-functional Pm21 alleles from four susceptible homozygous D. villosum lines (DvSus-1 to DvSus-4) and two susceptible wheat-D. villosum chromosome addition lines (DA6V#1 and DA6V#3). The genetic variations of non-functional Pm21 alleles involved point mutation, deletion and insertion, respectively. The results also showed that the non-functional Pm21 alleles in the two chromosome addition lines both came from the susceptible donors of D. villosum. This study gives a new insight into the evolutionary characteristics of Pm21 alleles and discusses how to sustainably utilize Pm21 in wheat production. This study also reveals the sequence variants and origins of non-functional Pm21 alleles in D. villosum populations.

Genome-Wide Identification and Evolutionary Analysis of NBS-LRR Genes From Dioscorea rotundata.

Dioscorea rotundata is an important food crop that is mainly cultivated in subtropical regions of the world. D. rotundata is frequently infected by various pathogens during its lifespan, which results in a substantial economic loss in terms of yield and quality. The disease resistance gene (R gene) profile of D. rotundata is largely unknown, which has greatly hampered molecular study of disease resistance in this species. Nucleotide-binding site–leucine-rich repeat (NBS-LRR) genes are the largest group of plant R genes, and they play important roles in plant defense responses to various pathogens. In this study, 167 NBS-LRR genes were identified from the D. rotundata genome. Subsequently, one gene was assigned to the resistance to powdery mildew8 (RPW8)-NBS-LRR (RNL) subclass and the other 166 genes to the coiled coil (CC)-NBS-LRR (CNL) subclass. None of the Toll/interleukin-1 receptor (TIR)-NBS-LRR (TNL) genes were detected in the genome. Among them, 124 genes are located in 25 multigene clusters and 43 genes are singletons. Tandem duplication serves as the major force for the cluster arrangement of NBS-LRR genes. Segmental duplication was detected for 18 NBS-LRR genes, although no whole-genome duplication has been documented for the species. Phylogenetic analysis revealed that D. rotundata NBS-LRR genes share 15 ancestral lineages with Arabidopsis thaliana genes. The NBS-LRR gene number increased by more than a factor of 10 during D. rotundata evolution. A conservatively evolved ancestral lineage was identified from D. rotundata, which is orthologs to the Arabidopsis RPM1 gene. Transcriptome analysis for four different tissues of D. rotundata revealed a low expression of most NBS-LRR genes, with the tuber and leaf displaying a relatively high NBS-LRR gene expression than the stem and flower. Overall, this study provides a complete set of NBS-LRR genes for D. rotundata, which may serve as a fundamental resource for mining functional NBS-LRR genes against various pathogens.

Discovery and analysis of NBS-LRR gene family in sweet potato genome

<p indent=0mm>The NBS-LRR gene families possess the most abundant resistance genes in plants. Members of the NBS-LRR gene families contain nucleotide-binding site (NBS) and leucine-leucine repeat (LRR) domains. The genome of sweet potato (<italic>Ipomoea batatas</italic>) cultivar has been sequenced but the genes have not been annotated yet. In this study, we predicted the exons of sweet potato genome and obtained the proteins sequences, which were then used to identify and analyze genes of NBS-LRR family. There were 379 genes within NBS-LRR family, amounting to 0.212% of the total genes of sweet potato. The number of the N type, NL type, CNL type, TNL type and PN type was 120, 103, 133, 22, and 1, respectively. All of the chromosomes had NBS-LRR family genes but varied in number and 60.9% of them were clustered. NBS-LRR genes included 15 conservative domains and the genes were conservative within N terminal domain. The results provide references for further studies on the function of NBS-LRR family genes and resistance breeding of sweet potato.

The N-terminal domain in TRPM2 channel is a conserved nucleotide binding site.

This study by Tóth et al. has defined that the N-terminal MHR1/2 domain is a conserved ADPR binding site in TRPM2 from ancient cnidarians to vertebrate, and that it is the key ligand binding site for invertebrate TRPM2 channel activation by ADPR, the same as observed in human and zebrafish TRPM2.

Genome-wide identification and expression analysis of rice NLR genes responsive to the infections of Xanthomonas oryzae pv. oryzae and Magnaporthe oryzae

Nucleotide-binding site, leucine-rich repeat (NLR) genes play a critical role in rice disease resistance. However, the transcriptional activities of rice NLR genes during pathogen invasions are still unclear. To uncover their activities, we identified 430 regular rice NLR genes with both NBS and LRR domains, consisting of 192 CNL and 238 XNL (without a CC motif) members. We performed individual and integrative analyses based on 69 samples from rice microarray after the infections of Xanthomonas oryzae pv. oryzae (Xoo) and Magnaporthe oryzae (Mor). Among the rice NLR genes, 397 were found to be expressed at low/medium level, while 10 NLR genes were observed to show high expression levels. A total of 400 NLR genes were discovered to be differentially expressed in at least one sample. Furthermore, 46 rice NLR genes were identified to be differentially expressed in response to the two pathogens and 38 of them could be validated using RNA-seq data. Six cis-regulatory elements (MYC, STRE, MYB, ABRE, G-box, and AS-1) were observed to occur frequently in the promoter regions of rice NLR genes. Ten NLR genes were selected for qPCR detection, and seven of the NLR genes verified the validity of the microarray and RNA-seq data. Our results are helpful in revealing expressions and regulations of NLR genes in rice resistance to Xoo and Mor.

Arf6 Can Trigger Wave Regulatory Complex-Dependent Actin Assembly Independent of Arno.

The small GTPase ADP-ribosylation factor 6 (Arf6) anchors at the plasma membrane to orchestrate key functions, such as membrane trafficking and regulating cortical actin cytoskeleton rearrangement. A number of studies have identified key players that interact with Arf6 to regulate actin dynamics in diverse cell processes, yet it is still unknown whether Arf6 can directly signal to the wave regulatory complex to mediate actin assembly. By reconstituting actin dynamics on supported lipid bilayers, we found that Arf6 in co-ordination with Rac1(Ras-related C3 botulinum toxin substrate 1) can directly trigger actin polymerization by recruiting wave regulatory complex components. Interestingly, we demonstrated that Arf6 triggers actin assembly at the membrane directly without recruiting the Arf guanine nucleotide exchange factor (GEF) ARNO (ARF nucleotide-binding site opener), which is able to activate Arf1 to enable WRC-dependent actin assembly. Furthermore, using labelled E. coli, we demonstrated that actin assembly by Arf6 also contributes towards efficient phagocytosis in THP-1 macrophages. Taken together, this study reveals a mechanism for Arf6-driven actin polymerization.